Philip Beachy

All Publications

• Cellular and molecular mechanisms of Hedgehog signalling. Nature reviews. Molecular cell biology Zhang, Y., Beachy, P. A. 2023

  Abstract

  The Hedgehog signalling pathway has crucial roles in embryonic tissue patterning, postembryonic tissue regeneration, and cancer, yet aspects of Hedgehog signal transmission and reception have until recently remained unclear. Biochemical and structural studies surprisingly reveal a central role for lipids in Hedgehog signalling. The signal - Hedgehog protein - is modified by cholesterol and palmitate during its biogenesis, thereby necessitating specialized proteins such as the transporter Dispatched and several lipid-binding carriers for cellular export and receptor engagement. Additional lipid transactions mediate response tothe Hedgehog signal, including sterol activation of the transducer Smoothened. Access of sterols to Smoothened is regulated by the apparent sterol transporter and Hedgehog receptor Patched, whose activity is blocked by Hedgehog binding. Alongside these lipid-centric mechanisms and their relevance to pharmacological pathway modulation, we discuss emerging roles of Hedgehog pathway activity in stem cells or their cellular niches, with translational implications for regeneration and restoration of injured or diseased tissues.

  View details for DOI 10.1038/s41580-023-00591-1

  View details for PubMedID 36932157

• Dispatched uses Na+ flux to power release of lipid-modified Hedgehog. Nature Wang, Q., Asarnow, D. E., Ding, K., Mann, R. K., Hatakeyama, J., Zhang, Y., Ma, Y., Cheng, Y., Beachy, P. A. 2021

  Abstract

  The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters1,2 and to H+-driven transporters of the RND family3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly-localized sites of embryonic expression5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na+ gradient across the plasma membrane. The transmembrane channel and Na+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartateresidues that each coordinate and neutralize the charge of one of the three Na+ ions. DISP1-NNN and variants that disrupt single Na+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminalsignallingdomain of theSonic hedgehog protein(ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na+-site occupancies, which suggests a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na+ flux powers their conformationally driven activities.

  View details for DOI 10.1038/s41586-021-03996-0

  View details for PubMedID 34707294

• Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit. Proceedings of the National Academy of Sciences of the United States of America Zhang, Y., Lu, W., Bulkley, D. P., Liang, J., Ralko, A., Han, S., Roberts, K. J., Li, A., Cho, W., Cheng, Y., Manglik, A., Beachy, P. A. 2020

  Abstract

  Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.

  View details for DOI 10.1073/pnas.2011560117

  View details for PubMedID 33139559

• Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature Deshpande, I., Liang, J., Hedeen, D., Roberts, K. J., Zhang, Y., Ha, B., Latorraca, N. R., Faust, B., Dror, R. O., Beachy, P. A., Myers, B. R., Manglik, A. 2019

  Abstract

  Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

  View details for DOI 10.1038/s41586-019-1355-4

  View details for PubMedID 31263273

• Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched. Cell Zhang, Y., Bulkley, D. P., Xin, Y., Roberts, K. J., Asarnow, D. E., Sharma, A., Myers, B. R., Cho, W., Cheng, Y., Beachy, P. A. 2018

  Abstract

  Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

  View details for PubMedID 30415841

• Cilia-Associated Oxysterols Activate Smoothened. Molecular cell Raleigh, D. R., Sever, N., Choksi, P. K., Sigg, M. A., Hines, K. M., Thompson, B. M., Elnatan, D., Jaishankar, P., Bisignano, P., Garcia-Gonzalo, F. R., Krup, A. L., Eberl, M., Byrne, E. F., Siebold, C., Wong, S. Y., Renslo, A. R., Grabe, M., McDonald, J. G., Xu, L., Beachy, P. A., Reiter, J. F. 2018; 72 (2): 316

  Abstract

  Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11beta2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.

  View details for PubMedID 30340023

• Neuronal delivery of Hedgehog directs spatial patterning of taste organ regeneration. Proceedings of the National Academy of Sciences of the United States of America Lu, W. J., Mann, R. K., Nguyen, A. n., Bi, T. n., Silverstein, M. n., Tang, J. Y., Chen, X. n., Beachy, P. A. 2018; 115 (2): E200–E209

  Abstract

  How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog (Shh), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh-expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration.

  View details for PubMedID 29279401

  View details for PubMedCentralID PMC5777079

• Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 2018; 562 (7727): 367–72

  Abstract

  Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3'-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

  View details for DOI 10.1038/s41586-018-0590-4

  View details for PubMedID 30283141

• Hedgehog mediated degradation of Ihog adhesion proteins modulates cell segregation in Drosophila wing imaginal discs NATURE COMMUNICATIONS Hsia, E. C., Zhang, Y., Tran, H., Lim, A., Chou, Y., Lan, G., Beachy, P. A., Zheng, X. 2017; 8: 1275

  Abstract

  The Drosophila Hedgehog receptor functions to regulate the essential downstream pathway component, Smoothened, and to limit the range of signaling by sequestering Hedgehog protein signal within imaginal disc epithelium. Hedgehog receptor function requires both Patched and Ihog activity, the latter interchangeably encoded by interference hedgehog (ihog) or brother of ihog (boi). Here we show that Patched and Ihog activity are mutually required for receptor endocytosis and degradation, triggered by Hedgehog protein binding, and causing reduced levels of Ihog/Boi proteins in a stripe of cells at the anterior/posterior compartment boundary of the wing imaginal disc. This Ihog spatial discontinuity may contribute to classically defined cell segregation and lineage restriction at the anterior/posterior wing disc compartment boundary, as suggested by our observations that Ihog activity mediates aggregation of otherwise non-adherent cultured cells and that loss of Ihog activity disrupts wing disc cell segregation, even with downstream genetic rescue of Hedgehog signal response.

  View details for PubMedID 29097673

• Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells SCIENCE Zhao, C., Cai, S., Shin, K., Lim, A., Kalisky, T., Lu, W., Clarke, M. F., Beachy, P. A. 2017; 356 (6335): 284-?

  View details for DOI 10.1126/science.aal3485

  View details for Web of Science ID 000399540100053

• The Stromal Niche for Epithelial Stem Cells: A Template for Regeneration and a Brake on Malignancy. Cancer cell Roberts, K. J., Kershner, A. M., Beachy, P. A. 2017; 32 (4): 404–10

  Abstract

  Stromal restraint of cancer growth and progression-emerging as a widespread phenomenon in epithelial cancers such as bladder, pancreas, colon, and prostate-appears rooted in stromal cell niche activity. During normal tissue repair, stromal niche signals, often Hedgehog-induced, promote epithelial stem cell differentiation as well as self-renewal, thus specifying a regenerating epithelial pattern. In the case of cancerous tissue, stromal cell-derived differentiation signals in particular may provide a brake on malignant growth. Understanding and therapeutic harnessing of the role of stroma in cancer restraint may hinge on our knowledge of the signaling programs elaborated by the stromal niche.

  View details for PubMedID 29017054

  View details for PubMedCentralID PMC5679442

• Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. Proceedings of the National Academy of Sciences of the United States of America Myers, B. R., Neahring, L. n., Zhang, Y. n., Roberts, K. J., Beachy, P. A. 2017; 114 (52): E11141–E11150

  Abstract

  Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na+ to regulate Smoothened in our assays, raising the possibility that a Na+ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na+ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.

  View details for PubMedID 29229834

  View details for PubMedCentralID PMC5748227

• Control of inflammation by stromal Hedgehog pathway activation restrains colitis. Proceedings of the National Academy of Sciences of the United States of America Lee, J. J., Rothenberg, M. E., Seeley, E. S., Zimdahl, B., Kawano, S., Lu, W., Shin, K., Sakata-Kato, T., Chen, J. K., Diehn, M., Clarke, M. F., Beachy, P. A. 2016

  Abstract

  Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4(+)Foxp3(+) regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.

  View details for PubMedID 27815529

• Hedgehog Signaling Restrains Bladder Cancer Progression by Eliciting Stromal Production of Urothelial Differentiation Factors CANCER CELL Shin, K., Lim, A., Zhao, C., Sahoo, D., Pan, Y., Spiekerkoetter, E., Liao, J. C., Beachy, P. A. 2014; 26 (4): 521-533

  Abstract

  Hedgehog (Hh) pathway inhibitors are clinically effective in treatment of basal cell carcinoma and medulloblastoma, but fail therapeutically or accelerate progression in treatment of endodermally derived colon and pancreatic cancers. In bladder, another organ of endodermal origin, we find that despite its initial presence in the cancer cell of origin Sonic hedgehog (Shh) expression is invariably lost during progression to invasive urothelial carcinoma. Genetic blockade of stromal response to Shh furthermore dramatically accelerates progression and decreases survival time. This cancer-restraining effect of Hh pathway activity is associated with stromal expression of BMP signals, which stimulate urothelial differentiation. Progression is dramatically reduced by pharmacological activation of BMP pathway activity with low-dose FK506, suggesting an approach to management of human bladder cancer.

  View details for DOI 10.1016/j.cce11.2014.09.001

  View details for Web of Science ID 000343343800012

• Stromal response to Hedgehog signaling restrains pancreatic cancer progression. Proceedings of the National Academy of Sciences of the United States of America Lee, J. J., Perera, R. M., Wang, H., Wu, D., Liu, X. S., Han, S., Fitamant, J., Jones, P. D., Ghanta, K. S., Kawano, S., Nagle, J. M., Deshpande, V., Boucher, Y., Kato, T., Chen, J. K., Willmann, J. K., Bardeesy, N., Beachy, P. A. 2014; 111 (30): E3091-100

  Abstract

  Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.

  View details for DOI 10.1073/pnas.1411679111

  View details for PubMedID 25024225

• Identification of recurrent SMO and BRAF mutations in ameloblastomas. Nature genetics Sweeney, R. T., McClary, A. C., Myers, B. R., Biscocho, J., Neahring, L., Kwei, K. A., Qu, K., Gong, X., Ng, T., Jones, C. D., Varma, S., Odegaard, J. I., Sugiyama, T., Koyota, S., Rubin, B. P., Troxell, M. L., Pelham, R. J., Zehnder, J. L., Beachy, P. A., Pollack, J. R., West, R. B. 2014; 46 (7): 722-725

  Abstract

  Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

  View details for DOI 10.1038/ng.2986

  View details for PubMedID 24859340

• Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma. Nature cell biology Shin, K., Lim, A., Odegaard, J. I., Honeycutt, J. D., Kawano, S., Hsieh, M. H., Beachy, P. A. 2014; 16 (5): 469-478

  Abstract

  Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumour progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma in situ. Shh-expressing basal cells within this precursor lesion become tumour-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumour cell phenotype can diverge significantly from that of the cancer cell of origin.

  View details for DOI 10.1038/ncb2956

  View details for PubMedID 24747439

• Simultaneous measurement of smoothened entry into and exit from the primary cilium. PloS one Kim, J., Hsia, E. Y., Kim, J., Sever, N., Beachy, P. A., Zheng, X. 2014; 9 (8)

  Abstract

  Ciliary accumulation of signaling proteins must result from a rate of ciliary entry that exceeds ciliary exit, but approaches for distinguishing ciliary entry vs. exit are lacking. Using a photoconvertible fluorescent protein tag, we establish an assay that allows a separate but simultaneous examination of ciliary entry and exit of the Hedgehog signaling protein Smoothened in individual cells. We show that KAAD-cyclopamine selectively blocks entry, whereas ciliobrevin interferes initially with exit and eventually with both entry and exit of ciliary Smoothened. Our study provides an approach to understanding regulation of ciliary entry vs. exit of Hedgehog signaling components as well as other ciliary proteins.

  View details for DOI 10.1371/journal.pone.0104070

  View details for PubMedID 25119726

  View details for PubMedCentralID PMC4132089

• Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response. Developmental cell Myers, B. R., Sever, N., Chong, Y. C., Kim, J., Belani, J. D., Rychnovsky, S., Bazan, J. F., Beachy, P. A. 2013; 26 (4): 346-357

  Abstract

  Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo) by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine-rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

  View details for DOI 10.1016/j.devcel.2013.07.015

  View details for PubMedID 23954590

• Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form GENES & DEVELOPMENT Creanga, A., Glenn, T. D., Mann, R. K., Saunders, A. M., Talbot, W. S., Beachy, P. A. 2012; 26 (12): 1312-1325

  Abstract

  Owing to their covalent modification by cholesterol and palmitate, Hedgehog (Hh) signaling proteins are localized predominantly to the plasma membrane of expressing cells. Yet Hh proteins are also capable of mobilizing to and eliciting direct responses from distant cells. The zebrafish you gene, identified genetically >15 years ago, was more recently shown to encode a secreted glycoprotein that acts cell-nonautonomously in the Hh signaling pathway by an unknown mechanism. We investigated the function of the protein encoded by murine Scube2, an ortholog of you, and found that it mediates release in soluble form of the mature, cholesterol- and palmitate-modified Sonic hedgehog protein signal (ShhNp) when added to cultured cells or purified detergent-resistant membrane microdomains containing ShhNp. The efficiency of Scube2-mediated release of ShhNp is enhanced by the palmitate adduct of ShhNp and by coexpression in ShhNp-producing cells of mDispatchedA (mDispA), a transporter-like protein with a previously defined role in the release of lipid-modified Hh signals. The structural determinants of Scube2 required for its activity in cultured cell assays match those required for rescue of you mutant zebrafish embryos, and we thus conclude that the role of Scube/You proteins in Hh signaling in vivo is to facilitate the release and mobilization of Hh proteins for distant action.

  View details for DOI 10.1101/gad.191866.112

  View details for Web of Science ID 000305485300006

  View details for PubMedID 22677548

  View details for PubMedCentralID PMC3387659

• Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, M., Saunders, A. M., Hamaoka, B. Y., Beachy, P. A., Leahy, D. J. 2011; 108 (32): 13112-13117

  Abstract

  Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

  View details for DOI 10.1073/pnas.1109877108

  View details for Web of Science ID 000293691400035

  View details for PubMedID 21828006

  View details for PubMedCentralID PMC3156181

• Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder NATURE Shin, K., Lee, J., Guo, N., Kim, J., Lim, A., Qu, L., Mysorekar, I. U., Beachy, P. A. 2011; 472 (7341): 110-U145

  Abstract

  Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show in mice that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Sonic hedgehog (Shh). On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a conceptual framework for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signalling pathways in cancer growth and metastasis.

  View details for DOI 10.1038/nature09851

  View details for PubMedID 21389986

• Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Monje, M., Mitra, S. S., Freret, M. E., Raveh, T. B., Kim, J., Masek, M., Attema, J. L., Li, G., Haddix, T., Edwards, M. S., Fisher, P. G., Weissman, I. L., Rowitch, D. H., Vogel, H., Wong, A. J., Beachy, P. A. 2011; 108 (11): 4453-4458

  Abstract

  Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

  View details for DOI 10.1073/pnas.1101657108

  View details for PubMedID 21368213

• Interactions between Hedgehog proteins and their binding partners come into view GENES & DEVELOPMENT Beachy, P. A., Hymowitz, S. G., Lazarus, R. A., Leahy, D. J., Siebold, C. 2010; 24 (18): 2001-2012

  Abstract

  Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.

  View details for DOI 10.1101/gad.1951710

  View details for Web of Science ID 000281815500003

  View details for PubMedID 20844013

  View details for PubMedCentralID PMC2939362

• Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, J., Lee, J. J., Kim, J., Gardner, D., Beachy, P. A. 2010; 107 (30): 13432-13437

  Abstract

  Aberrant Hedgehog (Hh) pathway activation has been implicated in cancers of diverse tissues and organs, and the tumor growth-inhibiting effects of pathway antagonists in animal models have stimulated efforts to develop pathway antagonists for human therapeutic purposes. These efforts have focused largely on cyclopamine derivatives or other compounds that mimic cyclopamine action in binding to and antagonizing Smoothened, a membrane transductory component. We report here that arsenicals, in contrast, antagonize the Hh pathway by targeting Gli transcriptional effectors; in the short term, arsenic blocks Hh-induced ciliary accumulation of Gli2, the primary activator of Hh-dependent transcription, and with prolonged incubation arsenic reduces steady-state levels of Gli2. Arsenicals active in Hh pathway antagonism include arsenic trioxide (ATO), a curative agent in clinical use for acute promyelocytic leukemia (APL); in our studies, ATO inhibited growth of Hh pathway-driven medulloblastoma allografts derived from Ptch+/-p53-/- mice within a range of serum levels comparable to those achieved in treatment of human APL. Arsenic thus could be tested rapidly as a therapeutic agent in malignant diseases associated with Hh pathway activation and could be particularly useful in such diseases that are inherently resistant or have acquired resistance to cyclopamine mimics.

  View details for DOI 10.1073/pnas.1006822107

  View details for PubMedID 20624968

• Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth CANCER CELL Kim, J., Tang, J. Y., Gong, R., Kim, J., Lee, J. J., Clemons, K. V., Chong, C. R., Chang, K. S., Fereshteh, M., Gardner, D., Reya, T., Liu, J. O., Epstein, E. H., Stevens, D. A., Beachy, P. A. 2010; 17 (4): 388-399

  Abstract

  In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

  View details for DOI 10.1016/j.ccr.2010.02.027

  View details for PubMedID 20385363

• Dally-like core protein and its mammalian homologues mediate stimulatory and inhibitory effects on Hedgehog signal response PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Williams, E. H., Pappano, W. N., Saunders, A. M., Kim, M., Leahy, D. J., Beachy, P. A. 2010; 107 (13): 5869-5874

  Abstract

  The distribution and activities of morphogenic signaling proteins such as Hedgehog (Hh) and Wingless (Wg) depend on heparan sulfate proteoglycans (HSPGs). HSPGs consist of a core protein with covalently attached heparan sulfate glycosaminoglycan (GAG) chains. We report that the unmodified core protein of Dally-like (Dlp), an HSPG required for cell-autonomous Hh response in Drosophila embryos, alone suffices to rescue embryonic Hh signaling defects. Membrane tethering but not specifically the glycosylphosphatidylinositol linkage characteristic of glypicans is critical for this cell-autonomous activity. Our studies further suggest divergence of the two Drosophila and six mammalian glypicans into two functional families, an activating family that rescues cell-autonomous Dlp function in Hh response and a family that inhibits Hh response. Thus, in addition to the previously established requirement for HSPG GAG chains in Hh movement, these findings demonstrate a positive cell-autonomous role for a core protein in morphogen response in vivo and suggest the conservation of a network of antagonistic glypican activities in the regulation of Hh response.

  View details for DOI 10.1073/pnas.1001777107

  View details for Web of Science ID 000276159500033

  View details for PubMedID 20231458

  View details for PubMedCentralID PMC2851888

• Genetic and biochemical definition of the Hedgehog receptor GENES & DEVELOPMENT Zheng, X., Mann, R. K., Sever, N., Beachy, P. A. 2010; 24 (1): 57-71

  Abstract

  Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. Here we show that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. We demonstrate that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, we conclude that Ihog and Ptc together constitute the Drosophila Hh receptor.

  View details for DOI 10.1101/gad.1870310

  View details for Web of Science ID 000273249300008

  View details for PubMedID 20048000

  View details for PubMedCentralID PMC2802192

• Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, J., Kato, M., Beachy, P. A. 2009; 106 (51): 21666-21671

  Abstract

  Stimulation by the extracellular Hedgehog (Hh) protein signal has been shown to alter ciliary localization of the mammalian Hh receptor components Smoothened (Smo) and Patched (Ptc), and mutations that disrupt the structure and function of the cilium also disrupt Hh-induced changes in gene expression. But how ciliary events affect gene expression in the nucleus is not known, and to address this question we have characterized the cellular trafficking of Gli2, the principal mediator of Hh-dependent transcriptional activation. From a combination of pharmacological and genetic manipulations we find in resting cells that both Gli2 and Smo appear to shuttle in and out of the cilium, with Gli2 but not Smo requiring intact cytoplasmic microtubules for ciliary entry and both requiring the ciliary retrograde motor, cytoplasmic dynein 2, for ciliary exit. We also find that changes in ciliary and nuclear trafficking of Gli2 are triggered by the Hh-dependent accumulation of activated Smo in the cilium, resulting in a shift from primarily cytoplasmic localization to accumulation at the distal tip of the cilium and within the nucleus. Gli2 thus functions as a dynamic monitor of Smo activity in the cilium and thereby links Hh pathway activation in the cilium to transcriptional activation in the nucleus.

  View details for DOI 10.1073/pnas.0912180106

  View details for Web of Science ID 000272994200038

  View details for PubMedID 19996169

  View details for PubMedCentralID PMC2790365

• The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla NATURE McLellan, J. S., Zheng, X., Hauk, G., Ghirlando, R., Beachy, P. A., Leahy, D. J. 2008; 455 (7215): 979-U62

  Abstract

  Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.

  View details for DOI 10.1038/nature07358

  View details for Web of Science ID 000260038300053

  View details for PubMedID 18794898

  View details for PubMedCentralID PMC2679680

• Oxysterols pathway in are novel activators of the hedgehog pluripotent mesenchymal cells JOURNAL OF BIOLOGICAL CHEMISTRY Dwyer, J. R., Sever, N., Carlson, M., Nelson, S. F., Beachy, P. A., Parhami, F. 2007; 282 (12): 8959-8968

  Abstract

  Pluripotent mesenchymal cells form a population of precursors to a variety of cell types, including osteoblasts and adipocytes. Aging tilts the balance in favor of adipocyte differentiation at the expense of osteoblast differentiation, resulting in reduced bone formation and osteopenic disorders, including osteoporosis, in humans and animals. Understanding the mechanisms involved in causing this apparent shift in differentiation and identifying factors that stimulate osteoblast formation while inhibiting adipogenesis are of great therapeutic interest. In this study we report that specific, naturally occurring oxysterols, previously shown to direct pluripotent mesenchymal cells toward an osteoblast lineage, exert their osteoinductive effects through activation of Hedgehog signaling pathway. This was demonstrated by 1) oxysterol-induced expression of the Hh target genes Gli-1 and Patched, 2) oxysterol-induced activation of a luciferase reporter driven by a multimerized Gli-responsive element, 3) inhibition of oxysterol effects by the hedgehog pathway inhibitor, cyclopamine, and 4) unresponsiveness of Smoothened-/- mouse embryonic fibroblasts to oxysterols. Using Patched-/- cells that possess high baseline Gli activity, we found that oxysterols did not dramatically shift the IC50 concentration of cyclopamine needed to inhibit Gli activity in these cells. Furthermore, binding studies showed that oxysterols did not compete with fluorescently labeled cyclopamine, BODIPY-cyclopamine, for direct binding to Smoothened. These findings demonstrate that oxysterols stimulate hedgehog pathway activity by indirectly activating the seven-transmembrane pathway component Smoothened. Osteoinductive oxysterols are, therefore, novel activators of the hedgehog pathway in pluripotent mesenchymal cells, and they may be important modulators of this critical signaling pathway that regulates numerous developmental and post-developmental processes.

  View details for DOI 10.1074/jbc.M611741200

  View details for Web of Science ID 000245780300042

  View details for PubMedID 17200122

• Structure of a heparin-dependent complex of hedgehog and Ihog PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McLellan, J. S., Yao, S., Zheng, X., Geisbrecht, B. V., Ghirlando, R., Beachy, P. A., Leahy, D. J. 2006; 103 (46): 17208-17213

  Abstract

  Hedgehog (Hh) signaling molecules mediate key tissue-patterning events during animal development, and inappropriate activation of Hh signaling in adults has been associated with human cancers. Recently, a conserved family of type I integral membrane proteins required for normal response to the Hh signal was discovered. One member of this family, Ihog (interference hedgehog), functions upstream or at the level of Patched (Ptc), but how Ihog participates in Hh signaling remains unclear. Here, we show that heparin binding induces Ihog dimerization and is required to mediate high-affinity interactions between Ihog and Hh. We also present crystal structures of a Hh-binding fragment of Ihog, both alone and complexed with Hh. Heparin is not well ordered in these structures, but a basic cleft in the first FNIII domain of Ihog (IhogFn1) is shown by mutagenesis to mediate heparin binding. These results establish that Hh directly binds Ihog and provide the first demonstration of a specific role for heparin in Hh responsiveness.

  View details for DOI 10.1073/pnas.0606738103

  View details for Web of Science ID 000242249400028

  View details for PubMedID 17077139

  View details for PubMedCentralID PMC1859911

• Prevalence of off-target effects in Drosophila RNA interference screens NATURE Ma, Y., Creanga, A., Lum, L., Beachy, P. A. 2006; 443 (7109): 359-363

  Abstract

  RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21-23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such 'off-target effects' (OTEs). Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.

  View details for DOI 10.1038/nature05179

  View details for Web of Science ID 000240622000051

  View details for PubMedID 16964239

• A genomewide screen for components of the RNAi pathway in Drosophila cultured cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dorner, S., Lum, L., Kim, M., Paro, R., Beachy, P. A., Green, R. 2006; 103 (32): 11880-11885

  Abstract

  Posttranscriptional silencing by RNAi is initiated by dsRNAs that are processed into siRNAs that ultimately target homologous mRNAs for degradation. We used luciferase reporter constructs and a cultured cell-based assay to perform a genomewide screen for components of the RNAi pathway in Drosophila melanogaster. The screen identified seven genes that affect the RNAi response, five with previously described function (AGO2, Tis11, Hsc70-3, Hsc70-4, and hdc) and two annotated genes (CG17265 and CG10883).

  View details for DOI 10.1073/pnas.0605210103

  View details for Web of Science ID 000239701900013

  View details for PubMedID 16882716

  View details for PubMedCentralID PMC1525048

• The ihog cell-surface proteins bind hedgehog and mediate pathway activation CELL Yao, S. Q., Lum, L., Beachy, P. 2006; 125 (2): 343-357

  Abstract

  The ihog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown here to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response.

  View details for DOI 10.1016/j.cell.2006.02.040

  View details for Web of Science ID 000237241500023

  View details for PubMedID 16630821

• Defective cerebellar response to mitogenic Hedgehog signaling in Down's syndrome mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Roper, R. J., Baxter, L. L., Saran, N. G., Klinedinst, D. K., Beachy, P. A., Reeves, R. H. 2006; 103 (5): 1452-1456

  Abstract

  Trisomy 21 is the cause of Down [corrected] syndrome (DS) which is characterized by a number of phenotypes, including a brain which is small and hypocellular compared to that of euploid individuals. The cerebellum is disproportionately reduced. Ts65Dn mice are trisomic for orthologs of about half of the genes on human chromosome 21 and provide a genetic model for DS. These mice display a number of developmental anomalies analogous to those in DS, including a small cerebellum with a significantly decreased number of both granule and Purkinje cell neurons. Here we trace the origin of the granule cell deficit to precursors in early postnatal development, which show a substantially reduced mitogenic response to Hedgehog protein signaling. Purified cultures of trisomic granule cell precursors show a reduced but dose-dependent response to the Sonic hedgehog protein signal in vitro, demonstrating that this is a cell-autonomous deficit. Systemic treatment of newborn trisomic mice with a small molecule agonist of Hedgehog pathway activity increases mitosis and restores granule cell precursor populations in vivo. These results demonstrate a basis for and a potential therapeutic approach to a fundamental aspect of CNS pathology in DS.

  View details for DOI 10.1073/pnas.0510750103

  View details for Web of Science ID 000235094300054

  View details for PubMedID 16432181

  View details for PubMedCentralID PMC1360600

• Molecular mechanisms of Sonic hedgehog mutant effects in holoprosencephaly PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maity, T., Fuse, N., Beachy, P. A. 2005; 102 (47): 17026-17031

  Abstract

  Holoprosencephaly (HPE), a human developmental brain defect, usually is also associated with varying degrees of midline facial dysmorphism. Heterozygous mutations in the Sonic hedgehog (SHH) gene are the most common genetic lesions associated with HPE, and loss of Shh function in the mouse produces cyclopia and alobar forebrain development. The N-terminal domain (ShhNp) of Sonic hedgehog protein, generated by cholesterol-dependent autoprocessing and modification at the C terminus and by palmitate addition at the N terminus, is the active ligand in the Shh signal transduction pathway. Here, we analyze seven reported missense mutations (G31R, D88V, Q100H, N115K, W117G, W117R, and E188Q) that alter the N-terminal signaling domain of Shh protein, and show that two of these mutations (Q100H and E188Q), which are questionably linked to HPE, produce no detectable effects on function. The remaining five alterations affect normal processing, Ptc binding, and signaling to varying degrees. These effects include introduction of a recognition site for furin-like proteases by the G31R alteration, resulting in cleavage of 11 amino acid residues from the N terminus of ShhNp and consequent reduced signaling potency. Two other alterations, W117G and W117R, cause temperature-dependent misfolding and retention in the sterol-poor endoplasmic reticulum, thus disrupting cholesterol-dependent autoprocessing.

  View details for DOI 10.1073/pnas.0507848102

  View details for Web of Science ID 000233463200023

  View details for PubMedID 16282375

  View details for PubMedCentralID PMC1282174

• Extensive phosphorylation of Smoothened in Hedgehog pathway activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, C., Williams, E. H., Guo, Y., Lum, L., Beachy, P. A. 2004; 101 (52): 17900-17907

  Abstract

  The transmembrane protein Smoothened (Smo) is activated in response to the extracellular protein signal, Hedgehog (Hh), and transmits this state of pathway activity into the cell. Previous studies in Drosophila have correlated pathway activation with Smo accumulation and increased phosphorylation. Using immunopurification and mass spectrometry, we identify here 26 serine/threonine residues within the Smo C-terminal cytoplasmic tail that are phosphorylated in Hh-stimulated cells. By systematically substituting alanine or glutamic acid to block or simulate phosphorylation, we provide evidence for a functional role of collective phosphorylation of a subset of phosphoresidues in pathway activation. This role is indicated by the ability of altered Smo proteins to produce changes in transcription of Hh-responsive genes in vivo and in cultured cells. These altered Smo proteins also affect biochemical indicators of pathway activity, such as Smo accumulation and phosphorylation of other pathway components. The prevalence and arrangement of phosphoresidues within the Smo cytoplasmic tail at recognition sites for cAMP-dependent protein kinase and casein kinase 1 suggest a role for these kinases in Smo phosphorylation, and such a role is supported by the effects of manipulating kinase activities in cultured cells. Our studies confirm and extend previous studies showing a positive effect for cAMP-dependent protein kinase and uncover a positive role for casein kinase 1alpha in Hh pathway activation.

  View details for DOI 10.1073/pnas.0408093101

  View details for Web of Science ID 000226102700006

  View details for PubMedID 15598741

  View details for PubMedCentralID PMC535705

• Tissue repair and stem cell renewal in carcinogenesis NATURE Beachy, P. A., Karhadkar, S. S., Berman, D. M. 2004; 432 (7015): 324-331

  Abstract

  Cancer is increasingly being viewed as a stem cell disease, both in its propagation by a minority of cells with stem-cell-like properties and in its possible derivation from normal tissue stem cells. But stem cell activity is tightly controlled, raising the question of how normal regulation might be subverted in carcinogenesis. The long-known association between cancer and chronic tissue injury, and the more recently appreciated roles of Hedgehog and Wnt signalling pathways in tissue regeneration, stem cell renewal and cancer growth together suggest that carcinogenesis proceeds by misappropriating homeostatic mechanisms that govern tissue repair and stem cell self-renewal.

  View details for DOI 10.1038/nature03100

  View details for Web of Science ID 000225161400042

  View details for PubMedID 15549094

• Mending and malignancy NATURE Beachy, P. A., Karhadkar, S. S., Berman, D. M. 2004; 431 (7007): 402-402

  View details for DOI 10.1038/431402a

  View details for Web of Science ID 000224000500020

  View details for PubMedID 15385990

• The Hedgehog response network: Sensors, switches, and routers SCIENCE Lum, L., Beachy, P. A. 2004; 304 (5678): 1755-1759

  Abstract

  The Hedgehog (Hh) signaling pathway is intimately linked to cell growth and differentiation, with normal roles in embryonic pattern formation and adult tissue homeostasis and pathological roles in tumor initiation and growth. Recent advances in our understanding of Hh response have resulted from the identification of new pathway components and new mechanisms of action for old pathway components. The most striking new finding is that signal transmission from membrane to cytoplasm proceeds through recruitment, by the seven-transmembrane protein Smoothened, of an atypical kinesin, which routes pathway activation by interaction with other components of a complex that includes the latent zinc finger transcription factor, Ci.

  View details for Web of Science ID 000222089500031

  View details for PubMedID 15205520

• Novel lipid modifications of secreted protein signals ANNUAL REVIEW OF BIOCHEMISTRY Mann, R. K., Beachy, P. A. 2004; 73: 891-923

  Abstract

  Secreted signaling proteins function in a diverse array of essential patterning events during metazoan development, ranging from embryonic segmentation in insects to neural tube differentiation in vertebrates. These proteins generally are expressed in a localized manner, and they may elicit distinct concentration-dependent responses in the cells of surrounding tissues and structures, thus functioning as morphogens that specify the pattern of cellular responses by their tissue distribution. Given the importance of signal distribution, it is notable that the Hedgehog (Hh) and Wnt proteins, two of the most important families of such signals, are known to be covalently modified by lipid moieties, the membrane-anchoring properties of which are not consistent with passive models of protein mobilization within tissues. This review focuses on the mechanisms underlying biogenesis of the mature Hh proteins, which are dually modified by cholesteryl and palmitoyl adducts, as well as on the relationship between Hh proteins and the self-splicing proteins (i.e., proteins containing inteins) and the Hh-like proteins of nematodes. We further discuss the cellular mechanisms that have evolved to handle lipidated Hh proteins in the spatial deployment of the signal in developing tissues and the more recent findings that implicate palmitate modification as an important feature of Wnt signaling proteins.

  View details for DOI 10.1146/annurev.biochem.73.011303.073933

  View details for Web of Science ID 000223246400028

  View details for PubMedID 15189162

• Hedgehog signal transduction via smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2 MOLECULAR CELL Lum, L., Zhang, C., Oh, S., Mann, R. K., von Kessler, D. P., Taipale, J., Weis-Garcia, F., Gong, R. Y., Wang, B. L., Beachy, P. A. 2003; 12 (5): 1261-1274

  Abstract

  The seven-transmembrane protein Smoothened (Smo) transduces extracellular activation of the Hedgehog (Hh) pathway by an unknown mechanism to increase transcriptional activity of the latent cytoplasmic transcription factor Ci (Cubitus interruptus). Here, we present evidence that Smo associates directly with a Ci-containing complex that is scaffolded and stabilized by the atypical kinesin, Costal-2 (Cos2). This complex constitutively suppresses pathway activity, but Hh signaling reverses its regulatory effect to promote Ci-mediated transcription. In response to Hh activation of Smo, Cos2 mediates accumulation and phosphorylation of Smo at the membrane as well as phosphorylation of the cytoplasmic components Fu and Su(fu). Positive response of Cos2 to Hh stimulation requires a portion of the Smo cytoplasmic tail and the Cos2 cargo domain, which interacts directly with Smo.

  View details for Web of Science ID 000186764700021

  View details for PubMedID 14636583

• A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis NATURE GENETICS Cooper, M. K., Wassif, C. A., Krakowiak, P. A., Taipale, J., Gong, R. Y., Kelley, R. I., Porter, F. D., Beachy, P. A. 2003; 33 (4): 508-513

  Abstract

  Smith-Lemli-Opitz syndrome (SLOS), desmosterolosis and lathosterolosis are human syndromes caused by defects in the final stages of cholesterol biosynthesis. Many of the developmental malformations in these syndromes occur in tissues and structures whose embryonic patterning depends on signaling by the Hedgehog (Hh) family of secreted proteins. Here we report that response to the Hh signal is compromised in mutant cells from mouse models of SLOS and lathosterolosis and in normal cells pharmacologically depleted of sterols. We show that decreasing levels of cellular sterols correlate with diminishing responsiveness to the Hh signal. This diminished response occurs at sterol levels sufficient for normal autoprocessing of Hh protein, which requires cholesterol as cofactor and covalent adduct. We further find that sterol depletion affects the activity of Smoothened (Smo), an essential component of the Hh signal transduction apparatus.

  View details for DOI 10.1038/ng1134

  View details for Web of Science ID 000181928600023

  View details for PubMedID 12652302

• Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells SCIENCE Lum, L., Yao, S. Q., Mozer, B., Rovescalli, A., Von Kessler, D., Nirenberg, M., Beachy, P. A. 2003; 299 (5615): 2039-2045

  Abstract

  Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.

  View details for Web of Science ID 000181834200046

  View details for PubMedID 12663920

• Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer NATURE Watkins, D. N., Berman, D. M., Burkholder, S. G., Wang, B. L., Beachy, P. A., BAYLIN, S. B. 2003; 422 (6929): 313-317

  Abstract

  Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.

  View details for DOI 10.1038/nature01493

  View details for Web of Science ID 000181637300040

  View details for PubMedID 12629553

• Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened GENES & DEVELOPMENT Chen, J. K., Taipale, J., Cooper, M. K., Beachy, P. A. 2002; 16 (21): 2743-2748

  Abstract

  The steroidal alkaloid cyclopamine has both teratogenic and antitumor activities arising from its ability to specifically block cellular responses to vertebrate Hedgehog signaling. We show here, using photoaffinity and fluorescent derivatives, that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened (Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum, presumably through binding-mediated effects on protein conformation. These observations reveal the mechanism of cyclopamine's teratogenic and antitumor activities and further suggest a role for small molecules in the physiological regulation of Smo.

  View details for DOI 10.1101/gad.1025302

  View details for Web of Science ID 000179027900002

  View details for PubMedID 12414725

  View details for PubMedCentralID PMC187469

• Small molecule modulation of Smoothened activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. 2002; 99 (22): 14071-14076

  Abstract

  Smoothened (Smo), a distant relative of G protein-coupled receptors, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Although the cellular mechanisms that regulate Smo function remain unclear, the direct inhibition of Smo by cyclopamine, a plant-derived steroidal alkaloid, suggests that endogenous small molecules may be involved. Here we demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle in a manner that antagonizes cyclopamine action. In addition, we have identified four small molecules that directly inhibit Smo activity but are structurally distinct from cyclopamine. Functional and biochemical studies of these compounds provide evidence for the small molecule modulation of Smo through multiple mechanisms and yield insights into the physiological regulation of Smo activity. The mechanistic differences between the Smo antagonists may be useful in the therapeutic manipulation of Hh signaling.

  View details for DOI 10.1073/pnas.182542899

  View details for Web of Science ID 000178967400021

  View details for PubMedID 12391318

  View details for PubMedCentralID PMC137838

• Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched CELL Ma, Y., Erkner, A., Gong, R. Y., Yao, S. Q., Taipale, J., Basler, K., Beachy, P. A. 2002; 111 (1): 63-75

  Abstract

  The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.

  View details for Web of Science ID 000178461900009

  View details for PubMedID 12372301

• Medulloblastoma growth inhibition by Hedgehog pathway blockade SCIENCE Berman, D. M., Karhadkar, S. S., Hallahan, A. R., Pritchard, J. I., Eberhart, C. G., Watkins, D. N., Chen, J. K., Cooper, M. K., Taipale, J., Olson, J. M., Beachy, P. A. 2002; 297 (5586): 1559-1561

  Abstract

  Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.

  View details for Web of Science ID 000177697300059

  View details for PubMedID 12202832

• Patched acts catalytically to suppress the activity of Smoothened NATURE Taipale, J., Cooper, M. K., Maiti, T., Beachy, P. A. 2002; 418 (6900): 892-897

  Abstract

  Mutations affecting the transmembrane proteins Patched (Ptc) or Smoothened (Smo) that trigger ligand-independent activity of the Hedgehog (Hh) signalling pathway are associated with human tumours such as basal cell carcinoma (BCC) and medulloblastoma. Despite extensive genetic studies demonstrating the importance of these receptor components in embryonic patterning and cancer, the mechanism by which Ptc regulates Smo is not understood. Here we report that Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, we show that free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; we demonstrate that the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumour suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule.

  View details for DOI 10.1038/nature00989

  View details for Web of Science ID 000177555600042

  View details for PubMedID 12192414

• Skinny Hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal SCIENCE Chamoun, Z., Mann, R. K., Nellen, D., von Kessler, D. P., Bellotto, M., Beachy, P. A., Basler, K. 2001; 293 (5537): 2080-2084

  Abstract

  One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog (Hh) family of secreted signaling proteins. Here, we identify a segment polarity gene in Drosophila melanogaster, skinny hedgehog (ski), and show that its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and we provide genetic and biochemical evidence that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. Our results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal.

  View details for DOI 10.1126/science.1064437

  View details for Web of Science ID 000171028700072

  View details for PubMedID 11486055

• The Hedgehog and Wnt signaling pathways in cancer NATURE Taipale, J., Beachy, P. A. 2001; 411 (6835): 349-354

  Abstract

  The Wnt and Hedgehog (Hh) signalling pathways have long been known to direct growth and patterning during embryonic development. Recent evidence also implicates these pathways in the postembryonic regulation of stem-cell number in epithelia such as those of the skin and intestine, which undergo constant renewal. A pathological role for the Wnt and Hh pathways has emerged from studies showing a high frequency of specific human cancers associated with mutations that constitutively activate the transcriptional response of these pathways. This article focuses on Hh and Wnt signal transduction and reviews evidence suggesting that tumorigenesis associated with pathway activation may result from mis-specification of cells towards stem-cell or stem cell-like fates.

  View details for Web of Science ID 000168710000057

  View details for PubMedID 11357142

• Cholesterol modification of proteins BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS Mann, R. K., Beachy, P. A. 2000; 1529 (1-3): 188-202

  Abstract

  The demonstration over 30 years ago that inhibitors of cholesterol biosynthesis disrupt animal development suggested an intriguing connection between fundamental cellular metabolic processes and the more global processes of embryonic tissue patterning. Adding a new dimension to this relationship is the more recent finding that the Hedgehog family of tissue patterning factors are covalently modified by cholesterol. Here we review the mechanism of the Hedgehog autoprocessing reaction that results in this modification, and compare this reaction to that undergone by other autoprocessing proteins. We also discuss the biological consequences of cholesterol modification, in particular the use of cholesterol as a molecular handle in the spatial deployment of the protein signal in developing tissues. Finally, the developmental consequences of chemical and genetic disruption of cholesterol homeostasis are summarized, along with the potential importance of cholesterol-rich lipid rafts in production of and response to the Hh signal.

  View details for Web of Science ID 000166141400017

  View details for PubMedID 11111088

• Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine NATURE Taipale, J., Chen, J. K., Cooper, M. K., Wang, B. L., Mann, R. K., Milenkovic, L., Scott, M. P., Beachy, P. A. 2000; 406 (6799): 1005-1009

  Abstract

  Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

  View details for Web of Science ID 000089020200048

  View details for PubMedID 10984056

• Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb CELL Wang, B. L., Fallon, J. F., Beachy, P. A. 2000; 100 (4): 423-434

  Abstract

  Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. We demonstrate that PKA-dependent processing of vertebrate Gli3 in developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation.

  View details for Web of Science ID 000085446900005

  View details for PubMedID 10693759

• Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched National Academy of Sciences Colloquium on Proteolytic Processing and Physiological Regulation Fuse, N., Maiti, T., Wang, B. L., Porter, J. A., Hall, T. M., Leahy, D. J., Beachy, P. A. NATL ACAD SCIENCES. 1999: 10992–99

  Abstract

  The amino-terminal signaling domain of the Sonic hedgehog secreted protein (Shh-N), which derives from the Shh precursor through an autoprocessing reaction mediated by the carboxyl-terminal domain, executes multiple functions in embryonic tissue patterning, including induction of ventral and suppression of dorsal cell types in the developing neural tube. An apparent catalytic site within Shh-N is suggested by structural homology to a bacterial carboxypeptidase. We demonstrate here that alteration of residues presumed to be critical for a hydrolytic activity does not cause a loss of inductive activity, thus ruling out catalysis by Shh-N as a requirement for signaling. We favor the alternative, that Shh-N functions primarily as a ligand for the putative receptor Patched (Ptc). This possibility is supported by new evidence for direct binding of Shh-N to Ptc and by a strong correlation between the affinity of Ptc-binding and the signaling potency of Shh-N protein variants carrying alterations of conserved residues in a particular region of the protein surface. These results together suggest that direct Shh-N binding to Ptc is a critical event in transduction of the Shh-N signal.

  View details for Web of Science ID 000082868500012

  View details for PubMedID 10500113

  View details for PubMedCentralID PMC34231

• Nuclear trafficking of cubitus interruptus in the transcriptional regulation of hedgehog target gene expression CELL Chen, C. H., von Kessler, D. P., Park, W. J., Beachy, P. A. 1999; 98 (3): 305-316

  Abstract

  Transcriptional activation of Hedgehog (Hh) target genes requires Cubitus interruptus, a 155 kDa cytoplasmic zinc finger protein (Ci155), which in the absence of Hh signaling is processed to form a nuclear repressor (Ci75). We show that Hh signaling reduces phosphorylation of Ci155, and this reduction in turn appears to decrease processing. Blocking processing with proteasome inhibitors or altered Ci proteins, however, is insufficient for activation of Hh targets. We find that Hh signaling increases the rate of Ci155 nuclear import, resulting in significant nuclear accumulation. Even in the absence of signaling, nuclear accumulation of Ci155 suffices for significant induction of Hh targets, and active nuclear export of Ci155 is an essential mechanism for maintenance of the unstimulated state.

  View details for Web of Science ID 000081950300006

  View details for PubMedID 10458606

• Teratogen-mediated inhibition of target tissue response to Shh signaling SCIENCE Cooper, M. K., Porter, J. A., Young, K. E., Beachy, P. A. 1998; 280 (5369): 1603-1607

  Abstract

  Veratrum alkaloids and distal inhibitors of cholesterol biosynthesis have been studied for more than 30 years as potent teratogens capable of inducing cyclopia and other birth defects. Here, it is shown that these compounds specifically block the Sonic hedgehog (Shh) signaling pathway. These teratogens did not prevent the sterol modification of Shh during autoprocessing but rather inhibited the response of target tissues to Shh, possibly acting through the sterol sensing domain within the Patched protein regulator of Shh response.

  View details for Web of Science ID 000074061200041

  View details for PubMedID 9616123

• Crystal structure of a hedgehog autoprocessing domain: Homology between hedgehog and self-splicing proteins CELL Hall, T. M., Porter, J. A., Young, K. E., Koonin, E. V., Beachy, P. A., Leahy, D. J. 1997; 91 (1): 85-97

  Abstract

  The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. We have identified a 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing and report here its crystal structure. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins.

  View details for Web of Science ID A1997XZ80900011

  View details for PubMedID 9335337

• Multiple roles of cholesterol in hedgehog protein biogenesis and signaling Cold Spring Harbor Symposium on Quantitative Biology - Pattern Formation During Development Beachy, P. A., Cooper, M. K., Young, K. E., von Kessler, D. P., Park, W. J., Hall, T. M., Leahy, D. J., Porter, J. A. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1997: 191–204

  View details for Web of Science ID 000073570200024

  View details for PubMedID 9598352

• Cholesterol modification of hedgehog signaling proteins in animal development SCIENCE Porter, J. A., Young, K. E., Beachy, P. A. 1996; 274 (5285): 255-259

  Abstract

  Hedgehog (Hh) proteins comprise a family of secreted signaling molecules essential for patterning a variety of structures in animal embryogenesis. During biosynthesis, Hh undergoes an autocleavage reaction, mediated by its carboxyl-terminal domain, that produces a lipid-modified amino-terminal fragment responsible for all known Hh signaling activity. Here it is reported that cholesterol is the lipophilic moiety covalently attached to the amino-terminal signaling domain during autoprocessing and that the carboxyl-terminal domain acts as an intramolecular cholesterol transferase. This use of cholesterol to modify embryonic signaling proteins may account for some of the effects of perturbed cholesterol biosynthesis on animal development.

  View details for Web of Science ID A1996VM67100046

  View details for PubMedID 8824192

• Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function NATURE Chiang, C., Ying, L. T., Lee, E., Young, K. E., Corden, J. L., Westphal, H., Beachy, P. A. 1996; 383 (6599): 407-413

  Abstract

  Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

  View details for Web of Science ID A1996VL46300053

  View details for PubMedID 8837770

• Hedgehog patterning activity: Role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain CELL Porter, J. A., Ekker, S. C., Park, W. J., VONKESSLER, D. P., Young, K. E., Chen, C. H., Ma, Y., Woods, A. S., Cotter, R. J., Koonin, E. V., Beachy, P. A. 1996; 86 (1): 21-34

  Abstract

  Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

  View details for Web of Science ID A1996UX93400005

  View details for PubMedID 8689684

• A POTENTIAL CATALYTIC SITE REVEALED BY THE 1.7-ANGSTROM CRYSTAL-STRUCTURE OF THE AMINO-TERMINAL SIGNALING DOMAIN OF SONIC HEDGEHOG NATURE Hall, T. M., Porter, J. A., Beachy, P. A., Leahy, D. J. 1995; 378 (6553): 212-216

  Abstract

  Within the past few years, members of the hedgehog (hh) family of secreted signalling proteins have emerged as the primary signals generated by certain embryonic patterning centres. In vertebrate embryos, for example, sonic hedgehog expression in the notochord appears to be responsible for the local and long-range induction of ventral cell types within the neural tube and somites (reviewed in refs 1, 2). Protein products encoded by hh family members are synthesized as precursors that undergo autoprocessing to generate an amino-terminal domain that appears to be responsible for both local and long-range signalling activities, and a carboxy-terminal domain that contains the autoprocessing activity. As part of an effort to understand how hh family members participate in cell-to-cell signalling, we have determined and report here the crystal structure at 1.7 A of the amino-terminal domain of murine Sonic hedgehog (Shh-N). The structure revealed a tetrahedrally coordinated zinc ion that appears to be structurally analogous to the zinc coordination sites of zinc hydrolases, such as thermolysin and carboxypeptidase A. This previously unsuspected catalytic site represents a distinct activity from the autoprocessing activity that resides in the carboxy-terminal domain.

  View details for Web of Science ID A1995TD75900062

  View details for PubMedID 7477329

• CONTROL OF DROSOPHILA TRACHEAL BRANCHING BY THE NOVEL HOMEODOMAIN GENE UNPLUGGED, A REGULATORY TARGET FOR GENES OF THE BITHORAX COMPLEX DEVELOPMENT CHAING, C., Young, K. E., Beachy, P. A. 1995; 121 (11): 3901-3912

  Abstract

  We have identified a novel Drosophila homeodomain gene, unplugged (unp), whose function is required for formation of the tracheal branches that penetrate the CNS. In unp mutant embryos the segmentally repeated ganglionic branches stall and fail to penetrate the CNS and the segment-specific cerebral branch and associated cerebral anastomosis fail to form. Expression of unp in the founder cells for the cerebral branch within the first tracheal metamere is repressed in posterior segments by Ubx and other bithorax complex genes. This pattern of expression and homeotic gene regulation is reproduced by an unusual 2.6 kb cis-regulatory sequence located downstream of the unp transcription unit. Since the unp protein is localized to the nucleus of tracheal precursor cells as they migrate and extend, unp protein appears to play a regulatory role in neural branching of the tracheae, and the segment-specific aspects of these neural branching patterns appear to be generated by homeotic regulation of unp expression.

  View details for Web of Science ID A1995TF89600038

  View details for PubMedID 8582298

• PATTERNING ACTIVITIES OF VERTEBRATE HEDGEHOG PROTEINS IN THE DEVELOPING EYE AND BRAIN CURRENT BIOLOGY Ekker, S. C., Ungar, A. R., Greenstein, P., VONKESSLER, D. P., Porter, J. A., MOON, R. T., Beachy, P. A. 1995; 5 (8): 944-955

  Abstract

  The hedgehog (hh) family of secreted signaling proteins is responsible for developmental patterning in a variety of systems, including the neural tube, limbs and somites. Within the neural tube, at the level of the spinal cord, products of the vertebrate gene sonic hedgehog (shh) are proposed to function as a ventral patterning influence, with the capability of inducing floor plate and motor neurons.We report the isolation of tiggy-winkle hedgehog (twhh), a novel member of the zebrafish hh gene family. Both twhh and shh are expressed in the ventral midline of the embryonic zebrafish neural tube and brain, but twhh expression becomes limited to the neural tube, whereas shh is also expressed in the notochord. Both genes are expressed in the developing brain, in domains that include a discrete region in the floor of the diencephalon, located between the sites of the future optic stalks. Using pax-2 and pax-6 as markers of proximo-distal fate within the developing eye, we found that ectopic expression of either hh gene promoted proximal fates and suppressed distal fates. In contrast, proximal fates were lost in cyclops mutant embryos, which lack twhh- and shh-expressing forebrain cells. Both twhh and shh proteins undergo autoproteolytic processing in vivo; a fragment corresponding to the amino-terminal cleavage product was sufficient to carry out all signaling activities associated with twhh in eye and brain development.These findings suggest that secreted signals encoded by members of the hedgehog gene family, emanating from the ventral midline of the neural tube, not only play important roles in dorso-ventral patterning of the brain but also appear to constitute an early patterning activity along the proximo-distal axis of the developing eyes.

  View details for Web of Science ID A1995RN56900024

  View details for PubMedID 7583153

• PATTERNING OF THE NEURAL ECTODERM OF XENOPUS-LAEVIS BY THE AMINO-TERMINAL PRODUCT OF HEDGEHOG AUTOPROTEOLYTIC CLEAVAGE DEVELOPMENT Lai, C. J., Ekker, S. C., Beachy, P. A., MOON, R. T. 1995; 121 (8): 2349-2360

  Abstract

  The patterns of embryonic expression and the activities of Xenopus members of the hedgehog gene family are suggestive of role in neural induction and patterning. We report that these hedgehog polypeptides undergo autoproteolytic cleavage. Injection into embryos of mRNAs encoding Xenopus banded-hedgehog (X-bhh) or the amino-terminal domain (N) demonstrates that the direct inductive activities of X-bhh are encoded by N. In addition, both N and X-bhh pattern neural tissue by elevating expression of anterior neural genes. Unexpectedly, an internal deletion of X-bhh (delta N-C) was found to block the activity of X-bhh and N in explants and to reduce dorsoanterior structures in embryos. As elevated hedgehog activity increases the expression of anterior neural genes, and as delta N-C reduces dorsoanterior structures, these complementary data support a role for hedgehog in neural induction and anteroposterior patterning.

  View details for Web of Science ID A1995RN70300009

  View details for PubMedID 7671801

• DISTINCT EXPRESSION AND SHARED ACTIVITIES OF MEMBERS OF THE HEDGEHOG GENE FAMILY OF XENOPUS-LAEVIS DEVELOPMENT Ekker, S. C., McGrew, L. L., Lai, C. J., Lee, J. J., VONKESSLER, D. P., MOON, R. T., Beachy, P. A. 1995; 121 (8): 2337-2347

  Abstract

  The hedgehog family of signaling proteins is associated with a variety of spatial patterning activities in insects and vertebrates. Here we show that new members of this family isolated from Xenopus laevis are expressed embryonically in patterns suggestive of roles in patterning in the ectoderm, nervous system and somites. Banded hedgehog is expressed throughout the neural plate and subsequently in both the nervous system and in the dermatome of somites. Cephalic hedgehog is expressed in anterior ectoderm and endodermal structures, and sonic hedgehog is expressed in patterns which parallel those in other species. Injection of RNAs encoding Xenopus hedgehogs induces ectopic cement gland formation in embryos. Similar to reported activities of noggin and follistatin, Xenopus hedgehogs share a common ability to induce cement glands in animal cap explants. However, hedgehog activities in naive ectoderm appear capable of acting independently of noggin and follistatin since, although all three are induced by activin in animal cap explants, X-hh expression does not induce noggin or follistatin.

  View details for Web of Science ID A1995RN70300008

  View details for PubMedID 7671800

• INDUCTION OF MIDBRAIN DOPAMINERGIC-NEURONS BY SONIC HEDGEHOG NEURON Hynes, M., Porter, J. A., Chiang, C., Chang, D., TESSIERLAVIGNE, M., Beachy, P. A., Rosenthal, A. 1995; 15 (1): 35-44

  Abstract

  Midbrain dopaminergic neurons, whose loss in adults results in Parkinson's disease, can be specified during embryonic development by a contact-dependent signal from floor plate cells. Here we show that the amino-terminal product of Sonic hedgehog autoproteolysis (SHH-N), an inductive signal expressed by floor plate cells, can induce dopaminergic neurons in vitro. We show further that manipulations to increase the activity of cyclic AMP-dependent protein kinase A, which is known to antagonize hedgehog signaling, can block dopaminergic neuron induction by floor plate cells. Our results and those of other studies indicate that SHH-N can function in a dose-dependent manner to induce different cell types within the neural tube. Our results also provide the basis for a potential cell transplantation therapy for Parkinson's disease.

  View details for Web of Science ID A1995RL75600007

  View details for PubMedID 7619528

• LIMB-PATTERNING ACTIVITY AND RESTRICTED POSTERIOR LOCALIZATION OF THE AMINO-TERMINAL PRODUCT OF SONIC HEDGEHOG CLEAVAGE CURRENT BIOLOGY LOPEZMARTINEZ, A., Chang, D. T., Chiang, C., Porter, J. A., Ros, M. A., Simandl, B. K., Beachy, P. A., Fallon, J. F. 1995; 5 (7): 791-796

  Abstract

  Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH.We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud.Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

  View details for Web of Science ID A1995RL95300022

  View details for PubMedID 7583126

• FLOOR PLATE AND MOTOR-NEURON INDUCTION BY DIFFERENT CONCENTRATIONS OF THE AMINO-TERMINAL CLEAVAGE PRODUCT OF SONIC HEDGEHOG AUTOPROTEOLYSIS CELL Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A., Jessell, T. M. 1995; 81 (3): 445-455

  Abstract

  The differentiation of floor plate cells and motor neurons can be induced by Sonic hedgehog (SHH), a secreted signaling protein that undergoes autoproteolytic cleavage to generate amino- and carboxy-terminal products. We have found that both floor plate cells and motor neurons are induced by the amino-terminal cleavage product of SHH (SHH-N). The threshold concentration of SHH-N required for motor neuron induction is about 5-fold lower than that required for floor plate induction. Higher concentrations of SHH-N can induce floor plate cells at the expense of motor neuron differentiation. Our results suggest that the induction of floor plate cells and motor neurons by the notochord in vivo is mediated by exposure of neural plate cells to different concentrations of the amino-terminal product of SHH autoproteolytic cleavage.

  View details for Web of Science ID A1995QW89400016

  View details for PubMedID 7736596

• LONG-RANGE SCLEROTOME INDUCTION BY SONIC HEDGEHOG - DIRECT ROLE OF THE AMINO-TERMINAL CLEAVAGE PRODUCT AND MODULATION BY THE CYCLIC-AMP SIGNALING PATHWAY CELL Fan, C. M., Porter, J. A., Chiang, C., Chang, D. T., Beachy, P. A., TESSIERLAVIGNE, M. 1995; 81 (3): 457-465

  Abstract

  A long-range signal encoded by the Sonic hedgehog (Shh) gene has been implicated as the ventral patterning influence from the notochord that induces sclerotome and represses dermomyotome in somite differentiation. Long-range effects of hedgehog (hh) signaling have been suggested to result either from local induction of a secondary diffusible signal or from the direct action of the highly diffusible carboxy-terminal product of HH autoproteolytic cleavage. Here we provide evidence that the long-range somite patterning effects of SHH are instead mediated by a direct action of the amino-terminal cleavage product. We also show that pharmacological manipulations to increase the activity of cyclic AMP-dependent protein kinase A can selectively antagonize the effects of the amino-terminal cleavage product. Our results support the operation of a single evolutionarily conserved signaling pathway for both local and direct long-range inductive actions of HH family members.

  View details for Web of Science ID A1995QW89400017

  View details for PubMedID 7736597

• THE PRODUCT OF HEDGEHOG AUTOPROTEOLYTIC CLEAVAGE ACTIVE IN LOCAL AND LONG-RANGE SIGNALING NATURE Porter, J. A., VONKESSLER, D. P., Ekker, S. C., Young, K. E., Lee, J. J., Moses, K., Beachy, P. A. 1995; 374 (6520): 363-366

  Abstract

  The secreted protein products of the hedgehog (hh) gene family are associated with local and long-range signalling activities that are responsible for developmental patterning in multiple systems, including Drosophila embryonic and larval tissues and vertebrate neural tube, limbs and somites. In a process that is critical for full biological activity, the hedgehog protein (Hh) undergoes autoproteolysis to generate two biochemically distinct products, an 18K amino-terminal fragment, N, and a 25K carboxy-terminal fragment, C (ref. 16); mutations that block autoproteolysis impair Hh function. We have identified the site of autoproteolytic cleavage and find that it is broadly conserved throughout the hedgehog family. Knowing the site of cleavage, we were able to test the function of the N and C cleavage products in Drosophila assays. We show here that the N product is the active species in both local and long-range signalling. Consistent with this, all twelve mapped hedgehog mutations either affected the structure of the N product directly or otherwise blocked the release of N from the Hh precursor as a result of deletion or alteration of sequences in the C domain.

  View details for Web of Science ID A1995QN63000061

  View details for PubMedID 7885476

• ULTRABITHORAX PROTEIN IS NECESSARY BUT NOT SUFFICIENT FOR FULL ACTIVATION OF DECAPENTAPLEGIC EXPRESSION IN THE VISCERAL MESODERM EMBO JOURNAL Sun, B., Hursh, D. A., Jackson, D., Beachy, P. A. 1995; 14 (3): 520-535

  Abstract

  To elucidate the mechanisms by which homeotic selector (HOM) genes specify the unique features of Drosophila segments, we have analyzed the regulation of decapentaplegic (dpp), a transforming growth factor (TGF)-beta superfamily member, and have found that the Ultrabithorax (Ubx) HOM protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle (exd). The exd protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require Ubx protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element. Thus, even in an apparently simple case where visceral mesoderm expression of the dpp target gene mirrors that of the Ubx HOM protein, full activation by Ubx protein requires at least one additional factor. In addition, a distinct regulatory mode not directly involving Ubx protein also appears to contribute to PS7-specific expression.

  View details for Web of Science ID A1995QF71800012

  View details for PubMedID 7859741

  View details for PubMedCentralID PMC398110

• AUTOPROTEOLYSIS IN HEDGEHOG PROTEIN BIOGENESIS SCIENCE Lee, J. J., Ekker, S. C., VONKESSLER, D. P., Porter, J. A., Sun, B. I., Beachy, P. A. 1994; 266 (5190): 1528-1537

  Abstract

  Extracellular signaling proteins encoded by the hedgehog (hh) multigene family are responsible for the patterning of a variety of embryonic structures in vertebrates and invertebrates. The Drosophila hh gene has now been shown to generate two predominant protein species that are derived by an internal autoproteolytic cleavage of a larger precursor. Mutations that reduced the efficiency of autoproteolysis in vitro diminished precursor cleavage in vivo and also impaired the signaling and patterning activities of the HH protein. The two HH protein species exhibited distinctive biochemical properties and tissue distribution, and these differences suggest a mechanism that could account for the long- and short-range signaling activities of HH in vivo.

  View details for Web of Science ID A1994PV01500035

  View details for PubMedID 7985023

• THE NOVEL HOMEODOMAIN GENE BUTTONLESS SPECIFIES DIFFERENTIATION AND AXONAL GUIDANCE FUNCTIONS OF DROSOPHILA DORSAL MEDIAN CELLS DEVELOPMENT Chiang, C., Patel, N. H., Young, K. E., Beachy, P. A. 1994; 120 (12): 3581-3593

  Abstract

  We have identified a novel homeodomain gene, buttonless (btn), that is specifically expressed in 20 cells of a single type during Drosophila embryonic development. These cells, the dorsal median (DM) cells, are arranged as a single pair within each segment along the dorsal midline of the CNS. Distinctive features of the DM cells include a large cell body and a long thick process extending laterally to the muscle attachment site. In the absence of btn gene function the initial commitment to the DM cell fate is made but differentiation fails to occur and the DM cells are lost. The btn mutation thus specifically eliminates the DM cells, and this genetic ablation in turn reveals a requirement for DM cells as cellular cues for axonal guidance during transverse nerve outgrowth and bifurcation of the median nerve.

  View details for Web of Science ID A1994QB20000022

  View details for PubMedID 7821224

• PRODUCTS, GENETIC-LINKAGE AND LIMB PATTERNING ACTIVITY OF A MURINE HEDGEHOG GENE DEVELOPMENT Chang, D. T., Lopez, A., VONKESSLER, D. P., Chiang, C., Simandl, B. K., Zhao, R. B., Seldin, M. F., Fallon, J. F., Beachy, P. A. 1994; 120 (11): 3339-3353

  Abstract

  The hedgehog (hh) segmentation gene of Drosophila melanogaster encodes a secreted signaling protein that functions in the patterning of larval and adult structures. Using low stringency hybridization and degenerate PCR primers, we have isolated complete or partial hh-like sequences from a range of invertebrate species including other insects, leech and sea urchin. We have also isolated three mouse and two human DNA fragments encoding distinct hh-like sequences. Our studies have focused upon Hhg-1, a mouse gene encoding a protein with 46% amino acid identity to hh. The Hhg-1 gene, which corresponds to the previously described vhh-1 or sonic class, is expressed in the notochord, ventral neural tube, lung bud, hindgut and posterior margin of the limb bud in developing mouse embryos. By segregation analysis the Hhg-1 gene has been localized to a region in proximal chromosome 5, where two mutations affecting mouse limb development previously have been mapped. In Drosophila embryos, ubiquitous expression of the Hhg-1 gene yields effects upon gene expression and cuticle pattern similar to those observed for the Drosophila hh gene. We also find that cultured quail cells transfected with a Hhg-1 expression construct can induce digit duplications when grafted to anterior or mid-distal but not posterior borders within the developing chick limb; more proximal limb element duplications are induced exclusively by mid-distal grafts. Both in transgenic Drosophila embryos and in transfected quail cells, the Hhg-1 protein product is cleaved to yield two stable fragments from a single larger precursor. The significance of Hhg-1 genetic linkage, patterning activity and proteolytic processing in Drosophila and chick embryos is discussed.

  View details for Web of Science ID A1994PV97100026

  View details for PubMedID 7720571

• EXPRESSION OF A NOVEL TOLL-LIKE GENE SPANS THE PARASEGMENT BOUNDARY AND CONTRIBUTES TO HEDGEHOG FUNCTION IN THE ADULT EYE OF DROSOPHILA MECHANISMS OF DEVELOPMENT Chiang, C., Beachy, P. A. 1994; 47 (3): 225-239

  Abstract

  Many proteins involved in signal transduction and cell adhesion are characterized by the presence of an extracellular domain with repeated copies of a leucine-rich motif (LRR). Here we report the isolation and characterization of a novel gene, tlr (for Toll-like receptor), which encodes a protein containing multiple LRRs in its presumed extracellular domain, a single transmembrane segment and homology to the cytoplasmic domain of the interleukin 1 receptor in its presumed intracellular domain. The pattern of tlr expression at the extended germ band stage is characterized by 15 transverse stripes in the gnathal and trunk segments, with four patches of expression corresponding to head segments and an additional patch of expression in the presumptive hindgut. The segmentally repeated tlr stripes in the trunk overlap both the wingless and engrailed stripes and thus span the parasegment boundary. The tlr stripes require pair rule gene function for their establishment and later become dependent upon segment-polarity gene function for their maintenance. Segmental modulation of tlr expression later in the tracheal system is dependent upon the function of the homeotic genes of the bithorax complex. The tlr gene also is prominently expressed in the imaginal discs. In the eye disc, this expression occurs in two stripes at the anterior and posterior margins of the morphogenetic furrow; this expression is consistent with a genetic interaction between a tlr mutation and an eye-specific allele of hedgehog. All of these data combine to suggest a role for tlr in interactions between cells at critical boundaries during development.

  View details for Web of Science ID A1994PJ97200003

  View details for PubMedID 7848870

• THE DEGREE OF VARIATION IN DNA-SEQUENCE RECOGNITION AMONG 4 DROSOPHILA HOMEOTIC PROTEINS EMBO JOURNAL Ekker, S. C., Jackson, D. G., VONKESSLER, D. P., Sun, B. I., Young, K. E., Beachy, P. A. 1994; 13 (15): 3551-3560

  Abstract

  The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. We compare here the DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity. We further show that amino acid residues within the N-terminal arm are responsible for the sequence specificity differences between the Ultrabithorax and Abdominal-B homeodomains. Similarities and differences at the corresponding positions within the N-terminal arms are conserved in the vertebrate Abdominal-B-like HOM proteins, which play critical roles in limb specifications as well as in regional specification along the anterior-posterior axis. This and other patterns of residue conservation suggest that differential DNA sequence recognition may play a role in HOM protein function in a wide range of organisms.

  View details for Web of Science ID A1994PB90900017

  View details for PubMedID 7914870

  View details for PubMedCentralID PMC395259

• THE SEGMENT POLARITY GENE HEDGEHOG IS REQUIRED FOR PROGRESSION OF THE MORPHOGENETIC FURROW IN THE DEVELOPING DROSOPHILA EYE CELL Ma, C. Y., Zhou, Y., Beachy, P. A., Moses, K. 1993; 75 (5): 927-938

  Abstract

  Cell-type specification in the Drosophila compound eye begins at the morphogenetic furrow. The furrow sweeps across the developing eye epithelium and is coincident with four classes of cellular events: coordinated changes in cell shape, changes in gene expression, synchronization of the cell cycle, and the specification of a regular array of ommatidial founder cells. The molecular mechanisms that induce these events in the developing eye have hitherto been unknown. We identify here a gene specifically required for furrow progression, hedgehog (hh). We show that hh expression posterior to the morphogenetic furrow is continuously required for its progression. We propose that forward diffusion of hh protein induces anterior cells to enter the furrow.

  View details for Web of Science ID A1993MK96600012

  View details for PubMedID 8252628

• COOPERATIVE BINDING OF AN ULTRABITHORAX HOMEODOMAIN PROTEIN TO NEARBY AND DISTANT DNA SITES MOLECULAR AND CELLULAR BIOLOGY Beachy, P. A., Varkey, J., Young, K. E., VONKESSLER, D. P., Sun, B. I., Ekker, S. C. 1993; 13 (11): 6941-6956

  Abstract

  Cooperativity in binding of regulatory proteins to multiple DNA sites can heighten the sensitivity and specificity of the transcriptional response. We report here the cooperative DNA-binding properties of a developmentally active regulatory protein encoded by the Drosophila homeotic gene Ultrabithorax (Ubx). We show that naturally occurring binding sites for the Ubx-encoded protein contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more Ubx-encoded protein molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation in a simple yeast assay system. We found that cooperative binding is dependent on sequences outside the homeodomain, and we have identified regions predicted to form coiled coils carboxy terminal to the homeodomains of the Ubx-encoded protein and several other homeotic proteins. On the basis of our findings, we propose a multisite integrative model of homeotic protein action in which functional regulatory elements can be built from a few high-affinity sites, from many lower-affinity sites, or from sites of some intermediate number and affinity. An important corollary of this model is that even small differences in binding of homeotic proteins to individual sites could be summed to yield large overall differences in binding to multiple sites. This model is consistent with reports that homeodomain protein targets contain multiple individual binding site sequences distributed throughout sizable DNA regions. Also consistent is a recent report that sequences carboxy terminal to the Ubx homeodomain can contribute to segmental specificity.

  View details for Web of Science ID A1993MC84400033

  View details for PubMedID 8105373

  View details for PubMedCentralID PMC364756

• DIFFERENTIAL DNA-SEQUENCE RECOGNITION IS A DETERMINANT OF SPECIFICITY IN HOMEOTIC GENE-ACTION EMBO JOURNAL Ekker, S. C., VONKESSLER, D. P., Beachy, P. A. 1992; 11 (11): 4059-4072

  Abstract

  The homeotic genes of Drosophila encode transcriptional regulatory proteins that specify distinct segment identities. Previous studies have implicated the homeodomain as a major determinant of biological specificity within these proteins, but have not established the physical basis of this specificity. We show here that the homeodomains encoded by the Ultrabithorax and Deformed homeotic genes bind optimally to distinct DNA sequences and have mapped the determinants responsible for differential recognition. We further show that relative transactivation by these two proteins in a simple in vivo system can differ by nearly two orders of magnitude. Such differences in DNA sequence recognition and target activation provide a biochemical basis for at least part of the biological specificity of homeotic gene action.

  View details for Web of Science ID A1992JT32800028

  View details for PubMedID 1356765

  View details for PubMedCentralID PMC556916

• SECRETION AND LOCALIZED TRANSCRIPTION SUGGEST A ROLE IN POSITIONAL SIGNALING FOR PRODUCTS OF THE SEGMENTATION GENE HEDGEHOG CELL Lee, J. J., VONKESSLER, D. P., Parks, S., Beachy, P. A. 1992; 71 (1): 33-50

  Abstract

  The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.

  View details for Web of Science ID A1992JQ62300005

  View details for PubMedID 1394430

• OPTIMAL DNA-SEQUENCE RECOGNITION BY THE ULTRABITHORAX HOMEODOMAIN OF DROSOPHILA EMBO JOURNAL Ekker, S. C., Young, K. E., VONKESSLER, D. P., Beachy, P. A. 1991; 10 (5): 1179-1186

  Abstract

  The 61 amino acid homeodomain is conserved among members of a family of eukaryotic DNA-binding proteins that play regulatory roles in transcription and in development. We have refined a rapid method for determining optimal DNA binding sites and have applied it to a 72 amino acid peptide containing the homeodomain of the Ultrabithorax (Ubx) homeotic gene of Drosophila. The site (5'-TTAATGG-3') is tightly bound (KD approximately 7 x 10(-11) M) by the Ubx homeodomain peptide; the four central TAAT bases of this sequence play a primary role in determining the affinity of binding, with significant secondary contributions deriving from the flanking bases. Although previously defined genomic sites contain multiple TAAT sequences with flanking bases distinct from those in the optimal binding site, we have found a new binding site with seven near-perfect repeats of the optimal sequence; this site is located in the promoter region of decapentaplegic, a probable Ubx regulatory target. The presence of a TAAT motif in the binding sites for most other homeodomain proteins suggests the existence of a conserved mechanism for recognition of this core sequence, with further specificity conferred by interactions with bases flanking this core.

  View details for Web of Science ID A1991FJ82200018

  View details for PubMedID 1673656

  View details for PubMedCentralID PMC452771

• A MOLECULAR VIEW OF THE ULTRABITHORAX HOMEOTIC GENE OF DROSOPHILA TRENDS IN GENETICS Beachy, P. A. 1990; 6 (2): 46-51

  Abstract

  The pioneering work of E.B. Lewis drew attention early on to the developmental role of Ultrabithorax (Ubx), one of a group of homeotic genes that specify the distinguishing features of Drosophila segments. Recent molecular work sheds light on the genetic complexity discovered by Lewis and demonstrates that Ubx executes its developmental role by producing a family of closely related DNA-binding proteins that can function as transcription factors.

  View details for Web of Science ID A1990CM95100007

  View details for PubMedID 1970912

• AN ULTRABITHORAX PROTEIN BINDS SEQUENCES NEAR ITS OWN AND THE ANTENNAPEDIA P1 PROMOTERS CELL Beachy, P. A., Krasnow, M. A., Gavis, E. R., Hogness, D. S. 1988; 55 (6): 1069-1081

  Abstract

  The homeotic gene Ultrabithorax (Ubx), located in the bithorax complex of Drosophila, encodes a family of closely related proteins that direct the developmental fates of posterior thoracic and anterior abdominal metameres. We have purified a member of the Ubx protein family from an overproducing E. coli strain and have shown that it is sequence-specific DNA binding protein. The protein binds tightly to sequences near its own promoter and near the P1 promoter of Antenna-pedia (Antp), a homeotic gene Ubx is known to repress from genetic studies. The binding sites occur in clusters downstream of the transcription start sites, and far upstream at Antp P1. They range in size from 40 to 90 bp, and contain tandem repeats of the trinucleotide TAA or the related hexanucleotide TAA-TCG. These results suggest that the regulatory activities of Ubx are direct and are mediated by binding of Ubx proteins to promoter region sequences.

  View details for Web of Science ID A1988R562400015

  View details for PubMedID 2904838

• SEGMENTAL DISTRIBUTION OF BITHORAX COMPLEX PROTEINS DURING DROSOPHILA DEVELOPMENT NATURE Beachy, P. A., Helfand, S. L., Hogness, D. S. 1985; 313 (6003): 545-551

  Abstract

  The Ubx and bxd transcription units comprise a single functional domain in the bithorax complex of Drosophila melanogaster. The segmental distributions and nuclear localization of proteins encoded by the Ubx unit have been determined by immunofluorescence staining with antibodies raised against a fusion protein containing Ubx coding sequences. Wild-type and mutant distributions are consistent with a model in which the protein-coding functions of the domain derive from the Ubx unit and are regulated by the bxd unit.

  View details for Web of Science ID A1985ABQ6600040

  View details for PubMedID 3918274

• MOLECULAR-GENETICS OF THE BITHORAX COMPLEX IN DROSOPHILA-MELANOGASTER SCIENCE Bender, W., Akam, M., KARCH, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B., Hogness, D. S. 1983; 221 (4605): 23-29

  Abstract

  The bithorax complex in Drosophila melanogaster is a cluster of homeotic genes that specify developmental pathways for many of the body segments of the fly. The DNA of the bithorax complex has been isolated, and a region of 195,000 base pairs that covers the left half of the complex is described here. The lesions associated with many of the bithorax complex mutants have been identified, and most are due to DNA rearrangements. Most of the spontaneous mutants have insertions of a particular mobile element named "gypsy." This element affects the functions of sequences removed from the site of insertion. Mutant lesions for a given phenotypic class are distributed over large DNA distances of up to 73,000 base pairs.

  View details for Web of Science ID A1983QV59400006

  View details for PubMedID 17737996

• Probing restoration of taste receptor cell with engineered nanobodies Lu, W., Zhang, Y., Li, A., Beachy, P. A. OXFORD UNIV PRESS. 2022

  View details for Web of Science ID 000936532400047

• RNA splicing programs define tissue compartments and cell types at single-cell resolution ELIFE Olivieri, J., Dehghannasiri, R., Wang, P. L., Jang, S., de Morree, A., Tan, S. Y., Ming, J., Wu, A., Consortium, T., Quake, S. R., Krasnow, M. A., Salzman, J. 2021; 10

  View details for DOI 10.7554/eLife.70692.sa2

  View details for Web of Science ID 000715795700001

• Toward the restoration of damaged taste organs with a genetically encoded Hedgehog pathway agonist Lu, W., Sivakamasundari, V., Zhang, Y., Li, A., Beachy, P. A. OXFORD UNIV PRESS. 2021

  View details for Web of Science ID 000767787000273

• Deconstructing tumor heterogeneity: the stromal perspective. Oncotarget Vickman, R. E., Faget, D. V., Beachy, P., Beebe, D., Bhowmick, N. A., Cukierman, E., Deng, W., Granneman, J. G., Hildesheim, J., Kalluri, R., Lau, K. S., Lengyel, E., Lundeberg, J., Moscat, J., Nelson, P. S., Pietras, K., Politi, K., Pure, E., Scherz-Shouval, R., Sherman, M. H., Tuveson, D., Weeraratna, A. T., White, R. M., Wong, M. H., Woodhouse, E. C., Zheng, Y., Hayward, S. W., Stewart, S. A. 2020; 11 (40): 3621–32

  Abstract

  Significant advances have been made towards understanding the role of immune cell-tumor interplay in either suppressing or promoting tumor growth, progression, and recurrence, however, the roles of additional stromal elements, cell types and/or cell states remain ill-defined. The overarching goal of this NCI-sponsored workshop was to highlight and integrate the critical functions of non-immune stromal components in regulating tumor heterogeneity and its impact on tumor initiation, progression, and resistance to therapy. The workshop explored the opposing roles of tumor supportive versus suppressive stroma and how cellular composition and function may be altered during disease progression. It also highlighted microenvironment-centered mechanisms dictating indolence or aggressiveness of early lesions and how spatial geography impacts stromal attributes and function. The prognostic and therapeutic implications as well as potential vulnerabilities within the heterogeneous tumor microenvironment were also discussed. These broad topics were included in this workshop as an effort to identify current challenges and knowledge gaps in the field.

  View details for DOI 10.18632/oncotarget.27736

  View details for PubMedID 33088423

• A single-cell transcriptomic atlas characterizes ageing tissues in the mouse. Nature 2020

  Abstract

  Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence3, genomic instability4 and changes in the immune system2. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types.

  View details for DOI 10.1038/s41586-020-2496-1

  View details for PubMedID 32669714

• Ageing hallmarks exhibit organ-specific temporal signatures. Nature Schaum, N. n., Lehallier, B. n., Hahn, O. n., Pálovics, R. n., Hosseinzadeh, S. n., Lee, S. E., Sit, R. n., Lee, D. P., Losada, P. M., Zardeneta, M. E., Fehlmann, T. n., Webber, J. T., McGeever, A. n., Calcuttawala, K. n., Zhang, H. n., Berdnik, D. n., Mathur, V. n., Tan, W. n., Zee, A. n., Tan, M. n., Pisco, A. O., Karkanias, J. n., Neff, N. F., Keller, A. n., Darmanis, S. n., Quake, S. R., Wyss-Coray, T. n. 2020

  Abstract

  Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.

  View details for DOI 10.1038/s41586-020-2499-y

  View details for PubMedID 32669715

• Strategies for single-molecule tracking of Sonic Hedgehog delivery to the regenerative niche in adult taste buds Lu, W., Baghel, A., Beachy, P. A. OXFORD UNIV PRESS. 2019: E61–E62

  View details for Web of Science ID 000493389500165

• TARGETING THE HEDGEHOG PATHWAY IS A NOVEL THERAPEUTIC STRATEGY TO TREAT SCHISTOSOMIASIS FIBROSIS AND PORTAL HYPERTENSION Pereira, T., Vidigal, P., Voieta, I., Resende, V., Witek, R., Jegga, A., Arron, J., Madala, S., Lambertucci, J., Diehl, A., Wynn, T., Beachy, P. AMER SOC TROP MED & HYGIENE. 2019: 10–11

  View details for Web of Science ID 000507364502033

• Targeted disruption of transcriptional effector GLI2 attenuates breast tumor growth and metastasis Beachy, P., Cai, S., Ma, Y., Hatakeyama, J., Zhao, C., Stoffels, M., Verhaegh, W., van de Stolpe, A., Pegram, M. AMER ASSOC CANCER RESEARCH. 2018

  View details for Web of Science ID 000425489401310

• Role of KEAP1/NRF2 and TP53 Mutations in Lung Squamous Cell Carcinoma Development and Radiation Resistance. Cancer discovery Jeong, Y., Hoang, N. T., Lovejoy, A., Stehr, H., Newman, A. M., Gentles, A. J., Kong, W., Truong, D., Martin, S., Chaudhuri, A., Heiser, D., Zhou, L., Say, C., Carter, J. N., Hiniker, S. M., Loo, B. W., West, R. B., Beachy, P., Alizadeh, A. A., Diehn, M. 2016

  Abstract

  Lung squamous cell carcinoma (LSCC) pathogenesis remains incompletely understood, and biomarkers predicting treatment response remain lacking. Here, we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histologic and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in patients with non-small lung cancer (NSCLC) and could be noninvasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs.We developed an LSCC mouse model involving Trp53 and Keap1, which are frequently mutated in human LSCCs. In this model, ABSCs are the cell of origin of these tumors. KEAP1/NRF2 mutations increase radioresistance and predict local tumor recurrence in radiotherapy patients. Our findings are of potential clinical relevance and could lead to personalized treatment strategies for tumors with KEAP1/NRF2 mutations. Cancer Discov; 7(1); 86-101. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1.

  View details for PubMedID 27663899

• Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types CELL Loh, K. M., Chen, A., Koh, P. W., Deng, T. Z., Sinha, R., Tsai, J. M., Barkal, A. A., Shen, K. Y., Jain, R., Morganti, R. M., Shyh-Chang, N., Fernhoff, N. B., George, B. M., Wernig, G., Salomon, R. E., Chen, Z., Vogel, H., Epstein, J. A., Kundaje, A., Talbot, W. S., Beachy, P. A., Ang, L. T., Weissman, I. L. 2016; 166 (2): 451-467

  Abstract

  Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.cell.2016.06.011

  View details for PubMedID 27419872

• Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sever, N., Mann, R. K., Xu, L., Snell, W. J., Hernandez-Lara, C. I., Porter, N. A., Beachy, P. A. 2016; 113 (21): 5904-5909

  Abstract

  Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith-Lemli-Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-β-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3β,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith-Lemli-Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

  View details for DOI 10.1073/pnas.1604984113

  View details for Web of Science ID 000376779900052

  View details for PubMedID 27162362

  View details for PubMedCentralID PMC4889404

• Effects of Combined Treatment With Arsenic Trioxide and Itraconazole in Patients With Refractory Metastatic Basal Cell Carcinoma. JAMA dermatology Ally, M. S., Ransohoff, K., Sarin, K., Atwood, S. X., Rezaee, M., Bailey-Healy, I., Kim, J., Beachy, P. A., Chang, A. L., Oro, A., Tang, J. Y., Colevas, A. D. 2016; 152 (4): 452-456

  Abstract

  Tumor resistance is an emerging problem for Smoothened (SMO) inhibitor-treated metastatic basal cell carcinoma (BCC). Arsenic trioxide and itraconazole antagonize the hedgehog (HH) pathway at sites distinct from those treated by SMO inhibitors.To determine whether administration of intravenous arsenic trioxide and oral itraconazole in patients with metastatic BCC is associated with a reduction in GLI1 messenger RNA expression in tumor and/or normal skin biopsy samples.Five men with metastatic BCC who experienced relapse after SMO inhibitor treatment underwent intravenous arsenic trioxide treatment for 5 days, every 28 days, and oral itraconazole treatment on days 6 to 28. Data were collected from April 10 to November 14, 2013. Follow-up was completed on October 3, 2015, and data were analyzed from June 5 to October 6, 2015.The primary outcome was the change in messenger RNA levels of the GLI family zinc finger 1 (GLI1) gene (HH-pathway target gene) in biopsy specimens of normal skin or BCC before and after treatment. Secondary objectives were evaluation of tumor response and tolerability.Of the 5 patients (mean [SD] age, 52 [9] years; age range, 43-62 years), 3 completed 3 cycles of treatment and 2 discontinued treatment early owing to disease progression or adverse events. Adverse effects included grade 2 transaminitis and grade 4 leukopenia with a grade 3 infection. Overall, arsenic trioxide and itraconazole reduced GLI1 messenger RNA levels by 75% from baseline (P < .001). The best overall response after 3 treatment cycles was stable disease in 3 patients.Targeting the HH pathway with sequential arsenic trioxide and itraconazole treatment is a feasible treatment for metastatic BCC. Although some patients experienced stable disease for 3 months, none had tumor shrinkage, which may be owing to transient GLI1 suppression with sequential dosing. Continuous dosing may be required to fully inhibit the HH pathway and achieve clinical response.

  View details for DOI 10.1001/jamadermatol.2015.5473

  View details for PubMedID 26765315

• Localized JNK signaling regulates organ size during development ELIFE Willsey, H. R., Zheng, X., Pastor-Pareja, J. C., Willsey, A. J., Beachy, P. A., Xu, T. 2016; 5

  Abstract

  A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. Here we show that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control.

  View details for DOI 10.7554/eLife.11491

  View details for Web of Science ID 000375229600001

  View details for PubMedID 26974344

• The role of ciliary trafficking in Hedgehog receptor signaling SCIENCE SIGNALING Kim, J., Hsia, E. Y., Brigui, A., Plessis, A., Beachy, P. A., Zheng, X. 2015; 8 (379)

  Abstract

  Defects in the biogenesis of or transport through primary cilia affect Hedgehog protein signaling, and many Hedgehog pathway components traffic through or accumulate in cilia. The Hedgehog receptor Patched negatively regulates the activity and ciliary accumulation of Smoothened, a seven-transmembrane protein that is essential for transducing the Hedgehog signal. We found that this negative regulation of Smoothened required the ciliary localization of Patched, as specified either by its own cytoplasmic tail or by provision of heterologous ciliary localization signals. Surprisingly, given that Hedgehog binding promotes the exit of Patched from the cilium, we observed that an altered form of Patched that is retained in the cilium nevertheless responded to Hedgehog, resulting in Smoothened activation. Our results indicate that whereas ciliary localization of Patched is essential for suppression of Smoothened activation, the primary event enabling Smoothened activation is binding of Hedgehog to Patched, and Patched ciliary removal is secondary.

  View details for DOI 10.1126/scisignal.aaa5622

  View details for Web of Science ID 000356045100003

• Itraconazole Inhibits Enterovirus Replication by Targeting the Oxysterol-Binding Protein CELL REPORTS Strating, J. R., van der Linden, L., Albulescu, L., Bigay, J., Arita, M., Delang, L., Leyssen, P., van der Schaar, H. M., Lanke, K. H., Thibaut, H. J., Ulferts, R., Drin, G., Schlinck, N., Wubbolts, R. W., Sever, N., Head, S. A., Liu, J. O., Beachy, P. A., De Matteis, M. A., Shair, M. D., Olkkonen, V. M., Neyts, J., van Kuppeveld, F. J. 2015; 10 (4): 600-615

  View details for DOI 10.1016/j.celrep.2014.12.054

  View details for Web of Science ID 000349404100014

• Bifurcating action of Smoothened in Hedgehog signaling is mediated by Dlg5. Genes & development Chong, Y. C., Mann, R. K., Zhao, C., Kato, M., Beachy, P. A. 2015; 29 (3): 262-276

  Abstract

  Binding of the Hedgehog (Hh) protein signal to its receptor, Patched, induces accumulation of the seven-pass transmembrane protein Smoothened (Smo) within the primary cilium and of the zinc finger transcription factor Gli2 at the ciliary tip, resulting ultimately in Gli-mediated changes in nuclear gene expression. However, the mechanism by which pathway activation is communicated from Smo to Gli2 is not known. In an effort to elucidate this mechanism, we identified Dlg5 (Discs large, homolog 5) in a biochemical screen for proteins that preferentially interact with activated Smo. We found that disruption of Smo-Dlg5 interactions or depletion of endogenous Dlg5 leads to diminished Hh pathway response without a significant impact on Smo ciliary accumulation. We also found that Dlg5 is localized at the basal body, where it associates with another pathway component, Kif7. We show that Dlg5 is required for Hh-induced enrichment of Kif7 and Gli2 at the tip of the cilium but is dispensable for Gpr161 exit from the cilium and the consequent suppression of Gli3 processing into its repressor form. Our findings suggest a bifurcation of Smo activity in Hh response, with a Dlg5-independent arm for suppression of Gli repressor formation and a second arm involving Smo interaction with Dlg5 for Gli activation.

  View details for DOI 10.1101/gad.252676.114

  View details for PubMedID 25644602

  View details for PubMedCentralID PMC4318143

• Corrigendum: Identification of recurrent SMO and BRAF mutations in ameloblastomas. Nature genetics Sweeney, R. T., McClary, A. C., Myers, B. R., Biscocho, J., Neahring, L., Kwei, K. A., Qu, K., Gong, X., Ng, T., Jones, C. D., Varma, S., Odegaard, J. I., Sugiyama, T., Koyota, S., Rubin, B. P., Troxell, M. L., Pelham, R. J., Zehnder, J. L., Beachy, P. A., Pollack, J. R., West, R. B. 2014; 47 (1): 97-?

  View details for DOI 10.1038/ng0115-97b

  View details for PubMedID 25547605

• Spatially restricted Hedgehog signalling regulates HGF-induced branching of the adult prostate NATURE CELL BIOLOGY Lim, A., Shin, K., Zhao, C., Kawano, S., Beachy, P. A. 2014; 16 (12): 1135-U33

  Abstract

  Branching morphogenesis is thought to be governed by epithelial-stromal interactions, but the mechanisms underlying specification of branch location remain largely unknown. Prompted by the striking absence of Hedgehog (Hh) response at the sites of nascent buds in regenerating tubules of the adult prostate, we investigated the role of Hh signalling in adult prostate branching morphogenesis. We find that pathway activity is localized to stromal cells, and that its attenuation by genetic or pharmacologic manipulation leads to increased branching. Decreased pathway activity correlates with increased stromal production of hepatocyte growth factor (Hgf), and we show that Hgf induces epithelial tubule branching. Regulation of Hgf expression by Hh signalling is indirect, mediated by Hh-induced expression of the microRNAs miR-26a and miR-26b, which in turn downregulate expression of Hgf. Prostate tubule branching thus may be initiated from regions of low Hh pathway activity, with implications for the prostatic hyperplasia commonly observed in late adulthood.

  View details for DOI 10.1038/ncb3057

  View details for Web of Science ID 000345777300005

  View details for PubMedID 25362352

• Identification of recurrent SMO and BRAF mutations in ameloblastomas NATURE GENETICS Sweeney, R. T., McClary, A. C., Myers, B. R., Biscocho, J., Neahring, L., Kwei, K. A., Qu, K., Gong, X., Ng, T., Jones, C. D., Varma, S., Odegaard, J. I., Sugiyama, T., Koyota, S., Rubin, B. P., Troxell, M. L., Pelham, R. J., Zehnder, J. L., Beachy, P. A., Pollack, J. R., West, R. B. 2014; 46 (7): 722-725

  Abstract

  Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

  View details for DOI 10.1038/ng.2986

  View details for Web of Science ID 000338093800013

• Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma NATURE CELL BIOLOGY Shin, K., Lim, A., Odegaard, J. I., Honeycutt, J. D., Kawano, S., Hsieh, M. H., Beachy, P. A. 2014; 16 (5): 469-U194

  Abstract

  Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumour progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma in situ. Shh-expressing basal cells within this precursor lesion become tumour-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumour cell phenotype can diverge significantly from that of the cancer cell of origin.

  View details for DOI 10.1038/ncb2956

  View details for Web of Science ID 000335438400011

  View details for PubMedID 24747439

  View details for PubMedCentralID PMC4196946

• FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension. journal of clinical investigation Spiekerkoetter, E., Tian, X., Cai, J., Hopper, R. K., Sudheendra, D., Li, C. G., El-Bizri, N., Sawada, H., Haghighat, R., Chan, R., Haghighat, L., de Jesus Perez, V., Wang, L., Reddy, S., Zhao, M., Bernstein, D., Solow-Cordero, D. E., Beachy, P. A., Wandless, T. J., ten Dijke, P., Rabinovitch, M. 2013; 123 (8): 3600-3613

  Abstract

  Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

  View details for DOI 10.1172/JCI65592

  View details for PubMedID 23867624

• FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension. journal of clinical investigation Spiekerkoetter, E., Tian, X., Cai, J., Hopper, R. K., Sudheendra, D., Li, C. G., El-Bizri, N., Sawada, H., Haghighat, R., Chan, R., Haghighat, L., de Jesus Perez, V., Wang, L., Reddy, S., Zhao, M., Bernstein, D., Solow-Cordero, D. E., Beachy, P. A., Wandless, T. J., ten Dijke, P., Rabinovitch, M. 2013; 123 (8): 3600-3613

  Abstract

  Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

  View details for DOI 10.1172/JCI65592

  View details for PubMedID 23867624

• HEDGEHOG PATHWAY INHIBITION ERADICATES THE TUMOR-INITIATING CELL POPULATION IN DIPG, BUT DOES NOT PROLONG SURVIVAL 15th International Symposium on Pediatric Neuro-Oncology (ISPNO) Monje, M., Freret, M., Mitra, S., Mallick, S., Kim, J., Beachy, P. OXFORD UNIV PRESS INC. 2012: 28–28

  View details for Web of Science ID 000308394400104

• Identification of a cKit(+) Colonic Crypt Base Secretory Cell That Supports Lgr5(+) Stem Cells in Mice GASTROENTEROLOGY Rothenberg, M. E., Nusse, Y., Kalisky, T., Lee, J. J., Dalerba, P., Scheeren, F., Lobo, N., Kulkarni, S., Sim, S., Qian, D., Beachy, P. A., Pasricha, P. J., Quake, S. R., Clarke, M. F. 2012; 142 (5): 1195-?

  Abstract

  Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells.We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased.cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

  View details for DOI 10.1053/j.gastro.2012.02.006

  View details for Web of Science ID 000303113600038

  View details for PubMedID 22333952

  View details for PubMedCentralID PMC3911891

• COMPARISON OF DIPG NEUROSPHERE CELL LINES FROM THREE PATIENTS 16th Annual Scientific Meeting of the Society-for-Neuro-Oncology (SNO)/AANS/CNS Section on Tumors Monje, M., Mitra, S. S., Freret, M. E., Edwards, M. S., Weissman, I. L., Beachy, P. A. OXFORD UNIV PRESS INC. 2011: 165–165

  View details for Web of Science ID 000297026600650

• Hedgehogs, Flies, Wnts and MYCs: The Time Has Come for Many Things in Medulloblastoma JOURNAL OF CLINICAL ONCOLOGY Monje, M., Beachy, P. A., Fisher, P. G. 2011; 29 (11): 1395-1398

  View details for DOI 10.1200/JCO.2010.34.0547

  View details for Web of Science ID 000289276900016

  View details for PubMedID 21357776

• Patterning Axon Targeting of Olfactory Receptor Neurons by Coupled Hedgehog Signaling at Two Distinct Steps CELL Chou, Y., Zheng, X., Beachy, P. A., Luo, L. 2010; 142 (6): 954-966

  Abstract

  We present evidence for a coupled two-step action of Hedgehog signaling in patterning axon targeting of Drosophila olfactory receptor neurons (ORNs). In the first step, differential Hedgehog pathway activity in peripheral sensory organ precursors creates ORN populations with different levels of the Patched receptor. Different Patched levels in ORNs then determine axonal responsiveness to target-derived Hedgehog in the brain: only ORN axons that do not express high levels of Patched are responsive to and require a second step of Hedgehog signaling for target selection. Hedgehog signaling in the imaginal sensory organ precursors thus confers differential ORN responsiveness to Hedgehog-mediated axon targeting in the brain. This mechanism contributes to the spatial coordination of ORN cell bodies in the periphery and their glomerular targets in the brain. Such coupled two-step signaling may be more generally used to coordinate other spatially and temporally segregated developmental events.

  View details for DOI 10.1016/j.cell.2010.08.015

  View details for Web of Science ID 000281855000017

  View details for PubMedID 20850015

  View details for PubMedCentralID PMC3028148

• Truncating loss-of-function mutations of DISP1 contribute to holoprosencephaly-like microform features in humans HUMAN GENETICS Roessler, E., Ma, Y., Ouspenskaia, M. V., Lacbawan, F., Bendavid, C., Dubourg, C., Beachy, P. A., Muenke, M. 2009; 125 (4): 393-400

  Abstract

  Defective function of the Sonic Hedgehog (SHH) signaling pathway is the most frequent alteration underlying holoprosencephaly (HPE) or its various clinical microforms. We performed an extensive mutational analysis of the entire human DISP1 gene, required for secretion of all hedgehog ligand(s) and which maps to the HPE 10 locus of human chromosome 1q41, as a HPE candidate gene. Here, we describe two independent families with truncating mutations in human DISP1 that resemble the cardinal craniofacial and neuro-developmental features of a recently described microdeletion syndrome that includes this gene; therefore, we suggest that DISP1 function contributes substantially to both of these signs in humans. While these clinical features are consistent with common HPE microforms, especially those linked to defective signaling by Sonic Hedgehog, we have insufficient evidence so far that functionally abnormal DISP1 alleles will commonly contribute to the more severe features of typical HPE.

  View details for DOI 10.1007/s00439-009-0628-7

  View details for Web of Science ID 000265383500004

  View details for PubMedID 19184110

• Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia NATURE Zhao, C., Chen, A., Jamieson, C. H., Fereshteh, M., Abrahamsson, A., Blum, J., Kwon, H. Y., Kim, J., Chute, J. P., Rizzieri, D., Munchhof, M., VanArsdale, T., Beachy, P. A., Reya, T. 2009; 458 (7239): 776-U117

  Abstract

  Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established, its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the loss of Smoothened (Smo), an essential component of the Hh pathway, impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR-ABL1 oncoprotein. Loss of Smo causes depletion of CML stem cells--the cells that propagate the leukaemia--whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wild-type BCR-ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML might be avoided by targeting this essential stem cell maintenance pathway.

  View details for DOI 10.1038/nature07737

  View details for Web of Science ID 000265193600044

  View details for PubMedID 19169242

  View details for PubMedCentralID PMC2946231

• Targeted inhibition of hedgehog signaling by cyclopamine prodrugs for advanced prostate cancer BIOORGANIC & MEDICINAL CHEMISTRY Kumar, S. K., Roy, I., Anchoori, R. K., Fazli, S., Maitra, A., Beachy, P. A., Khan, S. R. 2008; 16 (6): 2764-2768

  Abstract

  A promising agent for use in prostate cancer therapy is the Hedgehog (Hh) signaling pathway inhibitor, cyclopamine. This compound, however, has the potential for causing serious side effects in non-tumor tissues. To minimize these bystander toxicities, we have designed and synthesized two novel peptide-cyclopamine conjugates as prostate-specific antigen (PSA)-activated prodrugs for use against prostate cancer. These prodrugs were composed of cyclopamine coupled to one of two peptides (either HSSKLQ or SSKYQ) that can be selectively cleaved by PSA, converting the mature prodrug into an active Hedgehog inhibitor within the malignant cells. Of the two prodrugs, Mu-SSKYQ-Cyclopamine was rapidly hydrolyzed, with a half-life of 3.2h, upon incubation with the PSA enzyme. Thus, modulating cyclopamine at the secondary amine with PSA-cleavable peptides is a promising strategy for developing prodrugs to target prostate cancer.

  View details for DOI 10.1016/j.bmc.2008.01.012

  View details for Web of Science ID 000255127700005

  View details for PubMedID 18249125

  View details for PubMedCentralID PMC2666345

• Disruption of the basal body comprises proteasomal function and perturbs intracellular Wnt response NATURE GENETICS Gerdes, J. M., Liu, Y., Zaghloul, N. A., Leitch, C. C., Lawson, S. S., Kato, M., Beachy, P. A., Beales, P. L., DeMartino, G. N., Fisher, S., Badano, J. L., Katsanis, N. 2007; 39 (11): 1350-1360

  Abstract

  Primary cilia and basal bodies are evolutionarily conserved organelles that mediate communication between the intracellular and extracellular environments. Here we show that bbs1, bbs4 and mkks (also known as bbs6), which encode basal body proteins, are required for convergence and extension in zebrafish and interact with wnt11 and wnt5b. Suppression of bbs1, bbs4 and mkks transcripts results in stabilization of beta-catenin with concomitant upregulation of T-cell factor (TCF)-dependent transcription in both zebrafish embryos and mammalian ciliated cells, a defect phenocopied by the silencing of the axonemal kinesin subunit KIF3A but not by chemical disruption of the cytoplasmic microtubule network. These observations are attributable partly to defective degradation by the proteasome; suppression of BBS4 leads to perturbed proteasomal targeting and concomitant accumulation of cytoplasmic beta-catenin. Cumulatively, our data indicate that the basal body is an important regulator of Wnt signal interpretation through selective proteolysis and suggest that defects in this system may contribute to phenotypes pathognomonic of human ciliopathies.

  View details for DOI 10.1038/ng.2007.12

  View details for Web of Science ID 000250575900019

  View details for PubMedID 17906624

• Misexpression of Sox9 in mouse limb bud mesenchyme induces polydactyly and rescues hypodactyly mice MATRIX BIOLOGY Akiyama, H., Stadler, H. S., Martin, J. F., Ishii, T. M., Beachy, P. A., Nakamura, T., de Crombrugghe, B. 2007; 26 (4): 224-233

  Abstract

  Our previous studies have demonstrated the essential roles of the transcription factor Sox9 in the commitment of mesenchymal cells to a chondrogenic cell lineage and in overt chondrogenesis during limb bud development. However, it remains unknown if Sox9 induces chondrogenesis in mesenchyme ectopically in vivo as a master regulator of chondrogenesis. In this study, we first generated mutant mice in which Sox9 was misexpressed in the limb bud mesenchyme. The mutant mouse embryos exhibited polydactyly in limb buds in association with ectopic expression of Sox5 and Sox6 although markers for the different axes of limb bud development showed a normal pattern of expression. Misexpression of Sox9 stimulated cell proliferation in limb bud mesenchyme, suggesting that Sox9 has a role in recruiting mesenchymal cells to mesenchymal condensation. Second, despite the facts that misexpression of Sonic hedgehog (Shh) induces polydactyly in a number of mutant mice and Shh-null mutants have severely defective cartilage elements in limb buds, misexpression of Sox9 did not restore limb bud phenotypes in Shh-null mutants. Rather, there was no expression of Sox9 in digit I of Hoxa13Hd mutant embryos, and Sox9 partially rescued hypodactyly in Hoxa13Hd mutant embryos. These results provide evidence that Sox9 induces ectopic chondrogenesis in mesenchymal cells and strongly suggest that its expression may be regulated by Hox genes during limb bud development.

  View details for DOI 10.1016/j.matbio.2006.12.002

  View details for Web of Science ID 000246755000003

  View details for PubMedID 17222543

• Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Peacock, C. D., Wang, Q., Gesell, G. S., Corcoran-Schwartz, I. M., Jones, E., Kim, J., Devereux, W. L., Rhodes, J. T., Huff, C. A., Beachy, P. A., Watkins, D. N., Matsui, W. 2007; 104 (10): 4048-4053

  Abstract

  The cancer stem cell hypothesis suggests that malignant growth depends on a subset of tumor cells with stem cell-like properties of self-renewal. Because hedgehog (Hh) signaling regulates progenitor cell fate in normal development and homeostasis, aberrant pathway activation might be involved in the maintenance of such a population in cancer. Indeed, mutational activation of the Hh pathway is associated with medulloblastoma and basal cell carcinoma; pathway activity is also critical for growth of other tumors lacking such mutations, although the mechanism of pathway activation is poorly understood. Here we study the role and mechanism of Hh pathway activation in multiple myeloma (MM), a malignancy with a well defined stem cell compartment. In this model, rare malignant progenitors capable of clonal expansion resemble B cells, whereas the much larger tumor cell population manifests a differentiated plasma cell phenotype that pathologically defines the disease. We show that the subset of MM cells that manifests Hh pathway activity is markedly concentrated within the tumor stem cell compartment. The Hh ligand promotes expansion of MM stem cells without differentiation, whereas the Hh pathway blockade, while having little or no effect on malignant plasma cell growth, markedly inhibits clonal expansion accompanied by terminal differentiation of purified MM stem cells. These data reveal that Hh pathway activation is heterogeneous across the spectrum of MM tumor stem cells and their more differentiated progeny. The potential existence of similar relationships in other adult cancers may have important biologic and clinical implications for the study of aberrant Hh signaling.

  View details for DOI 10.1073/pnas.0611682104

  View details for Web of Science ID 000244972400068

  View details for PubMedID 17360475

  View details for PubMedCentralID PMC1805487

• Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc. Natl. Acad. Sci. U.S.A. Peacock, C.D., Wang, Q., Gesell, G.S., Corcoran-Schwartz, I.M., Jones, E., Kim, J., Devereux, W.L., Rhodes, J.T., Huff, C.A., Beachy, P.A., Watkins, D.N., Matsui, W. 2007; 104: 4048-4053
• Minimizing the risk of reporting false positives in large-scale RNAi screens NATURE METHODS Echeverri, C. J., Beachy, P. A., Baum, B., Boutros, M., Buchholz, F., Chanda, S. K., Downward, J., Ellenberg, J., Fraser, A. G., Hacohen, N., Hahn, W. C., Jackson, A. L., Kiger, A., Linsley, P. S., Lum, L., Ma, Y., Mathey-Prevot, B., Root, D. E., Sabatini, D. M., Taipale, J., Perrimon, N., Bernards, R. 2006; 3 (10): 777-779

  Abstract

  Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

  View details for DOI 10.1038/nmeth1006-777

  View details for Web of Science ID 000240942600008

  View details for PubMedID 16990807

• Transcriptional activation by extradenticle in the Drosophila visceral mesoderm DEVELOPMENTAL BIOLOGY Stultz, B. G., Jackson, D. G., Mortin, M. A., Yang, X., Beachy, P. A., Hursh, D. A. 2006; 290 (2): 482-494

  Abstract

  decapentaplegic (dpp) is a direct target of Ultrabithorax (Ubx) in parasegment 7 (PS7) of the embryonic visceral mesoderm. We demonstrate that extradenticle (exd) and homothorax (hth) are also required for dpp expression in this location, as well as in PS3, at the site of the developing gastric caecae. A 420 bp element from dpp contains EXD binding sites necessary for expressing a reporter gene in both these locations. Using a specificity swap, we demonstrate that EXD directly activates this element in vivo. Activation does not require Ubx, demonstrating that EXD can activate transcription independently of homeotic proteins. Restoration is restricted to the domains of endogenous dpp expression, despite ubiquitous expression of altered specificity EXD. We demonstrate that nuclear EXD is more extensively phosphorylated than the cytoplasmic form, suggesting that EXD is a target of signal transduction by protein kinases.

  View details for DOI 10.1016/j.ydbio.2005.11.041

  View details for Web of Science ID 000235382500021

  View details for PubMedID 16403493

• Defective cerebellar response to mitogenic Hedgehog signaling in Down syndrome mice. Proc. Natl. Acad. Sci. U.S.A. Roper, R.J., Baxter, L.L., Saran, N.G., Klinedinst, D.K., Beachy, P.A., Reeves, R.H. 2006; 103: 1452-1456
• The ihog cell-surface proteins bind Hedgehog and mediate pathway activation Cell Yao, S., Lum, L., Beachy, P.A. 2006; 125: 343-357
• Structure of a heparin-dependent complex of Hedgehog and Ihog. Proc. Natl. Acad. Sci. U.S.A. McLellan, J.S., Yao, S., Zheng, X., Geisbrecht, B.V., Ghirlando, R., Beachy, P.A., Leahy, D.J. 2006; 103: 17208-17213
• Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2 SCIENCE Chen, W., Ren, X. R., Nelson, C. D., Barak, L. S., Chen, J. K., Beachy, P. A., de Sauvage, F., Lefkowitz, R. J. 2004; 306 (5705): 2257-2260

  Abstract

  Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

  View details for DOI 10.1126/science.1104135

  View details for Web of Science ID 000225950000047

  View details for PubMedID 15618519

• The SmoA1 mouse model reveals that notch signaling is critical for the growth and survival of sonic hedgehog-induced medulloblastomas CANCER RESEARCH Hallahan, A. R., Pritchard, J. I., Hansen, S., Benson, M., Stoeck, J., Hatton, B. A., Russell, T. L., Ellenbogen, R. G., Bernstein, I. D., Beachy, P. A., Olson, J. M. 2004; 64 (21): 7794-7800

  Abstract

  To develop a genetically faithful model of medulloblastoma with increased tumor incidence compared with the current best model we activated the Sonic Hedgehog (Shh) pathway by transgenically expressing a constitutively active form of Smoothened in mouse cerebellar granule neuron precursors (ND2:SmoA1 mice). This resulted in early cerebellar granule cell hyper-proliferation and a 48% incidence of medulloblastoma formation. Gene expression studies showed an increase in the known Shh targets Gli1 and Nmyc that correlated with increasing hyperplasia and tumor formation. Notch2 and the Notch target gene, HES5, were also significantly elevated in Smoothened-induced tumors showing that Shh pathway activation is sufficient to induce Notch pathway signaling. In human medulloblastomas reverse transcription-PCR for Shh and Notch targets revealed activation of both of these pathways in most tumors when compared with normal cerebellum. Notch pathway inhibition with soluble Delta ligand or gamma secretase inhibitors resulted in a marked reduction of viable cell numbers in medulloblastoma cell lines and primary tumor cultures. Treatment of mice with D283 medulloblastoma xenografts with a gamma secretase inhibitor resulted in decreased proliferation and increased apoptosis, confirming that Notch signaling contributes to human medulloblastoma proliferation and survival. Medulloblastomas in ND2:SmoA1 mice and humans have concomitant increase in Shh and Notch pathway activities, both of which contribute to tumor survival.

  View details for Web of Science ID 000224790600024

  View details for PubMedID 15520185

• Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis DEVELOPMENTAL BIOLOGY Berman, D. M., Desai, N., Wang, X., Karhadkar, S. S., Reynon, M., Abate-Shen, C., Beachy, P. A., Shen, M. M. 2004; 267 (2): 387-398

  Abstract

  Previous studies have demonstrated that the Hedgehog (Hh) signaling pathway plays a critical role in the development and patterning of many endodermally derived tissues. We have investigated the role of Sonic hedgehog (Shh) in formation of the prostate gland by examining the urogenital phenotype of Shh mutant fetuses. Consistent with earlier work reporting an essential role for Shh in prostate induction, we have found that Shh mutant fetuses display abnormal urogenital development and fail to form prostate buds. Unexpectedly, however, we have discovered that this prostate defect could be rescued by three different methods: renal grafting, explant culture in the presence of androgens, and administration of dihydrotestosterone (DHT) to pregnant mice, indicating that the prostate defect in Shh mutants is due to insufficient levels of androgens. Furthermore, we find that the inhibition of Hh pathway signaling by treatment with cyclopamine does not block prostate formation in explant culture, but instead produces morphological defects consistent with a role for Hh signaling in ductal patterning. Taken together, our studies indicate that the initial organogenesis of the prostate proceeds independently of Shh, but that Shh or other Hh ligands may play a role in subsequent events that pattern the prostate.

  View details for DOI 10.1016/j.ydbio.2003.11.018

  View details for Web of Science ID 000220351400009

  View details for PubMedID 15013801

• Differential requirements for Shh in mammary tissue and hair follicle morphogenesis DEVELOPMENTAL BIOLOGY Gallego, M. I., Beachy, P. A., Hennighausen, L., Robinson, G. W. 2002; 249 (1): 131-139

  Abstract

  Sonic Hedgehog (Shh) is a secreted morphogen that directs patterning and cellular differentiation through binding to its receptor Patched (Ptc). It is required for the development of skin-derived organs, such as hair, whiskers, and teeth. The mammary gland is a skin-derived organ that develops mainly during adult life in which Shh is expressed from puberty to lactation. We have investigated the role of Shh in mammary gland morphogenesis and differentiation by two transplantation approaches. Since Shh-null fetuses die at late embryogenesis, we transplanted Shh-null mammary anlagen into cleared fat pads and under the renal capsule of wild type host mice. Pregnancy-mediated functional differentiation of Shh-null mammary epithelium was indistinguishable from wild type transplants, while hair follicles derived from cotransplanted skin only developed in wild type transplants. Transplants of Ihh-null anlagen also developed normally. To assess the molecular consequences of Shh deletion in mammary tissue, we compared mRNA levels of patched 1, a target gene of Hedgehog signaling, in Shh-null and wild type mammary epithelial transplants. No reduction of Ptc1 transcripts was observed in Shh-null mammary tissues. Our results demonstrate that neither Shh nor Ihh is required for mammary gland morphogenesis and functional differentiation, suggesting that the two members of the Hedgehog family may have redundant function in activating the Ptc1 signaling pathway during mammary gland development.

  View details for DOI 10.1006/dbio.2002.0761

  View details for Web of Science ID 000177924600011

  View details for PubMedID 12217324

• Manifestation of the limb prepattern: Limb development in the absence of sonic hedgehog function DEVELOPMENTAL BIOLOGY Chiang, C., Litingtung, Y., Harris, M. P., Simandl, B. K., Li, Y., Beachy, P. A., Fallon, J. F. 2001; 236 (2): 421-435

  Abstract

  The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.

  View details for Web of Science ID 000170497400013

  View details for PubMedID 11476582

• Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 during craniofacial morphogenesis DEVELOPMENTAL DYNAMICS Munoz-Sanjuan, I., Cooper, M. K., Beachy, P. A., Fallon, J. F., Nathans, J. 2001; 220 (3): 238-245

  Abstract

  Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during chicken craniofacial development. cFHF-4 is expressed in the mesenchyme of the frontonasal process, and in the mesenchyme and ectoderm of the mandibular processes. The expression of cFHF-4 and other genes implicated in facial patterning have been analyzed in talpid(2) embryos or in the presence of exogenous retinoic acid. Talpid(2) mutants show abnormal patterns of gene expression, including up-regulation of cFHF-4 in the developing face, which correlate with defects in cartilage formation. By contrast, expression of cFHF-4 in the developing face is strongly downregulated by teratogenic doses of all-trans retinoic acid in a dose-dependent manner. Low levels of retinoic acid that produce distal upper beak truncations do not affect cShh, c-Patched-1, or c-Bmp-2 expression in the face, but downregulate cFHF-4 in the frontonasal process.

  View details for Web of Science ID 000167131400005

  View details for PubMedID 11241832

• Genetics of ventral forebrain development and holoprosencephaly CURRENT OPINION IN GENETICS & DEVELOPMENT Muenke, M., Beachy, P. A. 2000; 10 (3): 262-269

  Abstract

  The disease holoprosencephaly is the basis of the most common structural anomaly of the developing forebrain in humans. Numerous teratogens when administered during early gastrulation, have been associated with this condition. Recent studies have characterized molecules expressed in the prechordal plate which are critical for normal brain formation. Perturbation of signaling pathways involving these molecules have been shown to cause holoprosencephaly in humans and other organisms.

  View details for Web of Science ID 000087349400004

  View details for PubMedID 10826992

• Essential role for Sonic hedgehog during hair follicle morphogenesis DEVELOPMENTAL BIOLOGY Chiang, C., Swan, R. Z., Grachtchouk, M., Bolinger, M., Ying, L. T., Robertson, E. K., Cooper, M. K., Gaffield, W., Westphal, H., Beachy, P. A., Dlugosz, A. A. 1999; 205 (1): 1-9

  Abstract

  The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression of Gli1 and Ptc1 was reduced in Shh -/- dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development.

  View details for Web of Science ID 000078367800001

  View details for PubMedID 9882493

• Patterning of the embryonic forebrain CURRENT OPINION IN NEUROBIOLOGY Rubenstein, J. L., Beachy, P. A. 1998; 8 (1): 18-26

  Abstract

  The vertebrate forebrain is derived from the anterior neural plate, where anteroposterior, dorsoventral and local patterning mechanisms specify regional identify. The recent identification of genetic regulators of these processes has opened the way to elucidating how the major forebrain regions (i.e. cerebral cortex, basal ganglia, thalamus, and hypothalamus) are formed, and how molecular lesions in these processes cause human birth defects.

  View details for Web of Science ID 000072773400002

  View details for PubMedID 9568388

• Mediation of Sonic Hedgehog induced expression of COUP-TFII by a protein phosphatase SCIENCE Krishnan, V., Pereira, F. A., Qiu, Y., Chen, C. H., Beachy, P. A., Tsai, S. Y., Tsai, M. J. 1997; 278 (5345): 1947-1950

  Abstract

  A Sonic hedgehog (Shh) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter that binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway.

  View details for Web of Science ID A1997YL00200035

  View details for PubMedID 9395397

• pH-dependent enchancement of DNA binding by the ultrabithorax homeodomain BIOCHEMISTRY Li, L. K., VONKESSLER, D., Beachy, P. A., Matthews, K. S. 1996; 35 (30): 9832-9839

  Abstract

  Ultrabithorax (Ubx) and Deformed (Dfd) proteins of Drosophila melanogaster contain homeodomains (HD) that are structurally similar and recognize similar DNA sequences, despite functionally distinct genetic regulatory roles for Ubx and Dfd. We report in the present study that Ubx-HD binding to a single optimal target site displayed significantly increased affinity and higher salt concentration dependence at lower pH, while Dfd-HD binding to DNA was unaffected by pH. Results from studies of chimeric Ubx-Dfd homeodomains showed that the N- and C-terminal regions of the Ubx-HD are required for this pH dependence. The increase in binding affinity at lower pH was greater for the Ubx optimal binding site than for other DNA binding sites, indicating that subtle sequence alterations in DNA binding sites may influence pH-dependent behavior. These data demonstrate enhanced DNA binding affinity at lower pH for the Ubx-HD in vitro and suggest the potential for significant discrimination of DNA binding sites in vivo.

  View details for Web of Science ID A1996VA02500024

  View details for PubMedID 8703957

• Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Szakmary, A., Huang, S. M., Chang, D. T., Beachy, P. A., Sander, M. 1996; 93 (4): 1607-1612

  Abstract

  Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

  View details for Web of Science ID A1996TW69800048

  View details for PubMedID 8643678

• STRUCTURE AND EXPRESSION OF A FAMILY OF ULTRABITHORAX MESSENGER-RNAS GENERATED BY ALTERNATIVE SPLICING AND POLYADENYLATION IN DROSOPHILA GENES & DEVELOPMENT Kornfeld, K., Saint, R. B., Beachy, P. A., Harte, P. J., PEATTIE, D. A., Hogness, D. S. 1989; 3 (2): 243-258

  Abstract

  The 77-kb primary transcript of the homeotic Ultrabithorax (Ubx) gene is alternatively spliced to yield at least five different coding regions. Each is restricted to either a 3.2- or a 4.3-kb size class generated by alternative polyadenylation. The pathways for splicing and polyadenylation are therefore coordinately regulated, and because the relative abundance of the respective mRNAs varies throughout development, these pathways also appear to be developmentally regulated. Translation of these mRNAs yields a family of Ubx proteins characterized by constant amino- and carboxy-proximal regions of 247 and 99 amino acid residues, respectively. Members of this family are distinguished by a short variable region that links the constant regions and consists of different combinations of three optional elements of 9, 17, and 17 residues. Only four amino acid residues separate this variable region from the 60-residue homeo domain of the carboxy-terminal constant region. This proximity suggests that functional differences among the Ubx proteins derive from the differential effects of their variable regions on the DNA-binding capacity of the homeo domain. An argument is made that these functional differences are tissue specific.

  View details for Web of Science ID A1989T437900011

  View details for PubMedID 2565858

• REGULATION AND PRODUCTS OF THE UBX DOMAIN OF THE BITHORAX COMPLEX COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Hogness, D. S., Lipshitz, H. D., Beachy, P. A., PEATTIE, D. A., Saint, R. B., GOLDSCHMIDTCLERMONT, M., Harte, P. J., Gavis, E. R., Helfand, S. L. 1985; 50: 181-194

  View details for Web of Science ID A1985C628800023

  View details for PubMedID 3938361