Mark Krasnow

All Publications

• Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung. The Journal of experimental medicine Wu, T. T., Travaglini, K. J., Rustagi, A., Xu, D., Zhang, Y., Andronov, L., Jang, S., Gillich, A., Dehghannasiri, R., Martinez-Colon, G. J., Beck, A., Liu, D. D., Wilk, A. J., Morri, M., Trope, W. L., Bierman, R., Weissman, I. L., Shrager, J. B., Quake, S. R., Kuo, C. S., Salzman, J., Moerner, W. E., Kim, P. S., Blish, C. A., Krasnow, M. A. 2024; 221 (6)

  Abstract

  Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

  View details for DOI 10.1084/jem.20232192

  View details for PubMedID 38597954

• Neuroendocrinology of the lung revealed by single-cell RNA sequencing. eLife Kuo, C. S., Darmanis, S., Diaz de Arce, A., Liu, Y., Almanzar, N., Wu, T. T., Quake, S. R., Krasnow, M. A. 2022; 11

  Abstract

  Pulmonary neuroendocrine cells (PNECs) are sensory epithelial cells that transmit airway status to the brain via sensory neurons and locally via calcitonin gene-related peptide (CGRP) and γ- aminobutyric acid (GABA). Several other neuropeptides and neurotransmitters have been detected in various species, but the number, targets, functions, and conservation of PNEC signals are largely unknown. We used scRNAseq to profile hundreds of the rare mouse and human PNECs. This revealed over 40 PNEC neuropeptide and peptide hormone genes, most cells expressing unique combinations of 5-18 genes. Peptides are packaged in separate vesicles, their release presumably regulated by the distinct, multimodal combinations of sensors we show are expressed by each PNEC. Expression of the peptide receptors predicts an array of local cell targets, and we show the new PNEC signal angiotensin directly activates one subtype of innervating sensory neuron. Many signals lack lung targets so may have endocrine activity like those of PNEC-derived carcinoid tumors. PNECs are an extraordinarily rich and diverse signaling hub rivaling the enteroendocrine system.

  View details for DOI 10.7554/eLife.78216

  View details for PubMedID 36469459

• Molecularly defined circuits for cardiovascular and cardiopulmonary control. Nature Veerakumar, A., Yung, A. R., Liu, Y., Krasnow, M. A. 2022

  Abstract

  The sympathetic and parasympathetic nervous systems regulate the activities of internal organs1, but the molecular and functional diversity of their constituent neurons and circuits remains largely unknown. Here we use retrograde neuronal tracing, single-cell RNA sequencing, optogenetics and physiological experiments to dissect the cardiac parasympathetic control circuit in mice. We show that cardiac-innervating neurons in the brainstem nucleus ambiguus (Amb) are comprised of two molecularly, anatomically and functionally distinct subtypes. The first, which we call ambiguus cardiovascular (ACV) neurons (approximately 35 neurons per Amb), define the classical cardiac parasympathetic circuit. They selectively innervate a subset of cardiac parasympathetic ganglion neurons and mediate the baroreceptor reflex, slowing heart rate and atrioventricular node conduction in response to increased blood pressure. The other, ambiguus cardiopulmonary (ACP) neurons (approximately 15 neurons per Amb) innervate cardiac ganglion neurons intermingled with and functionally indistinguishable from those innervated by ACV neurons. ACP neurons also innervate most or all lung parasympathetic ganglion neurons-clonal labelling shows that individual ACP neurons innervate both organs. ACP neurons mediate the dive reflex, the simultaneous bradycardia and bronchoconstriction that follows water immersion. Thus, parasympathetic control of the heart is organized into two parallel circuits, one that selectively controls cardiac function (ACV circuit) and another that coordinates cardiac and pulmonary function (ACP circuit). This new understanding of cardiac control has implications for treating cardiac and pulmonary diseases and for elucidating the control and coordination circuits of other organs.

  View details for DOI 10.1038/s41586-022-04760-8

  View details for PubMedID 35650438

• The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science (New York, N.Y.) Jones, R. C., Karkanias, J., Krasnow, M. A., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Pisco, A. O., Tan, S. Y., Travaglini, K. J., Xu, C., Alcántara-Hernández, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Crasta, S., Culver, R. N., Cvijović, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kolluru, S., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W. J., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Morri, M., Mrouj, K., Mukherjee, S., Muser, T., Neuhöfer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Pisco, A. O., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L. C., Subramaniam, V. R., Swarup, A., Swift, M., Travaglini, K. J., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgó, E., Martin, B. A., Ozawa, M. G., Silva, O., Tan, S. Y., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Detweiler, A. M., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., de Morree, A., Olivieri, J. E., Park, M., Pisco, A. O., Ravikumar, N., Salzman, J., Stanley, G., Swift, M., Tan, M., Tan, W., Tarashansky, A. J., Vanheusden, R., Vorperian, S. K., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Alcántara-Hernández, M., Antony, J., Chan, C. K., Chang, C. A., Colville, A., Crasta, S., Culver, R., Dethlefsen, L., Ezran, C., Gillich, A., Hang, Y., Ho, P. Y., Irwin, J. C., Jang, S., Kershner, A. M., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Liu, S., Loeb, G. B., Lu, W. J., Maltzman, J. S., Metzger, R. J., de Morree, A., Neuhöfer, P., Perez, K., Phansalkar, R., Qi, Z., Rao, P., Raquer-McKay, H., Sasagawa, K., Scott, B., Sinha, R., Song, H., Spencer, S. P., Swarup, A., Swift, M., Travaglini, K. J., Trimm, E., Veizades, S., Vijayakumar, S., Wang, B., Wang, W., Winkler, J., Xie, J., Yung, A. R., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Krasnow, M., Kuo, C. S., Nguyen, P., Quake, S. R., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wang, B., Wu, A., Wu, S. M., Wyss-Coray, T. 2022; 376 (6594): eabl4896

  Abstract

  Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

  View details for DOI 10.1126/science.abl4896

  View details for PubMedID 35549404

• A molecular cell atlas of the human lung from single-cell RNA sequencing. Nature Travaglini, K. J., Nabhan, A. N., Penland, L., Sinha, R., Gillich, A., Sit, R. V., Chang, S., Conley, S. D., Mori, Y., Seita, J., Berry, G. J., Shrager, J. B., Metzger, R. J., Kuo, C. S., Neff, N., Weissman, I. L., Quake, S. R., Krasnow, M. A. 2020

  Abstract

  Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles1-9, it has been difficult to systematically identify and localize all molecularcell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution.

  View details for DOI 10.1038/s41586-020-2922-4

  View details for PubMedID 33208946

• Brain Circuit of Claustrophobia-like Behavior in Mice Identified by Upstream Tracing of Sighing. Cell reports Li, P. n., Li, S. B., Wang, X. n., Phillips, C. D., Schwarz, L. A., Luo, L. n., de Lecea, L. n., Krasnow, M. A. 2020; 31 (11): 107779

  Abstract

  Emotions are distinct patterns of behavioral and physiological responses triggered by stimuli that induce different brain states. Elucidating the circuits is difficult because of challenges in interrogating emotional brain states and their complex outputs. Here, we leverage the recent discovery in mice of a neural circuit for sighing, a simple, quantifiable output of various emotions. We show that mouse confinement triggers sighing, and this "claustrophobic" sighing, but not accompanying tachypnea, requires the same medullary neuromedin B (Nmb)-expressing neurons as physiological sighing. Retrograde tracing from the Nmb neurons identified 12 forebrain centers providing presynaptic input, including hypocretin (Hcrt)-expressing lateral hypothalamic neurons. Confinement activates Hcrt neurons, and optogenetic activation induces sighing and tachypnea whereas pharmacologic inhibition suppresses both responses. The effect on sighing is mediated by HCRT directly on Nmbneurons. We propose that this HCRT-NMB neuropeptide relay circuit mediates claustrophobic sighing and that activated Hcrt neurons are a claustrophobia brain state that directly controls claustrophobic outputs.

  View details for DOI 10.1016/j.celrep.2020.107779

  View details for PubMedID 32553161

• Capillary cell-type specialization in the alveolus. Nature Gillich, A. n., Zhang, F. n., Farmer, C. G., Travaglini, K. J., Tan, S. Y., Gu, M. n., Zhou, B. n., Feinstein, J. A., Krasnow, M. A., Metzger, R. J. 2020

  Abstract

  In the mammalian lung, an apparently homogenous mesh of capillary vessels surrounds each alveolus, forming the vast respiratory surface across which oxygen transfers to the blood1. Here we use single-cell analysis to elucidate the cell types, development, renewal and evolution of the alveolar capillary endothelium. We show that alveolar capillaries are mosaics; similar to the epithelium that lines the alveolus, the alveolar endothelium is made up of two intermingled cell types, with complex 'Swiss-cheese'-like morphologies and distinct functions. The first cell type, which we term the 'aerocyte', is specialized for gas exchange and the trafficking of leukocytes, and is unique to the lung. The other cell type, termed gCap ('general' capillary), is specialized to regulate vasomotor tone, and functions as a stem/progenitor cell in capillary homeostasis and repair. The two cell types develop from bipotent progenitors, mature gradually and are affected differently in disease and during ageing. This cell-type specialization is conserved between mouse and human lungs but is not found in alligator or turtle lungs, suggesting it arose during the evolution of the mammalian lung. The discovery of cell type specialization in alveolar capillaries transforms our understanding of the structure, function, regulation and maintenance of the air-blood barrier and gas exchange in health, disease and evolution.

  View details for DOI 10.1038/s41586-020-2822-7

  View details for PubMedID 33057196

• Rare Pulmonary Neuroendocrine Cells Are Stem Cells Regulated by Rb, p53, and Notch. Cell Ouadah, Y. n., Rojas, E. R., Riordan, D. P., Capostagno, S. n., Kuo, C. S., Krasnow, M. A. 2019; 179 (2): 403–16.e23

  Abstract

  Pulmonary neuroendocrine (NE) cells are neurosensory cells sparsely distributed throughout the bronchial epithelium, many in innervated clusters of 20-30 cells. Following lung injury, NE cells proliferate and generate other cell types to promote epithelial repair. Here, we show that only rare NE cells, typically 2-4 per cluster, function as stem cells. These fully differentiated cells display features of classical stem cells. Most proliferate (self-renew) following injury, and some migrate into the injured area. A week later, individual cells, often just one per cluster, lose NE identity (deprogram), transit amplify, and reprogram to other fates, creating large clonal repair patches. Small cell lung cancer (SCLC) tumor suppressors regulate the stem cells: Rb and p53 suppress self-renewal, whereas Notch marks the stem cells and initiates deprogramming and transit amplification. We propose that NE stem cells give rise to SCLC, and transformation results from constitutive activation of stem cell renewal and inhibition of deprogramming.

  View details for DOI 10.1016/j.cell.2019.09.010

  View details for PubMedID 31585080

• Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science (New York, N.Y.) Nabhan, A. n., Brownfield, D. G., Harbury, P. B., Krasnow, M. A., Desai, T. J. 2018

  Abstract

  Alveoli, the lung's respiratory units, are tiny sacs where oxygen enters the bloodstream. They are lined by flat AT1 cells, which mediate gas exchange, and AT2 cells, which secret surfactant. Rare AT2s also function as alveolar stem cells. We show that AT2 lung stem cells display active Wnt signaling and many of them are near single, Wnt-expressing fibroblasts. Blocking Wnt secretion depletes these stem cells. Daughter cells leaving the Wnt niche transdifferentiate into AT1s: maintaining Wnt signaling prevents transdifferentiation whereas abrogating Wnt signaling promotes it. Injury induces AT2 autocrine Wnts, recruiting 'bulk' AT2s as progenitors. Thus, individual AT2 stem cells reside in single cell fibroblast niches providing juxtacrine Wnts that maintain them, whereas injury induces autocrine Wnts that transiently expand the progenitor pool. This simple niche maintains the gas exchange surface, and is coopted in cancer.

  View details for PubMedID 29420258

• The Mouse Lemur, a Genetic Model Organism for Primate Biology, Behavior, and Health. Genetics Ezran, C., Karanewsky, C. J., Pendleton, J. L., Sholtz, A., Krasnow, M. R., Willick, J., Razafindrakoto, A., Zohdy, S., Albertelli, M. A., Krasnow, M. A. 2017; 206 (2): 651-664

  Abstract

  Systematic genetic studies of a handful of diverse organisms over the past 50 years have transformed our understanding of biology. However, many aspects of primate biology, behavior, and disease are absent or poorly modeled in any of the current genetic model organisms including mice. We surveyed the animal kingdom to find other animals with advantages similar to mice that might better exemplify primate biology, and identified mouse lemurs (Microcebus spp.) as the outstanding candidate. Mouse lemurs are prosimian primates, roughly half the genetic distance between mice and humans. They are the smallest, fastest developing, and among the most prolific and abundant primates in the world, distributed throughout the island of Madagascar, many in separate breeding populations due to habitat destruction. Their physiology, behavior, and phylogeny have been studied for decades in laboratory colonies in Europe and in field studies in Malagasy rainforests, and a high quality reference genome sequence has recently been completed. To initiate a classical genetic approach, we developed a deep phenotyping protocol and have screened hundreds of laboratory and wild mouse lemurs for interesting phenotypes and begun mapping the underlying mutations, in collaboration with leading mouse lemur biologists. We also seek to establish a mouse lemur gene "knockout" library by sequencing the genomes of thousands of mouse lemurs to identify null alleles in most genes from the large pool of natural genetic variants. As part of this effort, we have begun a citizen science project in which students across Madagascar explore the remarkable biology around their schools, including longitudinal studies of the local mouse lemurs. We hope this work spawns a new model organism and cultivates a deep genetic understanding of primate biology and health. We also hope it establishes a new and ethical method of genetics that bridges biological, behavioral, medical, and conservation disciplines, while providing an example of how hands-on science education can help transform developing countries.

  View details for DOI 10.1534/genetics.116.199448

  View details for PubMedID 28592502

• Breathing control center neurons that promote arousal in mice SCIENCE Yackle, K., Schwarz, L. A., Kam, K., Sorokin, J. M., Huguenard, J. R., Feldman, J. L., Luo, L., Krasnow, M. A. 2017; 355 (6332): 1411-1415

  Abstract

  Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9/Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain.

  View details for DOI 10.1126/science.aai7984

  View details for Web of Science ID 000397809500040

  View details for PubMedID 28360327

• The peptidergic control circuit for sighing NATURE Li, P., Janczewski, W. A., Yackle, K., Kam, K., Pagliardini, S., Krasnow, M. A., Feldman, J. L. 2016; 530 (7590): 293-?

  Abstract

  Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.

  View details for DOI 10.1038/nature16964

  View details for Web of Science ID 000370327100028

  View details for PubMedID 26855425

  View details for PubMedCentralID PMC4852886

• Oxygen regulation of breathing through an olfactory receptor activated by lactate NATURE Chang, A. J., Ortega, F. E., Riegler, J., Adison, D. V., Krasnow, M. A. 2015; 527 (7577): 240-?

  Abstract

  Animals have evolved homeostatic responses to changes in oxygen availability that act on different timescales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long-term responses to low oxygen (hypoxia) has been established, the pathway that mediates acute responses to hypoxia in mammals is not well understood. Here we show that the olfactory receptor gene Olfr78 is highly and selectively expressed in oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation that monitors blood oxygen and stimulates breathing within seconds when oxygen declines. Olfr78 mutants fail to increase ventilation in hypoxia but respond normally to hypercapnia. Glomus cells are present in normal numbers and appear structurally intact, but hypoxia-induced carotid body activity is diminished. Lactate, a metabolite that rapidly accumulates in hypoxia and induces hyperventilation, activates Olfr78 in heterologous expression experiments, induces calcium transients in glomus cells, and stimulates carotid sinus nerve activity through Olfr78. We propose that, in addition to its role in olfaction, Olfr78 acts as a hypoxia sensor in the breathing circuit by sensing lactate produced when oxygen levels decline.

  View details for DOI 10.1038/nature15721

  View details for Web of Science ID 000364396700046

  View details for PubMedID 26560302

  View details for PubMedCentralID PMC4765808

• Formation of a Neurosensory Organ by Epithelial Cell Slithering CELL Kuo, C. S., Krasnow, M. A. 2015; 163 (2): 394-405

  Abstract

  Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic.

  View details for DOI 10.1016/j.cell.2015.09.021

  View details for Web of Science ID 000362952700016

  View details for PubMedID 26435104

  View details for PubMedCentralID PMC4597318

• Subcellular Trafficking of FGF Controls Tracheal Invasion of Drosophila Flight Muscle CELL Peterson, S. J., Krasnow, M. A. 2015; 160 (1-2): 313-323

  Abstract

  To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell.

  View details for DOI 10.1016/j.cell.2014.11.043

  View details for Web of Science ID 000347923200027

  View details for PubMedID 25557078

• Mesenchymal cells. Defining a mesenchymal progenitor niche at single-cell resolution. Science Kumar, M. E., Bogard, P. E., Espinoza, F. H., Menke, D. B., Kingsley, D. M., Krasnow, M. A. 2014; 346 (6211)

  Abstract

  Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here, we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication.

  View details for DOI 10.1126/science.1258810

  View details for PubMedID 25395543

  View details for PubMedCentralID PMC4269943

• Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature Treutlein, B., Brownfield, D. G., Wu, A. R., Neff, N. F., Mantalas, G. L., Espinoza, F. H., Desai, T. J., Krasnow, M. A., Quake, S. R. 2014; 509 (7500): 371-375

  Abstract

  The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

  View details for DOI 10.1038/nature13173

  View details for PubMedID 24739965

• Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature Desai, T. J., Brownfield, D. G., Krasnow, M. A. 2014; 507 (7491): 190-194

  Abstract

  Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate.

  View details for DOI 10.1038/nature12930

  View details for PubMedID 24499815

• Progenitor Outgrowth from the Niche in Drosophila Trachea Is Guided by FGF from Decaying Branches SCIENCE Chen, F., Krasnow, M. A. 2014; 343 (6167): 186-189

  Abstract

  Although there has been progress identifying adult stem and progenitor cells and the signals that control their proliferation and differentiation, little is known about the substrates and signals that guide them out of their niche. By examining Drosophila tracheal outgrowth during metamorphosis, we show that progenitors follow a stereotyped path out of the niche, tracking along a subset of tracheal branches destined for destruction. The embryonic tracheal inducer branchless FGF (fibroblast growth factor) is expressed dynamically just ahead of progenitor outgrowth in decaying branches. Knockdown of branchless abrogates progenitor outgrowth, whereas misexpression redirects it. Thus, reactivation of an embryonic tracheal inducer in decaying branches directs outgrowth of progenitors that replace them. This explains how the structure of a newly generated tissue is coordinated with that of the old.

  View details for DOI 10.1126/science.1241442

  View details for Web of Science ID 000329440800043

  View details for PubMedID 24408434

• Radial Construction of an Arterial Wall DEVELOPMENTAL CELL Greif, D. M., Kumar, M., Lighthouse, J. K., Hum, J., An, A., Ding, L., Red-Horse, K., Espinoza, F. H., Olson, L., Offermanns, S., Krasnow, M. A. 2012; 23 (3): 482-493

  Abstract

  Some of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding of these diseases, but little is known about how concentric layers of muscle cells and the outer adventitial layer are assembled and patterned around endothelial tubes. Using histochemical, clonal, and genetic analysis in mice, here we show that the pulmonary artery wall is constructed radially, from the inside out, by two separate but coordinated processes. One is sequential induction of successive cell layers from surrounding mesenchyme. The other is controlled invasion of outer layers by inner layer cells through developmentally regulated cell reorientation and radial migration. We propose that a radial signal gradient controls these processes and provide evidence that PDGF-B and at least one other signal contribute. Modulation of such radial signaling pathways may underlie vessel-specific differences and pathological changes in arterial wall size and structure.

  View details for DOI 10.1016/j.devcel.2012.07.009

  View details for Web of Science ID 000308776400007

  View details for PubMedID 22975322

  View details for PubMedCentralID PMC3500096

• Coronary arteries form by developmental reprogramming of venous cells NATURE Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, M. A. 2010; 464 (7288): 549-U100

  Abstract

  Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus-the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart.

  View details for DOI 10.1038/nature08873

  View details for Web of Science ID 000275974200039

  View details for PubMedID 20336138

  View details for PubMedCentralID PMC2924433

• Dual origin of tissue-specific progenitor cells in Drosophila tracheal remodeling SCIENCE Weaver, M., Krasnow, M. A. 2008; 321 (5895): 1496-1499

  Abstract

  During Drosophila metamorphosis, most larval cells die. Pupal and adult tissues form from imaginal cells, tissue-specific progenitors allocated in embryogenesis that remain quiescent during embryonic and larval life. Clonal analysis and fate mapping of single, identified cells show that tracheal system remodeling at metamorphosis involves a classical imaginal cell population and a population of differentiated, functional larval tracheal cells that reenter the cell cycle and regain developmental potency. In late larvae, both populations are activated and proliferate, spread over and replace old branches, and diversify into various stalk and coiled tracheolar cells under control of fibroblast growth factor signaling. Thus, Drosophila pupal/adult tissue progenitors can arise both by early allocation of multipotent cells and late return of differentiated cells to a multipotent state, even within a single tissue.

  View details for DOI 10.1126/science.1158712

  View details for Web of Science ID 000259121800043

  View details for PubMedID 18669822

• The branching programme of mouse lung development NATURE Metzger, R. J., Klein, O. D., Martin, G. R., Krasnow, M. A. 2008; 453 (7196): 745-U1

  Abstract

  Mammalian lungs are branched networks containing thousands to millions of airways arrayed in intricate patterns that are crucial for respiration. How such trees are generated during development, and how the developmental patterning information is encoded, have long fascinated biologists and mathematicians. However, models have been limited by a lack of information on the normal sequence and pattern of branching events. Here we present the complete three-dimensional branching pattern and lineage of the mouse bronchial tree, reconstructed from an analysis of hundreds of developmental intermediates. The branching process is remarkably stereotyped and elegant: the tree is generated by three geometrically simple local modes of branching used in three different orders throughout the lung. We propose that each mode of branching is controlled by a genetically encoded subroutine, a series of local patterning and morphogenesis operations, which are themselves controlled by a more global master routine. We show that this hierarchical and modular programme is genetically tractable, and it is ideally suited to encoding and evolving the complex networks of the lung and other branched organs.

  View details for DOI 10.1038/nature07005

  View details for Web of Science ID 000256415300035

  View details for PubMedID 18463632

  View details for PubMedCentralID PMC2892995

• Social interactions among epithelial cells during tracheal branching morphogenesis NATURE Ghabrial, A. S., Krasnow, M. A. 2006; 441 (7094): 746-749

  Abstract

  Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Here we use genetic mosaic analysis in Drosophila melanogaster to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them.

  View details for DOI 10.1038/nature04829

  View details for Web of Science ID 000238095100053

  View details for PubMedID 16760977

• Tube morphogenesis: Making and shaping biological tubes CELL Lubarsky, B., Krasnow, M. A. 2003; 112 (1): 19-28

  Abstract

  Many organs are composed of epithelial tubes that transport vital fluids. Such tubular organs develop in many different ways and generate tubes of widely varying sizes and structures, but always with the apical epithelial surface lining the lumen. We describe recent progress in several diverse cell culture and genetic models of tube morphogenesis, which suggest apical membrane biogenesis, vesicle fusion, and secretion play central roles in tube formation and growth. We propose a unifying mechanism of tube morphogenesis that has been modified to create tube diversity and describe how defects in the tube size-sensing step can lead to polycystic kidney disease.

  View details for Web of Science ID 000181191500003

  View details for PubMedID 12526790

• Branching morphogenesis of the Drosophila tracheal system ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Ghabrial, A., Luschnig, S., Metzstein, M. M., Krasnow, M. A. 2003; 19: 623-647

  Abstract

  Many organs including the mammalian lung and vascular system consist of branched tubular networks that transport essential gases or fluids, but the genetic programs that control the development of these complex three-dimensional structures are not well understood. The Drosophila melanogaster tracheal (respiratory) system is a network of interconnected epithelial tubes that transports oxygen and other gases in the body and provides a paradigm of branching morphogenesis. It develops by sequential sprouting of primary, secondary, and terminal branches from an epithelial sac of approximately 80 cells in each body segment of the embryo. Mapping of the cell movements and shape changes during the sprouting process has revealed that distinct mechanisms of epithelial migration and tube formation are used at each stage of branching. Genetic dissection of the process has identified a general program in which a fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) are used repeatedly to control branch budding and outgrowth. At each stage of branching, the mechanisms controlling FGF expression and the downstream signal transduction pathway change, altering the pattern and structure of the branches that form. During terminal branching, FGF expression is regulated by hypoxia, ensuring that tracheal structure matches cellular oxygen need. A branch diversification program operates in parallel to the general budding program: Regional signals locally modify the general program, conferring specific structural features and other properties on individual branches, such as their substrate outgrowth preferences, differences in tube size and shape, and the ability to fuse to other branches to interconnect the network.

  View details for DOI 10.1146/annurev.cellbio.19.031403.160043

  View details for Web of Science ID 000186922500025

  View details for PubMedID 14570584

• Development - Genetic control of branching morphogenesis SCIENCE Metzger, R. J., Krasnow, M. A. 1999; 284 (5420): 1635-1639

  Abstract

  The genetic programs that direct formation of the treelike branching structures of two animal organs have begun to be elucidated. In both the developing Drosophila tracheal (respiratory) system and mammalian lung, a fibroblast growth factor (FGF) signaling pathway is reiteratively used to pattern successive rounds of branching. The initial pattern of signaling appears to be established by early, more global embryonic patterning systems. The FGF pathway is then modified at each stage of branching by genetic feedback controls and other signals to give distinct branching outcomes. The reiterative use of a signaling pathway by both insects and mammals suggests a general scheme for patterning branching morphogenesis.

  View details for Web of Science ID 000080668300034

• An organism-wide atlas of hormonal signaling based on the mouse lemur single-cell transcriptome. Nature communications Liu, S., Ezran, C., Wang, M. F., Li, Z., Awayan, K., Long, J. Z., De Vlaminck, I., Wang, S., Epelbaum, J., Kuo, C. S., Terrien, J., Krasnow, M. A., Ferrell, J. E. 2024; 15 (1): 2188

  Abstract

  Hormones mediate long-range cell communication and play vital roles in physiology, metabolism, and health. Traditionally, endocrinologists have focused on one hormone or organ system at a time. Yet, hormone signaling by its very nature connects cells of different organs and involves crosstalk of different hormones. Here, we leverage the organism-wide single cell transcriptional atlas of a non-human primate, the mouse lemur (Microcebus murinus), to systematically map source and target cells for 84 classes of hormones. This work uncovers previously-uncharacterized sites of hormone regulation, and shows that the hormonal signaling network is densely connected, decentralized, and rich in feedback loops. Evolutionary comparisons of hormonal genes and their expression patterns show that mouse lemur better models human hormonal signaling than mouse, at both the genomic and transcriptomic levels, and reveal primate-specific rewiring of hormone-producing/target cells. This work complements the scale and resolution of classical endocrine studies and sheds light on primate hormone regulation.

  View details for DOI 10.1038/s41467-024-46070-9

  View details for PubMedID 38467625

  View details for PubMedCentralID 1540572

• A cluster of neuropeptide S neurons regulates breathing and arousal. Current biology : CB Angelakos, C. C., Girven, K. S., Liu, Y., Gonzalez, O. C., Murphy, K. R., Jennings, K. J., Giardino, W. J., Zweifel, L. S., Suko, A., Palmiter, R. D., Clark, S. D., Krasnow, M. A., Bruchas, M. R., de Lecea, L. 2023

  Abstract

  Neuropeptide S (NPS) is a highly conserved peptide found in all tetrapods that functions in the brain to promote heightened arousal; however, the subpopulations mediating these phenomena remain unknown. We generated mice expressing Cre recombinase from the Nps gene locus (NpsCre) and examined populations of NPS+ neurons in the lateral parabrachial area (LPBA), the peri-locus coeruleus (peri-LC) region of the pons, and the dorsomedial thalamus (DMT). We performed brain-wide mapping of input and output regions of NPS+ clusters and characterized expression patterns of the NPS receptor 1 (NPSR1). While the activity of all three NPS+ subpopulations tracked with vigilance state, only NPS+ neurons of the LPBA exhibited both increased activity prior to wakefulness and decreased activity during REM sleep, similar to the behavioral phenotype observed upon NPSR1 activation. Accordingly, we found that activation of the LPBA but not the peri-LC NPS+ neurons increased wake and reduced REM sleep. Furthermore, given the extended role of the LPBA in respiration and the link between behavioral arousal and breathing rate, we demonstrated that the LPBA but not the peri-LC NPS+ neuronal activation increased respiratory rate. Together, our data suggest that NPS+ neurons of the LPBA represent an unexplored subpopulation regulating breathing, and they are sufficient to recapitulate the sleep/wake phenotypes observed with broad NPS system activation.

  View details for DOI 10.1016/j.cub.2023.11.018

  View details for PubMedID 38056461

• A brainstem circuit for phonation and volume control in mice. Nature neuroscience Veerakumar, A., Head, J. P., Krasnow, M. A. 2023

  Abstract

  Mammalian vocalizations are critical for communication and are produced through the process of phonation, in which expiratory muscles force air through the tensed vocal folds of the larynx, which vibrate to produce sound. Despite the importance of phonation, the motor circuits in the brain that control it remain poorly understood. In this study, we identified a subpopulation of ~160 neuropeptide precursor Nts (neurotensin)-expressing neurons in the mouse brainstem nucleus retroambiguus (RAm) that are robustly activated during both neonatal isolation cries and adult social vocalizations. The activity of these neurons is necessary and sufficient for vocalization and bidirectionally controls sound volume. RAm Nts neurons project to all brainstem and spinal cord motor centers involved in phonation and activate laryngeal and expiratory muscles essential for phonation and volume control. Thus, RAm Nts neurons form the core of a brain circuit for making sound and controlling its volume, which are two foundations of vocal communication.

  View details for DOI 10.1038/s41593-023-01478-2

  View details for PubMedID 37996531

  View details for PubMedCentralID 6687542

• An integrated cell atlas of the lung in health and disease. Nature medicine Sikkema, L., Ramírez-Suástegui, C., Strobl, D. C., Gillett, T. E., Zappia, L., Madissoon, E., Markov, N. S., Zaragosi, L. E., Ji, Y., Ansari, M., Arguel, M. J., Apperloo, L., Banchero, M., Bécavin, C., Berg, M., Chichelnitskiy, E., Chung, M. I., Collin, A., Gay, A. C., Gote-Schniering, J., Hooshiar Kashani, B., Inecik, K., Jain, M., Kapellos, T. S., Kole, T. M., Leroy, S., Mayr, C. H., Oliver, A. J., von Papen, M., Peter, L., Taylor, C. J., Walzthoeni, T., Xu, C., Bui, L. T., De Donno, C., Dony, L., Faiz, A., Guo, M., Gutierrez, A. J., Heumos, L., Huang, N., Ibarra, I. L., Jackson, N. D., Kadur Lakshminarasimha Murthy, P., Lotfollahi, M., Tabib, T., Talavera-López, C., Travaglini, K. J., Wilbrey-Clark, A., Worlock, K. B., Yoshida, M., van den Berge, M., Bossé, Y., Desai, T. J., Eickelberg, O., Kaminski, N., Krasnow, M. A., Lafyatis, R., Nikolic, M. Z., Powell, J. E., Rajagopal, J., Rojas, M., Rozenblatt-Rosen, O., Seibold, M. A., Sheppard, D., Shepherd, D. P., Sin, D. D., Timens, W., Tsankov, A. M., Whitsett, J., Xu, Y., Banovich, N. E., Barbry, P., Duong, T. E., Falk, C. S., Meyer, K. B., Kropski, J. A., Pe'er, D., Schiller, H. B., Tata, P. R., Schultze, J. L., Teichmann, S. A., Misharin, A. V., Nawijn, M. C., Luecken, M. D., Theis, F. J. 2023

  Abstract

  Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

  View details for DOI 10.1038/s41591-023-02327-2

  View details for PubMedID 37291214

  View details for PubMedCentralID 5762154

• Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF signaling. Nature communications Brownfield, D. G., de Arce, A. D., Ghelfi, E., Gillich, A., Desai, T. J., Krasnow, M. A. 2022; 13 (1): 7137

  Abstract

  The lung's gas exchange surface is comprised of alveolar AT1 and AT2 cells that are corrupted in several common and deadly diseases. They arise from a bipotent progenitor whose differentiation is thought to be dictated by differential mechanical forces. Here we show the critical determinant is FGF signaling. Fgfr2 is expressed in the developing progenitors in mouse then restricts to nascent AT2 cells and remains on throughout life. Its ligands are expressed in surrounding mesenchyme and can, in the absence of exogenous mechanical cues, induce progenitors to form alveolospheres with intermingled AT2 and AT1cells. FGF signaling directly and cell autonomously specifies AT2 fate; progenitors lacking Fgfr2 in vitro and in vivo exclusively acquire AT1 fate. Fgfr2 loss in AT2 cells perinatally results in reprogramming to AT1 identity, whereas loss or inhibition later in life triggers AT2 apoptosis and compensatory regeneration. We propose that Fgfr2 signaling selects AT2 fate during development, induces a cell non-autonomous AT1 differentiation signal, then continuously maintains AT2 identity and survival throughout life.

  View details for DOI 10.1038/s41467-022-34059-1

  View details for PubMedID 36414616

• Molecular hallmarks of heterochronic parabiosis at single-cell resolution. Nature Palovics, R., Keller, A., Schaum, N., Tan, W., Fehlmann, T., Borja, M., Kern, F., Bonanno, L., Calcuttawala, K., Webber, J., McGeever, A., Tabula Muris Consortium, Luo, J., Pisco, A. O., Karkanias, J., Neff, N. F., Darmanis, S., Quake, S. R., Wyss-Coray, T., Almanzar, N., Antony, J., Baghel, A. S., Bakerman, I., Bansal, I., Barres, B. A., Beachy, P. A., Berdnik, D., Bilen, B., Brownfield, D., Cain, C., Chan, C. K., Chen, M. B., Clarke, M. F., Conley, S. D., Demers, A., Demir, K., de Morree, A., Divita, T., du Bois, H., Ebadi, H., Espinoza, F. H., Fish, M., Gan, Q., George, B. M., Gillich, A., Gomez-Sjoberg, R., Green, F., Genetiano, G., Gu, X., Gulati, G. S., Hahn, O., Haney, M. S., Hang, Y., Harris, L., He, M., Hosseinzadeh, S., Huang, A., Huang, K. C., Iram, T., Isobe, T., Ives, F., Jones, R. C., Kao, K. S., Karnam, G., Kershner, A. M., Khoury, N., Kim, S. K., Kiss, B. M., Kong, W., Krasnow, M. A., Kumar, M. E., Kuo, C. S., Lam, J., Lee, D. P., Lee, S. E., Lehallier, B., Leventhal, O., Li, G., Li, Q., Liu, L., Lo, A., Lu, W., Lugo-Fagundo, M. F., Manjunath, A., May, A. P., Maynard, A., McKay, M., McNerney, M. W., Merrill, B., Metzger, R. J., Mignardi, M., Min, D., Nabhan, A. N., Ng, K. M., Nguyen, P. K., Noh, J., Nusse, R., Patkar, R., Peng, W. C., Penland, L., Pollard, K., Puccinelli, R., Qi, Z., Rando, T. A., Rulifson, E. J., Segal, J. M., Sikandar, S. S., Sinha, R., Sit, R. V., Sonnenburg, J., Staehli, D., Szade, K., Tan, M., Tato, C., Tellez, K., Torrez Dulgeroff, L. B., Travaglini, K. J., Tropini, C., Tsui, M., Waldburger, L., Wang, B. M., van Weele, L. J., Weinberg, K., Weissman, I. L., Wosczyna, M. N., Wu, S. M., Xiang, J., Xue, S., Yamauchi, K. A., Yang, A. C., Yerra, L. P., Youngyunpipatkul, J., Yu, B., Zanini, F., Zardeneta, M. E., Zee, A., Zhao, C., Zhang, F., Zhang, H., Zhang, M. J., Zhou, L., Zou, J. 2022

  Abstract

  The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

  View details for DOI 10.1038/s41586-022-04461-2

  View details for PubMedID 35236985

• Serum proteome analysis of systemic JIA and related lung disease identifies distinct inflammatory programs and biomarkers. Arthritis & rheumatology (Hoboken, N.J.) Chen, G., Deutsch, G. H., Schulert, G., Zheng, H., Jang, S., Trapnell, B., Lee, P., Macaubas, C., Ho, K., Schneider, C., Saper, V. E., de Jesus, A. A., Krasnow, M., Grom, A., Goldbach-Mansky, R., Khatri, P., Mellins, E. D., Canna, S. W. 2022

  Abstract

  OBJECTIVES: Recent observations in systemic Juvenile Idiopathic Arthritis (sJIA) suggest an increasing incidence of high-mortality interstitial lung disease (sJIA-LD) often characterized by a variant of pulmonary alveolar proteinosis (PAP). Co-occurrence of macrophage activation syndrome (MAS) and PAP in sJIA suggested a shared pathology, but sJIA-LD patients also commonly experience features of drug reaction such as atypical rashes and eosinophilia. We sought to investigate immunopathology and identify biomarkers in sJIA, MAS, and sJIA-LD.METHODS: We used SOMAscan to measure >1300 analytes in sera from healthy controls and patients with sJIA, MAS, sJIA-LD and other related diseases. We verified selected findings by ELISA and lung immunostaining. Because the proteome of a sample may reflect multiple states (sJIA, MAS, sJIA-LD), we used regression modeling to identify subsets of altered proteins associated with each state. We tested key findings in a validation cohort.RESULTS: Proteome alterations in active sJIA and MAS overlapped substantially, including known sJIA biomarkers like SAA and S100A9, and novel elevations of heat shock proteins and glycolytic enzymes. IL-18 was elevated in all sJIA groups, particularly MAS and sJIA-LD. We also identified an MAS-independent sJIA-LD signature notable for elevated ICAM5, MMP7, and allergic/eosinophilic chemokines, which have been previously associated with lung damage. Immunohistochemistry localized ICAM5 and MMP7 in sJIA-LD lung. ICAM5's ability to distinguish sJIA-LD from sJIA/MAS was independently validated.CONCLUSION: Serum proteins support an sJIA-to-MAS continuum, help distinguish sJIA, sJIA/MAS, and sJIA-LD and suggest etiologic hypotheses. Select biomarkers, such as ICAM5, could aid in early detection and management of sJIA-LD.

  View details for DOI 10.1002/art.42099

  View details for PubMedID 35189047

• Cell types of origin of the cell-free transcriptome. Nature biotechnology Vorperian, S. K., Moufarrej, M. N., Tabula Sapiens Consortium, Quake, S. R., Jones, R. C., Karkanias, J., Krasnow, M., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Tan, S. Y., Travaglini, K. J., Xu, C., Alcantara-Hernandez, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Culver, R. N., Cvijovic, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Mrouj, K., Mukherjee, S., Muser, T., Neuhofer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L., Subramaniam, V. R., Swarup, A., Swift, M., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgo, E., Martin, B. A., Ozawa, M. G., Silva, O., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Bierman, R., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., Olivieri, J. E., Park, M., Ravikumar, N., Stanley, G., Tan, W., Tarashansky, A. J., Vanheusden, R., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Culver, R., Dethlefsen, L., Ho, P., Liu, S., Maltzman, J. S., Metzger, R. J., Sasagawa, K., Sinha, R., Song, H., Wang, B., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Kuo, C. S., Nguyen, P., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wu, A., Wu, S. M., Wyss-Coray, T. 2022

  Abstract

  Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases.

  View details for DOI 10.1038/s41587-021-01188-9

  View details for PubMedID 35132263

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

  Abstract

  The extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach, to detect cell-type-specific splicing in >110K cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type-specifically spliced, including ubiquitously expressed genes MYL6 and RPS24. These results are validated with RNA FISH, single-cell PCR, and Smart-seq2. SpliZ analysis reveals 170 genes with regulated splicing during human spermatogenesis, including examples conserved in mouse and mouse lemur. The SpliZ allows model-based identification of subpopulations indistinguishable based on gene expression, illustrated by subpopulation-specific splicing of classical monocytes involving an ultraconserved exon in SAT1. Together, this analysis of differential splicing across multiple organs establishes that splicing is regulated cell-type-specifically.

  View details for DOI 10.7554/eLife.70692

  View details for PubMedID 34515025

• 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

• Fibrous Osteodystrophy, Chronic Renal Disease, and Uterine Adenocarcinoma in Aged Gray Mouse Lemurs (Microcebus murinus). Comparative medicine Casey, K. M., Karanewsky, C. J., Pendleton, J. L., Krasnow, M. R., Albertelli, M. A. 2021; 71 (3): 256-266

  Abstract

  The gray mouse lemur (Microcebus murinus, GML) is a nocturnal, arboreal, prosimian primate that is native to Madagascar. Captive breeding colonies of GMLs have been established primarily for noninvasive studies on questions related to circadian rhythms and metabolism. GMLs are increasingly considered to be a strong translational model for neurocognitive aging due to overlapping histopathologic features shared with aged humans. However, little information is available describing the clinical presentations, naturally occurring diseases, and histopathology of aged GMLs. In our colony, a 9 y-old, male, GML was euthanized after sudden onset of weakness, lethargy, and tibial fracture. Evaluation of this animal revealed widespread fibrous osteodystrophy (FOD) of the mandible, maxilla, cranium, appendicular, and vertebral bones. FOD and systemic metastatic mineralization were attributed to underlying chronic renal disease. Findings in this GML prompted periodic colony-wide serum biochemical screenings for azotemia and electrolyte abnormalities. Subsequently, 3 additional GMLs (2 females and 1 male) were euthanized due to varying clinical and serum biochemical presentations. Common to all 4 animals were FOD, chronic renal disease, uterine adenocarcinoma (females only), cataracts, and osteoarthritis. This case study highlights the concurrent clinical and histopathologic abnormalities that are relevant to use of GMLs in the expanding field of aging research.

  View details for DOI 10.30802/AALAS-CM-20-000078

  View details for PubMedID 34082858

• Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics. Nature medicine Muus, C., Luecken, M. D., Eraslan, G., Sikkema, L., Waghray, A., Heimberg, G., Kobayashi, Y., Vaishnav, E. D., Subramanian, A., Smillie, C., Jagadeesh, K. A., Duong, E. T., Fiskin, E., Triglia, E. T., Ansari, M., Cai, P., Lin, B., Buchanan, J., Chen, S., Shu, J., Haber, A. L., Chung, H., Montoro, D. T., Adams, T., Aliee, H., Allon, S. J., Andrusivova, Z., Angelidis, I., Ashenberg, O., Bassler, K., Becavin, C., Benhar, I., Bergenstrahle, J., Bergenstrahle, L., Bolt, L., Braun, E., Bui, L. T., Callori, S., Chaffin, M., Chichelnitskiy, E., Chiou, J., Conlon, T. M., Cuoco, M. S., Cuomo, A. S., Deprez, M., Duclos, G., Fine, D., Fischer, D. S., Ghazanfar, S., Gillich, A., Giotti, B., Gould, J., Guo, M., Gutierrez, A. J., Habermann, A. C., Harvey, T., He, P., Hou, X., Hu, L., Hu, Y., Jaiswal, A., Ji, L., Jiang, P., Kapellos, T. S., Kuo, C. S., Larsson, L., Leney-Greene, M. A., Lim, K., Litvinukova, M., Ludwig, L. S., Lukassen, S., Luo, W., Maatz, H., Madissoon, E., Mamanova, L., Manakongtreecheep, K., Leroy, S., Mayr, C. H., Mbano, I. M., McAdams, A. M., Nabhan, A. N., Nyquist, S. K., Penland, L., Poirion, O. B., Poli, S., Qi, C., Queen, R., Reichart, D., Rosas, I., Schupp, J. C., Shea, C. V., Shi, X., Sinha, R., Sit, R. V., Slowikowski, K., Slyper, M., Smith, N. P., Sountoulidis, A., Strunz, M., Sullivan, T. B., Sun, D., Talavera-Lopez, C., Tan, P., Tantivit, J., Travaglini, K. J., Tucker, N. R., Vernon, K. A., Wadsworth, M. H., Waldman, J., Wang, X., Xu, K., Yan, W., Zhao, W., Ziegler, C. G., NHLBI LungMap Consortium, Human Cell Atlas Lung Biological Network, Deutsch, G. H., Dutra, J., Gaulton, K. J., Holden-Wiltse, J., Huyck, H. L., Mariani, T. J., Misra, R. S., Poole, C., Preissl, S., Pryhuber, G. S., Rogers, L., Sun, X., Wang, A., Whitsett, J. A., Xu, Y., Alladina, J., Banovich, N. E., Barbry, P., Beane, J. E., Bhattacharyya, R. P., Black, K. E., Brazma, A., Campbell, J. D., Cho, J. L., Collin, J., Conrad, C., de Jong, K., Desai, T., Ding, D. Z., Eickelberg, O., Eils, R., Ellinor, P. T., Faiz, A., Falk, C. S., Farzan, M., Gellman, A., Getz, G., Glass, I. A., Greka, A., Haniffa, M., Hariri, L. P., Hennon, M. W., Horvath, P., Hubner, N., Hung, D. T., Huyck, H. L., Janssen, W. J., Juric, D., Kaminski, N., Koenigshoff, M., Koppelman, G. H., Krasnow, M. A., Kropski, J. A., Kuhnemund, M., Lafyatis, R., Lako, M., Lander, E. S., Lee, H., Lenburg, M. E., Marquette, C., Metzger, R. J., Linnarsson, S., Liu, G., Lo, Y. M., Lundeberg, J., Marioni, J. C., Mazzilli, S. A., Medoff, B. D., Meyer, K. B., Miao, Z., Misharin, A. V., Nawijn, M. C., Nikolic, M. Z., Noseda, M., Ordovas-Montanes, J., Oudit, G. Y., Pe'er, D., Powell, J. E., Quake, S. R., Rajagopal, J., Tata, P. R., Rawlins, E. L., Regev, A., Reid, M. E., Reyfman, P. A., Rieger-Christ, K. M., Rojas, M., Rozenblatt-Rosen, O., Saeb-Parsy, K., Samakovlis, C., Sanes, J. R., Schiller, H. B., Schultze, J. L., Schwarz, R. F., Segre, A. V., Seibold, M. A., Seidman, C. E., Seidman, J. G., Shalek, A. K., Shepherd, D. P., Sinha, R., Spence, J. R., Spira, A., Sun, X., Sundstrom, E., Teichmann, S. A., Theis, F. J., Tsankov, A. M., Vallier, L., van den Berge, M., Van Zyl, T. A., Villani, A., Weins, A., Xavier, R. J., Yildirim, A. O., Zaragosi, L., Zerti, D., Zhang, H., Zhang, K., Zhang, X. 2021

  Abstract

  Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

  View details for DOI 10.1038/s41591-020-01227-z

  View details for PubMedID 33654293

• Integrating Health Systems and Science to Respond to COVID-19 in a Model District of Rural Madagascar. Frontiers in public health Rakotonanahary, R. J., Andriambolamanana, H., Razafinjato, B., Raza-Fanomezanjanahary, E. M., Ramanandraitsiory, V., Ralaivavikoa, F., Tsirinomen'ny Aina, A., Rahajatiana, L., Rakotonirina, L., Haruna, J., Cordier, L. F., Murray, M. B., Cowley, G., Jordan, D., Krasnow, M. A., Wright, P. C., Gillespie, T. R., Docherty, M., Loyd, T., Evans, M. V., Drake, J. M., Ngonghala, C. N., Rich, M. L., Popper, S. J., Miller, A. C., Ihantamalala, F. A., Randrianambinina, A., Ramiandrisoa, B., Rakotozafy, E., Rasolofomanana, A., Rakotozafy, G., Andriamahatana Vololoniaina, M. C., Andriamihaja, B., Garchitorena, A., Rakotonirina, J., Mayfield, A., Finnegan, K. E., Bonds, M. H. 2021; 9: 654299

  Abstract

  There are many outstanding questions about how to control the global COVID-19 pandemic. The information void has been especially stark in the World Health Organization Africa Region, which has low per capita reported cases, low testing rates, low access to therapeutic drugs, and has the longest wait for vaccines. As with all disease, the central challenge in responding to COVID-19 is that it requires integrating complex health systems that incorporate prevention, testing, front line health care, and reliable data to inform policies and their implementation within a relevant timeframe. It requires that the population can rely on the health system, and decision-makers can rely on the data. To understand the process and challenges of such an integrated response in an under-resourced rural African setting, we present the COVID-19 strategy in Ifanadiana District, where a partnership between Malagasy Ministry of Public Health (MoPH) and non-governmental organizations integrates prevention, diagnosis, surveillance, and treatment, in the context of a model health system. These efforts touch every level of the health system in the district-community, primary care centers, hospital-including the establishment of the only RT-PCR lab for SARS-CoV-2 testing outside of the capital. Starting in March of 2021, a second wave of COVID-19 occurred in Madagascar, but there remain fewer cases in Ifanadiana than for many other diseases (e.g., malaria). At the Ifanadiana District Hospital, there have been two deaths that are officially attributed to COVID-19. Here, we describe the main components and challenges of this integrated response, the broad epidemiological contours of the epidemic, and how complex data sources can be developed to address many questions of COVID-19 science. Because of data limitations, it still remains unclear how this epidemic will affect rural areas of Madagascar and other developing countries where health system utilization is relatively low and there is limited capacity to diagnose and treat COVID-19 patients. Widespread population based seroprevalence studies are being implemented in Ifanadiana to inform the COVID-19 response strategy as health systems must simultaneously manage perennial and endemic disease threats.

  View details for DOI 10.3389/fpubh.2021.654299

  View details for PubMedID 34368043

• New approaches to small cell lung cancer therapy : from the laboratory to the clinic. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer Poirier, J. T., George, J. n., Owonikoko, T. K., Berns, A. n., Brambilla, E. n., Byers, L. A., Carbone, D. n., Chen, H. J., Christensen, C. L., Dive, C. n., Farago, A. F., Govindan, R. n., Hann, C. n., Hellmann, M. D., Horn, L. n., Johnson, J. E., Ju, Y. S., Kang, S. n., Krasnow, M. n., Lee, J. n., Lee, S. H., Lehman, J. n., Lok, B. n., Lovly, C. n., MacPherson, D. n., McFadden, D. n., Minna, J. n., Oser, M. n., Park, K. n., Park, K. S., Pommier, Y. n., Quaranta, V. n., Ready, N. n., Sage, J. n., Scagliotti, G. n., Sos, M. L., Sutherland, K. D., Travis, W. D., Vakoc, C. R., Wait, S. J., Wistuba, I. n., Wong, K. K., Zhang, H. n., Daigneault, J. n., Wiens, J. n., Rudin, C. M., Oliver, T. G. 2020

  Abstract

  Small cell lung cancer patient outcomes have not yet been significantly impacted by the revolution in precision oncology, primarily due to a paucity of genetic alterations in actionable driver oncogenes. Nevertheless, systemic therapies that include immunotherapy are beginning to show promise in the clinic. While these results are encouraging, many patients do not respond to or rapidly recur after current regimens, necessitating alternative or complementary therapeutic strategies. In this review, we discuss ongoing investigations into the pathobiology of this recalcitrant cancer and the therapeutic vulnerabilities that are exposed by the disease state. Included within this discussion is a snapshot of the current biomarker and clinical trial landscapes for small cell lung cancer. Finally, we identify key knowledge gaps that should be addressed in order to advance the field in pursuit of reduced small cell lung cancer mortality. This review largely summarizes work presented at the Third Biennial IASLC Small Cell Lung Cancer Meeting.

  View details for DOI 10.1016/j.jtho.2020.01.016

  View details for PubMedID 32018053

• 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

• 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

• Adult stem cells and regenerative medicine-a symposium report. Annals of the New York Academy of Sciences Cable, J., Fuchs, E., Weissman, I., Jasper, H., Glass, D., Rando, T. A., Blau, H., Debnath, S., Oliva, A., Park, S., Passegue, E., Kim, C., Krasnow, M. A. 2019

  Abstract

  Adult stem cells are rare, undifferentiated cells found in all tissues of the body. Although normally kept in a quiescent, nondividing state, these cells can proliferate and differentiate to replace naturally dying cells within their tissue and to repair its wounds in response to injury. Due to their proliferative nature and ability to regenerate tissue, adult stem cells have the potential to treat a variety of degenerative diseases as well as aging. In addition, since stem cells are often thought to be the source of malignant tumors, understanding the mechanisms that keep their proliferative abilities in check can pave the way for new cancer therapies. While adult stem cells have had limited practical and clinical applications to date, several clinical trials of stem cell-based therapies are underway. This report details recent research presented at the New York Academy of Sciences on March 14, 2019 on understanding the factors that regulate stem cell activity and differentiation, with the hope of translating these findings into the clinic.

  View details for DOI 10.1111/nyas.14243

  View details for PubMedID 31655007

• Genetic Identification of Vagal Sensory Neurons That Control Feeding. Cell Bai, L. n., Mesgarzadeh, S. n., Ramesh, K. S., Huey, E. L., Liu, Y. n., Gray, L. A., Aitken, T. J., Chen, Y. n., Beutler, L. R., Ahn, J. S., Madisen, L. n., Zeng, H. n., Krasnow, M. A., Knight, Z. A. 2019; 179 (5): 1129–43.e23

  Abstract

  Energy homeostasis requires precise measurement of the quantity and quality of ingested food. The vagus nerve innervates the gut and can detect diverse interoceptive cues, but the identity of the key sensory neurons and corresponding signals that regulate food intake remains unknown. Here, we use an approach for target-specific, single-cell RNA sequencing to generate a map of the vagal cell types that innervate the gastrointestinal tract. We show that unique molecular markers identify vagal neurons with distinct innervation patterns, sensory endings, and function. Surprisingly, we find that food intake is most sensitive to stimulation of mechanoreceptors in the intestine, whereas nutrient-activated mucosal afferents have no effect. Peripheral manipulations combined with central recordings reveal that intestinal mechanoreceptors, but not other cell types, potently and durably inhibit hunger-promoting AgRP neurons in the hypothalamus. These findings identify a key role for intestinal mechanoreceptors in the regulation of feeding.

  View details for DOI 10.1016/j.cell.2019.10.031

  View details for PubMedID 31730854

• The role of Olfr78 in the breathing circuit of mice Reply NATURE Chang, A. J., Kim, N. S., Hireed, H., de Arce, A., Ortega, F. E., Riegler, J., Madison, D. V., Krasnow, M. A. 2018; 561 (7724): E41

  View details for PubMedID 30258152

• Profile of an unknown airway cell NATURE Travaglini, K. J., Krasnow, M. A. 2018; 560 (7718): 313–14

  View details for DOI 10.1038/d41586-018-05813-7

  View details for Web of Science ID 000441673400026

  View details for PubMedID 30097657

• 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

• MicroRNA-9 Couples Brain Neurogenesis and Angiogenesis. Cell reports Madelaine, R. n., Sloan, S. A., Huber, N. n., Notwell, J. H., Leung, L. C., Skariah, G. n., Halluin, C. n., Paşca, S. P., Bejerano, G. n., Krasnow, M. A., Barres, B. A., Mourrain, P. n. 2017; 20 (7): 1533–42

  Abstract

  In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment.

  View details for PubMedID 28813666

• Developmental origin of lung macrophage diversity DEVELOPMENT Tan, S. Y., Krasnow, M. A. 2016; 143 (8): 1318-1327

  Abstract

  Macrophages are specialized phagocytic cells, present in all tissues, which engulf and digest pathogens, infected and dying cells, and debris, and can recruit and regulate other immune cells and the inflammatory response and aid in tissue repair. Macrophage subpopulations play distinct roles in these processes and in disease, and are typically recognized by differences in marker expression, immune function, or tissue of residency. Although macrophage subpopulations in the brain have been found to have distinct developmental origins, the extent to which development contributes to macrophage diversity between tissues and within tissues is not well understood. Here, we investigate the development and maintenance of mouse lung macrophages by marker expression patterns, genetic lineage tracing and parabiosis. We show that macrophages populate the lung in three developmental waves, each giving rise to a distinct lineage. These lineages express different markers, reside in different locations, renew in different ways, and show little or no interconversion. Thus, development contributes significantly to lung macrophage diversity and targets each lineage to a different anatomical domain.

  View details for DOI 10.1242/dev.129122

  View details for Web of Science ID 000385261300010

  View details for PubMedID 26952982

  View details for PubMedCentralID PMC4852511

• Small Cell Lung Cancer: Can Recent Advances in Biology and Molecular Biology Be Translated into Improved Outcomes? Journal of thoracic oncology Bunn, P. A., Minna, J. D., Augustyn, A., Gazdar, A. F., Ouadah, Y., Krasnow, M. A., Berns, A., Brambilla, E., Rekhtman, N., Massion, P. P., Niederst, M., Peifer, M., Yokota, J., Govindan, R., Poirier, J. T., Byers, L. A., Wynes, M. W., McFadden, D. G., Macpherson, D., Hann, C. L., Farago, A. F., Dive, C., Teicher, B. A., Peacock, C. D., Johnson, J. E., Cobb, M. H., Wendel, H., Spigel, D., Sage, J., Yang, P., Pietanza, M. C., Krug, L. M., Heymach, J., Ujhazy, P., Zhou, C., Goto, K., Dowlati, A., Christensen, C. L., Park, K., Einhorn, L. H., Edelman, M. J., Giaccone, G., Gerber, D. E., Salgia, R., Owonikoko, T., Malik, S., Karachaliou, N., Gandara, D. R., Slotman, B. J., Blackhall, F., Goss, G., Thomas, R., Rudin, C. M., Hirsch, F. R. 2016; 11 (4): 453-474

  View details for DOI 10.1016/j.jtho.2016.01.012

  View details for PubMedID 26829312

• Defining a mesenchymal progenitor niche at single-cell resolution SCIENCE Kumar, M. E., Bogard, P. E., Espinoza, F. H., Menke, D. B., Kingsley, D. M., Krasnow, M. A. 2014; 346 (6211): 827-?

  Abstract

  Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here, we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication.

  View details for DOI 10.1126/science.1258810

  View details for Web of Science ID 000349771600003

  View details for PubMedCentralID PMC4269943

• Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature Treutlein, B., Brownfield, D. G., Wu, A. R., Neff, N. F., Mantalas, G. L., Espinoza, F. H., Desai, T. J., Krasnow, M. A., Quake, S. R. 2014; 509 (7500): 371-375

  View details for DOI 10.1038/nature13173

  View details for PubMedID 24739965

• Two nested developmental waves demarcate a compartment boundary in the mouse lung NATURE COMMUNICATIONS Alanis, D. M., Chang, D. R., Akiyama, H., Krasnow, M. A., Chen, J. 2014; 5

  Abstract

  The lung is a branched tubular network with two distinct compartments--the proximal conducting airways and the peripheral gas exchange region--separated by a discrete boundary termed the bronchoalveolar duct junction (BADJ). Here we image the developing mouse lung in three-dimensions (3D) and show that two nested developmental waves demarcate the BADJ under the control of a global hormonal signal. A first wave of branching morphogenesis progresses throughout embryonic development, generating branches for both compartments. A second wave of conducting airway differentiation follows the first wave but terminates earlier, specifying the proximal compartment and setting the BADJ. The second wave is terminated by a glucocorticoid signalling: premature activation or loss of glucocorticoid signalling causes a proximal or distal shift, respectively, in BADJ location. The results demonstrate a new mechanism of boundary formation in complex, 3D organs and provide new insights into glucocorticoid therapies for lung defects in premature birth.

  View details for DOI 10.1038/ncomms4923

  View details for PubMedID 24879355

• Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature Desai, T. J., Brownfield, D. G., Krasnow, M. A. 2014; 507 (7491): 190-194

  View details for DOI 10.1038/nature12930

  View details for PubMedID 24499815

• Stem cells: Differentiated cells in a back-up role. Nature Desai, T. J., Krasnow, M. A. 2013; 503 (7475): 204-205

  View details for DOI 10.1038/nature12706

  View details for PubMedID 24196710

• Myb promotes centriole amplification and later steps of the multiciliogenesis program DEVELOPMENT Tan, F. E., Vladar, E. K., Ma, L., Fuentealba, L. C., Hoh, R., Espinoza, F. H., Axelrod, J. D., Alvarez-Buylla, A., Stearns, T., Kintner, C., Krasnow, M. A. 2013; 140 (20): 4277-4286

  Abstract

  The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

  View details for DOI 10.1242/dev.094102

  View details for Web of Science ID 000325153200017

  View details for PubMedID 24048590

  View details for PubMedCentralID PMC3787764

• Myb promotes centriole amplification and later steps of the multiciliogenesis program. Development Tan, F. E., Vladar, E. K., Ma, L., Fuentealba, L. C., Hoh, R., Espinoza, F. H., Axelrod, J. D., Alvarez-Buylla, A., Stearns, T., Kintner, C., Krasnow, M. A. 2013; 140 (20): 4277-4286

  Abstract

  The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

  View details for DOI 10.1242/dev.094102

  View details for PubMedID 24048590

• A Genome-Wide Association Study (GWAS) for Bronchopulmonary Dysplasia. Pediatrics Wang, H., St Julien, K. R., Stevenson, D. K., Hoffmann, T. J., Witte, J. S., Lazzeroni, L. C., Krasnow, M. A., Quaintance, C. C., Oehlert, J. W., Jelliffe-Pawlowski, L. L., Gould, J. B., Shaw, G. M., O'Brodovich, H. M. 2013; 132 (2): 290-297

  Abstract

  Twin studies suggest that heritability of moderate-severe bronchopulmonary dysplasia (BPD) is 53% to 79%, we conducted a genome-wide association study (GWAS) to identify genetic variants associated with the risk for BPD.The discovery GWAS was completed on 1726 very low birth weight infants (gestational age = 25(0)-29(6/7) weeks) who had a minimum of 3 days of intermittent positive pressure ventilation and were in the hospital at 36 weeks' postmenstrual age. At 36 weeks' postmenstrual age, moderate-severe BPD cases (n = 899) were defined as requiring continuous supplemental oxygen, whereas controls (n = 827) inhaled room air. An additional 795 comparable infants (371 cases, 424 controls) were a replication population. Genomic DNA from case and control newborn screening bloodspots was used for the GWAS. The replication study interrogated single-nucleotide polymorphisms (SNPs) identified in the discovery GWAS and those within the HumanExome beadchip.Genotyping using genomic DNA was successful. We did not identify SNPs associated with BPD at the genome-wide significance level (5 × 10(-8)) and no SNP identified in previous studies reached statistical significance (Bonferroni-corrected P value threshold .0018). Pathway analyses were not informative.We did not identify genomic loci or pathways that account for the previously described heritability for BPD. Potential explanations include causal mutations that are genetic variants and were not assayed or are mapped to many distributed loci, inadequate sample size, race ethnicity of our study population, or case-control differences investigated are not attributable to underlying common genetic variation.

  View details for DOI 10.1542/peds.2013-0533

  View details for PubMedID 23897914

• High quality genome-wide genotyping from archived dried blood spots without DNA amplification. PloS one St Julien, K. R., Jelliffe-Pawlowski, L. L., Shaw, G. M., Stevenson, D. K., O'Brodovich, H. M., Krasnow, M. A. 2013; 8 (5)

  Abstract

  Spots of blood are routinely collected from newborn babies onto filter paper called Guthrie cards and used to screen for metabolic and genetic disorders. The archived dried blood spots are an important and precious resource for genomic research. Whole genome amplification of dried blood spot DNA has been used to provide DNA for genome-wide SNP genotyping. Here we describe a 96 well format procedure to extract DNA from a portion of a dried blood spot that provides sufficient unamplified genomic DNA for genome-wide single nucleotide polymorphism (SNP) genotyping. We show that SNP genotyping of the unamplified DNA is more robust than genotyping amplified dried blood spot DNA, is comparable in cost, and can be done with thousands of samples. This procedure can be used for genome-wide association studies and other large-scale genomic analyses that require robust, high-accuracy genotyping of dried blood spot DNA.

  View details for DOI 10.1371/journal.pone.0064710

  View details for PubMedID 23737996

• Integrin Beta 1 Suppresses Multilayering of a Simple Epithelium PLOS ONE Chen, J., Krasnow, M. A. 2012; 7 (12)

  Abstract

  Epithelia are classified as either simple, a single cell layer thick, or stratified (multilayered). Stratified epithelia arise from simple epithelia during development, and transcription factor p63 functions as a key positive regulator of epidermal stratification. Here we show that deletion of integrin beta 1 (Itgb1) in the developing mouse airway epithelium abrogates airway branching and converts this monolayer epithelium into a multilayer epithelium with more than 10 extra layers. Mutant lung epithelial cells change mitotic spindle orientation to seed outer layers, and cells in different layers become molecularly and functionally distinct, hallmarks of normal stratification. However, mutant lung epithelial cells do not activate p63 and do not switch to the stratified keratin profile of epidermal cells. These data, together with previous data implicating Itgb1 in regulation of epidermal stratification, suggest that the simple-versus-stratified developmental decision may involve not only stratification inducers like p63 but suppressors like Itgb1 that prevent simple epithelia from inappropriately activating key steps in the stratification program.

  View details for DOI 10.1371/journal.pone.0052886

  View details for Web of Science ID 000313158800104

  View details for PubMedID 23285215

  View details for PubMedCentralID PMC3528644

• A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System PLOS GENETICS Ghabrial, A. S., Levi, B. P., Krasnow, M. A. 2011; 7 (7)

  Abstract

  Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general "house-keeping" genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system.

  View details for DOI 10.1371/journal.pgen.1002087

  View details for Web of Science ID 000293338600001

  View details for PubMedID 21750678

  View details for PubMedCentralID PMC3131284

• Targeting Robo4-Dependent Slit Signaling to Survive the Cytokine Storm in Sepsis and Influenza SCIENCE TRANSLATIONAL MEDICINE London, N. R., Zhu, W., Bozza, F. A., Smith, M. C., Greif, D. M., Sorensen, L. K., Chen, L., Kaminoh, Y., Chan, A. C., Passi, S. F., Day, C. W., Barnard, D. L., Zimmerman, G. A., Krasnow, M. A., Li, D. Y. 2010; 2 (23)

  Abstract

  The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host's innate immune response may provide a therapeutic strategy for treating multiple infectious agents.

  View details for DOI 10.1126/scitranslmed.3000678

  View details for Web of Science ID 000277304000004

  View details for PubMedID 20375003

  View details for PubMedCentralID PMC2875996

• Circulating blood cells function as a surveillance system for damaged tissue in Drosophila larvae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Babcock, D. T., Brock, A. R., Fish, G. S., Wang, Y., Perrin, L., Krasnow, M. A., Galko, M. J. 2008; 105 (29): 10017-10022

  Abstract

  Insects have an open circulatory system in which the heart pumps blood (hemolymph) into the body cavity, where it directly bathes the internal organs and epidermis. The blood contains free and tissue-bound immune cells that function in the inflammatory response. Here, we use live imaging of transgenic Drosophila larvae with fluorescently labeled blood cells (hemocytes) to investigate the circulatory dynamics of larval blood cells and their response to tissue injury. We find that, under normal conditions, the free cells rapidly circulate, whereas the tissue-bound cells are sessile. After epidermal wounding, tissue-bound cells around the wound site remain sessile and unresponsive, whereas circulating cells are rapidly recruited to the site of damage by adhesive capture. After capture, these cells distribute across the wound, appear phagocytically active, and are subsequently released back into circulation by the healing epidermis. The results demonstrate that circulating cells function as a surveillance system that monitors larval tissues for damage, and that adhesive capture, an important mechanism of recruitment of circulating cells to inflammatory sites in vertebrates, is shared by insects and vertebrates despite the vastly different architectures of their circulatory systems.

  View details for DOI 10.1073/pnas.0709951105

  View details for Web of Science ID 000257913200032

  View details for PubMedID 18632567

  View details for PubMedCentralID PMC2474562

• Circulating blood cells function as a surveillance system for damaged tissue in Drosophila larvae Proceedings of the National Academy of Sciences Babcock, D.T., Fish, G., Wang, Y., Krasnow, M.A., Galko, M.J. 2008; 105: 10017-10022
• Functions of the nonsense-mediated mRNA decay pathway in Drosophila development PLOS GENETICS Metzstein, M. M., Krasnow, M. A. 2006; 2 (12): 2143-2154

  Abstract

  Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila "photoshop" mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3' UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.

  View details for DOI 10.1371/journal.pgen.0020180

  View details for Web of Science ID 000243482100017

  View details for PubMedID 17196039

  View details for PubMedCentralID PMC1756896

• Drosophila talin and integrin genes are required for maintenance of tracheal terminal branches and luminal organization (vol 133, pg 2383, 2006) DEVELOPMENT Levi, B. P., Ghabrial, A. S., Krasnow, M. A. 2006; 133 (12): 2383-U13

  Abstract

  Epithelial tubes that compose many organs are typically long lasting, except under specific developmental and physiological conditions when network remodeling occurs. Although there has been progress elucidating mechanisms of tube formation, little is known of the mechanisms that maintain tubes and destabilize them during network remodeling. Here, we describe Drosophila tendrils mutations that compromise maintenance of tracheal terminal branches, fine gauge tubes formed by tracheal terminal cells that ramify on and adhere tightly to tissues in order to supply them with oxygen. Homozygous tendrils terminal cell clones have fewer terminal branches than normal but individual branches contain multiple convoluted lumens. The phenotype arises late in development: terminal branches bud and form lumens normally early in development, but during larval life lumens become convoluted and mature branches degenerate. Their lumens, however, are retained in the remaining branches, resulting in the distinctive multi-lumen phenotype. Mapping and molecular studies demonstrate that tendrils is allelic to rhea, which encodes Drosophila talin, a large cytoskeletal protein that links integrins to the cytoskeleton. Terminal cells mutant for myospheroid, the major Drosophila beta-integrin, or doubly mutant for multiple edematous wings and inflated alpha-integrins, also show the tendrils phenotype, and localization of myospheroid beta-integrin protein is disrupted in tendrils mutant terminal cells. The results provide evidence that integrin-talin adhesion complexes are necessary to maintain tracheal terminal branches and luminal organization. Similar complexes may stabilize other tubular networks and may be targeted for inactivation during network remodeling events.

  View details for DOI 10.1242/dev.02484

  View details for Web of Science ID 000238474600010

  View details for PubMedID 16720877

• Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gerber, A. P., Luschnig, S., Krasnow, M. A., Brown, P. O., Herschlag, D. 2006; 103 (12): 4487-4492

  Abstract

  Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.

  View details for DOI 10.1073/pnas.0509260103

  View details for Web of Science ID 000236362600031

  View details for PubMedID 16537387

  View details for PubMedCentralID PMC1400586

• Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases DEVELOPMENT Jarvis, L. A., Toering, S. J., Simon, M. A., Krasnow, M. A., Smith-Bolton, R. K. 2006; 133 (6): 1133-1142

  Abstract

  Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. Here, we show that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. We propose that this double-negative feedback circuit shapes the output profile of RTK signaling events.

  View details for DOI 10.1242/dev.02255

  View details for Web of Science ID 000236676900014

  View details for PubMedID 16481357

• serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila CURRENT BIOLOGY Luschnig, S., Batz, T., Armbruster, K., Krasnow, M. A. 2006; 16 (2): 186-194

  Abstract

  Many organs contain epithelial tubes that transport gases or liquids . Proper tube size and shape is crucial for organ function, but the mechanisms controlling tube diameter and length are poorly understood. Recent studies of tracheal (respiratory) tube morphogenesis in Drosophila show that chitin synthesis genes produce an expanding chitin cylinder in the apical (luminal) extracellular matrix (ECM) that coordinates the dilation of the surrounding epithelium . Here, we describe two genes involved in chitin modification, serpentine (serp) and vermiform (verm), mutations in which cause excessively long and tortuous tracheal tubes. The genes encode similar proteins with an LDL-receptor ligand binding motif and chitin binding and deacetylation domains. Both proteins are expressed and secreted during tube expansion and localize throughout the lumen in a chitin-dependent manner. Unlike previously characterized chitin pathway genes, serp and verm are not required for chitin synthesis or secretion but rather for its normal fibrillar structure. The mutations also affect structural properties of another chitinous matrix, epidermal cuticle. Our work demonstrates that chitin and the matrix proteins Serp and Verm limit tube elongation, and it suggests that tube length is controlled independently of diameter by modulating physical properties of the chitin ECM, presumably by N-deacetylation of chitin and conversion to chitosan.

  View details for DOI 10.1016/j.cub.2005.11.072

  View details for Web of Science ID 000235105900024

  View details for PubMedID 16431371

• Requirement for chitin biosynthesis in epithelial tube morphogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Devine, W. P., Lubarsky, B., Shaw, K., Luschnig, S., Messina, L., Krasnow, M. A. 2005; 102 (47): 17014-17019

  Abstract

  Many organs are composed of branched networks of epithelial tubes that transport vital fluids or gases. The proper size and shape of tubes are crucial for their transport function, but the molecular processes that govern tube size and shape are not well understood. Here we show that three genes required for tracheal tube morphogenesis in Drosophila melanogaster encode proteins involved in the synthesis and accumulation of chitin, a polymer of N-acetyl-beta-D-glucosamine that serves as a scaffold in the rigid extracellular matrix of insect cuticle. In all three mutants, developing tracheal tubes bud and extend normally, but the epithelial walls of the tubes do not expand uniformly, and the resultant tubes are grossly misshapen, with constricted and distended regions all along their lengths. The genes are expressed in tracheal cells during the expansion process, and chitin accumulates in the lumen of tubes, forming an expanding cylinder that we propose coordinates the behavior of the surrounding tracheal cells and stabilizes the expanding epithelium. These findings show that chitin regulates epithelial tube morphogenesis, in addition to its classical role protecting mature epithelia.

  View details for DOI 10.1073/pnas.0506676102

  View details for Web of Science ID 000233463200021

  View details for PubMedID 16287975

  View details for PubMedCentralID PMC1283532

• Cellular and genetic analysis of wound healing in Drosophila larvae PLOS BIOLOGY Galko, M. J., Krasnow, M. A. 2004; 2 (8): 1114-1126

  Abstract

  To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution.

  View details for DOI 10.1371/journal.pbio.0020239

  View details for Web of Science ID 000223647100012

  View details for PubMedCentralID PMC479041

• Gene expression during the life cycle of Drosophila melanogaster SCIENCE Arbeitman, M. N., Furlong, E. E., Imam, F., JOHNSON, E., Null, B. H., Baker, B. S., Krasnow, M. A., Scott, M. P., Davis, R. W., White, K. P. 2002; 297 (5590): 2270-2275

  Abstract

  Molecular genetic studies of Drosophila melanogaster have led to profound advances in understanding the regulation of development. Here we report gene expression patterns for nearly one-third of all Drosophila genes during a complete time course of development. Mutations that eliminate eye or germline tissue were used to further analyze tissue-specific gene expression programs. These studies define major characteristics of the transcriptional programs that underlie the life cycle, compare development in males and females, and show that large-scale gene expression data collected from whole animals can be used to identify genes expressed in particular tissues and organs or genes involved in specific biological and biochemical processes.

  View details for PubMedID 12351791

• Tube morphogenesis TRENDS IN CELL BIOLOGY Krasnow, M. A., Nelson, W. J. 2002; 12 (8): 351-351

  View details for Web of Science ID 000176998700001

  View details for PubMedID 12191903

• Developmental control of blood cell migration by the Drosophila VEGF pathway CELL Cho, N. K., Keyes, L., JOHNSON, E., Heller, J., Ryner, L., Karim, F., Krasnow, M. A. 2002; 108 (6): 865-876

  Abstract

  We show that a vascular endothelial growth factor (VEGF) pathway controls embryonic migrations of blood cells (hemocytes) in Drosophila. The VEGF receptor homolog is expressed in hemocytes, and three VEGF homologs are expressed along hemocyte migration routes. A receptor mutation arrests progression of blood cell movement. Mutations in Vegf17E or Vegf27Cb have no effect, but simultaneous inactivation of all three Vegf genes phenocopied the receptor mutant, and ectopic expression of Vegf27Cb redirected migration. Genetic experiments indicate that the VEGF pathway functions independently of pathways governing hemocyte homing on apoptotic cells. The results suggest that the Drosophila VEGF pathway guides developmental migrations of blood cells, and we speculate that the ancestral function of VEGF pathways was to guide blood cell movement.

  View details for Web of Science ID 000174563000014

  View details for PubMedID 11955438

• The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis DEVELOPMENT Shim, K., Blake, K. J., Jack, J., Krasnow, M. A. 2001; 128 (23): 4923-4933

  Abstract

  During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. We show that mutations in the Drosophila ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia, many of which are also affected in rib mutants. We propose that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements.

  View details for Web of Science ID 000172740900024

  View details for PubMedID 11731471

• A nuclear lamin is required for cytoplasmic organization and egg polarity in Drosophila NATURE CELL BIOLOGY Guillemin, K., Williams, T., Krasnow, M. A. 2001; 3 (9): 848-851

  Abstract

  Nuclear lamins are intermediate filaments that compose the nuclear lamina--the filamentous meshwork underlying the inner nuclear membrane--and are required for nuclear assembly, organization and maintenance. Here we present evidence that a nuclear lamin is also required for cytoplasmic organization in two highly polarized cell types. Zygotic loss-of-function mutations in the Drosophila gene encoding the principal lamin (Dm(0)) disrupt the directed outgrowth of cytoplasmic extensions from terminal cells of the tracheal system. Germline mutant clones disrupt dorsal-ventral polarity of the oocyte. In mutant oocytes, transcripts of the dorsal determinant Gurken, a transforming growth factor-alpha homologue, fail to localize properly around the anterodorsal surface of the oocyte nucleus; their ventral spread results in dorsalized eggs that resemble those of the classical dorsalizing mutations squid and fs(1)K10. The requirement of a nuclear lamin for cytoplasmic as well as nuclear organization has important implications for both the cellular functions of lamins and the pathogenesis of human diseases caused by lamin mutations.

  View details for Web of Science ID 000170979600020

  View details for PubMedID 11533666

• Genetic dissection of epithelial branching and oxygen response pathways in Drosophila. JOHNSON, E., Chiu, S. K., Jarecki, J., Krasnow, M. A. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2001: 172–72

  View details for Web of Science ID 000169701100014

• Inhibition of angiogenesis by a mouse sprouty protein JOURNAL OF BIOLOGICAL CHEMISTRY Lee, S. H., Schloss, D. J., Jarvis, L., Krasnow, M. A., Swain, J. L. 2001; 276 (6): 4128-4133

  Abstract

  Sprouty negatively modulates branching morphogenesis in the Drosophila tracheal system. To address the role of mammalian Sprouty homologues in angiogenesis, another form of branching morphogenesis, a recombinant adenovirus engineered to express murine Sprouty-4 selectively in endothelial cells, was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Sprouty-4 expression inhibited branching and sprouting of small vessels, resulting in abnormal embryonic development. In vitro, Sprouty-4 inhibited fibroblast growth factor and vascular endothelial cell growth factor-mediated cell proliferation and migration and prevented basic fibroblast growth factor and vascular endothelial cell growth factor-induced MAPK phosphorylation in endothelial cells, indicating inhibition of tyrosine kinase-mediated signaling pathways. The ability of constitutively activated mutant Ras(L61) to rescue Sprouty-4 inhibition of MAPK phosphorylation suggests that Sprouty inhibits receptor tyrosine kinase signaling upstream of Ras. Thus, Sprouty may regulate angiogenesis in normal and disease processes by modulating signaling by endothelial tyrosine kinases.

  View details for PubMedID 11053436

• Genetic control of epithelial tube size in the Drosophila tracheal system DEVELOPMENT Beitel, G. J., Krasnow, M. A. 2000; 127 (15): 3271-3282

  Abstract

  The proper size of epithelial tubes is critical for the function of the lung, kidney, vascular system and other organs, but the genetic and cellular mechanisms that control epithelial tube size are unknown. We investigated tube size control in the embryonic and larval tracheal (respiratory) system of Drosophila. A morphometric analysis showed that primary tracheal branches have characteristic sizes that undergo programmed changes during development. Branches grow at different rates and their diameters and lengths are regulated independently: tube length increases gradually throughout development, whereas tube diameter increases abruptly at discrete times in development. Cellular analysis and manipulation of tracheal cell number using cell-cycle mutations demonstrated that tube size is not dictated by the specific number or shape of the tracheal cells that constitute it. Rather, tube size appears to be controlled by coordinately regulating the apical (lumenal) surface of tracheal cells. Genetic analysis showed that tube sizes are specified early by branch identity genes, and the subsequent enlargement of branches to their mature sizes and maintenance of the expanded tubes involves a new set of genes described here, which we call tube expansion genes. This work establishes a genetic system for investigating tube size regulation, and provides an outline of the genetic program and cellular events underlying tracheal tube size control.

  View details for Web of Science ID 000088854100008

  View details for PubMedID 10887083

• The Drosophila genome sequence: Implications for biology and medicine SCIENCE Kornberg, T. B., Krasnow, M. A. 2000; 287 (5461): 2218-2220

  Abstract

  The 120-megabase euchromatic portion of the Drosophila melanogaster genome has been sequenced. Because the genome is compact and many genetic tools are available, and because fly cell biology and development have much in common with mammals, this sequence may be the Rosetta stone for deciphering the human genome.

  View details for Web of Science ID 000086049100037

  View details for PubMedID 10731136

• Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF CELL Jarecki, J., JOHNSON, E., Krasnow, M. A. 1999; 99 (2): 211-220

  Abstract

  The Drosophila tracheal (respiratory) system is a tubular epithelial network that delivers oxygen to internal tissues. Sprouting of the major tracheal branches is stereotyped and controlled by hard-wired developmental cues. Here we show that ramification of the fine terminal branches is variable and regulated by oxygen, and that this process is controlled by a local signal or signals produced by oxygen-starved cells. We provide evidence that the critical signal is Branchless (Bnl) FGF, the same growth factor that patterns the major branches during embryogenesis. During larval life, oxygen deprivation stimulates expression of Bnl, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. Thus, a single growth factor is reiteratively used to pattern each level of airway branching, and the change in branch patterning results from a switch from developmental to physiological control of its expression.

  View details for Web of Science ID 000083159700011

  View details for PubMedID 10535739

• Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed DEVELOPMENT Minowada, G., Jarvis, L. A., Chi, C. L., Neubuser, A., Sun, X., Hacohen, N., Krasnow, M. A., Martin, G. R. 1999; 126 (20): 4465-4475

  Abstract

  The Drosophila sprouty gene encodes an antagonist of FGF and EGF signaling whose expression is induced by the signaling pathways that it inhibits. Here we describe a family of vertebrate Sprouty homologs and demonstrate that the regulatory relationship with FGF pathways has been conserved. In both mouse and chick embryos, Sprouty genes are expressed in intimate association with FGF signaling centers. Gain- and loss-of-function experiments demonstrate that FGF signaling induces Sprouty gene expression in various tissues. Sprouty overexpression obtained by infecting the prospective wing territory of the chick embryo with a retrovirus containing a mouse Sprouty gene causes a reduction in limb bud outgrowth and other effects consistent with reduced FGF signaling from the apical ectodermal ridge. At later stages of development in the infected limbs there was a dramatic reduction in skeletal element length due to an inhibition of chondrocyte differentiation. The results provide evidence that vertebrate Sprouty proteins function as FGF-induced feedback inhibitors, and suggest a possible role for Sprouty genes in the pathogenesis of specific human chondrodysplasias caused by activating mutations in Fgfr3.

  View details for Web of Science ID 000083683300007

  View details for PubMedID 10498682

• Genetic control of branching morphogenesis. Science Metzger, R. J., Krasnow, M. A. 1999; 284 (5420): 1635-1639

  Abstract

  The genetic programs that direct formation of the treelike branching structures of two animal organs have begun to be elucidated. In both the developing Drosophila tracheal (respiratory) system and mammalian lung, a fibroblast growth factor (FGF) signaling pathway is reiteratively used to pattern successive rounds of branching. The initial pattern of signaling appears to be established by early, more global embryonic patterning systems. The FGF pathway is then modified at each stage of branching by genetic feedback controls and other signals to give distinct branching outcomes. The reiterative use of a signaling pathway by both insects and mammals suggests a general scheme for patterning branching morphogenesis.

  View details for PubMedID 10383344

• Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila DEVELOPMENT Kramer, S., Okabe, M., Hacohen, N., Krasnow, M. A., Hiromi, Y. 1999; 126 (11): 2515-2525

  Abstract

  Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.

  View details for Web of Science ID 000081019500020

  View details for PubMedID 10226010

• stumps, a Drosophila gene required for fibroblast growth factor (FGF)-directed migrations of tracheal and mesodermal cells GENETICS Imam, F., Sutherland, D., Huang, W., Krasnow, M. A. 1999; 152 (1): 307-318

  Abstract

  Fibroblast growth factors (FGFs) bind to FGF receptors, transmembrane tyrosine kinases that activate mitogenic, motogenic, and differentiative responses in different tissues. While there has been substantial progress in elucidating the Ras-MAP kinase pathway that mediates the differentiative responses, the signal transduction pathways that lead to directed cell migrations are not well defined. Here we describe a Drosophila gene called stumps that is required for FGF-dependent migrations of tracheal and mesodermal cells. These migrations are controlled by different FGF ligands and receptors, and they occur by different cellular mechanisms: the tracheal migrations occur as part of an epithelium whereas the mesodermal migrations are fibroblast-like. In the stumps mutant, tracheal cells fail to move out from the epithelial sacs, and only rudimentary tracheal branches form. Mesodermal cells fail in their dorsal migrations after gastrulation. The stumps mutation does not block all FGF signaling effects in these tissues: both random cell migrations and Ras-MAP kinase-mediated induction of FGF-specific effector genes occurred upon ectopic expression of the ligand or upon expression of a constitutively activated Ras protein in the migrating cells. The results suggest that stumps function promotes FGF-directed cell migrations, either by potentiating the FGF signaling process or by coupling the signal to the cellular machinery required for directed cell movement.

  View details for Web of Science ID 000080219100026

  View details for PubMedID 10224263

  View details for PubMedCentralID PMC1460608

• Adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS DEVELOPMENT Englund, C., Uv, A. E., Cantera, R., Mathies, L. D., Krasnow, M. A., Samakovlis, C. 1999; 126 (7): 1505-1514

  Abstract

  Neurons and glial cells provide guidance cues for migrating neurons. We show here that migrating epithelial cells also contact specific neurons and glia during their pathfinding, and we describe the first gene required in the process. In wild-type Drosophila embryos, the ganglionic tracheal branch navigates a remarkably complex path along specific neural and glial substrata, switching substrata five times before reaching its ultimate target in the CNS. In adrift mutants, ganglionic branches migrate normally along the intersegmental nerve, but sporadically fail to switch to the segmental nerve and enter the CNS; they wind up meandering along the ventral epidermis instead. adrift encodes a novel nuclear protein with an evolutionarily conserved motif. The gene is required in the trachea and is expressed in the leading cells of migrating ganglionic branches where it is induced by the branchless FGF pathway. We propose that Adrift regulates expression of tracheal genes required for pathfinding on the segmental nerve, and FGF induction of adrift expression in migrating tracheal cells promotes the switch from the intersegmental to the segmental nerve.

  View details for Web of Science ID 000079848200015

  View details for PubMedID 10068643

• Oxygen regulation of tracheal branching is mediated by the branchless fibroblast growth factor JOHNSON, E., Krasnow, M. ACADEMIC PRESS INC ELSEVIER SCIENCE. 1998: 204–

  View details for Web of Science ID 000074281900274

• sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways CELL Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y., Krasnow, M. A. 1998; 92 (2): 253-263

  Abstract

  Antagonists of several growth factor signaling pathways play important roles in developmental patterning by limiting the range of the cognate inducer. Here, we describe an antagonist of FGF signaling that patterns apical branching of the Drosophila airways. In wild-type embryos, the Branchless FGF induces secondary branching by activating the Breathless FGF receptor near the tips of growing primary branches. In sprouty mutants, the FGF pathway is overactive and ectopic branches are induced on the stalks of primary branches. We show that FGF signaling induces sprouty expression in the nearby tip cells, and sprouty acts nonautonomously and in a competitive fashion to block signaling to the more distant stalk cells. sprouty encodes a novel cysteine-rich protein that defines a new family of putative signaling molecules that may similarly function as FGF antagonists in vertebrate development.

  View details for Web of Science ID 000071672600014

  View details for PubMedID 9458049

• The hypoxic response: Huffing and HIFing CELL Guillemin, K., Krasnow, M. A. 1997; 89 (1): 9-12

  View details for Web of Science ID A1997WR68500003

  View details for PubMedID 9094708

• Genes that control organ form: Lessons from bone and branching morphogenesis Cold Spring Harbor Symposium on Quantitative Biology - Pattern Formation During Development Krasnow, N. A. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1997: 235–240

  View details for Web of Science ID 000073570200028

  View details for PubMedID 9598356

• Branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching CELL Sutherland, D., Samakovlis, C., Krasnow, M. A. 1996; 87 (6): 1091-1101

  Abstract

  The molecular basis for patterning of complex organ structures like the lung and insect tracheal system is unknown. Here, we describe the Drosophila gene branchless (bnl) and demonstrate that it is a key determinant of the tracheal branching pattern. bnl is required for tracheal branching and is expressed dynamically in clusters of cells surrounding the developing tracheal system at each position where a new branch will form and grow out. Localized misexpression of bnl can direct branch formation and outgrowth to new positions. Generalized misexpression activates later programs of tracheal gene expression and branching, resulting in massive networks of branches. bnl encodes a homolog of mammalian fibroblast growth factors (FGFs) and appears to function as a ligand for the breathless receptor tyrosine kinase, an FGF receptor homolog expressed on developing tracheal cells. The results suggest that this FGF pathway specifies the tracheal branching pattern by guiding tracheal cell migration during primary branch formation and then activating later programs of finer branching at the ends of growing primary branches.

  View details for Web of Science ID A1996VY44700014

  View details for PubMedID 8978613

• Regulated Breathless receptor tyrosine kinase activity required to pattern cell migration area branching in the Drosophila tracheal system GENES & DEVELOPMENT Lee, T., Hacohen, N., Krasnow, M., Montell, D. J. 1996; 10 (22): 2912-2921

  Abstract

  Receptor tyrosine kinases (RTKs) are members of a diverse class of signaling molecules well known for their roles in cell fate specification, cell differentiation, and oncogenic transformation. Recently several RTKs have been implicated in cell and axon motility, and RTKs are known to mediate chemotactic guidance of tissue culture cells. We have investigated whether the Drosophila FGF receptor homolog, Breathless (BTL), whose activity is necessary for each phase of branching morphogenesis in the embryonic tracheal system, might play a role in guiding the directed migration of tracheal cells. We found that expression of a constitutively active receptor during tracheal development interfered with directed tracheal cell migration and led to extra secondary and terminal branch-forming cells. Reduction in endogenous BTL signaling enhanced the cell migration defects while suppressing the ectopic branching defects. These results are consistent with a model for tracheal development in which spatially regulated BTL activity guides tracheal cell migration and quantitatively regulated BTL activity determines the patterns of secondary and terminal branching cell fates.

  View details for Web of Science ID A1996VV15600009

  View details for PubMedID 8918892

• Genetic control of epithelial tube fusion during Drosophila tracheal development DEVELOPMENT Samakovlis, C., Manning, G., Steneberg, P., Hacohen, N., Cantera, R., Krasnow, M. A. 1996; 122 (11): 3531-3536

  Abstract

  During development of tubular networks such as the mammalian vascular system, the kidney and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous network. Little is known of the cellular mechanisms or molecular control of epithelial tube fusion. We describe the cellular dynamics of a tracheal fusion event in Drosophila and identify a gene regulatory hierarchy that controls this extraordinary process. A tracheal cell located at the developing fusion point expresses a sequence of specific markers as it grows out and contacts a similar cell from another tube; the two cells adhere and form an intercellular junction, and they become doughnut-shaped cells with the lumen passing through them. The early fusion marker Fusion-1 is identified as the escargot gene. It lies near the top of the regulatory hierarchy, activating the expression of later fusion markers and repressing genes that promote branching. Ectopic expression of escargot activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans. This establishes a simple genetic system to study fusion of epithelial tubes.

  View details for Web of Science ID A1996VV37300018

  View details for PubMedID 8951068

• The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered DEVELOPMENT Montagne, J., Groppe, J., Guillemin, K., Krasnow, M. A., Gehring, W. J., Affolter, M. 1996; 122 (9): 2589-2597

  Abstract

  The adult Drosophila wing is formed by an epithelial sheet, which differentiates into two non-neural tissues, vein or intervein. A large number of genes, many of them encoding components of an EGF-receptor signaling pathway, have previously been shown to be required for differentiation of vein tissue. Much less is known about the molecular control of intervein differentiation. Here we report that the Drosophila homolog of the mammalian Serum Response Factor gene (DSRF), which encodes a MADS-box containing transcriptional regulator, is expressed in the future intervein tissue of wing imaginal discs. In adult flies carrying only one functional copy of the DSRF gene, additional vein tissue develops in the wing, indicating that DSRF is required to spatially restrict the formation of veins. In mitotic clones lacking DSRF, intervein tissue fails to differentiate and becomes vein-like in appearance. Genetic and molecular evidence demonstrates that DSRF is encoded by the blistered locus, which produces ectopic veins and blistered wings when mutant. Our results show that DSRF plays a dual role during wing differentiation. It acts in a dosage-dependent [correction of dosage-dependant] manner to suppress the formation of wing veins and is required cell-autonomously to promote the development of intervein cells. We propose that DSRF acts at a key step between regulatory genes that define the early positional values in the developing wing disc and the subsequent localized expression of intervein-specific structural genes.

  View details for Web of Science ID A1996VJ07500003

  View details for PubMedID 8787734

• Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events DEVELOPMENT Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D. C., Guillemin, K., Krasnow, M. A. 1996; 122 (5): 1395-1407

  Abstract

  The tracheal (respiratory) system of Drosophila melanogaster is a branched network of epithelial tubes that ramifies throughout the body and transports oxygen to the tissues. It forms by a series of sequential branching events in each hemisegment from T2 to A8. Here we present a cellular and initial genetic analysis of the branching process. We show that although branching is sequential it is not iterative. The three levels of branching that we distinguish involve different cellular mechanisms of tube formation. Primary branches are multicellular tubes that arise by cell migration and intercalation; secondary branches are unicellular tubes formed by individual tracheal cells; terminal branches are subcellular tubes formed within long cytoplasmic extensions. Each level of branching is accompanied by expression of a different set of enhancer trap markers. These sets of markers are sequentially activated in progressively restricted domains and ultimately individual tracheal cells that are actively forming new branches. A clonal analysis demonstrates that branching fates are not assigned to tracheal cells until after cell division ceases and branching begins. We further show that the breathless FGF receptor, a tracheal gene required for primary branching, is also required to activate expression of markers involved in secondary branching and that the pointed ETS-domain transcription factor is required for secondary branching and also to activate expression of terminal branch markers. The combined morphological, marker expression and genetic data support a model in which successive branching events are mechanistically and genetically distinct but coupled through the action of a tracheal gene regulatory hierarchy.

  View details for Web of Science ID A1996UM55800006

  View details for PubMedID 8625828

• The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system DEVELOPMENT Guillemin, K., Groppe, J., Ducker, K., Treisman, R., Hafen, E., Affolter, M., Krasnow, M. A. 1996; 122 (5): 1353-1362

  Abstract

  We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.

  View details for Web of Science ID A1996UM55800002

  View details for PubMedID 8625824

• A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression EMBO JOURNAL VANGELDER, R. N., Krasnow, M. A. 1996; 15 (7): 1625-1631

  Abstract

  The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila.

  View details for Web of Science ID A1996UF65800017

  View details for PubMedID 8612586

  View details for PubMedCentralID PMC450072

• Partners in time. Circadian rhythms. Current biology Van Gelder, R. N., Krasnow, M. A. 1996; 6 (3): 244-246

  Abstract

  The timeless gene is a second essential component of the circadian clock in Drosophila; its product interacts physically with the only other known clock component, the period gene product. Together they control the daily cycle of expression of their own and other loci.

  View details for PubMedID 8805241

• Extent and character of circadian gene expression in Drosophila melanogaster: Identification of twenty oscillating mRNAs in the fly head CURRENT BIOLOGY VANGELDER, R. N., Bae, H., Palazzolo, M. J., Krasnow, M. A. 1995; 5 (12): 1424-1436

  Abstract

  Although mRNAs expressed with a circadian rhythm have been isolated from many species, the extent and character of circadianly regulated gene expression is unknown for any animal. In Drosophila melanogaster, only the period (per) gene, an essential component of the circadian pacemaker, is known to show rhythmic mRNA expression. Recent work suggests that the encoded Per protein controls its own transcription by an autoregulatory feedback loop. Per might also control the rhythmic expression of other genes to generate circadian behavior and physiology. The goals of this work were to evaluate the extent and character of circadian control of gene expression in Drosophila, and to identify genes dependent on per for circadian expression.A large collection of anonymous, independent cDNA clones was used to screen for transcripts that are rhythmically expressed in the fly head. 20 of the 261 clones tested detected mRNAs with a greater than two-fold daily change in abundance. Three mRNAs were maximally expressed in the morning, whereas 17 mRNAs were most abundant in the evening--when per mRNA is also maximally expressed (but when the flies are inactive). Further analysis of the three 'morning' cDNAs showed that each has a unique dependence on the presence of a light-dark cycle, on timed feeding, and on the function of the per gene for its oscillation. These dependencies were different from those determined for per and for a novel 'evening' gene. Sequence analysis indicated that all but one of the 20 cDNAs identified previously uncloned genes.Diurnal control of gene expression is a significant but limited phenomenon in the fly head, which involves many uncharacterized genes. Diurnal control is mediated by multiple endogenous and exogenous mechanisms, even at the level of individual genes. A subset of circadianly expressed genes are predominantly or exclusively dependent on per for their rhythmic expression. The per gene can therefore influence the expression of genes other than itself, but for many rhythmically expressed genes, per functions in conjunction with external inputs to control their daily expression patterns.

  View details for Web of Science ID A1995TL10200020

  View details for PubMedID 8749395

• EXTRADENTICLE PROTEIN IS A SELECTIVE COFACTOR FOR THE DROSOPHILA HOMEOTICS - ROLE OF THE HOMEODOMAIN AND YPWM AMINO-ACID MOTIF IN THE INTERACTION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johnson, F. B., Parker, E., Krasnow, M. A. 1995; 92 (3): 739-743

  Abstract

  The Drosophila homeotic selector (HOM) genes encode a family of DNA binding transcription factors that specify developmental fates of different body segments by differentially regulating the activity of downstream target genes. A central question is how the HOM proteins achieve their developmental specificity despite the very similar DNA binding specificities of isolated HOM proteins in vitro. Specificity could be achieved by differential interactions with protein cofactors. The extradenticle gene might encode such a cofactor since it interacts genetically in parallel with Ultrabithorax, abdominal-A, and perhaps other HOM genes. By using a yeast two-hybrid system, we demonstrate selective interaction of the extradenticle homeodomain protein with certain Ultrabithorax and abdominal-A proteins but not with an Antennapedia protein or a more distant homeodomain protein. Strong interaction with Ultrabithorax proteins requires only the Ultrabithorax homeodomain and a 15-residue N-terminal extension that includes Tyr-Pro-Trp-Met (YPWM), a tetrapeptide motif found near the homeodomain in most HOM proteins and their mammalian Hox counterparts. The size and sequence of the region between the YPWM element and the homeodomain differ among Ultrabithorax isoforms, and this variable region appears to affect the interaction detected in the assay.

  View details for Web of Science ID A1995QF18500020

  View details for PubMedID 7846045

  View details for PubMedCentralID PMC42695

• A DIFFERENTIAL RESPONSE ELEMENT FOR THE HOMEOTICS AT THE ANTENNAPEDIA P1 PROMOTER OF DROSOPHILA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Saffman, E. E., Krasnow, M. A. 1994; 91 (16): 7420-7424

  Abstract

  Homeotic genes encode DNA-binding transcription factors that specify the identity of a segment or segments in particular body regions of Drosophila. The developmental specificity of these proteins results from their differential regulation of various target genes. This specificity could be achieved by use of different regulatory elements by the homeoproteins or by use of the same elements in different ways. The Ultrabithorax (UBX), abdominal-A (ABD-A), and Antennapedia (ANTP) homeoproteins differentially regulate the Antennapedia P1 promoter in a cell culture cotransfection assay: UBX and ABD-A repress, whereas ANTP activates P1. Either of two regions of P1 can confer this pattern of differential regulation. One of the regions lies downstream and contains homeoprotein-binding sites flanking a 37-bp region called BetBS. ANTP protein activates transcription through the binding sites, whereas UBX and ABD-A both activate transcription through BetBS and use the flanking binding sites to prevent this effect. Thus, homeoproteins can use the same regulatory element but in very different ways. Chimeric UBX-ANTP proteins and UBX deletion derivatives demonstrate that functional specificity in P1 regulation is dictated mainly by sequences outside the homeodomain, with important determinants in the N-terminal region of the proteins.

  View details for Web of Science ID A1994PA37600009

  View details for PubMedID 7914367

  View details for PubMedCentralID PMC44412

• PREPARATION AND ANALYSIS OF PURE CELL-POPULATIONS FROM DROSOPHILA METHODS IN CELL BIOLOGY, VOL 44 Cumberledge, S., Krasnow, M. A. 1994; 44: 143-159

  View details for Web of Science ID A1994BC27U00009

  View details for PubMedID 7707949

• INTERCELLULAR SIGNALING IN DROSOPHILA SEGMENT FORMATION RECONSTRUCTED INVITRO NATURE Cumberledge, S., Krasnow, M. A. 1993; 363 (6429): 549-552

  Abstract

  Genetic studies show that intercellular signalling is involved in key steps in Drosophila melanogaster development, but it has not previously been possible to investigate these processes in simplified in vitro systems. Analysis of engrailed (en) and wingless (wg) and other segment polarity genes suggests that two or more intercellular signalling processes may be involved in intrasegmental patterning. Expression of en and wg begins about three hours after egg laying, in adjacent rows of cells in the posterior half of each segmental primordium. In wg- embryos and in conditional mutants in which wg function is inactivated during a critical period between three and five hours after egg laying, early en expression begins normally but then disappears within several hours. The wg gene encodes a protein highly similar to the product of the mouse Wnt-1 proto-oncogene, a secreted glycoprotein; wg protein is proposed to function as an extracellular signal, maintaining en expression and activating other molecular and morphogenetic processes in nearby cells. Several lines of evidence support the model, including the secretion of wg protein in the embryo, genetic mosaic experiments and cell lineage studies. We tested this model using purified embryonic cells isolated by whole animal cell sorting, and validated three key predictions: (1) when en-expressing cells from early embryos are grown alone in culture, they rapidly and selectively lose en expression; (2) purified wg-expressing cells provide a locally active signal that prevents this loss; (3) heterologous cells engineered to express wg also show signalling activity, indicating that wg protein alone, or in conjunction with more generally expressed factors, is the signal.

  View details for Web of Science ID A1993LF93900052

  View details for PubMedID 8505983

• Development of the Drosophila tracheal system. In the Development of Drosophila (Eds A. Martinez-Arias and M. Bate), Cold Spring Harbor Press (COld Spring Harbor, New York), Krasnow MA., Manning G 1993: 609-68
• DIFFERENTIAL REGULATION OF TRANSCRIPTION PREINITIATION COMPLEX ASSEMBLY BY ACTIVATOR AND REPRESSOR HOMEO DOMAIN PROTEINS GENES & DEVELOPMENT Johnson, F. B., Krasnow, M. A. 1992; 6 (11): 2177-2189

  Abstract

  Different eukaryotic transcription factors can act through the same upstream binding site to differentially regulate target gene expression, but little is known of the underlying mechanisms. Here, we show that Ultrabithorax and even-skipped homeo domain proteins (UBX and EVE) of Drosophila melanogaster exert active and opposite effects on in vitro transcription when bound to a common site upstream of a core promoter. Both the activator UBX and the repressor EVE affect the extent but not the rate constant of preinitiation complex (preIC) formation. Both regulators act early in preIC assembly and are dispensable later. Assembling complexes become resistant to regulation by the bound proteins, but activation by UBX is restored upon ATP or dATP addition, and regulation by both proteins is restored after the addition of all four nucleoside triphosphates and transcription initiation. The results establish that upstream activators and repressors can function by fundamentally similar mechanisms, by differentially regulating an early step in preIC assembly, leading to formation of functionally distinct transcription complexes. A subsequent step renders mature complexes transiently refractory to activation and repression. Implications for the mechanism of transcription complex assembly and turnover and its regulation are discussed, including a new role for ATP in turnover.

  View details for Web of Science ID A1992JX84600014

  View details for PubMedID 1358759

• WHOLE ANIMAL-CELL SORTING OF DROSOPHILA EMBRYOS SCIENCE Krasnow, M. A., Cumberledge, S., Manning, G., Herzenberg, L. A., Nolan, G. P. 1991; 251 (4989): 81-85

  Abstract

  Use of primary culture cells has been limited by the inability to purify most types of cells, particularly cells from early developmental stages. In whole animal cell sorting (WACS), live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and fluorescence-activated cell sorting. With WACS, incipient posterior compartment cells that express the engrailed gene were purified from early Drosophila embryos. Neuronal precursor cells were also purified, and they differentiated into neurons with high efficiency in culture. Because there are many lacZ strains, it may be possible to purify most types of Drosophila cells. The same approach is also applicable to other organisms for which germ-line transformation is possible.

  View details for Web of Science ID A1991EQ60300032

  View details for PubMedID 1898782

• STIMULATION OF TRANSCRIPTION BY AN ULTRABITHORAX PROTEIN INVITRO GENES & DEVELOPMENT Johnson, F. B., Krasnow, M. A. 1990; 4 (6): 1044-1052

  Abstract

  The Ultrabithorax (Ubx) gene of Drosophila melanogaster encodes a family of UBX proteins that are thought to specify the developmental fates of segments in the posterior thorax and anterior abdomen by controlling the expression of a set of target genes. UBX proteins bind DNA in vitro, and they activate or repress different natural and synthetic target promoters in cultured cells. Here it is shown that a purified UBX protein can stimulate transcription of a synthetic target gene in extracts of cultured D. melanogaster cells. Stimulation is dependent on the presence of upstream, promoter-region binding sites but is independent of binding site orientation. A naturally occurring binding site cluster and a binding site consensus sequence consisting of TAA trinucleotide repeats can mediate this activation. A minimal promoter fused to such sites is activated by UBX, suggesting that transcriptional stimulation could result from an interaction between the promoter-bound protein and the general transcriptional machinery.

  View details for Web of Science ID A1990DJ09500015

  View details for PubMedID 1974540

• TRANSCRIPTIONAL ACTIVATION BY THE ANTENNAPEDIA AND FUSHI-TARAZU PROTEINS IN CULTURED DROSOPHILA CELLS CELL Winslow, G. M., Hayashi, S., Krasnow, M., Hogness, D. S., Scott, M. P. 1989; 57 (6): 1017-1030

  Abstract

  Drosophila homeodomain proteins bind to specific DNA sequences in vitro and are hypothesized to regulate the transcription of other genes during development. Using a cotransfection assay, we have shown that homeodomain proteins encoded by the homeotic gene Antennapedia (Antp) and the segmentation gene fushi tarazu, as well as a hybrid homeodomain protein, are activators of transcription from specific promoters in cultured Drosophila cells. Sequences downstream of the Antp P1 and Ultrabithorax transcription start sites mediate the observed activation. A TAA-rich DNA sequence to which the Antp protein binds in vitro is sufficient to confer regulation on a heterologous promoter. The results demonstrate that homeodomain proteins are transcriptional regulators in vivo and that in cultured cells, different homeodomain-containing proteins can act upon a common sequence to modulate gene transcription.

  View details for Web of Science ID A1989AC40300015

  View details for PubMedID 2567631

• TRANSCRIPTIONAL ACTIVATION AND REPRESSION BY ULTRABITHORAX PROTEINS IN CULTURED DROSOPHILA CELLS CELL Krasnow, M. A., Saffman, E. E., Kornfeld, K., Hogness, D. S. 1989; 57 (6): 1031-1043

  Abstract

  Homeotic genes of Drosophila melanogaster such as Ultrabithorax (Ubx) and Antennapedia (Antp) have long been thought to select metameric identity during development by controlling the expression of various target genes. Here we describe a cotransfection assay in cultured D. melanogaster cells that is used to demonstrate that Ubx proteins (UBX) can repress an Antp promoter fusion and activate a Ubx promoter fusion, activities predicted from genetic studies. We show (a) that UBX proteins regulated the level of accurately initiated Antp P1 and Ubx transcripts, (b) that activation of the Ubx promoter required a downstream cluster of UBX binding sites, and (c) that binding site sequences were sufficient to confer regulation on a heterologous promoter, regardless of their orientation or precise position. We conclude that UBX proteins are transcriptional repressors and activators, and that their actions are mediated by binding to promoter region sequences. Each member of the UBX protein family has similar regulatory abilities, but the properties of synthetic mutant forms suggest that UBX proteins may have a modular design similar to other transcriptional regulators.

  View details for Web of Science ID A1989AC40300016

  View details for PubMedID 2567632

• Transcriptional regulation by homeotic gene products in cultured Drosophila cells and in vitro Current Communications in Molecular Biology, Cold Spring Harbor Press Parker, E., Johnson, B., Saffman, E.E., Krasnow, M.A. 1989: 23-29
• 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

• RECOMBINATION SITE SELECTION BY TN3 RESOLVASE - TOPOLOGICAL TESTS OF A TRACKING MECHANISM CELL Benjamin, H. W., Matzuk, M. M., Krasnow, M. A., Cozzarelli, N. R. 1985; 40 (1): 147-158

  Abstract

  In vitro recombination by Tn3 resolvase of plasmids containing two directly repeated recombination (res) sites generates two singly interlinked catenated rings. This simple product catenane structure was maintained over a wide range of substrate supercoil densities and in a reaction mixture in which phage lambda Int-mediated recombination generated its characteristic multiply interlinked forms. Using substrates containing four res sites, we found that resolvase recombined neighboring res sites with high preference. This position effect implies that resolvase searches systematically along the DNA for a partner site. Intervening res sites in the opposite orientation did not prevent translocation. We analyzed the geometric arrangement of the interlocked rings after multiple recombination events in a four-site substrate and the pattern of segregation of nonspecific reporter rings catenated to the standard substrate. The results of these novel topological tests imply that the translocating enzyme may not make continuous contact with the DNA.

  View details for Web of Science ID A1985AAZ2900018

  View details for PubMedID 2981625

• A TOPOLOGICAL TREATMENT OF RECOMBINATION AND TOPOISOMERASES COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Cozzarelli, N. R., Krasnow, M. A., GERRARD, S. P., White, J. H. 1984; 49: 383-400

  View details for Web of Science ID A1984AEJ8000044

  View details for PubMedID 6099248

• Site-specific recombination by Tn3 resolvase: models for pairing of recombination sites UCLA Symposia on Molecular and Cellular Biology Krasnow, M.A., Matzuk, M.M., Dugan, J.M., Benjamin, H.W., Cozzarelli, N.R. 1983; X: 637-659
• DETERMINATION OF THE ABSOLUTE HANDEDNESS OF KNOTS AND CATENANES OF DNA NATURE Krasnow, M. A., Stasiak, A., Spengler, S. J., Dean, F., Koller, T., Cozzarelli, N. R. 1983; 304 (5926): 559-560

  Abstract

  DNA winds about itself in a right-handed or left-handed fashion at several structural levels. The double helix is generally right-handed and is given a (+) sign by convention, whereas supercoiling of the helix axis is always (-) in the cell. The winding in higher -order forms such as knots and catenanes is unknown, and this has impeded elucidation of the mechanisms of their formation and resolution by replication, recombination and topoisomerase action. We introduce here a procedure for determining the handedness of DNA winding by inspection of electron micrographs of DNA molecules coated with Escherichia coli RecA protein. We demonstrate the validity of the method and show that DNA topoisomerase I of E. coli generates an equal mixture of (+) and (-) duplex DNA knots, and that one product of recombination by resolvase of transposon Tn3 (refs 8, 9) is a catenane of uniquely (+) sign.

  View details for Web of Science ID A1983RC16500056

  View details for PubMedID 6308470

• Escherichia coli type-1 topoisomerases: identification, mechanism, and role in recombination. Cold Spring Harbor symposia on quantitative biology Dean, F., Krasnow, M. A., Otter, R., Matzuk, M. M., Spengler, S. J., Cozzarelli, N. R. 1983; 47: 769-777

  View details for PubMedID 6305585

• SITE-SPECIFIC RELAXATION AND RECOMBINATION BY THE TN3 RESOLVASE - RECOGNITION OF THE DNA PATH BETWEEN ORIENTED RES SITES CELL Krasnow, M. A., Cozzarelli, N. R. 1983; 32 (4): 1313-1324

  Abstract

  We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.

  View details for Web of Science ID A1983QN11800033

  View details for PubMedID 6301692

• Catenation of DNA rings by Topoisomerases: mechanism of control by spermidine Journal of Biological Chemistry Krasnow, M.A., Cozzarelli, N.R. 1982; 257: 2687-2693
• ESCHERICHIA-COLI TYPE-1 TOPOISOMERASES - IDENTIFICATION, MECHANISM, AND ROLE IN RECOMBINATION COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Dean, F., Krasnow, M. A., Otter, R., Matzuk, M. M., Spengler, S. J., Cozzarelli, N. R. 1982; 47: 769-777

  View details for Web of Science ID A1982QR19100021

• CATENATION OF DNA RINGS BY TOPOISOMERASES - MECHANISM OF CONTROL BY SPERMIDINE JOURNAL OF BIOLOGICAL CHEMISTRY Krasnow, M. A., Cozzarelli, N. R. 1982; 257 (5): 2687-2693

  View details for Web of Science ID A1982NE46000089

  View details for PubMedID 6277910