Honors & Awards
K99/R00 Pathway to Independence Award (NIH/NIGMS), Stanford University (2019-2022)
American Heart Association Postdoctoral Fellowship, Stanford University (2019)
School of Medicine Dean's Postdoctoral Fellowship, Stanford University (2016-2017)
Jump Start Award for Excellence in Research, Stanford University (2016)
Dr. Eva Mary Kavan Prize for Excellence in Research on the Brain, UCLA Brain Research Institute (2015)
Dissertation Year Fellowship, UCLA Graduate Division (2013-2014)
Distinction in Teaching Award, UCLA Life Sciences (2010)
Graduation with honors in Biology, Willamette University (2007)
Carson Undergraduate Research Grant, Willamette University (2006)
Philip C. Armstrong Biology Scholarship, Willamette University (2006)
Bachelor of Arts, Willamette University (2007)
Doctor of Philosophy, University of California Los Angeles (2015)
Rajat Rohatgi, Postdoctoral Faculty Sponsor
Gene-teratogen interactions influence the penetrance of birth defects by altering Hedgehog signaling strength.
Development (Cambridge, England)
Birth defects result from interactions between genetic and environmental factors, but the mechanisms remain poorly understood. We find that mutations and teratogens interact in predictable ways to cause birth defects by changing target cell sensitivity to Hedgehog (Hh) ligands. These interactions converge on a membrane protein complex, the MMM complex, that promotes degradation of the Hh transducer Smoothened (SMO). Deficiency of the MMM component MOSMO results in elevated SMO and increased Hh signaling, causing multiple birth defects. In utero exposure to a teratogen that directly inhibits SMO reduces the penetrance and expressivity of birth defects in Mosmo-/- embryos. Additionally, tissues that develop normally in Mosmo-/- embryos are refractory to the teratogen. Thus, changes in the abundance of the protein target of a teratogen can change birth defect outcomes by quantitative shifts in Hh signaling. Consequently, small molecules that re-calibrate signaling strength could be harnessed to rescue structural birth defects.
View details for DOI 10.1242/dev.199867
View details for PubMedID 34486668
- Ten simple rules for writing compelling recommendation letters. PLoS computational biology 2021; 17 (2): e1008656
A Membrane-Tethered Ubiquitination Pathway Regulates Hedgehog Signaling and Heart Development.
The etiology of congenital heart defects (CHDs), which are among the most common human birth defects, is poorly understood because of its complex genetic architecture. Here, we show that two genes implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a receptor-like ubiquitin ligase complex that catalyzes the ubiquitination and degradation of the Hedgehog pathway transducer Smoothened. Homozygous Megf8 and Mgrn1 mutations increased Smoothened abundance and elevated sensitivity to Hedgehog ligands. While mice heterozygous for loss-of-function Megf8 or Mgrn1 mutations were normal, double heterozygous embryos exhibited an incompletely penetrant syndrome of CHDs with heterotaxy. Thus, genetic interactions can arise from biochemical mechanisms that calibrate morphogen signaling strength, a conclusion broadly relevant for the many human diseases in which oligogenic inheritance is emerging as a mechanism for heritability.
View details for DOI 10.1016/j.devcel.2020.08.012
View details for PubMedID 32966817
- Advice for the worried. Science (New York, N.Y.) 2020; 368 (6489): 438
Biochemical mechanisms of vertebrate hedgehog signaling.
Development (Cambridge, England)
2019; 146 (10)
Signaling pathways that mediate cell-cell communication are essential for collective cell behaviors in multicellular systems. The hedgehog (HH) pathway, first discovered and elucidated in Drosophila, is one of these iconic signaling systems that plays many roles during embryogenesis and in adults; abnormal HH signaling can lead to birth defects and cancer. We review recent structural and biochemical studies that have advanced our understanding of the vertebrate HH pathway, focusing on the mechanisms by which the HH signal is received by patched on target cells, transduced across the cell membrane by smoothened, and transmitted to the nucleus by GLI proteins to influence gene-expression programs.
View details for PubMedID 31092502
Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling.
Previously we proposed that transmission of the Hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.
View details for DOI 10.7554/eLife.50051
View details for PubMedID 31657721
Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics
2018; 16 (2): e2003127
During tissue development, multipotent progenitors differentiate into specific cell types in characteristic spatial and temporal patterns. We addressed the mechanism linking progenitor identity and differentiation rate in the neural tube, where motor neuron (MN) progenitors differentiate more rapidly than other progenitors. Using single cell transcriptomics, we defined the transcriptional changes associated with the transition of neural progenitors into MNs. Reconstruction of gene expression dynamics from these data indicate a pivotal role for the MN determinant Olig2 just prior to MN differentiation. Olig2 represses expression of the Notch signaling pathway effectors Hes1 and Hes5. Olig2 repression of Hes5 appears to be direct, via a conserved regulatory element within the Hes5 locus that restricts expression from MN progenitors. These findings reveal a tight coupling between the regulatory networks that control patterning and neuronal differentiation and demonstrate how Olig2 acts as the developmental pacemaker coordinating the spatial and temporal pattern of MN generation.
View details for DOI 10.1371/journal.pbio.2003127
View details for Web of Science ID 000426253300003
View details for PubMedID 29389974
View details for PubMedCentralID PMC5811045
G protein-coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog.
2018; 11 (516)
The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαsand PKA.
View details for PubMedID 29438014
View details for PubMedCentralID PMC5828112
CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog.
2018; 44 (1): 113–29.e8
To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspan protein we named Atthog. The function of these negative regulators converged on Smoothened (SMO), an oncoprotein that transduces the Hh signal across the membrane. In the absence of Atthog, SMO was stabilized at the cell surface and concentrated in the ciliary membrane, boosting cell sensitivity to the ligand Sonic Hedgehog (SHH) and consequently altering SHH-guided neural cell-fate decisions. Thus, we uncovered genes that modify the interpretation of morphogen signals by regulating protein-trafficking events in target cells.
View details for PubMedID 29290584
View details for PubMedCentralID PMC5792066
Netrin1 Produced by Neural Progenitors, Not Floor Plate Cells, Is Required for Axon Guidance in the Spinal Cord
2017; 94 (4): 790-?
Netrin1 has been proposed to act from the floor plate (FP) as a long-range diffusible chemoattractant for commissural axons in the embryonic spinal cord. However, netrin1 mRNA and protein are also present in neural progenitors within the ventricular zone (VZ), raising the question of which source of netrin1 promotes ventrally directed axon growth. Here, we use genetic approaches in mice to selectively remove netrin from different regions of the spinal cord. Our analyses show that the FP is not the source of netrin1 directing axons to the ventral midline, while local VZ-supplied netrin1 is required for this step. Furthermore, rather than being present in a gradient, netrin1 protein accumulates on the pial surface adjacent to the path of commissural axon extension. Thus, netrin1 does not act as a long-range secreted chemoattractant for commissural spinal axons but instead promotes ventrally directed axon outgrowth by haptotaxis, i.e., directed growth along an adhesive surface.
View details for DOI 10.1016/j.neuron.2017.03.007
View details for PubMedID 28434801
Cholesterol activates the G-protein coupled receptor Smoothened to promote morphogenetic signaling.
Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.
View details for DOI 10.7554/eLife.20304
View details for PubMedID 27705744
View details for PubMedCentralID PMC5123864
Notch Activity Modulates the Responsiveness of Neural Progenitors to Sonic Hedgehog Signaling
2015; 33 (4): 373-387
Throughout the developing nervous system, neural stem and progenitor cells give rise to diverse classes of neurons and glia in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges through the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh gradient depends on both the amount of ligand and duration of exposure, but the mechanisms permitting prolonged responses to Shh are not well understood. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to attain sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates subcellular localization of the Shh receptor Patched1, gating the translocation of the key effector Smoothened to primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch shaping the interpretation of the Shh morphogen gradient and influencing cell fate determination.
View details for DOI 10.1016/j.devcel.2015.03.005
View details for Web of Science ID 000355151900004
View details for PubMedID 25936505
View details for PubMedCentralID PMC4449290
Gli protein activity is controlled by multisite phosphorylation in vertebrate hedgehog signaling.
2014; 6 (1): 168-181
Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity.
View details for DOI 10.1016/j.celrep.2013.12.003
View details for PubMedID 24373970
View details for PubMedCentralID PMC3915062
- My Brain Told Me to Do It DEVELOPMENTAL CELL 2013; 25 (5): 436-438
Retinoid Acid Specifies Neuronal Identity through Graded Expression of Ascl1
2013; 23 (5): 412-418
Cell diversity and organization in the neural tube depend on the integration of extrinsic signals acting along orthogonal axes. These are believed to specify distinct cellular identities by triggering all-or-none changes in expression of combinations of transcription factors. Under the influence of a common dorsoventral signal, sonic hedgehog, and distinct anterior-posterior (A-P) inductive signals, two topographically related progenitor pools that share a common transcriptional code produce serotonergic and V3 neurons in the hindbrain and spinal cord, respectively. These neurons have different physiological properties, functions, and connectivity. Serotonergic involvement in neuropsychiatric diseases has prompted greater characterization of their postmitotic repertoire of fate determinants, which include Gata2, Lmx1b, and Pet1, whereas V3 neurons express Sim1. How distinct serotonergic and V3 neuronal identities emerge from progenitors that share a common transcriptional code is not understood. Here, we show that changes in retinoid activity in these two progenitor pools determine their fates. Retinoids, via Notch signaling, control the expression level in progenitors of the transcription factor Ascl1, which selects serotonergic and V3 neuronal identities in a dose-dependent manner. Therefore, quantitative differences in the expression of a single component of a transcriptional code can select distinct cell fates.
View details for DOI 10.1016/j.cub.2013.01.046
View details for Web of Science ID 000315764000025
View details for PubMedID 23416099
Transcription factor Olig2 defines subpopulations of retinal progenitor cells biased toward specific cell fates
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (20): 7882-7887
Previous lineage analyses have shown that retinal progenitor cells (RPCs) are multipotent throughout development, and expression-profiling studies have shown a great deal of molecular heterogeneity among RPCs. To determine if the molecular heterogeneity predicts that an RPC will produce particular types of progeny, clonal lineage analysis was used to investigate the progeny of a subset of RPCs, those that express the basic helix-loop-helix transcription factor, Olig2. The embryonic Olig2(+) RPCs underwent terminal divisions, producing small clones with primarily two of the five cell types being made by the pool of RPCs at that time. The later, postnatal Olig2(+) RPCs also made terminal divisions, which were biased toward production of rod photoreceptors and amacrine cell interneurons. These data indicate that the multipotent progenitor pool is made up of distinctive types of RPCs, which have biases toward producing subsets of retinal neurons in a terminal division, with the types of neurons produced varying over time. This strategy is similar to that of the developing Drosophila melanogaster ventral nerve cord, with the Olig2(+) cells behaving as ganglion mother cells.
View details for DOI 10.1073/pnas.1203138109
View details for Web of Science ID 000304369800066
View details for PubMedID 22543161