Academic Appointments

Administrative Appointments

  • Department of Ophthalmology, Stanford University School of Medicine (2017 - Present)

Honors & Awards

  • Pathway to Stop Diabetes Career Initiator Award, American Diabetes Association (2016)
  • McCormick and Gabilan Faculty Award, McCormick and Gabilan Foundation (2017)
  • E. Matilda Ziegler Foundation for the Blind Award, E. Matilda Ziegler Foundation (2018)
  • Doris Duke Pilot Funding, Doris Duke Foundation (2022)

Professional Education

  • Postdoc, Harvard Medical School, Genetics (2016)
  • PhD, Vanderbilt University, Cell and Developmental Biology (2009)
  • BS, Fudan University, Biological Sciences (2004)

Current Research and Scholarly Interests

Our research focuses on unraveling the molecular mechanisms underlying retinal development and diseases. We employ genetic and genomic tools to explore how various retinal cell types, including neurons, glia, and the vasculature, respond to developmental cues and disease insults at the epigenomic and transcriptional levels. In addition, we investigate their interactions and collective contributions to maintain retinal integrity.

1. Investigating retinal development:
We utilize genetic tools and methods such as in vivo plasmid electroporation and CRISPR to dissect the roles of cis-regulatory elements and transcription factors in controlling retinal development.

2. Understanding diabetes-induced cell-type-specific responses in the retina:
Diabetes triggers a range of multicellular responses in the retina, such as vascular lesions, glial dysfunction, and neurodegeneration, all of which contribute to retinopathy. We delve into the detailed molecular mechanisms underlying these diabetes-induced cell-type-specific responses and the pathogenesis of diabetic retinopathy.

3. Developing molecular tools for labeling and manipulation of specific cell types in vivo:
Cis-regulatory elements, particularly enhancers, play pivotal roles in directing tissue- and cell-type-specific expression. Our interest lies in identifying enhancers that can drive cell type-specific expression in the retina and brain. We incorporate these enhancers into plasmid or AAV-based delivery systems, enabling precise labeling and manipulation of specific cell types in vivo.

Stanford Advisees

  • Doctoral Dissertation Reader (AC)
    Emma Costa, Cheyanne Lewis
  • Postdoctoral Faculty Sponsor
    Boxiong Deng
  • Doctoral Dissertation Advisor (AC)
    Chelsea Nnebe
  • Doctoral Dissertation Co-Advisor (AC)
    Duo Sun

Graduate and Fellowship Programs

All Publications

  • iGWAS: Image-based genome-wide association of self-supervised deep phenotyping of retina fundus images. PLoS genetics Xie, Z., Zhang, T., Kim, S., Lu, J., Zhang, W., Lin, C. H., Wu, M. R., Davis, A., Channa, R., Giancardo, L., Chen, H., Wang, S., Chen, R., Zhi, D. 2024; 20 (5): e1011273


    Existing imaging genetics studies have been mostly limited in scope by using imaging-derived phenotypes defined by human experts. Here, leveraging new breakthroughs in self-supervised deep representation learning, we propose a new approach, image-based genome-wide association study (iGWAS), for identifying genetic factors associated with phenotypes discovered from medical images using contrastive learning. Using retinal fundus photos, our model extracts a 128-dimensional vector representing features of the retina as phenotypes. After training the model on 40,000 images from the EyePACS dataset, we generated phenotypes from 130,329 images of 65,629 British White participants in the UK Biobank. We conducted GWAS on these phenotypes and identified 14 loci with genome-wide significance (p<5×10-8 and intersection of hits from left and right eyes). We also did GWAS on the retina color, the average color of the center region of the retinal fundus photos. The GWAS of retina colors identified 34 loci, 7 are overlapping with GWAS of raw image phenotype. Our results establish the feasibility of this new framework of genomic study based on self-supervised phenotyping of medical images.

    View details for DOI 10.1371/journal.pgen.1011273

    View details for PubMedID 38728357

  • Dendritic mGluR2 and perisomatic Kv3 signaling regulate dendritic computation of mouse starburst amacrine cells. Nature communications Acarón Ledesma, H., Ding, J., Oosterboer, S., Huang, X., Chen, Q., Wang, S., Lin, M. Z., Wei, W. 2024; 15 (1): 1819


    Dendritic mechanisms driving input-output transformation in starburst amacrine cells (SACs) are not fully understood. Here, we combine two-photon subcellular voltage and calcium imaging and electrophysiological recording to determine the computational architecture of mouse SAC dendrites. We found that the perisomatic region integrates motion signals over the entire dendritic field, providing a low-pass-filtered global depolarization to dendrites. Dendrites integrate local synaptic inputs with this global signal in a direction-selective manner. Coincidental local synaptic inputs and the global motion signal in the outward motion direction generate local suprathreshold calcium transients. Moreover, metabotropic glutamate receptor 2 (mGluR2) signaling in SACs modulates the initiation of calcium transients in dendrites but not at the soma. In contrast, voltage-gated potassium channel 3 (Kv3) dampens fast voltage transients at the soma. Together, complementary mGluR2 and Kv3 signaling in different subcellular regions leads to dendritic compartmentalization and direction selectivity, highlighting the importance of these mechanisms in dendritic computation.

    View details for DOI 10.1038/s41467-024-46234-7

    View details for PubMedID 38418467

    View details for PubMedCentralID 4371705

  • Gut-liver axis calibrates intestinal stem cell fitness. Cell Kim, G., Chen, Z., Li, J., Luo, J., Castro-Martinez, F., Wisniewski, J., Cui, K., Wang, Y., Sun, J., Ren, X., Crawford, S. E., Becerra, S. P., Zhu, J., Liu, T., Wang, S., Zhao, K., Wu, C. 2024


    The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/β-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

    View details for DOI 10.1016/j.cell.2024.01.001

    View details for PubMedID 38280375

  • EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Muller cells. Molecular vision Liu, L., Jiang, Y., Al-Shabrawey, M., Ren, X., Wang, S., Steinle, J. J. 2024; 30: 167-174


    Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Muller cells.Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Muller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Muller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo.Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Muller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs.Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Muller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.

    View details for PubMedID 38601015

  • A positively tuned voltage indicator for extended electrical recordings in the brain. Nature methods Evans, S. W., Shi, D., Chavarha, M., Plitt, M. H., Taxidis, J., Madruga, B., Fan, J. L., Hwang, F., van Keulen, S. C., Suomivuori, C., Pang, M. M., Su, S., Lee, S., Hao, Y. A., Zhang, G., Jiang, D., Pradhan, L., Roth, R. H., Liu, Y., Dorian, C. C., Reese, A. L., Negrean, A., Losonczy, A., Makinson, C. D., Wang, S., Clandinin, T. R., Dror, R. O., Ding, J. B., Ji, N., Golshani, P., Giocomo, L. M., Bi, G., Lin, M. Z. 2023; 20 (7): 1104-1113


    Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

    View details for DOI 10.1038/s41592-023-01913-z

    View details for PubMedID 37429962

  • α A-crystallin's role in the regulation of Muller cell trophic support Gregolynskyj, A., Wang, S., Fort, P. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2023
  • Postsynaptic synucleins mediate endocannabinoid signaling. Nature neuroscience Albarran, E., Sun, Y., Liu, Y., Raju, K., Dong, A., Li, Y., Wang, S., Sudhof, T. C., Ding, J. B. 2023


    Endocannabinoids are among the most powerful modulators of synaptic transmission throughout the nervous system, and yet little is understood about the release of endocannabinoids from postsynaptic compartments. Here we report an unexpected finding that endocannabinoid release requires synucleins, key contributors to Parkinson's disease. We show that endocannabinoids are released postsynaptically by a synuclein-dependent and SNARE-dependent mechanism. Specifically, we found that synuclein deletion blocks endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wild-type but not of mutant alpha-synuclein. Whole-cell recordings and direct optical monitoring of endocannabinoid signaling suggest that the synuclein deletion specifically blocks endocannabinoid release. Given the presynaptic role of synucleins in regulating vesicle lifecycle, we hypothesize that endocannabinoids are released via a membrane interaction mechanism. Consistent with this hypothesis, postsynaptic expression of tetanus toxin light chain, which cleaves synaptobrevin SNAREs, also blocks endocannabinoid-dependent signaling. The unexpected finding that endocannabinoids are released via a synuclein-dependent mechanism is consistent with a general function of synucleins in membrane trafficking and adds a piece to the longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.

    View details for DOI 10.1038/s41593-023-01345-0

    View details for PubMedID 37248337

  • Mettl14-mediated m6A modification ensures the cell-cycle progression of late-born retinal progenitor cells. Cell Reports Li, L., Sun, Y., Davis, A. E., Shah, S. H., Hamed, L. K., Wu, M., Lin, C., Ding, J. B., Wang, S. 2023
  • Multiplexed genome regulation in vivo with hyper-efficient Cas12a. Nature cell biology Guo, L. Y., Bian, J., Davis, A. E., Liu, P., Kempton, H. R., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D. A., Xu, X., Jamiolkowski, R. M., Hu, Y., Wang, S., Qi, L. S. 2022


    Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

    View details for DOI 10.1038/s41556-022-00870-7

    View details for PubMedID 35414015

  • A protocol to inject ocular drug implants into mouse eyes. STAR protocols Lin, C., Sun, Y. J., Lee, S. H., Mujica, E. M., Kunchur, C. R., Wu, M., Yang, J., Jung, Y. S., Chiang, B., Wang, S., Mahajan, V. B. 2022; 3 (1): 101143


    Ocular drug implants (ODIs) are beneficial for treating ocular diseases. However, the lack of a robust injection approach for small-eyed model organisms has been a major technical limitation in developing ODIs. Here, we present a cost-effective, minimally invasive protocol to deliver ODIs into the mouse vitreous called Mouse Implant Intravitreal Injection (MI3). MI3 provides two alternative surgical approaches (air-pressure or plunger) to deliver micro-scaled ODIs into milli-scaled eyes, and expands the preclinical platforms to determine ODIs' efficacy, toxicity, and pharmacokinetics. For complete details on the use and execution of this protocol, please refer to Sun etal. (2021).

    View details for DOI 10.1016/j.xpro.2022.101143

    View details for PubMedID 35141566

  • Identification of cis-regulatory modules for adeno-associated virus-based cell type-specific targeting in the retina and brain. The Journal of biological chemistry Lin, C. H., Sun, Y., Chan, C. S., Wu, M. R., Gu, L., Davis, A. E., Gu, B., Zhang, W., Tanasa, B., Zhong, L. R., Emerson, M. M., Chen, L., Ding, J., Wang, S. 2022: 101674


    Adeno Associated Viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which have then been incorporated into AAVs and tested for cell type-specificity in the CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs, as many chromatin-accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, here we explored strategies to increase efficiency in identifying active CRMs for AAV-based cell type-specific labeling and manipulation. We found that pre-screening of chromatin-accessible putative CRMs based on the density of cell type-specific transcription factor binding sites (TFBSs) can significantly increase efficiency in identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin-accessible regions flanking cell type-specific genes can render cell type-specificity in many cases. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain. Both strategies utilize available genomic datasets and can be employed to generate AAVs targeting specific cell types in CNS without conducting comprehensive screening and sequencing experiments, making a step forward in cell type-specific research.

    View details for DOI 10.1016/j.jbc.2022.101674

    View details for PubMedID 35148987

  • An intravitreal implant injection method for sustained drug delivery into mouse eyes. Cell reports methods Sun, Y. J., Lin, C., Wu, M., Lee, S. H., Yang, J., Kunchur, C. R., Mujica, E. M., Chiang, B., Jung, Y. S., Wang, S., Mahajan, V. B. 2021; 1 (8)


    Using small molecule drugs to treat eye diseases carries benefits of specificity, scalability, and transportability, but their efficacy is significantly limited by a fast intraocular clearance rate. Ocular drug implants (ODIs) present a compelling means for the slow and sustained release of small molecule drugs inside the eye. However, methods are needed to inject small molecule ODIs into animals with small eyes, such as mice, which are the primary genetic models for most human ocular diseases. Consequently, it has not been possible to fully investigate efficacy and ocular pharmacokinetics of ODIs. Here, we present a robust, cost-effective, and minimally invasive method called "mouse implant intravitreal injection" (MI3) to deliver ODIs into mouse eyes. This method will expand ODI research to cover the breadth of human eye diseases modeled in mice.

    View details for DOI 10.1016/j.crmeth.2021.100125

    View details for PubMedID 35128514

  • Foxo1 controls gut homeostasis and commensalism by regulating mucus secretion. The Journal of experimental medicine Chen, Z., Luo, J., Li, J., Kim, G., Chen, E. S., Xiao, S., Snapper, S. B., Bao, B., An, D., Blumberg, R. S., Lin, C. H., Wang, S., Zhong, J., Liu, K., Li, Q., Wu, C., Kuchroo, V. K. 2021; 218 (9)


    Mucus produced by goblet cells in the gastrointestinal tract forms a biological barrier that protects the intestine from invasion by commensals and pathogens. However, the host-derived regulatory network that controls mucus secretion and thereby changes gut microbiota has not been well studied. Here, we identify that Forkhead box protein O1 (Foxo1) regulates mucus secretion by goblet cells and determines intestinal homeostasis. Loss of Foxo1 in intestinal epithelial cells (IECs) results in defects in goblet cell autophagy and mucus secretion, leading to an impaired gut microenvironment and dysbiosis. Subsequently, due to changes in microbiota and disruption in microbiome metabolites of short-chain fatty acids, Foxo1 deficiency results in altered organization of tight junction proteins and enhanced susceptibility to intestinal inflammation. Our study demonstrates that Foxo1 is crucial for IECs to establish commensalism and maintain intestinal barrier integrity by regulating goblet cell function.

    View details for DOI 10.1084/jem.20210324

    View details for PubMedID 34287641

  • Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and cis-regulatory modules in the retina. Development (Cambridge, England) Chan, C. S., Lonfat, N., Zhao, R., Davis, A. E., Li, L., Wu, M., Lin, C., Ji, Z., Cepko, C. L., Wang, S. 2020


    Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, Orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells utilize a cohort of TFs with different expression patterns, and multiple CRMs with different chromatin configurations, to precisely regulate the expression of Otx2.

    View details for DOI 10.1242/dev.187922

    View details for PubMedID 32631829

  • Enhancer transcription identifies cis-regulatory elements for photoreceptor cell types. Development (Cambridge, England) Perez-Cervantes, C., Smith, L. A., Nadadur, R. D., Hughes, A. E., Wang, S., Corbo, J. C., Cepko, C., Lonfat, N., Moskowitz, I. P. 2020


    Identification of cell-type specific cis-regulatory elements (CREs) is critical for understanding development and disease, although identification of functional regulatory elements remains challenging. We hypothesized that context-specific CREs could be identified by context-specific non-coding RNA (ncRNA) profiling, based on the observation that active CREs produce ncRNAs. We applied ncRNA profiling to identify rod and cone photoreceptor CREs from wild-type and mutant mouse retinas, defined by presence or absence of the rod-specific transcription factor (TF), Nrl, respectively. Nrl-dependent ncRNA expression strongly correlated with epigenetic profiles of rod and cone photoreceptors, identified thousands of candidate rod- and cone-specific CREs, and identified motifs for rod- and cone-specific TFs. Colocalization of NRL and the retinal TF CRX correlated with rod-specific ncRNA expression, whereas CRX alone favored cone-specific ncRNA expression, providing quantitative evidence that heterotypic TF interactions distinguish cell type-specific CRE activity. We validated the activity of novel Nrl-dependent ncRNA-defined CREs in developing cones. This work supports differential ncRNA profiling as a platform for the identification of cell-type specific CREs and discovery of molecular mechanisms underlying TF-dependent CRE activity.

    View details for DOI 10.1242/dev.184432

    View details for PubMedID 31915147

  • Neurog3-Independent Methylation Is the Earliest Detectable Mark Distinguishing Pancreatic Progenitor Identity. Developmental cell Liu, J., Banerjee, A., Herring, C. A., Attalla, J., Hu, R., Xu, Y., Shao, Q., Simmons, A. J., Dadi, P. K., Wang, S., Jacobson, D. A., Liu, B., Hodges, E., Lau, K. S., Gu, G. 2019; 48 (1): 49


    In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: alpha, beta, delta, and gamma; when and how the Neurog3+ cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3+ cells co-expressing Myt1 (i.e., Myt1+Neurog3+) were biased toward beta cell fate, while those not simultaneously expressing Myt1 (Myt1-Neurog3+) favored alpha fate. Myt1 manipulation only marginally affected alpha versus beta cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1+Neurog3+ cells displayed higher Dnmt1 expression and enhancer methylation at Arx, an alpha-fate-promotinggene. Inhibiting Dnmts in pancreatic progenitors promoted alpha cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored beta cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs.

    View details for PubMedID 30620902

  • The THO Complex Coordinates Transcripts for Synapse Development and Dopamine Neuron Survival. Cell Maeder, C. I., Kim, J., Liang, X., Kaganovsky, K., Shen, A., Li, Q., Li, Z., Wang, S., Xu, X. Z., Li, J. B., Xiang, Y. K., Ding, J. B., Shen, K. 2018


    Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export complex (THOC) as an important regulator of presynapse development in C.elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are retained in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses, and compromised dopamine function. CRE binding protein (CREB) interacts with THOC to mark synaptic transcripts for efficient nuclear export. Deletion of Thoc5, a THOC subunit, in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the substantia nigra compacta. These cellular defects lead to abrogated dopamine release, ataxia, and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for neuronal differentiation and survival.

    View details for PubMedID 30146163

  • Synaptotagmin 4 Regulates Pancreatic beta Cell Maturation by Modulating the Ca2+ Sensitivity of Insulin Secretion Vesicles DEVELOPMENTAL CELL Huang, C., Walker, E. M., Dadi, P. K., Hu, R., Xu, Y., Zhang, W., Sanavia, T., Mun, J., Liu, J., Nair, G. G., Tan, H., Wang, S., Magnuson, M. A., Stoeckert, C. J., Hebrok, M., Gannon, M., Han, W., Stein, R., Jacobson, D. A., Gu, G. 2018; 45 (3): 347-+


    Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the β cell Ca2+ sensor Syt7, increased by ∼8-fold during β cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating β cell maturation.

    View details for PubMedID 29656931

    View details for PubMedCentralID PMC5962294

  • Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury. Nature Lin, S. n., Nascimento, E. M., Gajera, C. R., Chen, L. n., Neuhöfer, P. n., Garbuzov, A. n., Wang, S. n., Artandi, S. E. 2018


    Hepatocytes are replenished gradually during homeostasis and robustly after liver injury1, 2. In adults, new hepatocytes originate from the existing hepatocyte pool3-8, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases9-11. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERTHighhepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERTHighhepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERTHighhepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERTHighhepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a 'distributed model' of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.

    View details for PubMedID 29618815

  • Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies ELIFE Tang, J. C., Drokhlyansky, E., Etemad, B., Rudolph, S., Guo, B., Wang, S., Ellis, E. G., Li, J. Z., Cepko, C. L. 2016; 5


    The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes.

    View details for DOI 10.7554/eLife.15312

    View details for Web of Science ID 000379852400001

    View details for PubMedID 27205882

  • Photoreceptor Fate Determination in the Vertebrate Retina INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Wang, S., Cepko, C. L. 2016; 57 (5)


    Photoreceptors are highly specialized primary sensory neurons that sense light and initiate vision. This critical role is well demonstrated by the fact that visual impairment accompanies photoreceptor loss or dysfunction in many human diseases. With the remarkable advances in stem cell research, one therapeutic approach is to use stem cells to generate photoreceptors and then engraft them into diseased eyes. Knowledge of the molecular mechanisms that control photoreceptor genesis during normal development can greatly aid in the production of photoreceptor cells for this approach. This article will discuss advances in our understanding of the molecular mechanisms that regulate photoreceptor fate determination during development. Recent lineage studies have shown that there are distinct retinal progenitor cells (RPCs) that produce specific combinations of daughter cell types, including photoreceptors and other types of retinal cells. Gene regulatory networks, in which transcription factors interact via cis-regulatory DNA elements, have been discovered that operate within distinct RPCs, and/or newly postmitotic cells, to direct the choice of photoreceptor fate.

    View details for DOI 10.1167/iovs.15-17672

    View details for Web of Science ID 000378039300005

    View details for PubMedID 27116662

  • A Gene Regulatory Network Controls the Binary Fate Decision of Rod and Bipolar Cells in the Vertebrate Retina DEVELOPMENTAL CELL Wang, S., Sengel, C., Emerson, M. M., Cepko, C. L. 2014; 30 (5): 513-527


    Gene regulatory networks (GRNs) regulate critical events during development. In complex tissues, such as the mammalian central nervous system (CNS), networks likely provide the complex regulatory interactions needed to direct the specification of the many CNS cell types. Here, we dissect a GRN that regulates a binary fate decision between two siblings in the murine retina, the rod photoreceptor and bipolar interneuron. The GRN centers on Blimp1, one of the transcription factors (TFs) that regulates the rod versus bipolar cell fate decision. We identified a cis-regulatory module (CRM), B108, that mimics Blimp1 expression. Deletion of genomic B108 by CRISPR/Cas9 in vivo using electroporation abolished the function of Blimp1. Otx2 and RORβ were found to regulate Blimp1 expression via B108, and Blimp1 and Otx2 were shown to form a negative feedback loop that regulates the level of Otx2, which regulates the production of the correct ratio of rods and bipolar cells.

    View details for DOI 10.1016/j.devcel.2014.07.018

    View details for Web of Science ID 000341296100007

    View details for PubMedID 25155555

  • NeuroD Factors Regulate Cell Fate and Neurite Stratification in the Developing Retina JOURNAL OF NEUROSCIENCE Cherry, T. J., Wang, S., Bormuth, I., Schwab, M., Olson, J., Cepko, C. L. 2011; 31 (20): 7365-7379


    Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to control critical aspects of development in many tissues. To identify bHLH genes that might regulate specific aspects of retinal cell development, we investigated the expression of bHLH genes in single, developing mouse retinal cells, with particular emphasis on the NeuroD family. Two of these factors, NeuroD2 and NeuroD6/NEX, had not been previously reported as expressed in the retina. A series of loss- and gain-of-function experiments was performed, which suggested that NeuroD genes have both similarities and differences in their activities. Notably, misexpression of NeuroD genes can direct amacrine cell processes to two to three specific sublaminae in the inner plexiform layer. This effect is specific to cell type and NeuroD gene, as the AII amacrine cell type is refractory to the effects of NeuroD1 and NeuroD6, but uniquely sensitive to the effect of NeuroD2 on neurite targeting. Additionally, NeuroD2 is endogenously expressed in AII amacrine cells, among others, and loss of NeuroD2 function results in a partial loss of AII amacrine cells. The effects of misexpressing NeuroD genes on retinal cell fate determination also suggested shared and divergent functions. Remarkably, NeuroD2 misexpression induced ganglion cell production even after the normal developmental window of ganglion cell genesis. Together, these data suggest that members of the NeuroD family are important for neuronal cell type identity and may be involved in several cell type-specific aspects of retinal development, including fate determination, differentiation, morphological development, and circuit formation.

    View details for DOI 10.1523/JNEUROSCI.2555-10.2011

    View details for Web of Science ID 000290716600016

    View details for PubMedID 21593321

  • Neurog3 gene dosage regulates allocation of endocrine and exocrine cell fates in the developing mouse pancreas DEVELOPMENTAL BIOLOGY Wang, S., Yan, J., Anderson, D. A., Xu, Y., Kanal, M. C., Cao, Z., Wright, C. V., Gu, G. 2010; 339 (1): 26-37


    The basic helix-loop-helix transcription factor Neurog3 (Neurogenin3 or Ngn3) actively drives endodermal progenitor cells towards endocrine islet cell differentiation during embryogenesis. Here, we manipulate Neurog3 expression levels in endocrine progenitor cells without altering its expression pattern using heterozygosity and a hypomorph. Lowered Neurog3 gene dosage in the developing pancreatic epithelium reduces the overall production of endocrine islet cells without significantly affecting the proportions of various islet cell types that do form. A reduced Neurog3 production level in the endocrine-directed pancreatic progenitor population activates the expression of Neurog3 in an increased number of epithelial progenitors. Yet a significant number of these Neurog3+ cells detected in heterozygous and hypomorphic pancreata, possibly those that express low levels of Neurog3, move on to adopt pancreatic ductal or acinar fates. These data directly demonstrate that achieving high levels of Neurog3 expression is a critical step for endocrine commitment from multipotent pancreatic progenitors. These findings also suggest that a high level of Neurog3 expression could mediate lateral inhibition or other unknown feedback mechanisms to regulate the number of cells that initiate Neurog3 transcription and protein production. The control of Neurog3+ cell number and the Neurog3 threshold-dependent endocrine differentiation mechanism combine to select a specific proportion of pancreatic progenitor cells to adopt the islet cell fate.

    View details for DOI 10.1016/j.ydbio.2009.12.009

    View details for Web of Science ID 000274870700003

    View details for PubMedID 20025861

  • Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, S., Jensen, J. N., Seymour, P. A., Hsu, W., Dor, Y., Sander, M., Magnuson, M. A., Serup, P., Gu, G. 2009; 106 (24): 9715-9720


    Neurog3 (Neurogenin 3 or Ngn3) is both necessary and sufficient to induce endocrine islet cell differentiation from embryonic pancreatic progenitors. Since robust Neurog3 expression has not been detected in hormone-expressing cells, Neurog3 is used as an endocrine progenitor marker and regarded as dispensable for the function of differentiated islet cells. Here we used 3 independent lines of Neurog3 knock-in reporter mice and mRNA/protein-based assays to examine Neurog3 expression in hormone-expressing islet cells. Neurog3 mRNA and protein are detected in hormone-producing cells at both embryonic and adult stages. Significantly, inactivating Neurog3 in insulin-expressing beta cells at embryonic stages or in Pdx1-expressing islet cells in adults impairs endocrine function, a phenotype that is accompanied by reduced expression of several Neurog3 target genes that are essential for islet cell differentiation, maturation, and function. These findings demonstrate that Neurog3 is required not only for initiating endocrine cell differentiation, but also for promoting islet cell maturation and maintaining islet function.

    View details for DOI 10.1073/pnas.0904247106

    View details for Web of Science ID 000267045500032

    View details for PubMedID 19487660

  • Myt1 and Ngn3 form a feed-forward expression loop to promote endocrine islet cell differentiation DEVELOPMENTAL BIOLOGY Wang, S., Hecksher-Sorensen, J., Xu, Y., Zhao, A., Dor, Y., Rosenberg, L., Serup, P., Gu, G. 2008; 317 (2): 531-540


    High levels of Ngn3 expression in pancreatic progenitor cells are both necessary and sufficient to initiate endocrine differentiation. While it is clear that the Notch-Hes1-mediated signals control the number of Ngn3-expressing cells in the developing pancreas, it is not known what factors control the level of Ngn3 expression in individual pancreatic cells. Here we report that Myt1b and Ngn3 form a feed-forward expression loop that regulates endocrine differentiation. Myt1b induces glucagon expression by potentiating Ngn3 transcription in pancreatic progenitors. Vice versa, Ngn3 protein production induces the expression of Myt1. Furthermore, pancreatic Myt1 expression largely, but not totally, relies on Ngn3 activity. Surprisingly, a portion of Myt1 expressing pancreatic cells express glucagon and other alpha cell markers in Ngn3 nullizygous mutant animals. These results demonstrate that Myt1b and Ngn3 positively regulate each other's expression to promote endocrine differentiation. In addition, the data uncover an unexpected Ngn3 expression-independent endocrine cell production pathway, which further bolsters the notion that the seemingly equivalent endocrine cells of each type, as judged by hormone and transcription factor expression, are heterogeneous in their origin.

    View details for DOI 10.1016/j.ydbio.2008.02.052

    View details for Web of Science ID 000255898600014

    View details for PubMedID 18394599

  • Loss of Myt1 function partially compromises endocrine islet cell differentiation and pancreatic physiological function in the mouse MECHANISMS OF DEVELOPMENT Wang, S., Zhang, J., Zhao, A., Hipkens, S., Magnuson, M. A., Gu, G. 2007; 124 (11-12): 898-910


    Myelin transcription factor 1 (Myt1) is one of the three vertebrate C2HC-type zinc finger transcription factors that include Myt1 (Nzf1), Myt1L (Png1), and Myt3 (Nzf3, St18). All three paralogs are widely expressed in developing neuronal cells. Yet their function for mammalian development has not been investigated directly. Here we report that only Myt1 is expressed in the embryonic pancreas, in both endocrine progenitors and differentiated islet cells. Myt1(-/-) animals die postnatally, likely due to confounding effects in multiple tissues. The endocrine tissues in the embryonic Myt1(-/-) pancreas contained abnormal islet cells that expressed multiple hormones; although hormone levels were normal. We also created pancreas-specific Myt1 knockout mice. These mutant animals had no obvious physical defects from their wild-type littermates. Male mutant animals had reduced glucose-clearing abilities and abnormal multi-hormone-expressing cells present in their endocrine islets. In addition, they also had reduced Glut2 expression, and attenuated glucose-induced insulin secretion in the adult islets. Surprisingly, the expression of the Myt1 paralogs, Myt1l and Myt3, was induced in the embryonic Myt1(-/-) pancreas. The consequences of Myt1 inactivation in the developing pancreas could be masked by activation of its paralogs, Myt1l and Myt3. These findings suggest Myt1 is involved in proper endocrine differentiation and function.

    View details for DOI 10.1016/j.mod.2007.08.004

    View details for Web of Science ID 000251644500007

    View details for PubMedID 17928203

  • The fringe molecules induce endocrine differentiation in embryonic endoderm. by activating cMyt1/cMyt3 DEVELOPMENTAL BIOLOGY Xu, Y., Wang, S., Zhang, H., Zhao, A., Stanger, B. Z., Gu, G. 2006; 297 (2): 340-349


    Endocrine differentiation in the early embryonic pancreas is regulated by Notch signaling. Activated Notch signaling maintains pancreatic progenitor cells in an undifferentiated state, whereas suppression of Notch leads to endocrine cell differentiation. Yet it is not known what mechanism is employed to inactivate Notch in a correct number of precursor cells to balance progenitor proliferation and differentiation. We report that an established Notch modifier, Manic Fringe (Mfng), is expressed in the putative endocrine progenitors, but not in exocrine pancreatic tissues, during early islet differentiation. Using chicken embryonic endoderm as an assaying system, we found that ectopic Mfng expression is sufficient to induce endodermal cells to differentiate towards an endocrine fate. This endocrine-inducing activity depends on inactivation of Notch. Furthermore, ectopic Mfng expression induces the expression of basic helix-loop-helix gene, Ngn3, and two zinc finger genes, cMyt1 and cMyt3. These results suggest that Mfng-mediated repression of Notch signaling could serve as a trigger for endocrine islet differentiation.

    View details for DOI 10.1016/j.ydbio.2006.04.456

    View details for Web of Science ID 000240836100004

    View details for PubMedID 16920096