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)
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 understanding the molecular mechanisms that underlie retinal development and diseases. We utilize genetic and genomic tools to uncover how different types of retinal cells, including retinal neurons, glia and the vasculature, respond to developmental cues and disease insults at the epigenomic and transcriptional levels, and how they interact and collectively contribute to the integrity of the retina.
1. Retinal cell fate specification.
We are using 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 cell fate specification.
2. The multicellular responses elicited by diabetes in the retina.
Diabetes can induce multicellular responses in the retina, including vascular lesions, glial dysfunction and neurodegeneration, all of which contribute to retinopathy. We are using diabetic rats as models to investigate the detailed molecular mechanisms underlying the diabetes-induced multicellular responses, and the disease mechanisms of diabetic retinopathy.
3. Molecular tools that allow for cell type-specific labeling and manipulation in vivo.
Cis-regulatory elements, such as enhancers, play essential roles in directing tissue/cell type-specific and stage-specific expression. We are interested in identifying enhancers that can drive cell type-specific expression in the retina and brain, and incorporating them into plasmid or AAV based delivery systems.
Synaptotagmin 4 Regulates Pancreatic beta Cell Maturation by Modulating the Ca2+ Sensitivity of Insulin Secretion Vesicles
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 DOI 10.1016/j.devcel.2018.03.013
View details for Web of Science ID 000432461400010
View details for PubMedID 29656931
View details for PubMedCentralID PMC5962294
Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies
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
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
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
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
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
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
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
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
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