2014 — 2015 |
Lapan, Sylvain |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Transcriptional Control of Retinal Bipolar Neuron Diversification
DESCRIPTION (provided by applicant): Much of the neuronal diversity in the central nervous system (CNS) exists at the level of neuronal subtypes. The vertebrate retina is composed of greater than 60 classifiable subtypes, and has long served as a model to study the generation of diverse cell types from multipotent progenitors. Retinal bipolar neurons are emerging as a particularly strong system for studying subtype diversification. They are born in the postnatal period in mouse, and are therefore a uniquely accessible population for perturbation by electroporation and retroviral infection. Retinal bipolar neurons are represented by approximately 12 subtypes characterized to different extents by immunohistochemical properties, morphology, and electrophysiology. An understanding of the mechanisms by which these subtypes are distinguished from one another can emerge from studying the specification of a single subtype in detail. The type 2 cone bipolar neuron (CBP2s) is an excellent candidate for further investigation. It is defined by several unique markers and expression of a transcription factor, Bhlhb5, which is expressed in no other bipolar neurons and is maintained after differentiation. Bhlhb5 knockout mice lack CBP2s, but the function of this gene in bipolar subtype determination is otherwise unexplored. Bhlhb5 is a central factor in determination of interneuron subtypes in the spinal cord. The objective of this proposal is to explore the gene regulatory network that defines a single bipolar subtype, CBP2, by focusing on the role of Bhlhb5. The following aims are proposed: 1) Identify differentially expressed genes in CBP2 targeted by Bhlhb5, 2) Determine the role of Bhlhb5 in maintaining subtype-specific gene expression and 3) Determine the ability of Bhlhb5 to alter the fates of bipolar precursors. The principles that emerge from studying bipolar neuron diversification are likely to have relevance to the formation of neuronal subtypes throughout the CNS. Furthermore, this work has relevance to human health, as the diversity of bipolar neurons observed in the mouse retina is largely consistent with what has been observed in human retina. In particular, cis-regulatory analyses proposed here can be used to generate new drivers for use in gene therapies based on expression of light-activated channels in bipolar neurons. Furthermore, the mechanisms of cell fate regulation discovered may improve directed differentiation of stem cells into retinal tissue, with the ultimate goal of regenerating functional visual circuits.
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0.934 |
2017 — 2018 |
Lapan, Sylvain |
K99Activity Code Description: To support the initial phase of a Career/Research Transition award program that provides 1-2 years of mentored support for highly motivated, advanced postdoctoral research scientists. |
Regulation of Cell Type Identity in Retinal Bipolar Cell Precursors
PROJECT SUMMARY / ABSTRACT Bipolar cells of the retina are a highly diverse class of interneurons that receive direct input from photoreceptors, with 15 distinct types displaying unique gene expression patterns and functions in the visual circuitry. The comprehensive morphological and molecular information available, and powerful tools for genetic perturbation, make bipolar cells an ideal population for investigating how retinal interneurons become distinct during development. This project outlines a mentored (K99) phase during which the candidate can enhance existing expertise in genetic analysis of bipolar cell development through additional training, while applying these skills to address hypotheses emerging from new single-cell RNA-seq (scRNA-seq) datasets of mature and developing bipolar neurons. Specifically, this project is designed to interrogate how the combination of temporal differences in onset of proneural gene expression and extrinsic signaling mechanisms influence the decision of precursors of bipolar cells to become specific types. Aim 1 proposes to determine how the timing of proneural gene expression during postnatal development relates to the generation of distinct bipolar cell types, employing temporally controlled fate mapping of Neurog2-expressing cells and transient inhibition of proneural gene expression. In Aim 2, extrinsic cues regulating bipolar cell type identity during postnatal development will be investigated using a retinal explant system and pharmacological inhibitors of signaling pathway receptors identified as candidates from scRNA-seq expression datasets. A set of markers for all 15 bipolar cell types will be used for comprehensive phenotypic analysis of types. In Aim 3, enhancers of genes that are required for generating specific bipolar cell types, starting with Fezf2, will be analyzed for the binding of signal transduction pathway effectors and other transcriptional regulators. The K99 phase of the award will focus on parts of Aims 1-3, specifically temporally controlled fate mapping (Aim 1A), establishing the explant assay and applying inhibitors (Aim 2), and performing motif prediction and binding analysis for a known enhancer of Fezf2 (Aim 3), in addition to career development and training activities. In the R00 phase, the functional consequence of delaying proneural gene expression will be analyzed using transient Hes1 misexpression (Aim 1B), hits from the small-molecule inhibition experiment will be validated genetically and a temporal requirement for signaling will be further investigated (Aim 2), and enhancer analysis will be extended beyond Fezf2, to other genes required for generating particular bipolar cell types, including Isl1, Ebf1 and Prdm8 (Aim 3). The mentored phase will be conducted under the supervision of Dr. Constance Cepko, with additional advisory contributions from Dr. Paola Arlotta (Harvard) and faculty in the Genetics Department at Harvard Medical School. The extensive resources and career development opportunities available at Harvard Medical School, Dr. Cepko's mentorship, and the research activities planned in the K99 period will enable the candidate to achieve the long- term goal of becoming an independent investigator dedicated to the study of retinal neurogenesis.
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0.934 |