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.

Public Health Relevance

Retinal bipolar neurons convey light information from photoreceptors, and are present as 12 distinct subtypes whose individual features enable the retina to interpret visual information before sending signals to the brain. The molecular mechanisms that allow any given subtype to acquire an identity different from its neighbors remain a mystery, and this proposal will address this topic using innovative approaches that target the cells that produce neurons during postnatal development. The proposed research can lead to knowledge and reagents that facilitate therapies for blindness, including introduction of therapeutic genes into bipolar neurons (gene therapy), as well as stem cell-based approaches to repairing retinal tissue.

National Institute of Health (NIH)
National Eye Institute (NEI)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F05-R (20))
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Agarwal, Neeraj
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Harvard Medical School
Schools of Medicine
United States
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Shekhar, Karthik; Lapan, Sylvain W; Whitney, Irene E et al. (2016) Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics. Cell 166:1308-1323.e30