Diversification of the closely related bipolar cell types in retinal development

The National Eye Institute awarded Duke University $163,160 on February 8, 2027, under the Vision Research program (CFDA 93.867) to support research on the diversification of closely related bipolar cell types in retinal development.

The funded research uses bipolar cells of the mouse retina as a model system to determine the developmental timing when bipolar cell types first become molecularly distinct and how these distinctions confer unique functional properties in mature types. The work explores two aspects of bipolar cell identity: early fate specification through the proneural basic helix-loop-helix transcription factor target gene Vexin, and mature expression of type-specific cell surface proteins controlling axon terminal tiling. The research employs single-cell long-read sequencing and addresses a gap in understanding the genetic mechanisms that differentiate closely related neuronal cell types within the central nervous system.

The period of performance extends from February 8, 2027, through June 30, 2029, with work performed in Durham, North Carolina. This is a project grant, a non-renewable assistance type under the NEI Vision Research program.

Abstract

During development, progenitor cells adopt a spectrum of cell fates via differential expression of cell type-specific proteins. The difficulty of specifying cell fate is particularly high in the central nervous system (CNS), due to the remarkable variety of different neuron types. While many CNS studies have focused on gene-level expression differences in broad categories of neuronal cell types, many of these broad families can be further subdivided into closely related cell types that differ in key ways despite sharing many overall similarities. Little is known about the genetic mechanisms responsible for differentiating these closely related cell types. This GAP in knowledge hinders our basic understanding of developmental neurobiology. Here i address this knowledge GAP using bipolar cells of the mouse retina as a model system. The objective of this proposal is to ascertain the time during development when bipolar types first become molecularly distinct, and how these molecular distinctions impart unique functional properties in mature types. My central hypothesis is that choices during the early postmitotic period bring about gene- and mrna isoform-level differences at maturity that distinguish individual bipolar cell types. This hypothesis was formulated based on my own data using single cell long-read sequencing which uncovered a novel bhlh transcription factor target expressed in bipolar cell precursors, as well as published bulk long-read data from our lab which demonstrated that alternate mrna isoforms of cell surface genes are expressed in unique cell types. In this study i will explore two aspects of bipolar cell identity: early fate specification of cell type by the proneural bhlh target gene vexin, and mature expression of type-specific cell surface proteins controlling tiling of axon terminals. Aim 1: based on anatomical criteria, differing bipolar cell types are already specified from postmitotic bipolar cell precursors days before known type-specific gene markers are expressed. My working hypothesis is that there must be molecular factors acting between these times to specify each type, and that vexin is one such proneural factor. This hypothesis will be tested using gain- and loss-of-function tools in vivo, which are expected to reveal a role for vexin in specifying bipolar cell type identity. Aim 2: one key functional property of bipolar types is the phenomenon known as tiling, where each type of bipolar cell has arbors that cover the retina independently. This independent tiling requires the existence of type-specific cell-surface molecules driving homotypic arbor repulsion, but gene-level analysis has not identified such molecules. My working hypothesis is that isoforms of cell adhesion genes provide the necessary molecular variety for bipolar cell tiling. My preliminary data suggests candidate adhesion molecule isoforms could have the appropriate type-specific expression to mediate tiling. These molecules will be tested for effects on tiling using misexpression in developing retina. This work is significant because IT is expected to identify previously unknown molecular markers that distinguish closely related bipolar cell types at early and late stages of development, which make way for future studies understanding how cell fate emerges to establish type-specific function.