The increasing availability of transcriptomic technologies within the last decade has facilitated high throughput identification of gene expression differences that define distinct cell types as well as the molecular pathways that drive their specification. The retinal projection neurons, retinal ganglion cells (RGCs), can be categorized into distinct morphological and functional subtypes and by the laterality of their projections. Here, we present a method for purifying the sparse population of ipsilaterally projecting RGCs in mouse retina from their contralaterally projecting counterparts during embryonic development through rapid retrograde labeling followed by fluorescence-activated cell sorting (FACS). Through microarray analysis, we uncovered the distinct molecular signatures that define and distinguish ipsilateral and contralateral RGCs during the critical period of axonal outgrowth and decussation, with over three hundred genes differentially expressed within these two cell populations. Amongst the differentially expressed genes confirmed through in vivo expression validation, several genes that mark “immaturity” are expressed within postmitotic ipsilateral RGCs. Moreover, at least one complementary pair, Igf1 and Igfbp5, is upregulated in contralateral or ipsilateral RGCs, respectively, and may represent signaling pathways that determine ipsilateral vs. contralateral RGC identity. Importantly, the cell cycle regulator cyclin D2 is highly expressed in peripheral ventral retina with a dynamic expression pattern that peaks during the period of ipsilateral RGC production. Thus, the molecular signatures of ipsilateral and contralateral RGCs and the mechanisms that regulate their differentiation are more diverse than previously expected.
Significance Statement: This study presents a new method for isolating ipsilaterally and contralaterally projecting retinal ganglion cells (RGCs) via retrograde labeling and fluorescence-activated cell sorting. The subsequent transcriptomic analysis of these purified populations by microarray, followed by in vivo expression validation, revealed that ipsilateral RGCs have a distinct set of genes, which govern neurogenesis, differentiation, and axon guidance when compared to contralateral RGCs. Elucidating these gene programs contribute to our understanding of how decussating systems, in particular, the binocular circuit, are established. This information is critical for directing the appropriate RGC subtype differentiation and axon regeneration for repair after injury.
The authors declare no competing financial interests.
Supported by NIH grants R01 EY012736 and EY015290 (CM), T32 GM07367 and EY013933 (QW), and Fight for Sight (FM).