Mechanistic logic and timing of retinal bipolar cell class and type specification

Underlying the five neuronal cell classes that comprise the mammalian retina is a tremendous amount of cell type diversity. Within the bipolar cell class of retinal interneurons, which mediate visual signalling from photoreceptor cells to amacrine and ganglion cells, at least 15 distinct types have been classified based on gene expression and morphology. How bipolar cell types are specified and to what extent intrinsic and extrinsic factors are involved in this process is poorly understood. To address these issues, we developed an in vitro strategy using retinal cell dissociation and metabolic cell cycle labelling to track the progression of retinal progenitor cells and determine the timing and requirement of cell–cell interactions during bipolar cell class/type specification. Consistent with the idea that bipolar cell development unfolds in a stepwise manner, the expression of Tg.BC-Vsx2:CRE, a marker for bipolar cell class specification, appeared about 36 h after the last retinal progenitor cell S-phase and preceded the expression of type-specific markers, VSX1, PKCα, Tg.mGluR6:βgal, which appeared 12–24 h later. Tg.BC-Vsx2:CRE-expressing bipolar cells were generated, albeit at reduced levels, when postmitotic retinal progenitor cells were plated ~ 24 h after their last S-phase and cultured with little to no cell–cell contact, however, none of these bipolar cells exhibited type-specific marker expression. In contrast, cells cultured in the same manner but with abundant cell–cell contact, or cells dissociated ~ 48 after their last S-phase and cultured with little to no cell–cell contact, gave rise to bipolar cells expressing type-specific markers. The requirement for cell–cell contact was not indiscriminate, as co-culturing with non-retinal HEK293T cells did not rescue the appearance of bipolar cell-type specific marker expression. Together, these results support a stepwise model for bipolar cell development in which intrinsic and extrinsic factors specify bipolar cells as a class through the formation of a pan-bipolar cell intermediate precursor within the first 36 h following retinal progenitor cell cycle exit. Bipolar cell type specification appears dissociable from class specification and requires extrinsic factors within the first 48 h after progenitor cell cycle exit.