Inhibitor of differentiation 2A influences growth and differentiation of the developing vertebrate retina upstream of the notch signaling pathway

Inhibitor of differentiation (Id) family helix-loop-helix proteins regulate the proliferation, survival and differentiation of numerous cell types during development, however their function during retinal development has not been analyzed. Using loss-of-function and overexpression assays in zebrafish, I demonstrate that Id2a levels modulate retinoblast cell cycle kinetics and thereby influence neuron and glia formation in the retina. Id2a-deficient retinas possess increased numbers of cells occupying S phase, at the expense of mitotic cells, and kinetic analyses demonstrate that Id2a is required for S-phase progression and/or the transition from S to M phase. Id2a-dependent defects in retinoblast proliferation lead to microphthalmia and to an absence of nearly all differentiated inner and outer nuclear layer cell types. Overexpression of id2a has the opposite effect on retinoblast cell cycle kinetics: id2a-overexpressing retinoblasts progress from S to M phase more rapidly and they undergo mitosis more frequently, which results in macrophthalmia. Mosaic analyses reveal that Id2a function in facilitating both cell cycle progression and neuronal differentiation in the retina is non-cell-autonomous, suggesting that Id2a functions upstream of the extrinsic pathways that regulate retinogenesis. In an effort to identify which extrinsic pathways function downstream of Id2a, I discovered that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through an RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, I identified a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis.