Abstract
Maintaining neurogenesis in growing tissues requires a tight balance between progenitor cell proliferation and differentiation. In the zebrafish retina, neuronal differentiation proceeds in two stages with embryonic retinal progenitor cells (RPCs) of the central retina accounting for the first rounds of differentiation, and stem cells from the ciliary marginal zone (CMZ) being responsible for late neurogenesis and growth of the eye. In this study, we analyse two mutants with small eyes that display defects during both early and late phases of retinal neurogenesis. These mutants carry lesions in gdf6a, a gene encoding a BMP family member previously implicated in dorsoventral patterning of the eye. We show that gdf6a mutant eyes exhibit expanded retinoic acid (RA) signalling and demonstrate that exogenous activation of this pathway in wild-type eyes inhibits retinal growth, generating small eyes with a reduced CMZ and fewer proliferating progenitors, similar to gdf6a mutants. We provide evidence that RA regulates the timing of RPC differentiation by promoting cell cycle exit. Furthermore, reducing RA signalling in gdf6a mutants re-establishes appropriate timing of embryonic retinal neurogenesis and restores putative stem and progenitor cell populations in the CMZ. Together, our results support a model in which dorsally expressed gdf6a limits RA pathway activity to control the transition from proliferation to differentiation in the growing eye.
Highlights
The balance between cell proliferation and differentiation is spatially and temporally regulated during development, ensuring the generation of tissues with the correct proportion of differentiated cells (Schmidt et al, 2013; Urbán and Guillemot, 2014)
We investigated whether pharmacological activation of the RA pathway using AM580, a retinoic acid receptor alpha (RARα) agonist (Gianni et al, 1996), affects eye development to gdf6a mutants
Precocious RA-mediated differentiation depletes retinal progenitors Because RA pathway activity is enhanced in gdf6a mutant retinae, we examined whether RA could modulate cell cycle exit and neurogenesis of retinal progenitor cells (RPCs) by pharmacologically activating or inhibiting RA signalling and examining atoh7 and Tg[atoh7:GFP] expression
Summary
The balance between cell proliferation and differentiation is spatially and temporally regulated during development, ensuring the generation of tissues with the correct proportion of differentiated cells (Schmidt et al, 2013; Urbán and Guillemot, 2014). To gauge the balance between proliferation and differentiation after the onset of retinal neurogenesis in gdf6a mutants, we labelled all S-phase cells with BrdU for 10 h beginning at ∼50 hpf With this labelling regime, only a small proportion of cells in wild-type eyes ( primarily RGCs) are BrdU negative and are likely to have exited the cell cycle prior to BrdU treatment (Fig. 6C, red cells; Ohnuma et al, 1999). Precocious RA-mediated differentiation depletes retinal progenitors Because RA pathway activity is enhanced in gdf6a mutant retinae, we examined whether RA could modulate cell cycle exit and neurogenesis of RPCs by pharmacologically activating or inhibiting RA signalling and examining atoh and Tg[atoh7:GFP] expression. These observations suggest that RA can promote cell cycle exit and the production of atoh7-positive neurons in developing retinae
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