Abstract

The intrinsic and extrinsic factors that regulate vertebrate photoreceptor specification and differentiation are complex, and our understanding of all the players is far from complete. Her9, the zebrafish ortholog of human HES4, is a basic helix-loop-helix-orange transcriptional repressor that regulates neurogenesis in several developmental contexts. We have previously shown that her9 is upregulated during chronic rod photoreceptor degeneration and regeneration in adult zebrafish, but little is known about the role of her9 during retinal development. To better understand the function of Her9 in the retina, we generated zebrafish her9 CRISPR mutants. Her9 homozygous mutants displayed striking retinal phenotypes, including decreased numbers of rods and red/green cones, whereas blue and UV cones were relatively unaffected. The reduction in rods and red/green cones correlated with defects in photoreceptor subtype lineage specification. The remaining rods and double cones displayed abnormal outer segments, and elevated levels of apoptosis. In addition to the photoreceptor defects, her9 mutants also possessed a reduced proliferative ciliary marginal zone, and decreased and disorganized Müller glia. Mutation of her9 was larval lethal, with no mutants surviving past 13 days post fertilization. Our results reveal a previously undescribed role for Her9/Hes4 in photoreceptor differentiation, maintenance, and survival.

Highlights

  • The intrinsic and extrinsic factors that regulate vertebrate photoreceptor specification and differentiation are complex, and our understanding of all the players is far from complete

  • The neural retina is composed of three layers; the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL); the light-sensitive rod and cone photoreceptors reside in the ONL

  • We used fluorescent in situ hybridization (FISH) to detect her[9] mRNA in the developing zebrafish retina between 24 and 72 hpf, a time period which encompasses the progression from a pseudostratified proliferative retinal neuro-epithelium to a fully laminated, functional retina containing all retinal cell types

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Summary

Introduction

The intrinsic and extrinsic factors that regulate vertebrate photoreceptor specification and differentiation are complex, and our understanding of all the players is far from complete. The zebrafish genome contains 19 hairy-related (her) genes which have been shown to play essential roles in developmental processes such as somitogenesis, neural tube and nervous system development, floor plate development, cell cycle exit, and a­ poptosis[6,7] Her proteins often function as downstream effectors of the NotchDelta signaling pathway and mediate cross-talk between Notch and other p­ athways[8]. Her[9] expression is upregulated during the specific degeneration and regeneration of rod photoreceptors in the zebrafish retina, suggesting a role for this transcription factor in retinal ­regeneration[18] These studies strongly implicate her[9] in regulating continual neurogenesis and injury-induced regeneration in the post-embryonic retina, the function of her[9] during embryonic retinal development has not been thoroughly investigated

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