Overexpression of NeuroD in the Developing Retina of the Zebrafish

Abstract

Abstract: : Purpose: In the retinas of larval and adult teleosts, the bHLH transcription factor, neuroD, is expressed in proliferating cells of the rod lineage and differentiating cone photoreceptors. The purpose of this study is to investigate the function of neuroD in these cells. Methods: The open reading frame (ORF) of the zebrafish neuroD cDNA was subcloned into the Clontech promoterless enhanced green fluorescent protein (EGFP), pEGFP–1, expression vector containing the zebrafish heat shock promoter (HSP) 70/4. The neuroD ORF was inserted into the modified pHSP70/4–EGFP vector between the heat shock promoter and sequences encoding the enhanced green fluorescent protein. The functionality of this construct was tested in vitro in HEK293 cells and in vivo in transient transgenic embryos injected at the 2–4 cell stage and treated at 24 hrs post fertilization by heat shock (37°C for 60 min). Several hundred embryos were also injected and raised to sexual maturity to establish stable transgenic lines. Progeny of injected embryos were screened by heat shock to identify those displaying germline transmission. Cells and embryos were evaluated using fluorescence microscopy and in situ hybridization with probes to neuroD and islet1. Results: Examining EGFP expression in transfected HEK293 cells showed that the HSP promoter drives transcription, and the neuroD^EGFP fusion protein is trafficked to the nucleus. In situ hybridization on sections from transient transgenic animals shows that heat shock can induce ectopic expression of neuroD mRNA in vivo, including in the nervous system and retina. In these animals, ectopic mRNA was present by 24hrs after heat shock, and was maintained for at least 72hrs. A line of animals transgenic for HSP70/4–neuroD^EGFP was identified. Heat shock treatment of these animals resulted in robust EGFP fluorescence throughout the embryo, including the retina, and ectopic expression of islet1, a downstream target of neuroD. Retinas from transgenic animals heat shocked at 24hpf and sacrificed at 48hpf showed a developmental delay compared to heat shocked, age–matched controls. Conclusions: The HSP70/4–neuroD^EGFP construct gives rise to functional protein in vitro and in vivo, and a line of transgenic animals has been established. Conditional gain–of–function experiments will be used to investigate the role of neuroD in cells of the rod lineage and newly differentiated cone photoreceptors.

Keywords: transgenics/knock-outs • retinal development • photoreceptors