Abstract
Purpose:
Our long term objective is to identify genes regulating photoreceptor development. Previously, we reported the identification of a zebrafish homologue of Six3, a member of the six/sine oculis family of transcription factors, as a regulator of rod number and spatial patterning. The goal of this study is to characterize the mechanisms underlying the observed changes in rod patterning.
Methods:
The spatial and temporal expression patterns of six genes were tracked by in situ hybridization and quantified by real time PCR. six7 expression was manipulated by injection of splice blocking or translation blocking morpholinos into one cell-stage embryos or injection of in vitro transcribed mRNA. Cell proliferation was assayed by immunolabeling for PH3 or EdU incorporation. Other analysis included routine immunolabeling and histology.
Results:
In situ hybridization and RT-PCR revealed dynamic changes in the pattern of six7 expression during zebrafish embryogenesis. Following an initial peak of expression at 10-18 hpf in the presumptive eye field, labeling was virtually undetectable in the embryo by 24 hpf, but at 52 hpf low level expression was again detected. Injection of six7 morpholinos resulted in an increased number and the uniform distribution of rods across the larval retina. Knockdown of expression in the early embryo was confirmed by detection of an alternatively spliced mRNA utilizing a cryptic splice site 5’ to the ATG initiation codon. However, at 48 hpf, in situ hybridization and RT-PCR revealed increased expression of the six7 mRNA in the central retina of six7 morpholino-injected embryos coincident with photoreceptor genesis. Morpholino injection also led to significant increases in PH3 and EdU labeling in the neuroblastic layer, and EdU-labeling localized with six7 expression in the central retina. Lineage tracing indicated that these proliferative cells differentiate as photoreceptors. Injection of six7 mRNA altered early forebrain patterning but did not affect photoreceptor cell fate in six7 morpholino-injected and uninjected embryos.
Conclusions:
Our data suggest a novel role for six7 in photoreceptor cell patterning by regulating the proliferation of retinal progenitors. Future investigations are aimed at identifying molecular partners involved in the six7 regulation of photoreceptor cell numbers in zebrafish.
Keywords: 739 transcription factors •
698 retinal development •
648 photoreceptors