Negative regulation of Vsx1 by its paralog Chx10/Vsx2 is conserved in the vertebrate retina

Abstract

Chx10-and Vsx1 are the only Paired-like CVC (Prd-L:CVC) homeobox genes in the mouse genome. Both are expressed in the retina and have important but distinct roles in retinal development. Mutations in Chx10 cause reduced retinal progenitor cell (RPC) proliferation and an absence of bipolar cells, while mutations in Vsx1 impair differentiation of cone bipolar cells. Given their structural similarities and importance in retinal development, we sought to determine if a regulatory interaction exists between these genes and whether inactivation of both genes blocks initiation of retinal development. We found that Chx10 binds to a specific sequence in the Vsx1 5′-intergenic region and represses the activity of a luciferase reporter under the control of the Vsx1 promoter. This is consistent with our observation that there is an inverse relationship between the levels of Chx10 and Vsx1 immunostaining within the bipolar cell class. Furthermore, Vsx1 mRNA is upregulated in the RPCs of Chx10 deficient mice and zebrafish embryos injected with a chx10 morpholino. In mice deficient for both Chx10 and Vsx1 and zebrafish embryos coinjected with chx10 and vsx1 morpholinos, the changes in embryonic retinal development and marker expression are similar in magnitude to embryos with Chx10 loss of function only. From these studies, we propose that Vsx1 is a direct target of Chx10-mediated transcriptional repression. Although Vsx1 mRNA is upregulated in Chx10 deficient RPCs, Vsx1 does not genetically compensate for loss of Chx10, demonstrating that Prd-L:CVC genes, although important, are not absolutely required to initiate retinal development.

Analysis of opo cis-regulatory landscape uncovers Vsx2 requirement in early eye morphogenesis

“…5e). This enrichment in the ChIP sample was even higher than the observed for a fragment of the vsx1_promoter, previously identified as a Vsx2 direct target 48 ( Fig. 5e).…”

“…A number of knockdown studies in zebrafish have shown that the reported role for Vsx2 in mammals is, at least, partially conserved in teleosts. Thus, small eye phenotypes, abnormal bipolar cell specification and Mitf upregulation have been described in vsx2-knockdown embryos in zebrafish 15,16,48 . Interestingly, one of these reports 15 described abnormally shaped eyes displaying a flattened morphology at early stages in zebrafish (24 h.p.f.).…”

MicroRNA-20a is essential for normal embryogenesis by targetingvsx1mRNA in fish

“…18,[21][22][23][24][25][26][27] It is initially transcribed in a wide embryonic region but gradually restricted within distinct cell types at subsequent developmental stages. Therefore, post-transcriptional regulation might be required for the transition from one developmental stage to the next and the establishment of different cell identities.…”

“…18,23,24,26,27 To determine whether inhibition of Vsx1 expression by miR-20a is essential for normal embryogenesis, we examined whether releasing the inhibition by decoying miR-20a could elicit excessive generation of vsx1 marked neurons. Whole mount in situ hybridization showed that, at 18 hpf, wild type embryos generated 9 distinct vsx1 marked neurons in the central tier of spinal cord and the neurons were arranged in regular space corresponding to the somites ( Fig.…”

“…During neurogenesis, vsx1 is expressed in presumptive neurons of hindbrain and spinal cord in zebrafish and Xenopus, 23,24 suggesting that it might play a role in neurons formation in the central neural system. During retinogenesis of examined vertebrates, vsx1 is expressed weakly (in zebrafish and mouse) or strongly (in goldfish, chick and Xenopus) in undifferentiated, presumptive neural retina, then up-regulated selectively in presumptive bipolar cells, 18,23,[25][26][27] and has been demonstrated to play a role in regulating the proliferation and differentiation of retinal progenitors as well as the functional maintenance of bipolar cells. 18,[25][26][27] Therefore, post-transcriptional regulation might be required for the transition from one developmental stage to the next and the establishment of different cell identities.…”