Brief Report: Rx1 Defines Retinal Precursor Identity by Repressing Alternative Fates Through the Activation of TLE2 and Hes4

Abstract

The molecular mechanisms underlying the acquisition of retinal precursor identity are scarcely defined. Although the homeobox gene Rx1 (also known as Rax) plays a major role in specifying retinal precursors and maintaining their multipotent state, the involved mechanisms remain to be largely deciphered. Here, following a highthroughput screen for genes regulated by Rx1, we found that this transcription factor specifies the fate of retinal progenitors by repressing genes normally activated in adjacent ectodermal territories. Unexpectedly, we also observed that Rx1, mainly through the activation of the transcriptional repressors TLE2 and Hes4, is necessary and sufficient to inhibit endomesodermal gene expression in retinal precursors of the eye field. In particular, Rx1 knockdown leads retinogenic blastomeres to adopt an endomesodermal fate, indicating a previously undescribed function for Rx1 in preventing the expression of endomesoderm determinants known to inhibit retinal fate. Altogether these data suggest that an essential requirement to establish a retinal precursor identity is the active inhibition of pathways leading to alternative fates.

Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character

“…Our own observations also at least raise questions about the differences observed between these two methodologies and highlight potential advantages of using mutations for studying development. For example, although we have identified new targets downstream of rax in addition to confirming previously known targets as described above, our observations are not consistent with some interactions implied by experiments utilizing strategies to assay Rax function through MO-knockdown or expression of transcriptionally repressive forms of Rax (Andreazzoli et al, 1999, 2003; Giannaccini et al, 2013; Terada et al, 2006). Specific downstream genetic interactions inferred from such studies, such as placing xhmgb3 downstream of rax (Terada et al, 2006) or transcriptional repressors crx , tle2 and hes4 (Giannaccini et al, 2013), are not consistent with the RNA-Seq analyses presented here where we see that the expression levels of these transcripts are unaffected in rax mutant tissue at neural plate stages.…”

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation

“…To investigate whether the ocular phenotype upon Sipa1l3 or Epha4 LOF is mediated through the downregulation of rax, we performed rescue experiments using rax RNA (Giannaccini et al, 2013). These experiments indeed showed that rax overexpression partially but significantly rescued the eye phenotype induced by knocking down Sipa1l3 or Epha4 (Fig.…”

“…Proper injection was confirmed at stages 13-20 using a fluorescence microscope. For rescue experiments, 30 or 50 ng Sipa1l3 or 30 ng Epha4 MO were coinjected with the corresponding RNA in following amounts: 0.1-0.24 ng rat sipa1l3 RNA, 0.24 ng rat sipa1l3_R1491* RNA, 0.1-0.5 ng chicken epha4 RNA, 0.1 ng Xenopus dnLEF RNA, 0.1-0.5 ng Xenopus dshΔdix RNA (Miller et al, 1999) and 0.1-0.25 ng Xenopus rax RNA (Giannaccini et al, 2013). The hormone-inducible dnLEF construct (Deroo et al, 2004) was induced with 10 µM dexamethasone from stage 15 on.…”

The age-regulated zinc finger factor ZNF367 is a new modulator of embryonic neurogenesis

“…ISH on cryosections was performed as described 11 . The following plasmids were used for preparation of antisense RNA probes, enzyme used for linearization and polymerases are indicated: X. laevis Znf367 EST clone image (ID_6637026) was cloned in pBKS-(XhoI, T7); Pcna-pBSK (SalI ,T7); sox2-pCS2+ (EcoR1,T7); N-tubulin-pBKS(NotI,T3); elrC-pBKS (NOTI,T7); elrD-pBSK (XhoI,T3); rx1 12 . nrg1-pBKS (BamHI, T3); p27-Pbsk (BamHI, T7).…”