Sox11 Is Required to Maintain Proper Levels of Hedgehog Signaling during Vertebrate Ocular Morphogenesis

Abstract

Ocular coloboma is a sight-threatening malformation caused by failure of the choroid fissure to close during morphogenesis of the eye, and is frequently associated with additional anomalies, including microphthalmia and cataracts. Although Hedgehog signaling is known to play a critical role in choroid fissure closure, genetic regulation of this pathway remains poorly understood. Here, we show that the transcription factor Sox11 is required to maintain specific levels of Hedgehog signaling during ocular development. Sox11-deficient zebrafish embryos displayed delayed and abnormal lens formation, coloboma, and a specific reduction in rod photoreceptors, all of which could be rescued by treatment with the Hedgehog pathway inhibitor cyclopamine. We further demonstrate that the elevated Hedgehog signaling in Sox11-deficient zebrafish was caused by a large increase in shha transcription; indeed, suppressing Shha expression rescued the ocular phenotypes of sox11 morphants. Conversely, over-expression of sox11 induced cyclopia, a phenotype consistent with reduced levels of Sonic hedgehog. We screened DNA samples from 79 patients with microphthalmia, anophthalmia, or coloboma (MAC) and identified two novel heterozygous SOX11 variants in individuals with coloboma. In contrast to wild type human SOX11 mRNA, mRNA containing either variant failed to rescue the lens and coloboma phenotypes of Sox11-deficient zebrafish, and both exhibited significantly reduced transactivation ability in a luciferase reporter assay. Moreover, decreased gene dosage from a segmental deletion encompassing the SOX11 locus resulted in microphthalmia and related ocular phenotypes. Therefore, our study reveals a novel role for Sox11 in controlling Hedgehog signaling, and suggests that SOX11 variants contribute to pediatric eye disorders.

Sox4 regulates choroid fissure closure by limiting Hedgehog signaling during ocular morphogenesis

“…As mentioned previously, we have shown that knockdown of Sox11 causes coloboma due to an elevation of Hh signaling (Pillai-Kastoori et al, 2014). Moreover, the coloboma phenotype in both sox11 and sox4 morphants resembles other animal models of overactive Hh signaling (Lee et al, 2008; Bassett et al, 2010; Lee et al, 2012; Zhang et al, 2013).…”

“…Therefore, we asked whether cyclopamine, a pharmacological Hh inhibitor, could reduce the incidence of coloboma in sox4 morphants. We treated sox4 morphants with 2 μM cyclopamine, a concentration which did not produce any ocular defects on its own (Figure 5E), and which was shown previously to rescue the coloboma phenotype in zebrafish blowout mutants (which carry a mutation in the ptch2 receptor) and sox11 morphants (Lee et al, 2008; Pillai-Kastoori et al, 2014). Sox4 morphants were exposed to either cyclopamine or an equivalent amount of ethanol vehicle from 5.5 to 13 hpf, and then analyzed at 48 hpf for the presence of coloboma.…”

“…We recently demonstrated that the SoxC transcription factor Sox11 is one of the upstream regulators of Hh signaling, and that in the zebrafish, Sox11 regulates choroid fissure closure as well as retinal neurogenesis by inhibiting the expression of Shh (Pillai-Kastoori et al, 2014). The Sox proteins are named for a shared motif called the SRY box, a high-mobility-group (HMG) DNA binding domain homologous to the DNA-binding domain of the mammalian sex-determining gene SRY.…”

“…Like Sox11, Sox4 is expressed in multiple embryonic tissues, including the retina (Dy et al, 2008; Usui et al, 2013; Pillai-Kastoori et al, 2014). In the mouse, Sox4 expression initiates in the central retina near the optic nerve at E11.5.…”

Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network

“…These features could be mimicked in surface fish by shh overexpression, and eye development was restored partially in cavefish embryos by using cyclopamine (Yamamoto et al 2004). Recently, it has been demonstrated that mutations in SOX11 and SMAD7 may lead to various ODA through the activation of the SHH pathway (Zhang et al 2013;Pillai-Kastoori et al 2014). In humans, PTCH1 mutations have been associated previously with basal cell nevus syndrome (BCNS) (MIM#109400) and with holoprosencephaly (HPE7) (MIM#610828).…”