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

Abstract

SoxC transcription factors play critical roles in many developmental processes, including neurogenesis, cardiac formation, and skeletal differentiation. In vitro and in vivo loss-of-function studies have suggested that SoxC genes are required for oculogenesis, however the mechanism was poorly understood. Here, we have explored the function of the SoxC factor Sox4 during zebrafish eye development. We show that sox4a and sox4b are expressed in the forebrain and periocular mesenchyme adjacent to the optic stalk during early eye development. Knockdown of sox4 in zebrafish resulted in coloboma, a structural malformation of the eye that is a significant cause of pediatric visual impairment in humans, in which the choroid fissure fails to close. Sox4 morphants displayed altered proximo-distal patterning of the optic vesicle, including expanded pax2 expression in the optic stalk, as well as ectopic cell proliferation in the retina. We show that the abnormal ocular morphogenesis observed in Sox4-deficient zebrafish is caused by elevated Hedgehog (Hh) signaling, and this is due to increased expression of the Hh pathway ligand Indian hedgehog b (ihhb). Consistent with these results, coloboma in sox4 morphants could be rescued by pharmacological treatment with the Hh inhibitor cyclopamine, or by co-knockdown of ihhb. Conversely, overexpression of sox4 reduced Hh signaling and ihhb expression, resulting in cyclopia. Finally, we demonstrate that sox4 and sox11 have overlapping, but not completely redundant, functions in regulating ocular morphogenesis. Taken together, our data demonstrate that Sox4 is required to limit the extent of Hh signaling during eye development, and suggest that mutations in SoxC factors could contribute to the development of coloboma.

Zebrafish zic2 controls formation of periocular neural crest and choroid fissure morphogenesis

“…Zebrafish that lack the functional Hh receptor blowout/ptc 1 due to mutations show increased levels Hh signaling and develop with incompletely penetrant coloboma; this defect is efficiently rescued by exposure to low levels of cyclopamine, a small molecule that inhibits Hh signaling (Lee et al, 2008a). Cyclopamine treatment also rescues coloboma caused by knockdown of sox4 or sox11, transcription factors that function as inhibitory modulators of Hh signaling during retinal morphogenesis (Pillai-Kastoori et al, 2014; Wen et al, 2015). We reasoned that, if Hh signaling is expanded in zic2 mutants, inhibition of Hh signaling should reduce the penetrance and/or expressivity of coloboma.…”

The genomic basis of cichlid fish adaptation within the deepwater “twilight zone” of Lake Malawi

“…), which is expressed in early zebrafish eye development (Wen et al. ) and also plays an active role in Wnt signalling by stabilizing β‐catenin via a complex feedback loop (Bhattaram et al. ).…”

“…Knockdown of SOX4 in zebrafish resulted in structural malformations of the eye (Wen et al. ). SOX2 , also affects Wnt signalling via feedback inhibition, and has been shown to crucially regulate retina formation in Xenopus (Agathocleous et al.…”

“…Generally the Tcf/Lef family of molecules mediate canonical Wnt signalling by regulating downstream target gene expression (Behrens et al 1996). TCF7L1 has been demonstrated to directly repress SOX4 (Gribble et al 2009), which is expressed in early zebrafish eye development (Wen et al 2015) and also plays an active role in Wnt signalling by stabilizing β-catenin via a complex feedback loop (Bhattaram et al 2014). Knockdown of SOX4 in zebrafish resulted in structural malformations of the eye (Wen et al 2015).…”

Tracking the fate of her4 expressing cells in the regenerating retina using her4:Kaede zebrafish

“…Immunohistochemistry was performed on retinal cryosections as previously described (Fadool, 2003, Pillai-Kastoori et al, 2014, Wen et al, 2014). The following primary antibodies and dilutions were used: 4C12 (mouse, 1:100) generously provided by J. Fadool (Florida State University, Tallahassee, FL), which labels rod photoreceptor cell bodies; 1D1 (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL), which recognizes Rhodopsin; Zpr-1 (mouse, 1:20, ZIRC), which labels red-green double cones; Zrf-1 (mouse, 1:5000, ZIRC), which labels Müller glia; HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which recognizes retinal ganglion cells and amacrine cells; PKCα (rabbit, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes bipolar cells, Prox1 (rabbit, 1:2000, Millipore, Billerica, MA), which recognizes horizontal cells; Nr2e3 (rabbit, 1:100), generously provided by J. Nathans (Johns Hopkins, Baltimore, MD), which labels rod photoreceptor progenitor/precursor cells; anti-PCNA (mouse, 1:100, Santa Cruz Biotechnology, Dallas, TX), which labels proliferating cells; anti-BrdU (mouse, 1:500, Sigma, St. Louis, MO), which marks cells in S phase of the cell cycle; and anti-Kaede (rabbit, 1:500, MBL, Nagoya, Japan), which labels Kaede expressing cells.…”