Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation

Abstract

BackgroundUveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. ResultsWe find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. ConclusionThese results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant. Key FindingsO_LIOveractive Hedgehog signaling in the ptch2 mutant causes increased netrin expression C_LIO_LISpatiotemporally specific overexpression of netrin1a and netrin1b can cause coloboma C_LIO_LISpatiotemporally specific overexpression of netrin1a can disrupt optic fissure and stalk formation as well as optic stalk cell morphology, similar to the ptch2 mutant C_LIO_LILoss of netrin ligands in the ptch2 mutant does not rescue the phenotype C_LI