Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation

Abstract

AbstractBackgroundUveal 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 receptorptch2produce 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 zebrafishptch2mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find thatnetrin1aornetrin1boverexpression 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 theptch2mutant: loss ofnetringenes does not rescue theptch2mutant phenotype.ConclusionThese results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in theptch2mutant.Key FindingsOveractive Hedgehog signaling in theptch2mutant causes increased netrin expressionSpatiotemporally specific overexpression of netrin1a and netrin1b can cause colobomaSpatiotemporally specific overexpression of netrin1a can disrupt optic fissure and stalk formation as well as optic stalk cell morphology, similar to theptch2mutantLoss of netrin ligands in theptch2mutant does not rescue the phenotype