Mechanisms of Copper-Dependent Notochord Formation in Zebrafish

The interplay of genes and nutrition during early development profoundly impacts fetal outcome. Copper is an essential nutrient required for the redox activity of many enzymes, and recent work in our laboratory has elucidated the phenotype of copper deficiency in zebrafish, which includes a strikingly distorted notochord. The studies described here establish the specific genetic etiology of this distortion and reveal a number of gene-gene and gene-nutrient interactions critical to notochord morphogenesis. We first demonstrate that the notochord distortion observed in copper-deficient zebrafish results from lysyl oxidase cuproenzyme inhibition. Four lysyl oxidase family members are expressed throughout the developing zebrafish notochord, including loxl1 and loxl5b. Morpholino antisense experiments reveal that knockdown of loxl1 results in notochord distortion and also demonstrate overlapping roles for loxl1 and loxl5b in notochord formation. Furthermore, partial knockdown of either loxl1 or loxl5b sensitizes embryos to notochord distortion when combined with reduced copper availability, and any of these treatments alone sensitizes embryos to notochord distortion after partial disruption of the gene encoding a lysyl oxidase substrate, col2a1. To elucidate additional gene-nutrient interactions involved in notochord formation, we conducted a forward genetic screen for mutants that exhibit increased notochord distortion after partial lysyl oxidase inhibition. This screen was facilitated by the identification of a novel, highly potent lysyl oxidase inhibitor, 2-mercaptopyridine-N-oxide, and yielded a mutant with defects in notochord and vascular morphogenesis, puff daddygw1. Subsequent work demonstrated that the puff daddygw1 phenotype results from loss of zebrafish fibrillin-2. Importantly, the notochords of puff daddygw1 mutants are strikingly sensitized to distortion under conditions of suboptimal copper nutrition that do not affect wild-type embryos. This sensitization is also observed in a published notochord mutant, gulliverm208, and we demonstrate that the gulliverm208 phenotype arises from a missense mutation in the alpha 1 chain of type VIII collagen. Taken together, these studies demonstrate essential roles for copper and multiple extracellular matrix components in late notochord formation and suggest that nutritional status should be interpreted within the polymorphic genetic context of the developing embryo