UC San Diego

Forward genetic screens employing the zebrafish model organism are a powerful technique to study genes involved in vertebrate embryonic development. Through ethyl-nitrosourea (ENU)-induced mutagenesis, we identified zebrafish mutant embryos that were defective in hematopoietic stem cell (HSC) specification. These embryos also displayed moderate cardiac edema, tail deformities and craniofacial defects, specifically, neural necrosis and eye underdevelopment. Using RNA sequencing-based linkage mapping, we identified candidate single nucleotide polymorphisms (SNPs) including a missense mutation in the sh3pxd2b gene linked to our loss-of-hematopoiesis (loh) phenotype. To validate that the SNP within sh3pxd2b is sufficient to cause the loh phenotype, we performed rescue experiments by injecting wild-type sh3pxd2b mRNA into mutant embryos. Wild-type mRNA partially rescued HSC expression, but not the morphological defects of mutant embryos. In addition, we recapitulated the loss of HSC phenotype by injecting sh3pxd2b splice-block morpholino oligonucleotides into wild-type embryos. Sh3pxd2b morphant embryos exhibited similar phenotypes to our loh mutant embryos, including reduced HSC expression. Rescue and knock-down experiments confirm that sh3pxd2b is the causal gene that leads to rescued in hematopoiesis and prominent malformations of the head, eye, heart and tail. In conclusion, we have identified and characterized a novel, recessive mutant allele of sh3pxd2b, associated with a defect in HSC development through a forward genetic approach.