An atlas of seven zebrafish hox cluster mutants provides insights into sub/neofunctionalization of vertebrate Hox clusters.

Vertebrate Hox clusters are comprised of multiple Hox genes that control morphology and developmental timing along multiple body axes. Although results of genetic analyses using Hox-knockout mice have been accumulating, genetic studies in other vertebrates have not been sufficient for functional comparisons of vertebrate Hox genes. In this study, we isolated all of the seven hox cluster loss-of-function alleles in zebrafish using the CRISPR-Cas9 system. Comprehensive analysis of the embryonic phenotype and X-ray micro-computed tomography scan analysis of adult fish revealed several species-specific functional contributions of homologous Hox clusters along the appendicular axis, whereas important shared general principles were also confirmed, as exemplified by serial anterior vertebral transformations along the main body axis, observed in fish for the first time. Our results provide insights into discrete sub/neofunctionalization of vertebrate Hox clusters after quadruplication of the ancient Hox cluster. This set of seven complete hox cluster loss-of-function alleles provide a formidable resource for future developmental genetic analysis of the Hox patterning system in zebrafish.

Enhancer activity-based identification of functional enhancers using zebrafish embryos.

Chromatin immunoprecipitation (ChIP) against enhancer-associated marks with massive sequencing is a powerful approach to identify genome-wide distributions of putative enhancers. However, functional in vivo analysis is required to elucidate the activities of predicted enhancers. Using zebrafish embryos, we established a ChIP-Injection method that enables identification of functional enhancers based on their enhancer activities in embryos. Each reporter gene possessing the enhancer-associated genomic region enriched by ChIP was injected into zebrafish embryos to analyze the activity of putative enhancers. By using the ChIP-Injection, we identified 32 distinct putative enhancers that drove specific expression. Additionally, we generated transgenic lines that exhibit distributions of the EGFP signal as was observed in the screening. Furthermore, the expression pattern driven by the identified somite-specific enhancer resembled that of the gene acta2. The results indicate that ChIP-Injection provides an efficient approach for identification of active enhancers in a potentially wide variety of developmental tissues and stages.