Dissection of zebrafish shha function using site-specific targeting with a Cre-dependent genetic switch

Abstract

Despite the extensive use of zebrafish as a model organism in developmental biology and regeneration research, genetic techniques enabling conditional analysis of gene function are limited. In this study, we generated <i>Zwitch</i>, a Cre-dependent invertible gene-trap cassette, enabling the establishment of conditional alleles in zebrafish by generating intronic insertions via in vivo homologous recombination. To demonstrate the utility of <i>Zwitch</i>, we generated a conditional <i>sonic hedgehog a</i> (<i>shha</i>) allele. Homozygous <i>shha</i> mutants developed normally; however, <i>shha</i> mutant embryos globally expressing Cre exhibited strong reductions in endogenous <i>shha</i> and <i>shha</i> target gene mRNA levels and developmental defects associated with null <i>shha</i> mutations. Analyzing a conditional <i>shha</i> mutant generated using an epicardium-specific inducible Cre driver revealed unique roles for epicardium-derived Shha in myocardial proliferation during heart development and regeneration. <i>Zwitch</i> will extend the utility of zebrafish in organ development and regeneration research and might be applicable to other model organisms.