A transgene targeted to the zebrafish nkx2.4b locus drives specific green fluorescent protein expression and disrupts...

With the goal of labeling and manipulating the zebrafish hypothalamus, we sought to target a green fluorescent protein (gfp) transgene to the expression domains of nkx2.4b, a gene expressed during hypothalamic and thyroid development. We combined transcription activator-like effector nucleases (TALENs)-mediated mutagenesis with a targeting construct to enable insertion of a gfp transgene into the endogenous nkx2.4b genomic locus.

Injection of TALENs targeted to the first exon of nkx2.4b created a predicted null allele, and homozygous mutant embryos displayed loss of thyroid markers. From embryos injected with both TALENs and a targeting construct carrying a gfp transgene, we recovered a line in which GFP was expressed specifically in the hypothalamus and thyroid. Fish homozygous for this allele lacked exon 1 of nkx2.4b and exhibited hypothyroid phenotypes.

By combining TALENs injections with a targeting construct that contained a gfp transgene, we were able to recover an allele in which GFP is expressed in the nkx2.4b expression domains, with homozygous phenotypes suggesting the creation of a loss-of-function transgenic line. These results demonstrate the creation of a useful tool for studying hypothalamus and thyroid development.