Generation of the nkx2.4b zd20 null allele. A, Transcription activator‐like effector nucleases (TALENs) were targeted to exon 1 of the nkx2.4b gene, upstream of exon 2 which encodes the DNA‐binding homeodomain (HD). The 5‐bp deletion in zd20 is underlined in the spacer sequence located between the two TALENs binding sites. B, DNA alignment of nkx2.4b in wild‐type and zd20 mutant fish showing the 5‐bp deletion, identified with a red bar. C, Wild‐type Nkx2.4b amino acid sequence—serine 71 is labeled in magenta. An arrowhead marks the exon 1‐exon 2 boundary, and the homeodomain in exon 2 is underlined. D, The zd20 allele translation showing the predicted amino acid sequence resulting from the frameshift in grey, with a stop codon at position 114

nkx2.4bzd20/zd20 embryos fail to express markers of thyroid differentiation. Lateral views of 2 days postfertilization (dpf) embryos stained by in situ hybridization for thyroid primordium markers (hhex and pax8), and markers of thyroid function (tg and tpo). Yellow boxes indicate magnified regions

Targeting a gfp transgene to the nkx2.4b locus. A, Schematic showing the strategy for targeted insertion of a gfp transgene to replace the first exon of nkx2.4b. Genotyping primers for panel (G) are indicated by arrows. B,C, Whole mount image of a live embryo at 1 day postfertilization (dpf), B, and a confocal maximum‐Z projection of a dissected brain (ventral view) at 7 dpf, C, showing GFP expression in the hypothalamus of nkx2.4bzd21 fish. D,E, Live nkx2.4bzd21 embryos at 2 dpf, D, and 3 dpf, E, showing GFP expression in the thyroid primordium (arrows). Expression of GFP in the hypothalamus is obscured by the retinal pigment epithelium in panel D. F,G, Ventral views of live larvae at 5 dpf, showing GFP‐expressing thyroid follicular cells present in the jaw midline of nkx2.4bzd21/+ larvae (arrow in F), but absent in nkx2.4bzd21/zd21 larvae, G. In both genotypes, hypothalamic GFP expression is visible out of the plane of focus. H,I, Gross morphological comparison at 1 month postfertilization showing nkx2.4bzd21/zd21 fish with increased melanophore density and erythema, similar to previously described hypothyroid phenotypes. Yellow boxes indicate magnified regions. J, Genomic polymerase chain reaction analysis of GFP‐negative progeny from a nkx2.4bzd21/+ incross shows the presence of an amplicon for nxk2.4b exon 1 (137 bp product), and no amplicon for gfp (356 bp product, a smaller non‐specific amplicon is marked with asterisk). Analysis of GFP‐positive progeny with hypothyroid morphology shows the absence of the nxk2.4b exon 1 Amplicon and the presence of the gfp amplicon