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Fig. 2 Whole-mount ISH analysis of pituitary hormone-encoding transcripts in 48 hpf embryos. (A,C–E) Transcript levels measured by the integrated densities of the whole-mount-ISH-staining signals of tshb ((A); N = 10 homozygotes and 13 WTs), gh ((C); N = 18 homozygotes and 10 WTs), pomca ((D); N = 13 homozygotes and 11 WTs) and prl ((E); N = 10 homozygotes and 6 WTs) in 48 hpf lhx4 mutants and their WT siblings from heterozygous intercross. Homozygous lhx4 mutants expressed lower amounts of tshb compared with their WT siblings ((A); *** p < 0.001, Mann–Whitney test), while the expressions of gh, pomca and prl remained unaltered (C–E). Error bars indicate s.e.m. (B) Representative samples (heads of 48 hpf embryos, dorsal view) of WT sibling (left) and homozygous lhx4 mutant (right), analyzed by whole-mount ISH using the tshb probe. Tshb ISH signal (denoted by arrows) is substantially reduced in the pituitary of homozygous lhx4 mutant at 48 hpf as compared with its WT sibling. Scale bar = 100 µm.