bozozok mutant embryos lack hhex expression and boz mRNA overexpression induces ectopic hhex expression. (A–F) Expression of hhex at 40% epiboly was examined by in situ hybridization. Embryos derive from a cross of boz heterozygous fish. Genotypes of classes of embryos are indicated: wt for wild-type, +/- for boz heterozygous, boz for homozygous mutant. Embryos shown are representative for the classes of genotypes indicated. (A,B) Wild-type and heterozygous boz embryos express hhex in the dorsal YSL. (C,D) Homozygous boz mutant embryos lack hhex expression. (E,F) Injection of 5 pg boz mRNA induces hhex expression in the YSL of 97% of injected embryos derived from crosses of boz^+/- parents. (A,C,E: animal views; B,D,F: lateral views; A,B: dorsal to the right).

LiCl treatment induces hhex expression in wild-type and heterozygous boz mutant embryos, but not in homozygous boz mutant embryos. (A–F) Whole-mount in situ hybridization for hhex at 40% epiboly after LiCl treatment, which was performed as described in Materials and methods. Genotypes of classes of embryos for which the pictures are representative are indicated (see Fig. 1). (A–C) Following LiCl exposure, wild-type and heterozygous boz embryos exhibit ectopic hhex expression in the YSL. (D–F) Following LiCl exposure, homozygous boz embryos from the same clutch lack any hhex expression. (A,D: animal views; B,E: lateral views focusing on the embryonic midline; C,F: lateral views focused on the marginal surface).

swirl/bmp2b mutant embryos exhibit normal hhex expression and boz mRNA microinjection induces hhex expression in swirl embryos. (A–F) Expression of hhex at 50% epiboly. Genotypes of classes of embryos for which the pictures are representative are indicated (see Fig. 1). (A–C) hhex expression of a representative embryo from a swr^+/- incross. (D–F) A representative embryo from a swr^+/- intercross, injected with 5 pg boz mRNA. (A,D: animal view; B,E: lateral view, in B dorsal to the right; C: dorsal view, focussed at the marginal region; F: lateral view, focussed at the margin).

Expression of hhex in various Nodal-deficient mutant embryos is not different from hhex expression in wild-type embryos. (A–D) Expression of hhex at 50% epiboly. Definitive genotypes (wt, MZoep) as well as genotypes of classes of embryos for which the pictures are representative (sqt, cyc) are indicated in the panels: (A) Wild-type embryo. (B) Representative embryo from a sqt^+/- intercross. (C) Representative embryo from a cyc^+/- intercross. (D) A MZoep mutant embryo. (A–D: animal views, dorsal to the right).

VP16-vega1 and VP16-vega2 can induce ectopic hhex expression in homozygous boz mutant embryos, and hhex expression is expanded in Df^st7 mutants. (A,B) hhex expression in a noninjected wild-type embryo. (C–F) Expression of hhex in a wild-type (C,D) and a boz mutant (E,F) embryo after injection of 100 pg VP16-vega1 mRNA. (G–J) Expression of hhex in a wild-type (G,H) and a boz (I, J) embryo after injection of 100 pg VP16-vega2 mRNA. (K,L) Expression of hhex in a noninjected embryo (K) and in an embryo injected with 25 pg of vega2 mRNA (L). (M,N) Expression of boz in a noninjected embryo (M) and in an embryo injected with 100 pg VP16-vega2 mRNA (N). (O–R) hhex expression in wild-type and heterozygous Df^st7 embryos (O,P) and in a Df^st7 homozygous mutant embryo (Q–R). Pictures are either focussed to the midline (O,Q) or the lateral marginal region (P,R). All embryos were fixed at 50% epiboly stage. All embryos depicted in A–J and O–R were genotyped after photography. (A,C,E,G,I,K–N: animal views; B,D,F,H,J,O–R: lateral views; A,B,K,M,O,P: dorsal to the right, all others, except for L,Q,R, putative dorsal to the right; probes used are indicated in the bottom left corner of each panel).

hhex does not repress wnt8 expression at the dorsal margin. (A,B) Whole-mount in situ hybridization for hhex in a cyc^b16 mutant embryo at 50% epiboly. cyc^b16 is a deletion covering the hhex locus, such that mutant embryos lack hhex expression. (C,D) Expression of wnt8 in a wild-type embryo. (E,F) Expression of wnt8 in a representative embryo from a cyc^b16+/- intercross at shield stage: wt, deficiency heterozygous and cyc^b16 mutant embryos have indistinguishable wnt8 expression. (A,C,E: animal view, in C and E dorsal to the right; B,D,F: lateral view, in D and F dorsal to the front; probes used are indicated in the bottom left corner of each panel).