Alk8 is required for early liver development. Wild-type and alk8 mutant zebrafish embryos at 26 (A-H), 28 (I,J) and 48 (K,L) hpf were analyzed for hhex (A,B), prox1 (C,D), gata4 (E,F), gata6 (G,H), foxa3 (I,J) and cp (K,L) expression. (A,B) hhex expression in the liver region (arrows) is greatly reduced in the mutants, whereas its expression in the pancreatic islet appears unaffected (arrowheads). (C,D) prox1 expression in the liver region (arrows) is also greatly reduced in the mutants, whereas its expression in the interrenal primordium appears unaffected (arrowheads). To better visualize hepatic prox1 expression, a side-view image is shown in an inset. (E-H) gata4 and gata6 expression is reduced in the liver region (arrows), whereas their expression in the intestinal endoderm is only mildly affected (brackets). (I,J) The leftward bending of the gut appears unaffected in the mutants (brackets). (K,L) Hepatocyte expression of cp is completely absent in the mutants. All images, except insets, are dorsal views, anterior left.

Bmp signaling is not essential for the maintenance of specified liver progenitors. Embryos obtained from outcrossing a hemizygous Tg(hsp70l:dnBmpr-GFP) zebrafish were heat shocked at 22 (A-C,G-I,M-O) or 26 (D-F,J-L,P-R) hpf, harvested at 33 (A-C,G-I), 36 (D-F,J-L) or 38-40 (M-R) hpf, and examined for hhex (A-F), prox1 (G-L) and cp (M-R) expression. The percentage of hemizygous embryos exhibiting a similar expression pattern is indicated in the lower left corner (n=8-10). When embryos were heat shocked at 22 hpf, both hhex and prox1 expression in the liver region (arrows) were greatly reduced in the hemizygous embryos, whereas their expression in other regions (arrowheads) appeared unaffected (B,C,H,I). By contrast, when they were heat shocked at 26 hpf, both hhex and prox1 were clearly expressed in the liver region in all the hemizygous embryos (E,F,K,L, arrows). However, hhex and prox1 expression in the liver region (arrows) was somewhat reduced compared with wild-type siblings, whereas hhex expression in the pancreatic islet and prox1 expression in the interrenal primordium (arrowheads) appeared unaffected (D-F,J-L). Hepatocyte differentiation, assessed by cp expression, barely occurred in the hemizygous embryos heat shocked at 22 hpf (N,O, arrows), and was clearly reduced in those heat shocked at 26 hpf (Q,R, arrows). All images, except insets, are dorsal views, anterior left. Insets are side views, anterior left.

Overexpression of wild-type alk8 under a heat-shock promoter rescued liver defects in alk8 mutant zebrafish embryos. (A-R) Embryos obtained from crossing alk8^+/- females with Tg(hsp70:alk8);alk8^+/- males were heat shocked at 18 (A,C,G,I,M,O) or 34 (D,F,J,L,P,R) hpf, and harvested at 34 (A-C,G-I), 42 (D-F,J-L), 47 (M-O) or 54 (P-R) hpf. The expression of hhex (A-F), prox1 (G-L) and cp (M-R) was then examined (arrows). When alk8 was overexpressed at 18 hpf, the expression of hhex, prox and cp in the mutant embryos (C,I,O, arrows) was comparable to that in wild-type siblings (A,G,M, arrows), whereas their expression in the mutant embryos that were not heat shocked was strongly reduced (B,H,N, arrows). Even when alk8 was overexpressed at 34 hpf, hhex, prox and cp expression was substantial in the mutant embryos (F,L,R, arrows) compared with the mutant embryos that were not head shocked (E,K,Q, arrows).

Gata4 and Gata6 are required for the expansion and differentiation of liver progenitors. Wild-type zebrafish embryos were injected with 10 ng gata4 MO and/or 2.5 ng gata6 MO, harvested at 37-38 hpf and examined for hhex (A-E), prox1 (F-J) and cp (K-N) expression. Injections of gata4 or gata6 MO weakly reduced hhex and prox1 expression in the liver region (B,C,G,H); injections of both MOs together strongly reduced their expression (D,E,I,J), but did not completely abolish it. Injections of gata4, gata6 or both MOs severely affected hepatocyte differentiation, as shown by cp expression (L-N). All images are dorsal views, anterior left (n=13-20).

Fgf signaling is essential for hepatoblast specification. Embryos obtained from outcrossing a hemizygous Tg(hsp70l:dnfgfr1-EGFP) zebrafish were heat shocked at 18 hpf, harvested at 29-31 (A-N) or 38-40 (O,P) hpf, and examined for hhex (A-C), prox1 (D,E), gata4 (F-H), gata6 (I-K), foxa3 (L-N) and cp (O,P) expression. The percentage of hemizygous embryos exhibiting a similar expression pattern is indicated in the lower left corner (n=8-10). (A-C) hhex expression in the liver region (arrows) was greatly reduced or almost absent in the hemizygous embryos, whereas its expression in the pancreatic islet appeared unaffected (arrowheads). (D,E) prox1 expression in the liver region (arrows) was almost absent in the hemizygous embryos, whereas its expression in the interrenal primordium was less affected (arrowheads). To better visualize hepatic prox1 expression, a side-view image is shown in an inset. (F-K) gata4 and gata6 expression was greatly reduced in the liver region (arrows), whereas their expression in the intestinal endoderm was less affected (brackets). (L-N) The leftward bending of the gut did not occur in the hemizygous embryos (brackets). (P) Hepatocyte expression of cp was absent in the hemizygous embryos. All images, except insets, are dorsal views, anterior left.

Fgf signaling is not essential for the maintenance of specified liver progenitors. Embryos obtained from outcrossing a hemizygous Tg(hsp70l:dnfgfr1-EGFP) zebrafish were heat shocked at 22 (A-C,G,H,L-N) or 26 (D-F,I-K,O-Q) hpf, harvested at 35-36 (A-K) or 38-40 (L-Q) hpf and examined for hhex (A-F), prox1 (G-K), and cp (L-Q) expression. The percentage of the hemizygous embryos exhibiting a similar expression pattern is indicated in the lower left corner (n=8-10). When embryos were heat shocked at 22 hpf, hhex expression in the liver region (arrows) was greatly reduced (B) or almost absent (C) in the hemizygous embryos, whereas its expression in the pancreatic islet (arrowheads) appeared unaffected (B,C). prox1 expression in the liver (black arrows) and retina (white arrows) was also greatly reduced in the hemizygous embryos (H), whereas its expression in the interrenal primordium appeared unaffected (H, arrowheads). By contrast, when embryos were heat shocked at 26 hpf, both hhex and prox1 were clearly expressed in the liver region in all the hemizygous embryos (E,F,J,K, arrows). However, hhex and prox1 expression in the liver region was reduced compared with wild-type siblings, whereas hhex expression in the pancreatic islet and prox1 expression in the interrenal primordium appeared unaffected (E,F,J,K, arrowheads). Hepatocyte differentiation, assessed by cp expression, was severely defective in the hemizygous embryos heat shocked at 22 hpf (M,N, arrows) and weakly reduced in those heat shocked at 26 hpf (P,Q, arrows). All images, except insets, are dorsal views, anterior left. Insets are side views, anterior left.

Overexpression of Bmp2b under a heat-shock promoter partially rescued hepatoblast specification in zebrafish embryos lacking Fgf signaling. Embryos obtained from crossing a hemizygous Tg(hsp70:bmp2b)^fr13 female with a hemizygous Tg(hsp70l:dnfgfr1-EGFP) male were heat shocked at 18 hpf and harvested at 30-32 (A-K) or 38-40 (L-Q) hpf. The expression of hhex (A-F), prox1 (G-K) and cp (L-Q) was then examined. hhex, prox and cp expression in the liver region mostly recovered in a majority of the embryos overexpressing Bmp2b and lacking Fgf signaling (E,F,J,K,P,Q, arrows), whereas their expression in embryos lacking Fgf signaling was strongly reduced (C,D,I,N,O, arrows). Twenty-five percent of the double hemizygous embryos did not show recovery of prox1 expression. The expression of hhex and prox1 in the embryos overexpressing Bmp2b (B,H, arrows) was comparable to that in wild-type siblings (A,G, arrows); cp expression was enhanced in the embryos overexpressing Bmp2b (M) compared with wild-type siblings (L). The percentage of the embryos exhibiting a similar expression is indicated in the lower left corner (n=11-12). All images are dorsal views, anterior up.