Fig. S13

Fhl1b is an essential mediator of Bmp2b signaling directing the liver versus pancreas fate decision.

(A-D) Confocal images of control embryos (A), bmp2b-overexpressing embryos (B), fhl1b morphants (C), and bmp2b-overexpressing fhl1b morphants (D) at 72 hpf, stained for Islet (red; expression in the dorsal pancreatic bud is outlined by white dotted circles) and Prox1 (blue). (B) Prox1 expression in the liver was greatly expanded when bmp2b expression was induced at the 8-somite stage, whereas Islet expression in the mesenchymal cells surrounding the HPD system as well as in the pancreatic endocrine cells appeared unaffected. As in fhl1b morphants (C), the majority of bmp2b-overexpressing fhl1b morphants exhibited an enlarged Islet-positive pancreatic endocrine cell population with a reduced number of Prox1-positive cells in the liver (D, 80% (22 out of total 28 embryos analyzed)). A small portion of bmp2b-overexpressing fhl1b morphants restored the developmental defects of the liver and pancreatic endocrine formation (D, 20% (6 out of total 28 embryos analyzed)). (E-L) Whole-mount in situ hybridization showing the expression of hhex (E-H) and pdx1 (I-L), comparing control embryos (E and I), id2a morphants (F and J), fhl1b morphants (G and K), and double fhl1b/id2a morphants (H and L) at 30 hpf. hhex is expressed in the liver (black arrows) and the dorsal pancreatic bud (white dotted circles). pdx1 is expressed in the developing pancreas including the dorsal pancreatic bud (white dotted circles) and intestine (black brackets), but not in the liver. The hhex expression domain was reduced in the liver of id2a morphants (F, black arrow) but appeared unaffected in the dorsal pancreatic bud (F, white dotted circle). fhl1b morphants showed a reduced hhex expression domain in the liver (G, black arrow) with a concomitant expansion of its expression domain in the dorsal pancreatic bud (G, white dotted circle). Double fhl1b/id2a morphants showed a more severe reduction of hhex expression domain in the liver (H, black arrow), whereas its expression domain in the dorsal pancreatic bud was comparable to that in single fhl1b morphants (compare G and H, white dotted circles). pdx1 expression in id2a morphants was comparable to that in control embryos (J). pdx1 expression domain in double fhl1b/id2a morphants in the dorsal pancreatic bud was expanded (L, white dotted circle), whereas its expression in the intestinal bulb primordium appeared to be reduced (L, black bracket), and was comparable to that in fhl1b morphants (K, white dotted circle and black bracket, respectively). (M-N) Whole-mount in situ hybridization showing the expression of id2a. The expression of id2a in the liver biliary epithelial cells of fhl1b morphants (N) was comparable to that in control embryos (M) at 72 hpf. (O) Schematic model of the relationship between Bmp2b, Fhl1b, and Id2a in liver versus pancreas fate decision. Fhl1b is essentially required for suppressing pdx1 expression to keep progenitors competent to differentiate into the liver, while Id2a is required primarily for liver development. Solid lines indicate connections supported by the data previously reported and presented in this study, while dashed lines indicate potential connections by an unknown Bmp2b effector. A-D, confocal projection images, ventral views, anterior to the top. E-N, dorsal views, anterior to the left (n = 20 per each condition). Scale bars, 20 µm.