Fhl1b regulates the patterning and subsequent fate of the medial and lateral endodermal progenitors.

<p>(A-D) Whole-mount <i>in situ</i> hybridization showing the expression of <i>pdx1</i> (A and B) and <i>neurod</i> (C and D), comparing that of control embryos (A and C) and <i>fhl1b</i> morphants (B and D) at the 14-somite stage (A and B) and 18 hpf (C and D). <i>pdx1</i> is expressed at high levels in the most medial cells (white asterisks) and at low levels in the lateral cells (gray arrows). <i>neurod</i> is expressed in the high-level <i>pdx1</i>-expressing cells. In <i>fhl1b</i> morphants, high levels of <i>pdx1</i> (white asterisks) and <i>neurod</i> expression were expanded laterally (B and D). (E-F) Ventral confocal images showing <i>TgBAC(neurod</i>:EGFP<i>)</i>^<i>nl1</i>, β-catenin (white), and Topro (blue) at 18 hpf (the notochord is outlined by yellow dashed lines). Somites are numbered from anterior to posterior (S1-S4). (E) In control embryos, <i>TgBAC(neurod</i>:EGFP<i>)</i>^<i>nl1</i>-expressing cells are located close to the notochord. (F) Ectopic <i>TgBAC(neurod</i>:EGFP<i>)</i>^<i>nl1</i>-expressing cells were found in lateral endodermal regions in <i>fhl1b</i> morphants (white arrows). (G and H) Confocal images of <i>Tg(sox17</i>:<i>GFP)</i>^<i>s870</i> embryos at 55 hpf, stained for uncaged-Fluorescein (red) and Islet (blue). In control embryos (G), lateral 2 (L2) cells gave rise to the liver (white rhombi), intestine (white arrowhead), and exocrine pancreas (white double arrows), but rarely gave rise to the endocrine pancreas. In <i>fhl1b</i> morphants (H), L2 cells contributed to the Islet-positive pancreatic endocrine cells (white arrows), but not to the liver or exocrine pancreas. (I-L) Whole-mount <i>in situ</i> hybridization showing the expression of <i>pdx1</i> (I and J) and <i>neurod</i> (K and L) at 18 hpf, comparing control embryos (I and K) and <i>fhl1b</i>-overexpressing embryos (J and L, heat shock applied at the 8-somite stage). In embryos induced to overexpress <i>fhl1b</i> at the 8-somite stage (J and L), <i>neurod</i> and high levels of <i>pdx1</i> expression (white asterisks in J) were maintained, while low levels of <i>pdx1</i> expression (gray arrows) were reduced. (M-N’) Confocal images of <i>Tg(sox17</i>:<i>GFP)</i>^<i>s870</i> embryos at 50 hpf, stained for uncaged-Fluorescein (red) and Prox1 (blue in M and N; grey in M’ and N’). In control embryos (M and M’), lateral 1 (L1) cells gave rise to the exocrine pancreas (white double arrows) and the intestine (white arrowheads), but not to the liver. In embryos induced to overexpress <i>fhl1b</i> at the 8-somite stage (N and N’), L1 cells mostly contributed to the Prox1-positive liver cells (white rhombi). A-D and I-L, dorsal views, anterior to the top. G-H and M-N, confocal projection images; E-F, M’, and N’, confocal single-plane images, ventral views, anterior to the top. Scale bars, 20 μm.