Lineage-Tracing Evidence for Hepatopancreatic Progenitors

(A–C) Ventral confocal images of control embryos at the 10 somite stage illustrating the accuracy of single-cell targeting by the laser. Tg(sox17:GFP)^s870 embryos were stained for GFP (gray) and uncaged fluorescein (red). A single endodermal cell was labeled in the region between somites 1 and 4 at three specific positions in the medio-lateral axis: the most medial cells (medial, [A]), cells immediately adjacent to the medial cells (lateral 1, [B]), and cells one cell away from the medial cells (lateral 2, [C]).

(D–I) Confocal projections of Tg(sox17:GFP)^s870 embryos at 50 hpf showing the progeny of the medial (D and G), lateral 1 (E and H), and lateral 2 (F and I) cells. Tg(sox17:GFP)^s870 embryos were stained for GFP (green), Insulin (blue), and uncaged fluorescein (red) (triple-positive cells appear white). Medial cells (D and G) mostly gave rise to pancreatic endocrine cells (arrows). Lateral 1 cells (E and H) gave rise to exocrine cells (double arrows), a small number of endocrine cells (arrows), as well as intestinal tissue (arrowheads) adjacent to the pancreas. Lateral 2 cells (F and I) gave rise to liver (asterisks), intestine (arrowheads), and exocrine cells (double arrows).

(J) 3D column graph tabulating the lineage-tracing data. Data in each column (%) were obtained by summing the number of embryos that showed incorporation into a given tissue type and normalizing it to the total number of embryos examined in each specific position: M, L1, and L2.

(K) Schematic diagram of Tg(sox17:GFP)^s870 expressing cells in the region between somites 1 and 4 with the bilateral position (red boxes) of cells that can give rise to liver and pancreas.

bmp2b Is Expressed in the Lateral Plate Mesoderm from an Early Stage

(A–D) Dorsal views (A and C) and transverse sections (B and D) showing bmp2b expression (dotted squares and white arrows) in the lateral plate mesoderm starting at the 10 somite stage (A and B) and persisting through the 14 somite stage (C and D). At the 10-somite stage, bmp2b starts to be expressed in the lateral plate mesoderm (A and B). The expression becomes more pronounced by the 14 somite stage (C and D). Note that the dispersed bmp2b expression at the 10 somite stage becomes more discrete by the 14 somite stage.

(E) Dorsal view of two-color in situ hybridization (myod1 in red and bmp2b in blue) showing that bmp2b is expressed adjacent to somites 1 through 3 at the 10 somite stage (dotted square).

(F) Confocal image of a transverse section showing the location of the lateral 2 endodermal cells (L2) in relation to the LPM (yellow dotted circle). Embryos were stained for Fibronectin (green) and ZO1 (red) and counterstained with Topro (white). A yolk syncytial layer (YSL) nucleus is marked with an asterisk.

(G and H) Dorsal views of pdx1 expression, comparing wild-type (G) and smo mutant (H) at the 10 somite stage. pdx1 is expressed at high levels in the most medial cells (asterisks) and at low levels in the lateral cells (arrows) in wild-type (G). Low-level pdx1 expression (arrows) is detected in smo mutant starting at the 10 somite stage (H).

The pdx1 “Gradient” Correlates with the Medio-Lateral Position of Endodermal Cells

(A–C″) Confocal projections of Tg(sox17:GFP)^s870 embryos at the 14 somite stage stained for pdx1 (red), GFP (green), and uncaged-fluorescein (blue). A medial cell (A–A″) (arrow), which gives rise to the endocrine pancreas, expresses high levels of pdx1 (outlined by white dashed line in [A″]). A lateral 1 cell (B–B″) (arrow), which gives rise to the exocrine pancreas and intestine as well as a small number of pancreatic endocrine cells, expresses low levels of pdx1 (outlined by white dashed line in [B″]). A lateral 2 cell (C–C″) (arrow), which gives rise to the liver, intestine, and exocrine pancreas, does not express pdx1 (outlined by white dashed line in [C″]).

Decrease in Bmp Signaling Leads to a Decrease in Liver Tissue and Increase in pdx1 Expressing Cells

(A–D) Dorsal views of hhex (A and B) and pdx1 (C and D) expression, comparing wild-type (A and C) and alk8 mutants (B and D) at 28 hpf. hhex is expressed in the liver (black arrow) as well as the dorsal pancreatic bud (white arrow) (A). hhex expression is greatly reduced in the liver forming region (black arrow) of alk8 mutants but appears unaffected in the dorsal pancreatic bud (white arrow) (B). pdx1 expression is expanded in alk8 mutants (D) compared to wild-type (C).

(E–F′) Confocal projections of Tg(gutGFP)^s854 wild-type (E and E′) and alk8 mutants (F and F′) at 28 hpf, stained for Pdx1 (red, outlined by yellow dashed line in E′ and F′) and Prox1 (white). The dorsal pancreatic bud (arrows; outlined by white dashed line) is also Pdx1 positive. Compared to wild-type (E and E′), in alk8 mutants (F and F′), the Pdx1 expression domain has expanded into the Prox1 expression domain, which is itself reduced.

(G and H) Dorsal views of pdx1 expression, comparing wild-type (G) and alk8 mutants (H) at 18 hpf. pdx1 is expressed at high levels in the most medial cells (asterisks) and at low levels in lateral cells (arrows) (G). In alk8 mutants (H), low levels of pdx1 expression were expanded laterally (the percentage of alk8 mutants showing a substantial lateral expansion of pdx1 expression was 54%, n = 7/13).

(I and J) Dorsal views of hhex expression comparing wild-type (I) and bmp2b MO-injected alk8 heterozygous (J) embryos at 28 hpf. Compared to wild-type (I), hhex expression was greatly reduced in the liver-forming region (black arrows) of most (n = 16/23) alk8 heterozygous embryos injected with bmp2b MO (J). hhex expression in the dorsal pancreatic bud (white arrows) appears unaffected.

Bmp2b Overexpression Causes a Pancreatic-to-Liver Fate Switch

(A–F) Dorsal views of hhex (A–C) and pdx1 (D–F) expression at 44 hpf, comparing control (A and D) and Bmp2b-overexpressing embryos (heat shock applied at the 8 [B and E] or 14 [C and F] somite stage). The liver and the dorsal pancreatic bud are marked by black brackets and white dotted lines, respectively. hhex is expressed in the liver and dorsal pancreatic bud (A). pdx1 is expressed in the developing gut, including the pancreas and intestine, but not in the liver (D). When bmp2b expression was induced at the 8 somite stage, endodermal hhex expression was greatly expanded (B) while pdx1 expression was reduced (E). When bmp2b expression was induced at the 14 somite stage, endodermal hhex expression was only slightly expanded (C), while pdx1 expression was maintained (F).

(G–I) Ventral views of confocal projection images showing Prox1 (red), Islet1 (white), and Tg(ptf1a:GFP)^jh1 (green) expression at 50 hpf, comparing control (G) and Bmp2b-overexpressing embryos (heat shock applied at the 8 [H] or 14 [I] somite stage). Prox1 is highly expressed in the liver as well as in the ventral pancreas, which is also marked by Tg(ptf1a:GFP)^jh1 expression (arrows) (G). Islet1 is expressed in the mesenchymal cells surrounding the hepatopancreatic duct as well as in the pancreatic endocrine cells. Prox1 expression was greatly expanded when bmp2b expression was induced at the 8 somite stage (H), while Tg(ptf1a:GFP)^jh1 expression was completely lost. When bmp2b expression was induced at the 14 somite stage (I), Prox1 expression was only slightly expanded, while the ventral pancreas was specified and exhibited Tg(ptf1a:GFP)^jh1 expression (arrows).

(J) Schematic diagram of lineage tracing experiment. A single L1 cell at the level of somite 2, which normally gives rise to the pancreas and intestine, was uncaged (red boxes).

(K and L) Confocal projections of Tg(sox17:GFP)^s870 embryos at 50 hpf stained for GFP (green), Prox1 (blue), and uncaged-fluorescein (red). In control embryos (K), L1 cells gave rise to pancreas and intestine (arrows), but not to liver. In embryos induced to overexpress bmp2b at the 8 somite stage (L), L1 cells contributed to the liver (arrows), as marked by Prox1 (blue).

Expression Pattern of bmp2a, 2b, 4 and 7
(A-B) Dorsal views of bmp4 (A) and bmp7 (B) expression at 21 and 24 hpf, respectively. (A) bmp4 is mainly expressed in the cardiac mesoderm (arrow). (B) bmp7 is expressed in the head region and otic vesicles (arrowheads).
(C and D) Dorsal views of bmp2a (C) and bmp2b (D) expression at 24 hpf. bmp2a and bmp2b are both expressed in the lateral plate mesoderm (dotted square) and fin folds (arrows) at this stage. (D) bmp2b is also expressed in the otic vesicles (arrowheads).
(E and F) Lateral (E) and dorsal (F) views of bmp2b expression at 75% epiboly (E) and the 5-somite stage (F). (E) bmp2b is expressed ventrally as well as in the dorsal organizer at 75% epiboly. (F) At the 5-somite stage, bmp2b is mainly expressed in the epidermis.

Bmp Signaling Regulates pdx1 Expression
(A–D) Dorsal views of pdx1 expression, comparing wild-type (A and C), Tg(hsp70l:dnBmpr-GFP)^w30 (B) and Tg(hsp70l:bmp2b)^f13 (D) hemizygous embryos at 18 hpf. pdx1 is expressed at high levels in the most medial cells (asterisks) and at low levels in the lateral cells (arrows) in wild-type (A and C). (B) In Tg(hsp70l:dnBmpr-GFP)^w30 hemizygous embryos heat-shocked at the 6-somite stage, pdx1 expression was expanded laterally (the percentage of embryos showing a substantial lateral expansion of pdx1 expression was 60%, n = 18/31). (D) In Tg(hsp70l:bmp2b)^f13 hemizygous embryos heatshocked at the 8-somite stage, high levels of pdx1 expression (asterisks) were maintained, while low levels (arrows in A, B and C) were severely reduced (the percentage of bmp2b).