Intra-endodermal interactions are required for pancreatic beta cell induction

Abstract

The cellular origin of signals that regulate pancreatic beta cell induction is not clearly defined. Here, we investigate the seeming paradox that Hedgehog/Smoothened signaling functions during gastrulation to promote pancreatic beta cell development in zebrafish, yet has an inhibitory role during later stages of pancreas development in amniotes. Our cell transplantation experiments reveal that in zebrafish, Smoothened function is not required in beta cell precursors. At early somitogenesis stages, when the zebrafish endoderm first forms a sheet, pancreatic beta cell precursors lie closest to the midline; however, the requirement for Smoothened lies in their lateral neighbors, which ultimately give rise to the exocrine pancreas and intestine. Thus, pancreatic beta cell induction requires Smoothened function cell-nonautonomously during gastrulation, to allow subsequent intra-endodermal interactions. These results clarify the function of Hedgehog signaling in pancreas development, identify an unexpected cellular source of factors that regulate beta cell specification, and uncover complex patterning and signaling interactions within the endoderm.

Figure. 1. Initial position of Tg(ins:GFP) expressing cells

12- (A-C, F), 15- (D-E, I) and 18- (G-H) somite stage (corresponding to 15, 16.5 and 18 hpf, respectively) Tg(ins:GFP) embryos showing GFP (green; [A-B], [D-I]), TOPRO (white; [B], [E], [H]), β-catenin (red; [B], [E], [H]) and Tg(sox17:DsRed)^s903 (red; [F], [I]) staining. Dorsal views of fixed embryos (A, D, G), transverse (B, E, H) and ventral (F, I) confocal images. (A) One Tg(ins:GFP) expressing cell (arrow) is present in the endoderm of this 12-somite stage embryo. Cells in the anterior neural tube (arrowhead) also express the ins:GFP transgene. (B) The Tg(ins:GFP) expressing cell (arrow) is located in the medio-lateral axis between the notochord and left somite. (C) Schematic diagram of a transverse section showing Tg(ins:GFP) expressing cells (green) within the endodermal sheet (yellow), as well as the notochord (red), somites (blue), neural tube (gray) and LPM (purple) at the 12-somite stage. Note that the endoderm consists of a monolayer of cells located at the ventral-most side of the embryo. (D-E, G-H) At the 15- (D, E) and 18- (G, H) somite stage, Tg(ins:GFP) expressing cells (arrows) lie bilateral to the notochord. Cells in the anterior neural tube (arrowhead) also express the ins:GFP transgene. (E, H) Mesenchymal cells (arrowheads) have migrated towards the midline. (F, I) At the 12- (F) and 15- (I) somite stage, gaps (asterisks) can be observed in the midline of the fusing endodermal sheet (red). Most Tg(ins:GFP) expressing cells (green; arrows) are located bilateral to the notochord.

Figure. 2. Absence of dorsal pancreatic bud derived β-cells in smoothened(smo) mutants

(A-B) Ventral confocal images of Tg(sox17:DsRed)^s903 (red) and β-catenin (green) expression, comparing wildtype (A) and smo mutants (B) at 18 hpf (mid-trunk region). Tg(sox17:DsRed)^s903 expressing endodermal cells show a similar arrangement and distribution around the midline (asterisks) in wildtypes (A) and smo mutants (B). (C-E, D-F) Dorsal views of Tg(ins:GFP) embryos stained for GFP (green), comparing wildtypes (C, E) and smo mutants (D, F) at 18 (C, D) and 24 (E, F) hpf. (C, D) Tg(ins:GFP) expressing cells are completely missing in 18 hpf smo mutants. Cells in the anterior neural tube (arrowhead) also express the ins:GFP transgene. (E) Tg(ins:GFP) expressing cells form a cluster in 24 hpf wildtype embryos (arrow). (F) Tg(ins:GFP) expressing cells are completely missing in most smo mutants though in approximately 5% of the mutants (n >100 mutants examined), one or two Tg(ins:GFP) expressing cells can be observed (F, arrow). (G-J) Confocal projections of wildtype (G, I) and smo mutant (H, J) endoderm at 34 (G, H) and 96 (I, J) hpf. (G, H) Tg(gutGFP) ^s854 embryos were stained for Islet1 (red) and Insulin (blue). (G) The endodermal cells (green) form a solid multi-cellular rod, and pancreatic endocrine cells (arrow and inset) aggregate into one cluster in wildtype embryos. (H) In smo mutants, the endodermal cells fail to condense into a midline rod and pancreatic endocrine cells are completely absent. (I, J) Tg(ins:GFP) embryos were stained for pan-Cadherin (red) and 2F11 (blue) to mark the endoderm and ductal structures, respectively. (I) Tg(ins:GFP) expressing β-cells (arrow) form a cluster inside the wildtype pancreas. (J) Although the morphology of the pancreas and liver is disrupted, Tg(ins:GFP) expressing β-cells (arrows) appear in smo mutants in ventral pancreatic bud derived tissues. L, liver; P, pancreas; IB, intestinal bulb.

Figure. 3. Shh compensates for the loss of axial mesoderm in the formation of pancreatic endocrine cells

(A-C) Ventral confocal images of Tg(ins:GFP) (green) and Tg(sox17:DsRed)^s903 (red) expression, comparing wildtypes (A), flh mutants (B) and shh overexpressing flh mutants (C) at 18 hpf. (C) At 18 hpf, Tg(ins:GFP) expressing cells were rescued in shh overexpressing flh mutants and scattered within the endodermal sheet (red). Most endodermal cells failed to converge following shh overexpression leading to the presence of gaps (asterisks) within the endodermal sheet. (D-I) Lateral (D-F) and dorsal (G-I) views of Tg(ins:GFP) embryos comparing wildtypes (D, G), flh mutants (E, H) and shh overexpressing flh mutants (F, I) at 24 hpf. (D, G) Wildtype embryos show one cluster of Tg(ins:GFP) expressing cells (arrow). (E, H) Tg(ins:GFP) expression is completely missing in flh mutants. (F, I) shh overexpressing flh mutants show rescue of Tg(ins:GFP) expression (arrow). (J-O) Confocal projections of pancreatic endocrine cells comparing wildtypes (J, M), flh mutants (K, N) and shh overexpressing flh mutants (L, O) at 48 (J-L) and 60 (M-O) hpf. Tg(ins:GFP) embryos were stained for GFP (green), Somatostatin (blue) and Glucagon (red). flh mutants show dramatically reduced pancreatic endocrine cells at 48 (K) and 60 (N) hpf, compared to wildtypes (J, M). Tg(ins:GFP) and Somatostatin expression were rescued at 48 hpf (L) and Glucagon expression was also detectable at 60 hpf (N) in shh overexpressing flh mutants.

Figure. 4. Cell non-autonomous requirement of Smoothened function in the induction of dorsal pancreatic endocrine cells

(A) Schematic diagram of the cell transplantation protocol. cas overexpressing wildtype or smo mutant Tg(ins:GFP) donor cells were transplanted into wildtype hosts. (B-G) Wildtype hosts with wildtype (B, D, F) and smo mutant (C, E, G) Tg(ins:GFP) donor cells at 30 hpf: Dorsal views (B, C) and confocal projections (D-G). All donor cells are labeled with rhodamine dextran (red). Note that only donor cells can express the ins:GFP transgene. (B) Wildtype donor cells contribute to the endoderm and express the ins:GFP transgene in wildtype hosts (arrow; 52%, n = 77/147). (C) smo mutant donor cells contribute to the endoderm and express the ins:GFP transgene (arrow) in wildtype hosts as frequently as wildtype cells do (51%, n = 25/49). (D-G) Hosts were stained for Islet1 which marks all pancreatic endocrine cells (blue) and motoneurons (blue, asterisk). (D, F) Wildtype donor cells differentiate into Islet1 positive endocrine cells (red cytoplasm, blue nucleus; arrowhead) and many of the donor cells co-express the ins:GFP transgene (triple positive cells appear white; arrows). (E, G) smo mutant donor cells also differentiate into Islet1 positive endocrine cells (red cytoplasm, blue nucleus; arrowhead) and many of the donor cells co-express the ins:GFP transgene (triple positive cells appear white; arrows). Single and double channel images are shown in Figure S3.

Figure. 5. Smoothened function is required in the endoderm for the induction of dorsal pancreatic β-cells

(A) Schematic diagram of the cell transplantation protocol. cas overexpressing wildtype or smo mutant Tg(ins:GFP) donor cells were transplanted into cas MO injected wildtype hosts. (B-E) Dorsal views of the cas MO injected hosts with wildtype (B, D) and smo mutant (C, E) donor cells at 24 hpf. Donor cells are labeled with rhodamine dextran (red). All the endodermal cells of the hosts are replaced by donor cells. (B, D) Wildtype-like Tg(ins:GFP) expression in the endoderm derived from wildtype donor cells (95%, n = 74/78). (C, E) Tg(ins:GFP) expressing cells are completely absent (C, 92%, n = 23/25) or amount to one or two cells (E, arrow; 8%, n = 2/25) when smo mutant donor cells form the endoderm in cas MO injected wildtype hosts.

Figure. 6. Cell-cell interactions between endodermal cells are required for dorsal pancreatic β-cell induction

(A) Schematic diagram of the cell transplantation protocol. cas overexpressing wildtype Tg(ins:GFP) donor cells were transplanted into wildtype or smo mutant Tg(ins:GFP) hosts. (B, C) Dorsal views of wildtype (B) and smo mutant (C) hosts with the endodermal cells derived from wildtype donor cells labeled with rhodamine dextran (red) at 18 hpf. (B) Wildtype hosts show wildtype-like Tg(ins:GFP) expression after transplantation. (C) Tg(ins:GFP) expression (arrows) is restored in smo mutant hosts when wildtype cells contribute to the endoderm (44.5%, n = 48/108). (D-E) Transverse confocal images of smo mutant hosts stained for GFP (green) and β-catenin (white) at 18 hpf. Wildtype donor cells are labeled with rhodamine dextran (red). At this stage, parts of the endodermal tissue have become multilayered as cells are converging actively to the midline. (D) Most of the Tg(ins:GFP) expressing cells in smo mutant hosts are derived from wildtype donor cells (arrows). (E) However, in several embryos (n = 6/48), lateral endoderm derived from wildtype donor cells correlates with Tg(ins:GFP) expression in medially located smo mutant host endoderm (arrow). (F) Schematic diagram of the cell transplantation protocol. cas overexpressing wildtype donor cells were transplanted into wildtype or smo mutant Tg(ins:GFP) hosts. (G, H) Lateral views of live smo mutant hosts for Tg(ins:GFP) (green) and rhodamine dextran (red) at 30 hpf. (G) When wildtype donor cells were not incorporated into the endoderm, smo mutants showed a complete absence of Tg(ins:GFP) expression in most cases (48/50) or at most two Tg(ins:GFP) expressing cells (2/50). (H) When wildtype donor cells were incorporated into the endoderm, several experimental smo mutants (n = 7/26) showed four to seven rescued Tg(ins:GFP) expressing cells (arrows; five rescued cells in this figure). [In these experimental smo mutants, pancreatic β-cells likely comprise both wildtype and smo mutant cells].

Figure. 7. Lineage tracing experiments reveal that endodermal precursors contribute to distinct endodermal tissues based on their medio-lateral position

(A-B) Dorsal views of live Tg(sox17:GFP)^s870 embryos at the 6-8 somite stage. (A) Bright field image showing the region between somites 1 and 4. (B) Tg(sox17:GFP) expression shows that endodermal cells are spread in a monolayer and have not yet fused at the midline. (C) Schematic diagram of Tg(sox17:GFP) expressing cells with three specific positions in the medio-lateral axis: the most medial cells (medial; dark green), cells immediately adjacent to the medial cells (lateral 1, green) and cells one cell apart from the medial cells (lateral 2, light green). Brackets indicate the area where uncaging was performed. (D-F) Ventral confocal images of control embryos at the 10-somite stage, confirming the accuracy of endodermal targeting by the laser. Tg(sox17:GFP)^s870 embryos were stained for GFP (gray) and uncaged-fluorescein (red). Two endodermal cells in the medial (D), lateral 1 (E) and lateral 2 (F) positions were labeled in the region between somites 2 and 3. (G-L) Confocal projections of Tg(sox17:GFP)^s870 embryos at 50 hpf showing the progeny of the medial (G, J), lateral 1 (H, K) and lateral 2 (I, L) cells. Tg(sox17:GFP)^s870 embryos were stained for GFP (green; [G-I]), Insulin (blue; [G-L]) and uncaged-fluorescein (red; [G-L]). (G, J), (H, K), and (I, L) are the same embryos. (G, J) Medial cells gave rise exclusively to pancreatic endocrine cells (arrows). (H, K) Lateral 1 cells gave rise to exocrine cells (double arrows), a small number of endocrine cells (arrows) as well as intestinal tissue (arrowhead) adjacent to the pancreas. (I, L) Lateral 2 cells gave rise to liver cells (asterisks), intestine (arrowhead) and exocrine cells (double arrow). Lateral 2 cells showed very little, if any, contribution to pancreatic endocrine cells (arrow).