TGF-beta isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas

Abstract

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.

Figure 1

Phenotype of DNTβRII transgenic mouse embryonic pancreas: A) Embryonic pancreata at various gestational time points were analyzed by immunostaining for markers of pancreatic differentiation. Amylase (red), insulin (blue) and glucagon (green) in wild- type CD1 (a,d,g), transgenic DNTβRII (b,e,h) and transgene induced DNTβRII+Zn (c,f,i). At E14.5 (a–c), transgenic pancreas showed increased expansion of the “cord region” (defined as the more central region of the pancreas where typically the endocrine cells are found). At E16.5 (d–f), the transgenic phenotype diverged most markedly from controls. The transgenic pancreas had prominent expansion of the cord region with an increase in endocrine cells, most notably in Zn-treated embryos. At E18.5 (g–i), continued cord region expansion was seen. B) Transgene-induced DNTβRII+Zn pancreata at E16.5 (b) and E18.5 (d) showed increased and abnormally dilated blood vessels, as shown by CD31 (red) staining. C) Quantitative analysis of E16.5 pancreas showed a significant increase in the amount of insulin-positive area, and in the amount of CD31 positive endothelium in transgenic pancreas compared to wild-type. *P value <0.05 and scale bar 60μm

Figure 2

Transgenic phenotype: expansion of the cord region with periductal accumulation of endocrine cells. Immunohistochemical analysis of embryonic pancreas in A) with duct marker DBA (green), insulin (blue), PDX-1 (red) and in B) insulin (red) amylase (blue) and DBA (green) of wild-type CD1 (a,c) or transgene-induced DNTβRII+Zn (b,d) at E16.5 (a,b) and E18.5 (c,d) showed prominent expansion of cord region with increased accumulation of endocrine cells in periductal region. Scale bar 60μm

Figure 3

Increased proliferation of endocrine cells in the embryonic pancreas of transgenic mice: proliferating endocrine cells in the embryonic pancreas were examined through BrdU incorporation, and then staining for BrdU (red/green) and (A) insulin (green) or (C) glucagon (red). At E16.5 and E18.5, the control wild-type CD1 endocrine cells showed low proliferation with few BrdU and Insulin/Glucagon-positive cells (a,c). However, at E16.5 the proliferative status of the insulin or glucagon cells in transgene-induced pancreas was significantly increased (arrowhead b). Quantitative analysis of proliferating endocrine cells in transgene-induced pancreas showed a significant increase in proliferation of insulin-positive (B) and glucagon-positive cells (D) at E16.5. However, the level of proliferation became similar to wild-type by E18.5. *P value <0.05 and scale bar 60μm

Figure 4

Increased endocrine precursors in the embryonic pancreas of transgenic mice: immunohistochemical analysis showed increased expression of endocrine precursor (A) neurogenin-3 positive (red) and (C) neuroD1-positive (red) cells in the transgene-induced pancreas at E14.5 (a,b) and E16.5 (c,d). At E16.5, there were abundant neurogenin-3 positive cells in and around the DBA-positive ducts (green) of transgene-induced pancreas. Quantitative RT-PCR analysis of transgene-induced pancreas showed significant upregulation in (B) neurogenin-3 and (D) neuroD1 mRNA expression. *P value <0.05 and scale bar 60μm

Figure 5

Ontogeny of endogenous TGF-β isoform receptors: expression of TGF-βRII (A,D,G,J,M), and expression of the two potential type I receptor binding partners of TGF-βRII, i.e. TGF-βRI/ALK5 (B,E,H,K,N) and ALK1 (C,F,I,L,O) was examined in wild-type embryonic pancreases at various time points of gestation. The expression of TGF-βRII in epithelium (arrows) was stronger and more localized compared to mesenchyme (arrowhead) at E11.5 and E12.5. At E14.5 and E16.5, TGF-βRII appeared to be mainly expressed in the pro-endocrine cord region (arrowheads) and in the ducts (arrows), and then at E18.5 predominantly in ducts (arrows) and blood vessels (arrowheads). The expression pattern of TGF-βRI/ALK5 paralleled TGF-βRII. ALK1 expression showed a different pattern, with low levels in the epithelium (arrow) at E11.5 (C), low levels in the epithelium (arrow) and very low levels in mesenchyme (arrowhead) at both E12.5 (F) and E14.5 (G). At E16.5 and E18.5, ALK1 was mainly expressed in blood vessels (L,O,arrowheads). The overlapping expression of RII and ALK1 in vasculature at this stage may suggest a role for the TGF-β RII/ALK1 dimer in regulating blood vessel development. Scale bar 60 μm.

Figure 6

Ontogeny of the transgene in embryonic DNTβRII pancreas by immunohistochemistry: the expression was compared between zinc treated and untreated embryonic pancreas. At E11.5 the transgene was absent unless induced by zinc. Addition of zinc at E11.5 augmented expression in the epithelium (arrows) and weakly in the mesenchyme (arrowheads). At E12.5, transgene expression began to appear in the epithelium even without zinc (arrows). However, with zinc, strong expression was seen not only in the epithelium at E12.5 (arrows), but also very clearly in the mesenchyme (arrowheads). At E14.5 only the cord or pro-endocrine region (arrowheads) expressed the transgene, either with or without zinc, but expression appeared much stronger with zinc-treatment, with some additional weak expression in acinar cells (arrows). At E16.5, however, the transgene was expressed weakly in the cord region and strongly in the acinar cells, with or without zinc treatment. At E18.5 essentially all of the transgene expression was in the acini, and the level of expression did not appear different between zinc-treated or untreated groups. Scale bar 60μm.

Figure 7

Quantitative analysis: quantification of mRNA for endogenous TGF-β receptors (A–C), transgenic receptor (D) and TGF-β ligands (E–G) were performed at various gestational time points by semi-quantitative RT-PCR. The endogenous TGF-β RII mRNA level gradually increased over gestation (A). TGF-β RI/ALK5 mRNA showed its highest level of expression at E16.5, and then a sharp decline at E18.5 (B).The drop in ALK5 mRNA levels at E18.5 suggests that other ALKs, particularly ALK1, may instead dimerize with TGF-β RII to mediate actions such as blood vessel development. The mRNA level of ALK1 showed a sudden rise late in gestation, consistent with a possible role in blood vessel development and growth (C). Quantification of the transgenic receptor showed significant upregulation of expression in zinc-treated pancreas at E11.5, E12.5 and E14.5 compared to untreated embryos (D and inset). However, baseline expression of the transgene at E16.5 and E18.5 becomes very high even in the absence of zinc, probably due to zinc-independent expression of the transgene in acinar tissue. Addition of zinc at these later ages did not affect the expression level of the transgene. The relative level of TGF-β isoform ligand mRNA were similar between wild-type and transgenic mice through E16.5. However, the level of expression then differed selectively at E18.5. The level of TGF-β1 and TGF-β3 mRNA significantly increased in transgenic pancreas treated with zinc, whereas TGF-β2 mRNA was decreased. *P value <0.05

Figure 8

In vitro mix and match experiment: four different mix-and-match in vitro recombinations between transgenic (DNTβRII) and wild-type (CD1) E11.5 pancreatic epithelium (e) and mesenchyme (m) were made. The recombination of wild-type mesenchyme and wild- type epithelium was considered as a control (c,g,k). 100μM ZnCl₂ was supplemented in the media of all the cultures to enhance transgene expression. Immunohistochemical staining (A) and quantitative analysis (B) showed that control cultures had the lowest amount of insulin-positive cells, and the greatest amount of amylase staining. When transgenic epithelium was recombined with either wild-type or transgenic mesenchyme, there was an augmentation of insulin-positive cells. However, the greatest augmentation of insulin-positive area was seen with recombination of transgenic mesenchyme with wild-type epithelium (a,e,i and B). PDX-1 staining suggested that most or all of the insulin-positive endocrine cells were mature β-cells (i,j,k,l). Scale bar (a-d) 60μm and (e-l and a–d inset) 120μm

Figure 9

In vitro neutralizing antibody experiment: in order to inhibit TGF-β isoform signaling at the ligand level, TGF-β pan-neutralizing antibodies were used. Treatment with 80 μg/ml neutralizing antibody (e-h) showed a significant increase in insulin-positive cells (P value < 0.05) (B). PDX-1 (red) staining of these insulin-positive cells suggested that the cells were mature β-cells (d and h). Rabbit non-immune serum control at a similar concentration had no effect (a–d). *P value <0.05 and scale bar (b–d and f–h) 120μm