Dynamic signaling network for the specification of embryonic pancreas and liver progenitors

Abstract

Studies of the formation of pancreas and liver progenitors have focused on individual inductive signals and cellular responses. Here, we investigated how bone morphogenetic protein, transforming growth factor-beta (TGFbeta), and fibroblast growth factor signaling pathways converge on the earliest genes that elicit pancreas and liver induction in mouse embryos. The inductive network was found to be dynamic; it changed within hours. Different signals functioned in parallel to induce different early genes, and two permutations of signals induced liver progenitor domains, which revealed flexibility in cell programming. Also, the specification of pancreas and liver progenitors was restricted by the TGFbeta pathway. These findings may enhance progenitor cell specification from stem cells for biomedical purposes and can help explain incomplete programming in stem cell differentiation protocols.

Fig. 1

Dynamic signal transduction molecule activation in the foregut endoderm revealed by whole mount immunofluorescence (IF). A-C. Fate map of foregut endoderm at different stages (23). D-O. Images looking into foregut. Arrows indicate regions with positive signal; brackets indicate regions lacking signal. m.e., medial endoderm; l.e., lateral endoderm; d.e., dorsal endoderm; hf, head fold; ht, heart; fg, foregut; liv., liver; v.p., ventral pancreas.

Fig. 2

Genetic and pharmacologic perturbation of signaling pathways. A, B. Whole mount IF of designated genotypes. Red arrow indicates presence of SMAD4 in ventral foregut endoderm; dashed arrow indicates lack of SMAD4. C-E. Images of 5S half embryo prior to (C) or after (D,E) culture. F-J. qRT-PCR on endoderm from half-embryos +/- inhibitors or from FoxA3^cre;SMAD4^CA/Δ (Smad4 null) embryos at 11S compared to that from 11S wild type embryos. N=2 to 8, each sample analyzed in triplicate; an asterisk indicate p≤0.05 determined by t-test; two asterisks, p≤0.005. “nd”, not determined. Error bars indicate standard deviation (SD) calculated with ΔCt method.

Fig. 3

Defective ventral pancreatic development in embryos deficient for Smad4 in the endoderm. A-F. Transverse IF sections for Pdx1 or AFP (red), and DAPI. G, H. Whole mount IF for Pdx1/DAPI.

Fig. 4

TGFβ signaling restrains the specification of ventral pancreatic progenitors. A,C,E. Whole mount X-gal staining (blue) for pancreatic progenitors. B, D, F. Sagittal sections, counterstained for Ki67 (brown). G. Dynamic network inducing the earliest genes for liver and pancreas specification. Arrows, positive signaling; barred lines, negative. The thicker lines for BMP and thinner lines for TGFβ at 5-6S indicate BMP as the dominant Smad4-dependent pathway for alb1, Hnf6, and Pdx1.