Preservation of proliferating pancreatic progenitor cells by Delta-Notch signaling in the embryonic chicken pancreas

Abstract

Background: Genetic studies have shown that formation of pancreatic endocrine cells in mice is dependent on the cell autonomous action of the bHLH transcription factor Neurogenin3 and that the extent and timing of endocrine differentiation is controlled by Notch signaling. To further understand the mechanism by which Notch exerts this function, we have investigated pancreatic endocrine development in chicken embryos.

Results: In situ hybridization showed that expression of Notch signaling components and pro-endocrine bHLH factors is conserved to a large degree between chicken and mouse. Cell autonomous inhibition of Notch signal reception results in significantly increased endocrine differentiation demonstrating that these early progenitors are prevented from differentiating by ongoing Notch signaling. Conversely, activated Notch1 induces Hes5-1 expression and prevents endocrine development. Notably, activated Notch also prevents Ngn3-mediated induction of a number of downstream targets including NeuroD, Hes6-1, and MyT1 suggesting that Notch may act to inhibit both Ngn3 gene expression and protein function. Activated Notch1 could also block endocrine development and gene expression induced by NeuroD. Nevertheless, Ngn3- and NeuroD-induced delamination of endodermal cells was insensitive to activated Notch under these conditions. Finally, we show that Myt1 can partially overcome the repressive effect of activated Notch on endocrine gene expression.

Conclusion: We conclude that pancreatic endocrine development in the chicken relies on a conserved bHLH cascade under inhibitory control of Notch signaling. This lays the ground for further studies that take advantage of the ease at which chicken embryos can be manipulated. Our results also demonstrate that Notch can repress Ngn3 and NeuroD protein function and stimulate progenitor proliferation. To determine whether Notch in fact does act in Ngn3-expressing cells in vivo will require further studies relying on conditional mutagenesis. Lastly, our results demonstrate that expression of differentiation markers can be uncoupled from the process of delamination of differentiating cells from the epithelium.

Figure 1

Expression of bHLH genes in the chicken pancreas. (A-L) In situ hybridization on serial sections from HH st. 22 (A-D) and HH st. 31 (E-L) chick pancreas showing the expression patterns of Ngn3 (A, E, I), NeuroD (B, F; J), Hes1 (C, G, K), Hes6-1 (D, H, L). Dorsal (dp) and ventral pancreas (vp) and the bile duct (bd) are indicated in A-D. At HH st. 22, Ngn3 is expressed in a few cells scattered in the epithelium (A) whereas NeuroD expression is confined to endocrine cell clusters located at the most distal part of the dorsal pancreas (red dashed lines in B). Hes1 is expressed broadly in the dp and vp epithelium as well as in the bd epithelium (C). Hes6-1 is expressed in a pattern resembling Ngn3 (compare A to D). At HH st. 31, Ngn3 is expressed in scattered cells within the epithelium (E) where they co-localize with Nkx6.1 (I). Ngn3 is not expressed in glucagon positive cells adjacent to the Nkx6.1 positive epithelium (I). In contrast, NeuroD expressing cells are confined to endocrine cells and are excluded from the Nkx6.1 positive epithelium (F, J, area corresponding to boxed areas in E, H). Hairy-1 is still expressed broadly in the epithelium (G) and some of the forming amylase positive acini are also positive for Hes1 (black arrowhead in G, K) but amylase positive acini without Hes1 expression can also be found (red arrowhead in G, K). Hes6-1 is expressed in a pattern closely resembling Ngn3 (H) as it co-localizes with Nkx6.1 in the epithelium and is excluded from glucagon positive endocrine cells (L). * denotes Nkx6.1 positive β-cells

Figure 2

Inhibition of Notch signaling induce excess endocrinedifferentiation. (A-D). Confocal sections of chicken embryonic pancreata 72 hours after electroporation with control vector (A), Delta1 (B), Serrate1 (C), and Serrate1-d1 (D) subjected to immunohistochemical stainings against GFP (green) and glucagon plus insulin (red). Cells expressing GFP from the vector readily co-localize with endocrine markers. However, most electroporated cells are not endocrine (A). In contrast, electroporation with either Delta1, Serrate1, or Serrate-d1 results in increased endocrine differentiation of electroporated cells as compared to the control (compare B, C, D to A). E. Quantitative analysis of endocrine differentiation 72 hours after electroporation with the indicated expression plasmids at HH st. 10–12 or HH st. 13–15. (F, G).

Figure 3

Cell autonomous effects of ligand expression is reversed by Notch1^ICD. 3D projections of confocal optical sections of chicken embryos subjected to whole mount immunohistochemical stainings for GFP (green) and glucagon plus insulin (red) (A-C) or GFP (green), glucagon (red) and amylase (blue) (D, E) 72 hours after electroporation with the indicated constructs. The most extreme phenotype resulting from ectopic Delta1 expression is an almost complete conversion of the pancreas into a tubular structure composed almost entirely of endocrine cells (compare B to A). When Delta1 is co-expressed with Notch1^ICD most transfected cells do not express the hormones (C). D and E show the entire dorsal pancreas at the same magnification (scale bar represents 100 μm). Note the even distribution of electroporated cells in the control (D) and the clustering of electroporated cells in the endocrine population in the Delta1 electroporated pancreas.

Figure 4

Notch1^ICD directly regulates proliferation and blocks endocrine differentiation. (A-F). Confocal sections of chicken embryonic pancreata 52 hours after electroporation with empty vector (A-C) or Notch1^ICD (D-F) subjected to immunohistochemical stainings against glucagon (red), and insulin (blue) (A and D), Nkx6.1 (red) (B and E), and MPM2 (red) (C and F). GFP was detected by its endogenous fluorescence (A-F). Inserts in A-F show higher magnifications of the boxed areas in the cognate panels. (G) Bar graph illustrating the quantitative analysis of endocrine differentiation 52 hours after electroporation with control vector or Notch1^ICD plasmid at HH st. 13–15. Note the absence of insulin and glucagon and the presense of MPM2 in GFP-positive cells in the Notch1^ICD electroporated embryos.

Figure 5

Notch1^ICD inhibits the activity of Ngn3. (A-H). Confocal sections of chicken embryonic pancreata 52 hours after electroporation with Ngn3 (A-D) or Ngn3 and Notch1^ICD (E-H) subjected to immunohistochemical stainings against Nkx6.1 (red) (A, E), Pax6 (red) (B, F), glucagon (red) and insulin (blue) (C, G), or MPM2 (red) (D, H). GFP was detected by its endogenous fluorescence (A-H). Inserts show higher magnification of boxed areas in the cognate panels. (I) Bar graph illustrating the quantitative analysis of MPM2/GFP double-positive cells as a percentage of total GFP-positive cells 52 hours after electroporation with the indicated expression plasmids at HH st. 13–15. Note the absence of insulin and glucagon, the presense of MPM2, and persistent Nkx6.1 expression in GFP-positive cells in the Ngn3 plus Notch1^ICD electroporated, compared to Ngn3 electroporated embryos.

Figure 6

bHLH genes are inversely regulated by Ngn3 and Notch1^ICD. (A-T). In situ hybridization on serial sections from embryonic pancreata 52 hours after electroporation with control vector (A-E), Ngn3 (F-J), Notch1^ICD (K-O), or Ngn3 and Notch1^ICD (P-T). The expression patterns of Hes1 (B, G, L, Q)), Hes5-1 (C, H, M, R), Hes6-1 (D, I, N, S), and NeuroD (E, J, O, T) were examined. A, F, K, P show GFP expressing electroporated cells (arrowheads) and the corresponding area is indicated with arrowheads in the rest of the panels. Note that Notch1^ICD apparently leads to decreased Hes1 expression (red arrowhead in L and arrowheads in Q) and induces Hes5-1 expression (M, R) and repress Hes6-1 (N, S) and NeuroD (O, T) expression regardless of the presence of Ngn3.

Figure 7

Differentiation can be uncoupled from delamination. (A-H). Confocal sections of chicken embryonic pancreata 52 hours after electroporation with Ngn3 (A-B), Ngn3 and Notch1^ICD (C-D), NeuroD (E-F), or NeuroD and Notch1^ICD (G-H) subjected to immunohistochemical stainings against Pax6 (red) and laminin (blue) (A, C, E, G) and glucagon (red) and somatostatin (blue) (B, D, F, H). GFP was detected by its endogenous fluorescence (A-H). Inserts show higher magnification of boxed areas in the cognate panels. Note that Notch1^ICD inhibits Ngn3 and NeuroD induced expression of endocrine markers without affecting delamination of the cells from the endoderm.

Figure 8

Myt1 can partially rescue the inhibitory effect by Notch1^ICD on Ngn3 function. (A-K). Immunohistochemical stainings (A, E, I-K) and in situ hybridizations (B-D, F-H) on sections in the duodenum 52 hours after electroporation with Ngn3 (A-D, I), Ngn3 and Notch1^ICD (E-H, J) or Ngn3 with Notch1^ICD and MyT1 (K) showing expression of Ngn3 (A, E), NeuroD (B, F), Myt1 (C, G), Hes6-1 (D, H), and βIII-tubulin. Note that Ngn3 fails to induce expression of endocrine markers in the presence of Notch1ICD. βIII-tubulin expression is partially restored when Notch1^ICD and MyT1 are co-expressed with Ngn3 (compare K to J and I).