The transcriptional activity of Neurog3 affects migration and differentiation of ectopic endocrine cells in chicken endoderm.

Neurog3 is expressed transiently in pancreatic endocrine progenitors where it is responsible for activating a transcription factor cascade which eventually defines the mature endocrine cells. However, the mechanism by which Neurog3 regulates different aspects of the endocrine differentiation program is less clear. In this report we used in ovo electroporation to investigate how manipulation of Neurog3 protein activity affected migration, differentiation and fate determination. We found that changes in the onset of Neurog3 expression only had minor effect on differentiation. However increasing the transcriptional activity of Neurog3 by fusing it to VP16 or co-electroporating with Ep300 caused the electroporated cells to migrate rather than differentiate. In contrast, reducing the transcriptional activity of Neurog3 by deleting parts of the activation domain, by fusing Neurog3 to the engrailed repressor domain, or co-electroporating with Hdac1 greatly increased the proportion of glucagon expressing cells.

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

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.

Analysis of pancreatic endocrine development in GDF11-deficient mice.

Here, we examine the role of GDF11 in pancreatic development. Using in situ hybridization and reverse transcriptase-polymerase chain reaction analyses, we show that Gdf11 transcripts are expressed in embryonic pancreas epithelium before the secondary transition but decrease rapidly afterward. To determine the function of GDF11 during pancreas development, we analyzed Gdf11(-/-) mouse embryos. In such embryos, pancreas size is twofold reduced at embryonic day (E) 18 compared with wild-type littermates. Quantification of the different tissue compartments shows a specific hypoplasia of the exocrine compartment, while the endocrine and ductal compartments are unaffected. Notably, NGN3(+) endocrine precursor cells are increased fourfold at E18, although the amount of endocrine cells in the pancreas of these animals is unchanged compared with wild-type littermates. Similarly, the maturation of endocrine cells as well as the ratio between alpha- and beta-cells appears normal.