Louise Rosenberg Christ:
Downstream events of neurogenin 3 in pancreatic endocrine differentiation

Date: 15-10-2009    Supervisor: Olaf Nielsen

Diabetes affects more than 170 million people world wide and a prognosis suggests that the prevalence will increase over the next 30 year. In Denmark, the prevalence doubled from 1996 to 2007, where 240.000 people were affected. Diabetes is the manifestation of abnormalities regarding glucose homeostasis which may have several causes. The most prevalent types are a result of either an auto-immunological destruction of the insulin producing ß-cells (type 1) or a combination of insulin resistance of the tissues and impaired ß-cell function (type 2) in both cases leading to hyperglycaemia.

Research in the developmental biology of the pancreas and the mechanism behind differentiation of the pancreatic progenitor cells into mature ß-cells provide knowledge of the molecular events and signals of the process that may be applied to stem cell research. The aim is to be able to induce directed differentiation of stem cells into insulin producing cells in vitro. The hope is that successful production of ß-cells from stem cells combined with a good protocol for the transplantation of these cells into human beings would offer an opportunity for diabetes patients to obtain a normal glucose homeostasis and be relieved of the eventually debilitating long-term effects of diabetes and the trouble with insulin injections several times a day.

In pancreatic development the expression of the transcription factor Neurog3 is a key event, as it marks the transition from the stage in development where the pancreas progenitor cells proliferate to form the cell pool of the future organ to the stage in development where the endocrine precursor cells and the individual endocrine cell types are specified. Neurog3 specifically induces endocrine differentiation that eventually leads to the generation of the mature hormone producing cells of the islets of Langerhans, and among these are the insulin producing ß-cells. The function of Neurog3 as a transcription factor and an endocrine inducer is well established, but the immediate events regarding Neurog3 function are poorly understood.

The scope of this thesis was to investigate these immediate functions of Neurog3 in endocrine development. We applied a deletion analysis to Neurog3 and investigated the resulting truncated Neurog3 proteins with regard to their cellular localisation, DNA-binding capacity, activity and in vivo function applying the technique of in ovo electroporation. Furthermore, the role of the binding partner E12 was investigated with regard to Neurog3 function. Mutations in the human Neurog3 were investigated with regard to their DNA binding capacity and the effect of E12 on their function in vivo. Finally, the transcription factor Myt1 was investigated as a putative direct target of Neurog3.

The truncated Neurog3 proteins revealed that an intact bHLH domain and the region of amino acids flanking the bHLH on the N-terminal side may be involved in nuclear localisation as the proteins lacking the N-terminal domain or with a truncated bHLH domain were present to a larger extent in the cytoplasm.

The N-terminal domain was not required for Neurog3 to induce activation of the Neurod1 promoter to the same level as the wild type protein, and the protein lacking the N-terminal domain had a very good DNA binding capacity. Truncations in the C-terminal domain reduced the transcriptional activity of Neurog3 on the Neurod1 promoter. in vivo the truncated proteins showed that even low levels of Neurog3 activity induced differentiation of glucagon expressing cells, migration and clustering. Surprisingly, the lack of the N-terminal domain enabled Neurog3 to induce ectopic insulin expression in vivo in the endoderm of the early chicken gut tube endoderm. This suggests that Neurog3 may be involved in lineage specification in addition to the function as an inducer of general endocrine fate. However, further investigations are needed to support this hypothesis.

The Neurog3 binding partner E12 did not enhance Neurog3 transcriptional activity on the Neurod1 promoter and did not increase Neurog3 induced differentiation in vivo in early chicken gut tube endoderm. This was surprising, and it is unknown if this observation is a direct effect of E12 or if it is a result of Neurog3 independent effects of E12 in the endodermal cells. However, the observation that Neurog3 induced migration but not differentiation when co-electroporated with E12 supports previous findings that the processes of Neurog3 induced differentiation and migration are uncoupled. The same effect of E12 was observed when it was co-electroporated with one of the human Neurog3 mutants or the double mutant. These mutations were found to have reduced DNA binding.

Finally, Myt1 was shown to be a direct target of Neurog3, as it induced transcription from the Myt1 promoter. The truncated Neurog3 proteins showed a slightly different effect on the Myt1 promoter as the N-terminal domain was required for Neurog3 to induce a full transcriptional response, and truncations from the C-terminal end also seemed to have a greater impact on the response of the Myt1 promoter than of the Neurod1 promoter. The Myt1 protein variants were located to different cellular compartments although they were able to interact. The impact of this observation is unknown.

In conclusion, this thesis supports that Neurog3 induced endocrine differentiation is a diverse process that involves transcription of several target genes perhaps in a differential way, and that Neurog3 mediated differentiation and migration are uncoupled processes. It is also shown that the transcription factor Myt1 is a direct target of Neurog3. An intact bHLH domain and the amino acids flanking the bHLH domain on the N-terminal side are involved in nuclear localisation. It was unexpected that E12 did not enhance Neurog3 mediated differentiation or in vitro activity. The ability of the Neurog3 protein lacking the N-terminal domain to bind DNA more efficiently and induce ectopic insulin expression suggests that further investigations of Neurog3 may reveal nuances of regulation and function that may be of relevance for the application of developmental biology to directed differentiation of stem cells into insulin expressing cells.