Role of Neurogenin3 in migration of pancreatic endocrine cells during development

The pancreas is one of the major organs of the digestive tract. Its endocrine function, mainly regulating glucose blood level, is achieved by endocrine cells. These cells are organized in clusters, the so-called islets of Langerhans. During development, these cells are specified within the pancreatic epithelium, and will subsequently delaminate and migrate out of the epithelium in order to form the islets. Neurogenin3 (Ngn3) is a bHLH transcription factor that is responsible for the differentiation of all endocrine cell types, but whether or not it has a role in their migration is still an open question. By using the chicken embryo model organism, we found that the differentiation and migration programs are two different processes that are induced by Ngn3 and that can be uncoupled. Therefore, we tried to unravel the mechanisms by which Ngn3 can induce endocrine cell migration. We found that, in both chick and mouse models, over-expression of Ngn3 induces a loss of apico-basal polarity, a breakdown of basal lamina, and more importantly, a loss of the epithelial marker E-cadherin (E-cad). It seems that this is not a direct effect mediated by direct binding to E-boxes in the E-cad promoter. Therefore, we are currently trying to find targets of Ngn3 that could mediate repression of E-cad, focusing on the zinc-finger transcription factors Snail1 and Snail2. Moreover, we also used a pancreatic explant culture method, developed in the laboratory of Pr. Jonathan Slack, which allows us to do time-lapse imaging and describe precisely the migration of endocrine cells in a system similar to in vivo conditions. Taking advantage of our Pdx1::Ngn3ERTM transgenic line, we followed Ngn3 misexpressing cells to understand in a more physiological manner how they migrate in the developing pancreas. We found that endocrine cell migrate at an average speed of 11.35 µm/h and migrate through an average length of 132.30 µm in 12 hours. We also found that endocrine cells migrate in the three compartments we defined: in the mesenchyme, inside the epithelium and at the edge of the epithelium. Their average velocity significantly changes depending on the compartment cells are migrating in. We found that the fastest cells are the ones migrating at the edge of the epithelium. RNA profiling comparing Ngn3 knockouts rescued or not by the Pdx1::Ngn3ERTM transgene was also used to identify the genes that function in endocrine cell migration downstream of Ngn3. We identified new Ngn3 target genes, some of which are implicated in migration such as the Doublecortin family.

Neurogenin3 Initiates Stepwise Delamination of Differentiating Endocrine Cells During Pancreas Development

Mathieu Gouzi, Anne Grapin-Botton, Keiichi Katsumoto, Yung Hae Kim

During development, pancreatic endocrine cells are specified within the pancreatic epithelium. They subsequently delaminate out of the epithelium and cluster in the mesenchyme to form the islets of Langerhans. Neurogenin3 (Ngn3) is a transcription factor required for the differentiation of all endocrine cells and we investigated its role in their delamination. We observed in the mouse pancreas that most Ngn3-positive cells have lost contact with the lumen of the epithelium, showing that the delamination from the progenitor layer is initiated in endocrine progenitors. Subsequently, in both mouse and chick newly born endocrine cells at the periphery of the epithelium strongly decrease E-cadherin, break-down the basal lamina and cluster into islets of Langerhans. Repression of E-cadherin is sufficient to promote delamination from the epithelium. We further demonstrate that Ngn3 indirectly controls Snail2 protein expression post-transcriptionally to repress E-cadherin. In the chick embryo, Ngn3 independently controls epithelium delamination and differentiation programs. Developmental Dynamics 240:589-604, 2011. (C) 2011 Wiley-Liss, Inc.

2011

Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes

Anne Grapin-Botton

The mechanisms by which the epithelium of the digestive tract and its associated glands are specified are largely unknown. One clue is that several transcription factors are expressed in specific regions of the endoderm prior to and during organogenesis. Pdx-1, far example, is expressed in the duodenum and pancreas and Pdx-1 inactivation results in an arrest of pancreatic development after buds formation. Similarly, ngn3 is transiently expressed in the developing pancreas and a knockout results in the absence of endocrine cells. This paper focuses on the question of whether these and other transcription factors, known to be necessary for pancreatic development, are also sufficient to drive a program of pancreatic organogenesis. Using in ovo electroporation of chick embryos, we show that ectopic expression of Pdx-1 or ngn3 causes cells to bud out of the epithelium like pancreatic progenitors. The Pdx-1-expressing cells extinguish markers for other nonpancreatic regions of the endoderm and initiate, but do not complete, pancreatic cytodifferentiation. Ectopic expression of ngn3 is sufficient to turn endodermal cells of any region into endocrine cells that form islets expressing glucagon and somatostatin in the mesenchyme. The results suggest that simple gene combinations could be used in stem cells to achieve specific endodermal tissue differentiation.

2001

A conserved transcriptional enhancer that specifies Tyrp1 expression to melanocytes

Friedrich Beermann

Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. Previous studies have suggested that pigmentation genes are controlled by different regulatory networks in melanocytes and RPE. The promoter of the tyrosinase-related family gene Tyrp1 has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. We thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. We further focused on a sequence located at -15 kb, which we identified as a melanocyte-specific enhancer as shown by cell culture and transgenic mice experiments. In addition, we show that the transcription factor Sox10 can activate this conserved enhancer. The presence of a distal Tyrp1 regulatory element, which specifies melanocyte-specific expression, supports the idea that separate regulatory sequences can mediate differential gene expression in melanocytes and RPE.

2006