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

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.

Role of Neurogenin3 in migration of pancreatic endocrine cells during development

Mathieu Gouzi

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.

EPFL2010

Pancreas-specific deletion of beta-catenin reveals Wnt-dependent and Wnt-independent functions during development

Anne Grapin-Botton

Mutations and deregulation of adenomatous polyposis coli (APC) and beta-catenin are implicated in specific cancers of the pancreas, but the role of Wnt pathway in normal pancreas development and homeostasis is unknown [1-4]. This article reports a comprehensive investigation of the activity and the role of the Wnt pathway in pancreas organogenesis. We have used two reporter lines to monitor canonical Wnt pathway activity during development and after birth and demonstrate activity in endocrine cells and in the mesenchyme. We have specifically deleted the beta-catenin gene in the epithelium of the pancreas and duodenum by using Pdx1-Cre mice. In agreement with Wnt pathway activity in pancreatic endocrine cells, we find a reduction in endocrine islet numbers. Our study reveals that beta-catenin deletion also affects cells in which Wnt pathway activity is not detected. Indeed, beta-catenin mutant cells have a competitive disadvantage during development that also affects the exocrine compartment. Moreover, the conditional knockout (KO) mice develop acute edematous pancreatitis perinatally due to the disruption of the epithelial structure of acini. These effects are likely to be due to the function of beta-catenin at the membrane. Mice later recover from pancreatitis and regenerate normal pancreas and duodenal villi from the wild-type (wt) cells that escape beta-catenin deletion.

2005

Definition of in Vitro Microenvironments to Characterize and Control Pancreatic Progenitor Expansion and Differentiation

Chiara Greggio

The pancreas plays a central role in metabolism. The exocrine pancreas, composed of ductal and acinar cells, secretes and delivers digestive enzymes into the duodenum where they contribute to food digestion. The endocrine pancreas, constituted by the islets of Langerhans, secretes hormones in the blood to control glucose levels. In particular, insulin is the hormone produced by β-cells that instructs the peripheral tissues to uptake circulating glucose. Diabetes mellitus is a heterogeneous disease characterized by deregulated glucose homeostasis due to an impaired insulin function. The advancements in clinical practice have made of diabetes a chronic, instead of a lethal, disease; yet no definitive cure is available. Beta-cell transplantation offers promising results, especially for type 1 diabetes where β-cells are selectively eliminated by an autoimmune attack. Unfortunately its application suffers from the scarcity of transplantable pancreas/islets from cadaveric donors. Human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) would constitute unlimited sources of transplantable β-cells, but the available differentiation protocols are not yet optimal, especially with regards to the numbers and the functionality of the generated β-cells. It is anticipated that a stepwise protocol would succeed in generating mature β-cells in vitro if it is capable of fully mimicking the embryonic development of these cells. Many aspects of pancreatic organogenesis have been elucidated and could serve as a guide for driving the commitment to β-cells. Unfortunately, pancreatic progenitors in vitro do not behave as in the intricate context of an embryo. This severely limits both the understanding of pancreatic development at the single-cell level and our ability in manipulating the process. The aim of our work was to develop in vitro methodologies to sustain pancreatic progenitor culture and expansion and to establish conditions to manipulate their progeny. To this aim, we combined an informed approach based on developmental biology and an empirical one. We first observed that pancreatic epithelial explants from embryonic day 10.5 mice could be cultured in presence of Matrigel™ and exogenous FGFs even in the absence of the mesenchyme that normally surrounds the epithelium. Notably, the removal of mesenchyme did not affect the endocrine commitment pattern of pancreatic progenitors. We then defined culture conditions allowing for the expansion of dissociated embryonic pancreatic progenitors in a three-dimensional Matrigel™-based environment. Under these conditions, progenitors proliferated and self-organized to generate pancreatic organoids containing both progenitors and differentiated cells, mostly exocrine, after 7 days of culture. When grafted into recipient pancreatic explants, the cultured progenitors contributed to the three epithelial pancreatic lineages (ductal, endocrine, acinar), integrating seamlessly into the endogenous cellular network. Thus, cultured progenitors retained their potential to differentiate into endocrine cells, but expressed it only in the appropriate niche. Subsequent removal of single components from the culture system led to the identification of some essential requirements for the maintenance/expansion of dissociated pancreatic progenitors, such as a strong FGF signaling and the Rho-associated kinase (ROCK) inhibitor Y-27632. In addition to this, we observed that only small clusters of pancreatic progenitors, but not single cells, were able to generate pancreatic organoids, suggesting a pivotal role for intercellular signaling in progenitor maintenance and expansion. By manipulating the components of the medium, we could affect fate commitment. In particular, the removal of FGF1 increased the yield of endocrine cells generated in vitro. In our search for a full control over the in vitro niche, we explored chemically defined matrices to replace Matrigel™. We showed that pancreatic progenitors could be cultured on laminin-functionalized hydrogels, although less efficiently than in Matrigel™. Moreover, differentiation into exocrine and endocrine cells occurred spontaneously under these conditions. By comparing Matrigel™, hydrogel microwells and two-dimensional culture systems, we uncovered the importance of a tridimensional architecture for pancreatic progenitor maintenance. We showed that pancreatic progenitors lost their identity as they flattened onto 2D surfaces, whereas 3D culture systems maintained the epithelial character of pancreatic progenitors and allowed the acquisition of apical polarization. To conclude, our work provides the first detailed characterization of in vitro culture systems for expansion and manipulation of dissociated embryonic pancreatic progenitors. Further implementation will hopefully allow the establishment of a fully-defined in vitro niche for developing pancreas with potential fundamental implications for the expansion of ES-derived pancreatic progenitors and their differentiation into functional β-cells.