Endoderm and mesoderm reciprocal signaling mediated by CXCL12 and CXCR4 regulates the migration of angioblasts and establishes the pancreatic fate.

We have discovered that angioblasts trigger an early inductive event in pancreatic differentiation. This event occurs soon after gastrulation, before the formation of blood vessels. Morphological studies revealed that Lmo2-expressing angioblasts reside in proximity to the somitic mesoderm and the gut endoderm from which pancreatic progenitors arise. The chemokine ligand CXCL12 expressed in the gut endoderm functions to attract the angioblasts that express its receptor CXCR4. Angioblasts then signal back to the gut endoderm to induce Pdx1 expression. Gain-of-function and loss-of-function experiments for CXCL12 and CXCR4 were performed to test their function in blood vessel formation and pancreatic differentiation. The ectopic expression of Cxcl12 in the endoderm attracted the angioblasts and induced ectopic Pdx1 expression, resulting in an expanded pancreatic bud and an increased area of insulin-expressing cells. By contrast, in chick embryos treated with beads soaked in AMD3100, an inhibitor of CXCR4, the migration of angioblasts towards the Cxcl12-expressing gut endoderm was arrested, causing a malformation of blood vessels. This led to the generation of a smaller pancreatic bud and a reduced area of insulin-expressing cells. Taken together, these results indicate that the gut endoderm and angioblasts attract each other through reciprocal CXCL12 and CXCR4 signaling. This has a pivotal role in the fate establishment of the pancreatic progenitor cells and in the potentiation of further differentiation into endocrine ß-cells.

Neurogenin3 initiates stepwise delamination of differentiating endocrine cells during pancreas development.

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.

Wnt4 is heterogeneously activated in maturing ß-cells to control calcium signaling, metabolism and function.

Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic ß-cells. ß-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between ß-cells via gap junctions. Here, we identify the importance of signaling between ß-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4-positive cells, being more mature while Wnt4-negative cells are more proliferative. Knock-out in adult ß-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between ß-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating ß-cells.

Embryonic and adult stem cell systems in mammals: ontology and regulation.

Stem cells are defined as having the ability to self-renew and to generate differentiated cells. During embryogenesis, cells are initially proliferative and pluripotent and then they gradually become restricted to different cell fates. In the adult, tissue stem cells are normally quiescent, but become proliferative upon injury. Knowledge from developmental biology and insights into the properties of stem cells are keys to further understanding and successful manipulation. Here, we first focus on ES cells, then on embryonic development, and then on tissue stem cells of endodermally derived tissues, particularly the liver and pancreas.

Nutrients men-TOR ß-Cells to Adulthood.

A major trigger of adult ß-cell insulin secretion is glucose. In a recent issue of Cell Metabolism, Helman and colleagues show that in fetuses insulin secretion depends on the activation of mTOR by amino acids and that reducing amino acids promotes maturation of ß-cells derived from pluripotent stem cells.

The role of CXCL12-CXCR4 signaling pathway in pancreatic development.

Chemokine (C-X-C motif) receptor 4 (CXCR4) is the receptor for chemokine (C-X-C motif) ligand 12 (CXCL12, also known as stromal derived factor-1, Sdf1). CXCR4, a protein consisting 352 amino acids, is known to transduce various signals such as cell differentiation, cell survival, cell proliferation, cell chemotaxis and apoptosis [1, 2]. The expression of CXCR4 is observed in embryonic stem cells, blood cells, haematopoietic stem cells, endothelial cells, angioblasts and smooth muscle cells [3-9]. The CXCL12-CXCR4 signaling pathway has very important roles in the embryonic development. Mutant mice for CXCL12 or CXCR4 genes showed lethality due to defects in neurogenesis, angiogenesis, cardiogenesis, myelopoiesis, lymphopoiesis and germ cell development [10-13]. Recently, we reported that CXCL12-CXCR4 signaling pathway has a crucial role in regional specification of the gut endoderm during early development [14]. Here, we would like to focus on the role of CXCL12-CXCR4 signaling pathway in pancreatic development and summarize recent findings of its role in the induction of the pancreatic progenitor cells.

Origin of pancreatic precursors in the chick embryo and the mechanism of endoderm regionalization.

To study the developmental origin of the pancreas we used DiI crystals to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. Endodermal precursor cells for the stomach, pancreas and intestine were found to segregate immediately after completion of gastrulation. Transplantation experiments showed that region-specific endodermal fates are determined sequentially in the order stomach, intestine, and then pancreas. Non-pancreatic endoderm transplanted to the stomach region generated ectopic pancreas expressing both insulin and glucagon. These results imply that a pancreas-inducing signal is emitted from somitic mesoderm underlying the pre-pancreatic region, and this extends rostrally beyond the stomach endoderm region at the early somite stage. Transplantation experiments revealed that the endoderm responding to these pancreatic-inducing signals lies within the pre-pancreatic region and extends caudally beyond the region of the intestinal endoderm. The results indicate that pancreatic fate is determined in the area of overlap between these two regions.

Conserved origin of the ventral pancreas in chicken.

To determine the origin of the ventral pancreas, a fate map of the ventral pancreas was constructed using DiI crystal or CM-DiI to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. First, the region lateral to the 7- to 9-somite level, which has been reported to contribute to the ventral pancreas, was shown to contribute mainly to the intestine or the dorsal pancreas. At the 10 somite stage (ss), the ventral pre-pancreatic cells reside laterally at the 2-somite level, at the lateral boarder of the somite. At this stage, however, the fate of these cells has not yet segregated and they contribute to the ventral pancreas and to the intestine or bile duct. The ventral pancreas fate segregated at the 17 ss; the cells residing at the somite boarder at the 4-somite level at the 17 ss were revealed to contribute to the ventral pancreas. Interestingly, the dorsal and the ventral pancreatic buds are different in both origin and function. These two pancreatic buds begin to fuse at day 7 (HH 30) of embryonic development. However, whereas the dorsal pancreas gives rise to both Insulin-expressing endocrine and Amylase-expressing exocrine cells, the ventral pancreas gives rise to Amylase-expressing exocrine cells, but not insulin-expressing endocrine cells before day 7 (HH 30) of embryonic development.

Cytoplasmic and molecular reconstruction of Xenopus embryos: synergy of dorsalizing and endo-mesodermalizing determinants drives early axial patterning.

Ablation of vegetal cytoplasm from newly fertilized Xenopus eggs results in the development of permanent blastula-type embryos (PBEs). PBEs cleave normally and develop into a very simple tissue consisting only of atypical epidermis. We tried to restore complete embryonic development in PBEs by cytoplasmic transplantation or by mRNA injection. We show a two-step reconstruction of the body plan. In the first step, PBEs injected with either marginal cytoplasm or synthetic VegT RNA restored gastrulation and mesoderm formation, but not axial patterning. Injection of Xwnt8 mRNA (acting upstream of beta-catenin and thus substitutes for the dorsal determinant) did not restore axial development in PBEs. Simultaneous injections of Xwnt8 and VegT into PBEs resulted in dorsal axis development, showing the synergy of these molecules in axial development. These results suggest that the mixing of two cytoplasmic determinants, i.e. the dorsal determinant in the vegetal pole and the endo-mesodermal determinant in the whole vegetal half, triggers the early axial developmental process in Xenopus embryos.