Arx and Nkx2.2 compound deficiency redirects pancreatic alpha- and beta-cell differentiation to a somatostatin/ghrelin co-expressing cell lineage

<p>Abstract</p> <p>Background</p> <p>Nkx2.2 and Arx represent key transcription factors implicated in the specification of islet cell subtypes during pancreas development. Mice deficient for <it>Arx </it>do not develop any alpha-cells whereas beta- and delta-cells are found in considerably higher numbers. In <it>Nkx2.2 </it>mutant animals, alpha- and beta-cell development is severely impaired whereas a ghrelin-expressing cell population is found augmented.</p> <p>Notably, <it>Arx </it>transcription is clearly enhanced in <it>Nkx2.2</it>-deficient pancreata. Hence in order to precise the functional link between both factors we performed a comparative analysis of <it>Nkx2.2/Arx </it>single- and double-mutants but also of <it>Pax6</it>-deficient animals.</p> <p>Results</p> <p>We show that most of the ghrelin⁺cells emerging in pancreata of <it>Nkx2.2</it>- and <it>Pax6</it>-deficient mice, express the alpha-cell specifier Arx, but also additional beta-cell related genes. In <it>Nkx2.2</it>-deficient mice, Arx directly co-localizes with iAPP, PC1/3 and Pdx1 suggesting an Nkx2.2-dependent control of <it>Arx </it>in committed beta-cells. The combined loss of <it>Nkx2.2 </it>and <it>Arx </it>likewise results in the formation of a hyperplastic ghrelin⁺cell population at the expense of mature alpha- and beta-cells. Surprisingly, such <it>Nkx2.2^-/-Arx^-</it>ghrelin⁺cells also express the somatostatin hormone.</p> <p>Conclusions</p> <p>Our data indicate that Nkx2.2 acts by reinforcing the transcriptional networks initiated by Pax4 and Arx in early committed beta- and alpha-cell, respectively. Our analysis also suggests that one of the coupled functions of Nkx2.2 and Pax4 is to counteract <it>Arx </it>gene activity in early committed beta-cells.</p

The Homeodomain-Containing Transcription Factors Arx and Pax4 Control Enteroendocrine Subtype Specification in Mice

Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3+ endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3+) and in early differentiating (ChromograninA−) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4

Gastrin: A Distinct Fate of Neurogenin3 Positive Progenitor Cells in the Embryonic Pancreas

Neurogenin3+ (Ngn3+) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin+ cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin+ cells do not express any other islet hormone. Pancreatic gastrin+ cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells

Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors

BACKGROUND: PAX6 is a transcription factor playing a crucial role in the development of the eye and in the differentiation of the pancreatic endocrine cells as well as of enteroendocrine cells. Studies on the mouse Pax6 gene have shown that sequences upstream from the P0 promoter are required for expression in the lens and the pancreas; but there remain discrepancies regarding the precise location of the pancreatic regulatory elements. RESULTS: Due to genome duplication in the evolution of ray-finned fishes, zebrafish has two pax6 genes, pax6a and pax6b. While both zebrafish pax6 genes are expressed in the developing eye and nervous system, only pax6b is expressed in the endocrine cells of the pancreas. To investigate the cause of this differential expression, we used a combination of in silico, in vivo and in vitro approaches. We show that the pax6b P0 promoter targets expression to endocrine pancreatic cells and also to enteroendocrine cells, retinal neurons and the telencephalon of transgenic zebrafish. Deletion analyses indicate that strong pancreatic expression of the pax6b gene relies on the combined action of two conserved regulatory enhancers, called regions A and C. By means of gel shift assays, we detected binding of the homeoproteins PDX1, PBX and PREP to several cis-elements of these regions. In constrast, regions A and C of the zebrafish pax6a gene are not active in the pancreas, this difference being attributable to sequence divergences within two cis-elements binding the pancreatic homeoprotein PDX1. CONCLUSION: Our data indicate a conserved role of enhancers A and C in the pancreatic expression of pax6b and emphasize the importance of the homeoproteins PBX and PREP cooperating with PDX1, in activating pax6b expression in endocrine pancreatic cells. This study also provides a striking example of how adaptative evolution of gene regulatory sequences upon gene duplication progressively leads to subfunctionalization of the paralogous gene pair

Analysis of transgenic animals conditionally misexpressing Pax4 in pancreatic alpha-cells

Dans ce travail, nous démontrons que l expression ectopique de Pax4 dans les cellules glucagon+ adultes induit, indépendamment de l âge, leur néogenèse et transformation en cellules bêta-like , ce qui entraîne une hypertrophie des îlots et une néogenèse inattendue des îlots. Par l utilisation de plusieurs approches de traçage, nous démontrons que la conversion des cellules alpha en cellules bêta-like médiée par l expression de Pax4, induit également la mobilisation de précurseurs situés dans ou à proximité des canaux pancréatiques. Ces cellules ré-expriment le gène développemental Ngn3 et adoptent successivement une identité de cellules glucagon+ puis de cellules bêta-like , suggérant le réveil des mécanismes embryonnaires. Il et à noter que ces processus sont capables de régénérer la totalité de la masse de cellules bêta après plusieurs séries d induction chimique du diabète. Ces résultats offrent ainsi des perspectives prometteuses pour concevoir de nouvelles stratégies thérapeutiques et régénératrices dans le contexte du diabète du type I. Dans un deuxième chapitre, ce travail décrit nos résultats d'analyse par puce à ADN de pancréas transgénique d animaux exprimant conditionnellement le gène Pax4 dans les cellules alpha adultes. Cette approche nous permis d'identifier de potentiels gènes cibles de Pax4, qui pourraient jouer un rôle important dans les processus de régénération de la masse de cellules bêta. L analyse de la fonction de l un de ces gènes, le facteur de croissance indépendante 1 (Gfi1) est décrite.In this work we demonstrate that the inducible misexpression of Pax4 in glucagon+ cells age-independently provokes their conversion into beta-like cells and their glucagon shortage-mediated replacement, this process resulting in islet hypertrophy and in an unexpected islet neogenesis. Taking advantage of several lineage-tracing approaches, we show that, upon Pax4-mediated alpha-to-beta-like cell conversion, pancreatic duct-lining precursor cells are mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon+ and a beta-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition (EMT). It is worth mentioning, that these processes can repeatedly regenerate the entire beta-cell mass, and thereby reverse several rounds of streptozotocin-mediated chemically-induced Type I diabetes. This approach thereby provides promising perspectives to design novel therapeutic regenerative strategies. Aiming to gain further insight into the molecular mechanisms underlying these regeneration and reprogramming processes, and thereby identify new putative targets of interest, a thorough micro array analysis was performed using pancreata from transgenic mice conditionally misexpressing Pax4 in adult alpha-cells. We thereby identified several promising candidate genes, whose gene expression was significantly altered in induces animals. Among these was Growth factor independent 1 (Gfi1): its expression pattern and putative function in the murine pancreas will be described in this work.NICE-Bibliotheque electronique (060889901) / SudocSudocFranceF

Caractérisation fonctionnelle du rôle des gènes Arx et Pax4 dans les processus de développement du pancréas endocrine

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF