Activation of FGFR(IIIc) isoforms promotes activin-induced mesendoderm development in mouse embryonic stem cells and reduces Sox17 coexpression in EpCAM+ cells

AbstractActivin induces the formation of definitive endoderm from mouse ES cells dependent on active fibroblast growth factor (Fgf) signaling. Here we report that Fgf4 is dispensable for activin A-induced differentiation of mouse ES cells into endoderm. We find that Fgf4−/− cells readily differentiate into definitive endoderm without exogenous administration of Fgf4. Additionally, we investigate the spatio-temporal dynamics of Fgf receptor (FGFR) isoform distribution in activin A-treated ES cell cultures and find that FGFR(III)c isoforms are expressed in DE as well as non-DE populations, whereas FGFR2(III)b and FGFR4 are found specifically enriched in the DE fraction. Ligands that preferentially activate the FGFR(III)c isoforms induce mesendoderm markers T and Gsc, but reduce expression of the DE marker Sox17 in activin-induced EpCAM+ cells. In contrast, ligands specifically activating FGFR(III)b isoforms have no effect on either population. Activation of FGFR(III)c isoforms results in a strong mitogenic effect on activin A-induced ES cell progeny early in the differentiation period whereas activation of FGFR(III)b isoforms has only a moderate mitogenic effect confined to the late differentiation period. We conclude that FGFR(III)c-isoform activation selectively drives the differentiation of mES cells toward mesendoderm and that Fgf4 is dispensable for the differentiation into definitive endoderm

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

Glucose induced MAPK signalling influences NeuroD1-mediated activation and nuclear localization

AbstractThe helix–loop–helix transcription factor NeuroD1 (also known as Beta2) is involved in β-cell survival during development and insulin gene transcription in adults. Here we show NeuroD1 is primarily cytoplasmic at non-stimulating glucose concentrations (i.e. 3 mM) in MIN6 β-cells and nuclear under stimulating conditions (i.e. 20 mM). Quantification revealed that NeuroD1 was in 40–45% of the nuclei at 3 mM and 80–90% at 20 mM. Treatment with the MEK inhibitor PD98059 or substitution of a serine for an alanine at a potential mitogen-activated protein kinase phosphorylation site (S274) in NeuroD1 significantly increased the cytoplasmic level at 20 mM glucose. The rise in NeuroD1-mediated transcription in response to glucose also correlated with the change in sub-cellular localization, a response attenuated by PD98059. The data strongly suggest that glucose-stimulation of the MEK–ERK signalling pathway influences NeuroD1 activity at least partially through effects on sub-cellular localization

Retinoic Acid Signaling Organizes Endodermal Organ Specification along the Entire Antero-Posterior Axis

Background: Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer. Methodology/Principal Findings: RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA + cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically wit

Insulin gene regulation and islet development as studied in genetically modified tumors and transgenic laboratory animals

The pancreatic islet of Langerhans is composed of four highly distinct cell types specialized to mass produce a particular hormone. Insulin is thus the main product released from the islet B—cell in response to elevated glucose.The four cell types maturate during fetal development. Pluripotent rat islet tumors can to a certain degree undergo similar maturation processes when passaged in vivo. Such a model has been used to study the B—cell specific process of insulin gene activation. Transgenic mice have been instrumental in defining the functional regulatory elements involved in restricting the insulin gene activity to the pancreatic B-cell. The tissue-specific enhancer/promoter has thus been identified and used in combination with a series of other genes which in transgenic mice targets expression of the gene in question selectively to the B-cell. Important transacting factors have been identified and cloned which are in part responsible for mediating tissue specific insulin gene expression. One such factor when "knocked-out" results in a phenotype lacking the entire pancreas. Future developments in targeting "knockout" of genes to particular cell types will help dissecting out the multiple functions of such regulatory transacting factors

Preservation of proliferating pancreatic progenitor cells by Delta-Notch signaling in the embryonic chicken pancreas

<p>Abstract</p> <p>Background</p> <p>Genetic studies have shown that formation of pancreatic endocrine cells in mice is dependent on the cell autonomous action of the bHLH transcription factor Neurogenin3 and that the extent and timing of endocrine differentiation is controlled by Notch signaling. To further understand the mechanism by which Notch exerts this function, we have investigated pancreatic endocrine development in chicken embryos.</p> <p>Results</p> <p>In situ hybridization showed that expression of Notch signaling components and pro-endocrine bHLH factors is conserved to a large degree between chicken and mouse. Cell autonomous inhibition of Notch signal reception results in significantly increased endocrine differentiation demonstrating that these early progenitors are prevented from differentiating by ongoing Notch signaling. Conversely, activated Notch1 induces <it>Hes5-1 </it>expression and prevents endocrine development. Notably, activated Notch also prevents Ngn3-mediated induction of a number of downstream targets including <it>NeuroD</it>, <it>Hes6-1</it>, and <it>MyT1 </it>suggesting that Notch may act to inhibit both <it>Ngn3 </it>gene expression and protein function. Activated Notch1 could also block endocrine development and gene expression induced by NeuroD. Nevertheless, Ngn3- and NeuroD-induced delamination of endodermal cells was insensitive to activated Notch under these conditions. Finally, we show that Myt1 can partially overcome the repressive effect of activated Notch on endocrine gene expression.</p> <p>Conclusion</p> <p>We conclude that pancreatic endocrine development in the chicken relies on a conserved bHLH cascade under inhibitory control of Notch signaling. This lays the ground for further studies that take advantage of the ease at which chicken embryos can be manipulated.</p> <p>Our results also demonstrate that Notch can repress Ngn3 and NeuroD protein function and stimulate progenitor proliferation. To determine whether Notch in fact does act in Ngn3-expressing cells <it>in vivo </it>will require further studies relying on conditional mutagenesis.</p> <p>Lastly, our results demonstrate that expression of differentiation markers can be uncoupled from the process of delamination of differentiating cells from the epithelium.</p

Permanent Neonatal Diabetes and Enteric Anendocrinosis Associated With Biallelic Mutations in NEUROG3

Artículo de publicación ISIOBJECTIVE—NEUROG3 plays a central role in the development of both pancreatic islets and enteroendocrine cells. Homozygous hypomorphic missense mutations in NEUROG3 have been recently associated with a rare form of congenital malabsorptive diarrhea secondary to enteroendocrine cell dysgenesis. Interestingly, the patients did not develop neonatal diabetes but childhood-onset diabetes. We hypothesized that null mutations in NEUROG3 might be responsible for the disease in a patient with permanent neonatal diabetes and severe congenital malabsorptive diarrhea. RESEARCH DESIGN AND METHODS—The single coding exon of NEUROG3 was amplified and sequenced from genomic DNA. The mutant protein isoforms were functionally characterized by measuring their ability to bind to an E-box element in the NEUROD1 promoter in vitro and to induce ectopic endocrine cell formation and cell delamination after in ovo chicken endoderm electroporation. RESULTS—Two different heterozygous point mutations in NEUROG3 were identified in the proband [c.82G.T (p.E28X) and c.404T.C (p.L135P)], each being inherited from an unaffected parent. Both in vitro and in vivo functional studies indicated that the mutant isoforms are biologically inactive. In keeping with this, no enteroendocrine cells were detected in intestinal biopsy samples from the patient. CONCLUSIONS—Severe deficiency of neurogenin 3 causes a rare novel subtype of permanent neonatal diabetes. This finding confirms the essential role of NEUROG3 in islet development and function in humans

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

AbstractHere we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17+ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17+ cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP+ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17+ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro