von Hippel-Lindau gene disruption in mouse pancreatic progenitors and its consequences on endocrine differentiation in vivo: importance of HIF1-α and VEGF-A upregulation.

AIM/HYPOTHESIS: Different studies have linked hypoxia to embryonic development. Specifically, when embryonic pancreases are cultured ex vivo under hypoxic conditions (3% O2), beta cell development is impaired. Different cellular signalling pathways are involved in adaptation to hypoxia, including the ubiquitous hypoxia-inducible-factor 1-α (HIF1-α) pathway. We aimed to analyse the effects of HIF1-α stabilisation on fetal pancreas development in vivo. METHODS: We deleted the Vhl gene, which encodes von Hippel-Lindau protein (pVHL), a factor necessary for HIF1-α degradation, by crossing Vhl-floxed mice with Sox9-Cre mice. RESULTS: HIF1-α was stabilised in pancreatic progenitor cells in which the HIF pathway was induced. The number of neurogenin-3 (NGN3)-expressing cells was reduced and consequently endocrine development was altered in Vhl knockout pancreases. HIF1-α stabilisation induced Vegfa upregulation, leading to increased vascularisation. To investigate the impact of increased vascularisation on NGN3 expression, we used a bioassay in which Vhl mutant pancreases were cultured with or without vascular endothelial growth factor (VEGF) receptor 2 (VEGF-R2) inhibitors (e.g. Ki8751). Ex vivo analysis showed that Vhl knockout pancreases developed fewer NGN3-positive cells compared with controls. Interestingly, this effect was blocked when vascularisation was inhibited in the presence of VEGF-R2 inhibitors. CONCLUSIONS/INTERPRETATION: Our data demonstrate that HIF1-α negatively controls beta cell differentiation in vivo by regulating NGN3 expression, and that this effect is mediated by signals from blood vessels.

HIF1α and pancreatic ß-cell development.

During early embryogenesis, the pancreas shows a paucity of blood flow, and oxygen tension, the partial pressure of oxygen (pO(2)), is low. Later, the blood flow increases as ß-cell differentiation occurs. We have previously reported that pO(2) controls ß-cell development in rats. Here, we checked that hypoxia inducible factor 1α (HIF1α) is essential for this control. First, we demonstrated that the effect of pO(2) on ß-cell differentiation in vitro was independent of epitheliomesenchymal interactions and that neither oxidative nor energetic stress occurred. Second, the effect of pO(2) on pancreas development was shown to be conserved among species, since increasing pO(2) to 21 vs. 3% also induced ß-cell differentiation in mouse (7-fold, P<0.001) and human fetal pancreas. Third, the effect of hypoxia was mediated by HIF1α, since the addition of an HIF1α inhibitor at 3% O(2) increased the number of NGN3-expressing progenitors as compared to nontreated controls (9.2-fold, P<0.001). In contrast, when we stabilized HIF1α by deleting ex vivo the gene encoding pVHL in E13.5 pancreas from Vhl floxed mice, Ngn3 expression and ß-cell development decreased in such Vhl-deleted pancreas compared to controls (2.5 fold, P<0.05, and 6.6-fold, P<0.001, respectively). Taken together, these data demonstrate that HIF1α exerts a negative control over ß-cell differentiation.

Culture, differentiation, and transduction of mouse E12.5 pancreatic spheres: an in vitro model for the secondary transition of pancreas development.

During the secondary transition of rodent pancreatic development, mainly between E12.5 and E15.5 in mice, exocrine and endocrine populations differentiate from pancreatic progenitors. Here we describe an experimental system for its study in vitro. First, we show that spheres derived from dissociated E12.5 mouse pancreases differentiate within 7 days into most pancreatic exocrine and endocrine cell types, including beta cells. The proportion and spatial repartition of the different endocrine populations mirror those observed during normal development. Thus, dissociation and culture do not impair the developmental events affecting pancreatic progenitors during the secondary transition. Moreover, dissociated cells from mouse E12.5 pancreas were transduced with ecotropic MLV-based retroviral vectors or, though less efficiently, with a mixture of ALV(A)-based retroviral vectors and gesicles containing the TVA (Tumor Virus A) receptor. As an additional improvement, we also created a transgenic mouse line expressing TVA under the control of the 4.5 kB pdx1 promoter (pdx1-TVA). We demonstrate that pancreatic progenitors from dissociated pdx1-TVA pancreas can be specifically transduced by ALV(A)-based retroviral vectors. Using this model, we expressed an activated mutant of the YAP transcriptional co-activator in pancreatic progenitors. These experiments indicate that deregulated YAP activity reduces endocrine and exocrine differentiation in the resulting spheres, confirming and extending previously published data. Thus, our experimental model recapitulates in vitro the crucial developmental decisions arising at the secondary transition and provides a convenient tool to study their genetic control.