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. 2012 Jul;26(7):2734–2742. doi: 10.1096/fj.11-199224

HIF1α and pancreatic β-cell development

Mylène Heinis *, Andrea Soggia *, Camille Bechetoille *, Marie-Thérèse Simon *, Carole Peyssonnaux †,‡, Pierre Rustin §,‖, Raphael Scharfmann *, Bertrand Duvillié *,1
PMCID: PMC4046099  PMID: 22426121

Abstract

During early embryogenesis, the pancreas shows a paucity of blood flow, and oxygen tension, the partial pressure of oxygen (pO2), is low. Later, the blood flow increases as β-cell differentiation occurs. We have previously reported that pO2 controls β-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 pO2 on β-cell differentiation in vitro was independent of epitheliomesenchymal interactions and that neither oxidative nor energetic stress occurred. Second, the effect of pO2 on pancreas development was shown to be conserved among species, since increasing pO2 to 21 vs. 3% also induced β-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% O2 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 β-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 β-cell differentiation.—Heinis, M., Soggia, A., Bechetoille, C., Simon, M.-T., Peyssonnaux, C., Rustin, P., Scharfmann, R., Duvillié, B. HIF1α and pancreatic β-cell development.

Keywords: oxygen tension, pVHL, pancreas, development

Organogenesis is controlled by a hierarchy of transcription factors and by the local cellular environment. Recent reports have indicated that oxygen tension, the partial pressure of oxygen (pO2), regulates the embryonic development of several organs, including trachea, heart, lung, limb bud, bone, and pancreas (1–7). During embryogenesis, pO2 varies from 2 to 9% (14.4–64.8 mmHg) in thymus, kidney medulla, or bone marrow, and it is even lower in pancreas, where it reaches 1% (7.2 mmHg) (6, 8). The adaptive response of cells to hypoxia is known to be mediated by the hypoxia-inducible factor (HIF) complex (9), a heterodimer composed of an oxygen-sensitive α and an oxygen-insensitive β subunit. In normoxia, HIF1α subunits are hydroxylated by prolyl hydroxylases (PHDs), and then subsequent binding of the Von Hippel Lindau protein (pVHL) tumor suppressor factor leads to proteasomal degradation of HIF1α (10). In the absence of pVHL, HIF1α is constitutively stabilized and active. In humans, mutations of Vhl lead to the von Hippel-Lindau disease, a syndrome characterized by benign or malignant tumors of the kidney, central nervous system and pancreas (11). In a large variety of cells, HIF1α is also stabilized and active at low pO2, and HIF1α thus regulates several physiological processes that allow an adaptative response of the organism to hypoxia. These mechanisms are known to include angiogenesis, erythropoiesis, modulation of cellular energy metabolism, innate immunity, neutrophil survival, and skin oxygen sensing (12–15).

The mature pancreas contains endocrine islets composed of cells producing hormones, such as insulin (β cells), or glucagon (α cells), and exocrine tissue composed of acinar cells producing enzymes, which are secreted into the intestine, such as carboxypeptidase A (CPA) or amylase. During embryogenesis, the pancreas originates from endoderm, and a temporally regulated program of transcription factors controls the development of endocrine and exocrine cells (16). The early precursor cells express the transcription factor pancreatic and duodenal homeobox factor 1 (PDX1), and endocrine differentiation is driven by the transitory expression of the proendocrine transcription factor neurogenin 3 (NGN3) (17, 18). Cellular and environmental signals also control pancreas development (19). At early stages of embryogenesis, the pancreas is poorly vascularized and HIF1α is stabilized (2, 8). Later, blood flow increases and at the same time endocrine differentiation occurs (2, 8). Several recent studies have shown the importance of the HIF pathway for mature β-cell function (20–23). The role of HIF1α in pancreas development has also been investigated during late stages of fetal life or postnatally (20–22). The effect of HIF1α on embryonic β-cell differentiation, however, has not yet been elucidated.

Previously, we showed that oxygen tension controls β-cell differentiation (2). Indeed, β-cell differentiation was reduced when cultures were maintained under hypoxia and increased when pO2 was elevated. In the present study, we investigated the mechanism by which oxygen tension controls β-cell development. Our specific aim was to decipher the HIF1α-dependent and HIF1α-independent effects of pO2 on β-cell development. We found that the effects of pO2 on β-cell development did not require epitheliomesenchymal interactions. Moreover, neither energetic nor oxidative stress was implicated in these effects. Interestingly, the control of β-cell development by pO2, which we found previously for rat pancreas, was also observed for human and mouse fetal pancreas. Using a HIF1α inhibitor, we showed that the HIF1α signaling pathway was involved in the effect of hypoxia on NGN3 expression. To further determine the role of HIF1α stabilization on β-cell development, we investigated the effect of deleting Vhl. Our genetic manipulation of the pVHL/HIF pathway in vitro resulted in impaired β-cell differentiation and implicated an alteration of NGN3 induction.

MATERIALS AND METHODS

Human tissues

Human pancreatic tissue fragments were obtained during elective termination of pregnancy after 7–8 wk of gestation, in compliance with French bioethics legislation. Approval was obtained from the Agence de Biomedecine, the French competent authority, along with maternal written consent.

Animals

Pregnant Wistar rats were purchased from the Janvier breeding center (CERJ Janvier, Le Genest St-Isle, France). Vhl fl/fl mice on a C57BL/6 background (24) were used. The animals had free access to food pellets and water. The first day postcoitum was taken as embryonic day 0.5 (E0.5). At E13.5, pregnant rats were killed by asphyxiation with carbon dioxide, and pregnant mice were killed by cervical dislocation, in full compliance with guidelines established by the French Animal Care Committee.

Genotyping of mice

Mice with a conditional null allele of Vhl have been described previously (25). The 2-lox allele contains two loxP sites, which flank the Vhl promoter and first exon, thus Cre-mediated recombination of the 2-lox allele generates a null allele (1-lox allele) in which both promoter and exon 1 are deleted (25). The genotype and the successful excision of the Vhl-floxed allele was demonstrated by PCR on genomic DNA isolated from the tail, the stomach, and pancreas of wild-type (WT), Vhl (fl/+), and Vhl (fl/fl) embryos. According to the method described in Biju et al. (26), the following primers were used to detect 1-lox (excised) and 2-lox (floxed) alleles of Vhl: Fwd I (5′-CTGGTACCCACGAAACTGTC-3′), Fwd II (5′-CTAGGCACCGAGCTTAGAGGTTTGCG-3′), and Rev primer (5′-CTGACTTCCACTGATGCTTGT CACAG-3′). The Vhl 2-lox allele was identified as a 460-bp band, the 1-lox allele was detected as a 260-bp band, and the WT allele was detected as a 290-bp band.

Dissection of pancreatic rudiments and organ culture

Mouse and rat embryos were harvested at E13.5. The dorsal pancreatic bud was dissected, as described previously (27). For the depletion of the mesenchyme, the digestive tract was incubated with 0.5 mg/ml collagenase A (Roche, Meylan, France) at 37°C for 30 min, then washed several times with Hank's balanced solution (Invitrogen, Cergy-Pontoise, France) at 4°C, and the epithelium was mechanically separated from the mesenchyme using needles on 0.25% agar, 25% Hank's balanced solution, and 75% RPMI gel (Life Technologies, Invitrogen) in a Petri dish (28). Pancreatic rudiments were cultured at the air–medium interface on Millicell culture plate inserts (Millipore, Molsheim, France) in Petri dishes containing RPMI 1640 (Invitrogen) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES (10 mM), l-glutamine (2 mM), nonessential amino acids (1×; Invitrogen) and 10% heat-inactivated calf serum (Hyclone, South Logan, UT, USA) (29). The HIF1α inhibitor [3-(2-(4-Adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester; Calbiochem, Meudon, France; ref. 30] was added at 25 μm in the culture medium. Cultures were maintained at 37°C in humidified 5% CO2 in a hypoxia chamber at various levels of pO2.

Transferase-mediated dUTP nick-end labeling (TUNEL) experiments

The TUNEL procedure was performed using an in situ cell death detection kit (Roche, Meylan, France), according to the manufacturer's instructions. Subsequently, E-cadherin (1:100; BD Biosciences, Le Pont de Claix, France) immunostaining was used to visualize the epithelium.

Determination of cellular ATP levels

The detection of ATP levels in whole rat pancreases was assessed by using a luminescence-based assay kit (Calbiochem).

Determination of mitochondrial and oxidative enzymatic activities

Pools of 3 pancreases cultured at 21 or 3% O2 were washed and quickly frozen in liquid nitrogen. Tissues were homogenized in ice-cold buffer containing 5 mM HEPES, 1 mM EGTA, 5 mM MgCl2, 1 mM dithiothreitol, and 0.1% Triton-X100 (pH 8.7), and then incubated for 60 min at 0°C. Enzyme activities were assessed by standard spectrophotometric methods, as described previously (31).

Infection of Vhl fl/fl pancreases

pCMV-GFP-Cre adenoviruses (ref.pG3.42155; Genethon, Evry, France) were used to infect Vhl fl/fl epithelia. Tissues were incubated with viral particles at the multiplicity of infection of 1000 for 4 h at 37°C in RPMI 1640. After infection, tissues were washed twice in HBSS and cultured at the air–medium interface.

Immunohistochemistry

Tissues were fixed in 10% formalin and processed for immunohistochemistry, as described previously (29). The following antibodies were used: mouse anti-insulin (1:2000; Sigma-Aldrich, Saint Quentin Fallavier, France), rabbit anti-PDX1 (1:1000; ref. 29), rabbit anti-CPA (1:600; Biogenesis; Valbiotech AbCys, Paris, France), mouse anti-E-cadherin (1:100; BD Biosciences), rabbit anti-NGN3 (1:1000, ref. 32) and rabbit anti-HIF1α (33). The fluorescent secondary antibodies were fluorescein-isothiocyanate anti-rabbit and Texas-red anti-mouse antibodies (1:200; Jackson Immunoresearch, Soham, UK), and Alexa Fluor anti-rabbit antibody (1:400; Biogenex Immunotech/Beckman, Marseille, France). For NGN3, revelation was performed using the Vectastain ABC kit (Vector, Malakoff, France). For HIF1α, revelation was performed with a biotinylated anti-rabbit antibody (1:200; Vector) and a TSA Plus Cyanin 5 system (Perkin Elmer, Villebon-sur-Yvette, France). Photographs were taken using a fluorescence microscope (Leitz DMRB; Leica Microsystems, Wetzlar, Germany) and a Hamamatsu C5810 cooled 3CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). No signals were observed when the first antibodies were omitted.

Quantification

To quantify the absolute number of insulin-expressing cells, all sections of each pancreatic rudiment were digitized. On every image, the surface area of insulin staining was quantified using IPLab 3.2.4 (Scanalytics, Fairfax, VA, USA), and the stained areas were tallied as described previously (29). Three to five rudiments per condition were analyzed. To measure the proliferation of early progenitors expressing E-cadherin, we counted the frequency of BrdU+ nuclei among 1000 E-cadherin+ cells. Three rudiments per condition were analyzed. Statistical significance was determined using Student's t test.

In situ hybridization

Tissues were fixed at 4°C in 4% paraformaldehyde, cryoprotected in 15% sucrose at 4°C overnight, embedded in 15% sucrose-7.5% gelatin, and frozen in isopentane. Cryosections (14 μm thick) were prepared. A Ngn3 probe (726 bp) was used, and in situ hybridization was done as described previously (34). No signal was obtained using a sense riboprobe.

Real-time PCR

Total RNA was purified using the RNeasy microkit (Qiagen, Courtaboeuf, France). The cDNA was generated using Superscript reagents (Invitrogen), and the real-time PCR was performed on a 7300 real-time PCR system (Applied Biosystems, Villebon sur Yvette, France) with a SYBR Green PCR master mix, as described previously (32). The oligonucleotide sequences for RT-PCR were PGK1, forward 5′-AGCTCTCTTCGCTGTATGTAGCC-3′ and reverse 5′-ACCTACAGCTGGAATCTCAGCCTT-3′; LDHA, forward 5′-GGTGGTTGACAGTGCATACG-3′ and reverse 5′-ACCCGCCTAAGGTTCTTCAT-3′;GLUT1, forward 5′-GGCTCCATTTAGGATTCGCCCATT-3′ and reverse 5′-TCGAGTGGTTGGTTGAGTTGGAAG-3′; VEGF-A, forward 5′-TGAGTGGCTTACCCTTCCCCATTT-3′ and reverse 5′-CTGCCCCATTGCTCTGTACCT-3′; and REDD1, forward 5′-GGGGTGGGTGTTAGGATCATTTGG-3′ and reverse 5′-GGATGCAGCTCCCTGGTGTTATTT-3′.

Western blot analysis

For Western blotting analysis, cells were lysed in Laemmli buffer. Proteins (20 μg) were resolved by SDS/PAGE and electrophoretically transferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA). After blocking with milk, membranes were probed with rabbit anti-adenosine monophosphate-activated protein kinase (AMPK)α and anti-phospho Thr172 AMPKα (both from Cell Signaling Ozyme, Saint Quentin, France), and mouse anti-actin (Sigma-Aldrich). Immunoreactive bands were visualized with the SuperSignal System (Pierce; Fisher Scientific, Illkirch, France).

RESULTS

Effect of pO2 on β-cell development is dose dependent and does not require epitheliomesenchymal interactions

Previously, we found that pancreases developed β cells when cultured at 21% O2, but their development was considerably reduced at 3% O2 (2). To decipher this effect of hypoxia, we cultured pancreases at several physiological oxygen tensions: 1, 3, and 5% O2. As shown in Supplemental Fig. S1, pO2 controls β-cell development in a linear fashion. To determine whether the effect of hypoxia on endocrine differentiation is reversible, pancreases were first cultured for 1 d at 3% O2, followed by culture at 21% O2 for the next 7 d (Supplemental Fig. S2A). In parallel, pancreases were cultured at 21% O2 for 7 d. The surface of insulin-positive cells was identical in the two conditions, indicating that the repressive effect of hypoxia on β-cell development is reversible. The importance of epitheliomesenchymal interactions in pancreatic development has been recognized from the 1960s (35). To study whether the effects of pO2 on β-cell development requires epitheliomesenchymal interactions, E13.5 rat pancreases devoid of mesenchyme were cultured at 3 and 21% O2. After 7 d in culture at 3% O2, the size of the tissue and the surface of insulin-positive cells were decreased as compared to 21% O2 (Fig. 1A). Three possible oxygen-dependent mechanisms could regulate the development of β cells: apoptosis, proliferation, and differentiation. The frequency of apoptotic cells was found to be similar in these two conditions (Supplemental Fig. S2B). To evaluate the effect of hypoxia on the proliferation of the precursor cells, we compared BrdU incorporation in pancreatic epithelia cultured with 21 or 3% O2 for 1 d (Supplemental Fig. S2C). As previously shown, incorporation of BrdU in PDX1-positive progenitors at normoxia was low when compared to pancreatic epithelium cultured in the presence of its mesenchyme (29). It further decreased at hypoxia (3.04±0.39% compared with 5.4±0.57% at normoxia, P < 0.05), but no significant difference was found between the size of the explants at hypoxia and normoxia at d 1 of culture(data not shown). On the other hand, the number of NGN3-expressing cells (Fig. 1B) was decreased by 71 ± 17% (P<0.001) in pancreases cultured at hypoxia compared with normoxia. Taken together, these results indicate that pO2 controls not only progenitor cell proliferation but more importantly endocrine differentiation. Such a decrease in β-cell differentiation under hypoxia was similar to the effect of hypoxia previously observed on pancreases cultured in the presence of mesenchyme (2). This result demonstrates that pO2 has a direct effect on pancreatic epithelial progenitor cells and does not require the presence of mesenchyme.

Figure 1.

Figure 1.

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Effect of hypoxia on β-cell differentiation does not require the presence of mesenchyme. A) Insulin (red) and carboxy-peptidase A (green) were detected by immunohistochemistry in E13.5 rat pancreas cultured for 7 d without mesenchyme at 21 or 3% O2. Quantification of the surface of insulin staining at d 7. B) NGN3 was detected by immunohistochemistry (in brown) in E13.5 rat pancreas cultured for 1 d without mesenchyme at 21 or 3% O2. Arrows indicate NGN3-positive cells. Scale bars = 50 μm. **P < 0.01; ***P < 0.001.

Neither energy stress nor oxidative stress are involved in the effects of hypoxia

Cells often adapt to hypoxia by reversibly switching off ATP-producing catabolic events to remain viable and survive in unfavorable microenvironments (36). These adaptative changes are governed by the intracellular energy-sensing system that depends on activation of the evolutionary conserved 5′ AMPK (37). AMPK is a downstream target of a protein kinase network that is induced by an increase in the AMP/ATP ratio. To evaluate the energy status of the pancreases cultured in hypoxia vs. normoxia, we first quantified the total ATP content in embryonic pancreases. Under hypoxic conditions, cellular ATP was decreased by 34% as compared to the normoxic controls (99.2±5.5 pmol/pancreas at 21% O2 vs. 65.0±4.7 pmol/pancreas at 3% O2, P<0.001; Fig. 2A). Because of the small size of these tissue samples, the quantification of AMP was not feasible. To investigate whether ATP deprivation led to energetic stress, we determined the AMP kinase activity by immunoblot. The average of phospho-AMPKα to total AMPKα was unaffected by hypoxia as compared to normoxia (1.1±0.03 at hypoxia vs. 1.2±0.02 at normoxia, P=0.084; Fig. 2B, C). Overall, these results indicate that the decrease in ATP under hypoxia was an adaptation to low oxygen tension rather than an energy stress.

Figure 2.

Figure 2.

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AMP kinase is not activated in the rat pancreas by hypoxia. E13.5 rat pancreases were cultured 1 d at 21 or 3% O2. A) Quantification of ATP content in each pancreas (n=10). B) Phospho-AMPKα and total AMPKα were analyzed by Western blot. Average of 3 analyses of P-AMPKα/total AMPKα is indicated for each condition of oxygen tension. C) Quantification of the AMPK-P/AMPK ratio and the pancreatic protein content (μg). Average of 3 analyses of P-AMPKα/total AMPKα and protein content is indicated for each condition of oxygen tension. NS, not significant.

The effects of hypoxia on β-cell development may rely on several different and concurrent processes, which most probably involve mitochondria, the major location of oxygen utilization in the cell. Oxygen availability may directly govern the level of oxidizing mitochondrial activity through the respiratory chain. Accordingly, one could have predicted that respiratory chain activity would be increased under increased oxygen. Our data do not support this view, since levels of five key components of the respiratory chain were found to be similar in explants grown at 3 or 21% O2 (Fig. 3). Moreover, we assessed aconitase activity, which is often used as a marker for oxidative stress in mitochondria. Indeed, aconitase contains an iron-sulfur cluster in its active site that is sensitive to oxidation and leads to enzyme inactivation (38). We found a similar aconitase activity in the pancreatic explants cultured at 3 or 21% O2. (Fig. 3). Altogether, these results indicate that the effects of pO2 involve neither a change in the activity of mitochondrial enzymes nor oxidative stress.

Figure 3.

Figure 3.

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Variations of pO2 do not induce oxidative stress in the rat pancreas. Activities of mitochondrial enzymes cytochrome oxidase, succinate cytochrome c reductase, glycerophosphate dehydrogenase, decylubiquinone cytochrome c reductase, and activity of the oxidative stress-sensitive enzyme aconitase was quantified in E13.5 rat pancreases cultured for 1 d at 21% O2 (solid bars) or 3% O2 (shaded bars). Similar levels were found in the tissues (n=3).

Negative effect of hypoxia on β-cell differentiation involves the HIF signaling pathway

To investigate whether hypoxia controls β-cell differentiation through HIF1α, we cultured rat pancreases at E13.5 to 21 or 3% O2 for 24 h, in the presence or in the absence of an inhibitor of HIF1α. This cell-permeable amidophenolic compound inhibits hypoxia-induced HIF1α transcription activity. To validate the effect of the HIF1α inhibitor, we first compared expression of several HIF1α target genes by RT-qPCR. We found that expression of the HIF1α target genes were induced at 3% O2 compared to 21% O2 (Fig. 4A). In the hypoxic condition, the addition of the HIF1α inhibitor reduced the expression of the HIF target genes as compared to nontreated pancreases. NGN3 expression was dramatically reduced at hypoxia compared to normoxia (Fig. 4B). On the other hand, when pancreases were cultured at 3% O2 with the HIF1α inhibitor, the number of NGN3-positive cells was significantly increased as compared to nontreated pancreases (P<0.01). Thus, we conclude that hypoxia down-regulates endocrine differentiation through HIF1α.

Figure 4.

Figure 4.

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Regulation of NGN3 expression by hypoxia involves the HIF signaling pathway. A) Real-time PCR quantification of Pgk1, LdhA, Glut1, VEGF-A, and Redd1 mRNA in pancreases cultured at the air–medium interface at 21% O2 (solid bars), or 3% O2 with (shaded bars) or without (open bars) 25 μM HIF1α inhibitor. B) Detection of NGN3 (brown) by immunohistochemistry in pancreases cultured for 1 d at 21% O2, 3% O2 with or without HIF1α inhibitor treatment. Arrows indicate NGN3-positive cells. Absolute number of NGN3-positive cells was quantified in each condition. Scale bars = 50 μm. *P < 0.05; **P < 0.01.

Regulation of β-cell development by oxygen tension is conserved between rodents and human

In previous work, we showed that oxygen tension controls β-cell development in rats (2). To investigate whether this effect is conserved between species, we used mouse and human pancreatic tissue samples. We cultured E13.5 mouse pancreases at the air–liquid interface in the presence of 21%O2 (normoxia) or 3%O2 (hypoxia). In normoxia, Ngn3 expression was induced on d 1, and insulin-positive cells developed on d 3 (Fig. 5A, B). On the other hand, Ngn3 was not induced, and very few β cells were detected on d 3 when the culture was performed under hypoxia. This effect was specific for the endocrine pathway, since the presence of acinar cells, as revealed by immunological detection of amylase, was similar for 21 and 3% O2 (Fig. 5B). To determine whether the control of β-cell development by pO2 is similar for human pancreas, we used fetal pancreases at 7-8 wk of gestation. After 7 d of culture at 21% O2, Ngn3 mRNA was detected in numerous cells, while this Ngn3 signal was considerably reduced at 3% O2 (Fig. 6A). Such results indicate that oxygen is necessary for Ngn3 expression. On d 14 of culture under 21% O2, β cells developed (Fig. 6B). On the other hand, β cells did not develop under 3% O2. Altogether, these data demonstrate that hypoxia decreases β-cell differentiation in a conserved manner between rats, mice, and humans.

Figure 5.

Figure 5.

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Hypoxia decreases β-cell differentiation in the mouse pancreas. A) E13.5 mouse pancreases were cultured at the air–medium interface at 21% or 3% O2. NGN3 was detected by immunohistochemistry (in brown) after 1 d (day 1) in culture. B) Insulin (red) and amylase (green) immunostaining was performed at d 3, and the surface of insulin staining was quantified. Scale bars = 50 μm. ***P < 0.001.

Figure 6.

Figure 6.

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pO2 controls Ngn3 expression and β-cell development in the human pancreas. A) Human pancreases from fetuses at 7-8 wk of gestation were cultured at the air medium interface at 21 or 3% O2. NGN3 was detected by in situ hybridization before (8 WG) and after 7 d (day 7) in culture. Arrows indicate cells expressing Ngn3. B) Insulin (red) and Pdx1 (green) were detected at d 14 of culture by immunohistochemistry. Scale bar: 50 μm. (***P<0.001).

Deletion of Vhl in the pancreatic epithelium enhances HIF1α levels and impairs the differentiation of β cells

To define the role of HIF1α on β-cell differentiation, we infected E13.5 Vhl fl/fl pancreatic epithelia with adenoviruses expressing both the Cre recombinase and the GFP reporter genes (Ade-Cre; Supplemental Fig. S3A). Cre-mediated excision of the floxed allele, resulting in deletion of the promoter and the first exon of Vhl, has been described previously (20). In tissues infected with Ade-Cre, GFP was broadly expressed in the epithelial cells of the pancreas (Supplemental Fig. S3A). In WT/Ade-Cre pancreas (control), GFP expression was detected in the insulin-positive cells, indicating that adenoviral infection did not alter β-cell development (Supplemental Fig. S4). Recombination of the Vhl locus in Ade-Cre infected epithelia (Vhl/Ade-Cre) was demonstrated at the DNA level by the detection of the excised allele (Supplemental Fig. S3B and ref. 25). As anticipated, the level of Vhl expression was reduced in Vhl/Ade-Cre epithelia as compared to WT/Ade-Cre epithelia (Supplemental Fig. S3C). To investigate the role of HIF1α on pancreas development, we cultured WT/Ade-Cre and Vhl/Ade-Cre epithelia under 21% O2. The proliferation of epithelial cells on d 1 of culture, and the surface of Hoechst staining on d 3 of culture did not differ between the WT/Ade-Cre and Vhl/Ade-Cre epithelia (Fig. 7A). Acinar cell development was estimated by quantification of the surface of amylase-staining in WT/Ade-Cre and Vhl/Ade-Cre epithelia on d 3 postinfection. Acinar cells developed in a similar fashion in WT/Ade-Cre and Vhl/Ade-Cre epithelia (Fig. 7B). Notably, HIF1α was stabilized in Vhl/Ade-Cre but not in WT/Ade-Cre epithelia, HIF1α being observed in CPA-positive and -negative cells (Fig. 7C). To determine whether HIF1α controls β-cell differentiation, we examined the development of β cells in WT/Ade-Cre and Vhl/Ade-Cre epithelia. At d 1, stabilization of HIF1α was detected in PDX1-negative cells of Vhl/Ade-Cre epithelia, but also in PDX1-positive cells, indicating that HIF1α was stabilized in the pancreatic precursors (Supplemental Fig. S3D). On d 3 postinfection, β cells developed in WT/Ade-Cre epithelia, while HIF1α protein was not detected (Fig. 8A). In Vhl/Ade-Cre epithelia, the surface area of insulin staining was reduced by 85% as compared to WT/Ade-Cre epithelia (Fig. 8A, B for quantification). Moreover, in Vhl/Ade-Cre epithelia, HIF1α staining was detected only in cells that did not express insulin (Fig. 8A), suggesting that insulin-expressing cells escaped Ade-Cre infection. Finally, we found that expression of NGN3 was decreased by 58% in Vhl/Ade-Cre as compared to WT/Ade-Cre epithelia on d 1 postinfection (Fig. 8C). Altogether, these data demonstrate that the pVHL/HIF1α pathway negatively controls β-cell differentiation.

Figure 7.

Figure 7.

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Stabilization of HIF1α does not affect acinar development. A) Percentage of BrdU incorporation was quantified in epithelial cells positive for E-cadherin from WT/Ade-Cre and Vhl/Ade-Cre epithelia at d 1 of culture (n=3). Total surface of Hoechst staining was quantified in each WT and Vhl-floxed epithelium infected with Ade-Cre at d 3 of culture. B) To compare the development of acinar cells in WT/Ade-Cre and Vhl/Ade-Cre pancreases, immunohistochemistry with anti-amylase antibodies was performed. Quantification of the surface of amylase staining (green) in WT-Cre or Vhl-Cre epithelia did not reveal any difference. C) HIF1α (green) and CPA (red) immunostaining on Vhl/Ade-Cre and WT/Ade-Cre epithelia at d 3 of culture. Arrows indicate cells that coexpress CPA and HIF1α. Scale bars = 50 μm.

Figure 8.

Figure 8.

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Deletion of Vhl in the pancreatic epithelia leads to HIF1α stabilization, and decreased β-cell differentiation. A) Pancreases from WT and Vhl fl/fl mouse embryos were infected with Ade-Cre and cultured for 3 d at 21% O2. HIF1α (red), and insulin protein expression (green) were analyzed by immunohistochemistry. In WT/Ade-Cre tissues, HIF1α was absent, and numerous β-cells developed. In Vhl/Ade-Cre pancreases, HIF1α was stabilized, and insulin staining was reduced. B) Quantification of the surface of insulin staining in WT, Vhl floxed epithelia, infected (+) or not (−) by Ade-Cre. C) NGN3 was detected by immunohistochemistry (in brown) in WT/Ade-Cre and Vhl/Ade-Cre pancreases at d 1 of culture. Scale bars = 50 μm.

DISCUSSION

We have previously shown that, in vitro, pO2 controls β-cell development in the embryonic rat pancreas (16). Our present study indicates first that the level of hypoxia that was used in these studies (3% O2) induces neither energy nor oxidative stress. Using embryonic mouse and human pancreatic tissue samples, we then confirmed the effect of pO2 on β-cell development, showing a conserved role for pO2 among mammalian species.

Recent studies also indicate that pO2 efficiently controls the differentiation of cells from several organs. This is the case of adipocytes, corneal limbal epithelial cells, and neural progenitor cells (39–41). In the mouse pancreas, it was also found that hypoxia decreases β-cell development, but this effect was not completely characterized at the molecular level (1, 2, 8). A physiological determinant of oxygen tension in the embryo is vascularization. During development, a correlation was found between the increase of the blood flow in the pancreas and β-cell differentiation (8). This finding suggests that pO2 plays a permissive role for β-cell differentiation in vivo. To support this hypothesis, we previously exposed E13.5 pregnant rats to hypoxia for 24 h, and the embryonic pancreases failed to express Ngn3 (2). Moreover, the fact that hypoxia occurs in the rodent and human pancreas at early stages of development further supports this hypothesis (42, 43).

Our working hypothesis for the present study was that the effects of pO2 were mediated by HIF1α. Using an HIF1α inhibitor, we first demonstrated that hypoxia negatively regulates β-cell differentiation through HIF1α. Indeed, NGN3 expression was decreased at 3% O2 compared to 21% O2, and the addition of HIF1α inhibitor at hypoxia partially restored the number of NGN3-positive cells. The fact that the number of NGN3-positive cells was lower at 3% O2 with the HIF1α inhibitor than cultures at 21% O2 suggests that pO2 has both HIF1α-dependent and HIF1α-independent effects on NGN3 expression. One other possibility is that efficiency of HIF1α inhibitor is not sufficient to completely abrogate the HIF signaling pathway and to restore NGN3 expression at hypoxia. To further define the HIF1α-dependent effects of pO2, we used a genetic approach. We found that deletion of Vhl, a gene encoding a protein necessary for the proteasomal degradation of HIF1α, led to impaired β-cell differentiation in vitro. In these experiments, the proliferation of the precursor cells was not altered by the presence of HIF1α. Recently, it was shown that the deletion of the Vhl-floxed allele mediated by pdx1-Cre caused the activation of HIF1α in all pancreatic cell types and resulted in a normal β-cell mass at 12 wk of age (20). However, when using the pdx1-Cre mouse to delete Vhl, only 70% of β-cells at 12 wk of age showed HIF1α staining. Such a result suggests that the pdx1-Cre transgene brings about recombination of the loxP sites in only 70% of β cells during development and adulthood (44). As endogenous expression of Pdx1 is normally found both in progenitors and differentiated β-cells during embryogenesis, it would be important to determine whether the HIF1α protein was stabilized before or after differentiation of β-cells in such mice. Therefore, further studies will be necessary to characterize the exact role of HIF1α expression in the progenitor cells in vivo. Another concern is the possibility that Vhl regulates pathways other than HIF1α, or stabilizes HIF2α. Indeed, the best studied role of Vhl has been the regulation of HIF1α and HIF2α (10), but the protein encoded by Vhl, pVHL, interacts also with several protein partners (45). Thus, we cannot formally exclude the possibility that genetic deletion of Vhl would influence the differentiation of β cells by modulating the expression of other proteins, including HIF2α. However, in mice lacking pVHL in β cells, HIF2α was not detectable (20). Moreover, in several studies, the phenotype of mice lacking pVHL in β cells impaired insulin secretion in response to glucose. This phenotype was rescued by deletion of hif1α (22, 44). These data indicate that the main effects of pVHL in the pancreas are very likely mediated by HIF1α. In our previous study (2), we showed that expression of Hes1, a direct target of the Notch-activated receptor, which controls expression of Ngn3, was induced by hypoxia. Moreover, the effect of hypoxia on the silencing of Ngn3 was abolished in the presence of a γ-secretase inhibitor, which blocked the Notch pathway and Hes1 expression. In addition, the forced stabilization of HIF1α by dimethyloxalyllglycine (DMOG), an inhibitor of prolyl hydroxylases, also decreased Ngn3 expression. The expression of Ngn3 in the presence of DMOG was restored when γ-secretase inhibitor was added to the culture medium (2). These data indicated that the effects of hypoxia are mediated by the HIF/Notch pathway. Recently, a direct link between HIF1α and Notch or HIF2α and Notch were found in neural, myogenic, and pancreatic cells (2, 41, 46). Thus, we speculate that in our experiments, the reduction of Ngn3 expression in the Vhl/Ade Cre epithelia was caused by the activation of the Notch pathway.

Finally, it was found that the hypoxia/pVHL/HIF1α pathway plays an important role in the differentiation of stem cells from various organs (4, 5, 40, 47). Our results demonstrate that in vitro, inhibition of differentiation by hypoxia also occurs in the pancreatic progenitors and is mediated by HIF1α. These new findings will help to define tools for the production of β-cells in vitro, a limiting step for a cell-based therapy for diabetes.

Supplementary Material

Supplemental Data
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Acknowledgments

The authors thank Genethon (Evry, France) for providing Ade-GFP-Cre ref.pG3.42155 and Dr. Sylvie Fabrega for the virus amplification. The authors thank Dr. Susan Saint-Just for correcting the English of the manuscript. M.H. and A.S. received support from the French Ministry of Research and Technology.

This study received support from the U.S. National Institutes of Health β-Cell Biology Consortium, the 6th European Union Framework Program β-Cell Therapy Integrated Project, the bilateral program Bundesministerium fur Bildung und Forschung L'Agence Nationale de la Recherche, European Foundation for the Study of Diabetes/NovoNordisk/Juvenile Diabetes Research Foundation, and Société Francophone du Diabéte/Association Française des Diabétiques.

Author contributions: M.H. performed research and analyzed data; A.S., C.B., and M.-T.S., performed research; C.P. provided the transgenic mice and proofread the manuscript; P. R. performed research and proofread the manuscript; R. S. designed research and corrected the manuscript; B. D. designed and performed research, analyzed data, and wrote the manuscript.

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

Abbreviations:
AMPK
adenosine monophosphate-activated protein kinase
CPA
carboxypeptidase A
E
embryonic day
HIF
hypoxia-inducible factor
NGN3
neurogenin 3
PDX1
pancreatic duodenal homeobox 1
pO2
partial pressure of oxygen
pVHL
von Hippel Lindau protein

REFERENCES

  • 1. Fraker C. A., Alvarez S., Papadopoulos P., Giraldo J., Gu W., Ricordi C., Inverardi L., Dominguez-Bendala J. (2007) Enhanced oxygenation promotes beta-cell differentiation in vitro. Stem Cells 25, 3155–3164 [DOI] [PubMed] [Google Scholar]
  • 2. Heinis M., Simon M. T., Ilc K., Mazure N. M., Pouyssegur J., Scharfmann R., Duvillie B. (2010) Oxygen tension regulates pancreatic beta-cell differentiation through hypoxia-inducible factor 1alpha. Diabetes 59, 662–669 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Jarecki J., Johnson E., Krasnow M. A. (1999) Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF. Cell 99, 211–220 [DOI] [PubMed] [Google Scholar]
  • 4. Provot S., Zinyk D., Gunes Y., Kathri R., Le Q., Kronenberg H. M., Johnson R. S., Longaker M. T., Giaccia A. J., Schipani E. (2007) Hif-1alpha regulates differentiation of limb bud mesenchyme and joint development. J. Cell Biol. 177, 451–464 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Schipani E., Ryan H. E., Didrickson S., Kobayashi T., Knight M., Johnson R. S. (2001) Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev. 15, 2865–2876 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Simon M. C., Keith B. (2008) The role of oxygen availability in embryonic development and stem cell function. Nat. Rev. Mol. Cell. Biol. 9, 285–296 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Tian H., Hammer R. E., Matsumoto A. M., Russell D. W., McKnight S. L. (1998) The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development. Genes Dev. 12, 3320–3324 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Shah S. R., Esni F., Jakub A., Paredes J., Lath N., Malek M., Potoka D. A., Prasadan K., Mastroberardino P. G., Shiota C., Guo P., Miller K. A., Hackam D. J., Burns R. C., Tulachan S. S., Gittes G. K. (2011) Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation. Dev. Biol. 349, 342–349 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Semenza G. L. (2007) Life with oxygen. Science 318, 62–64 [DOI] [PubMed] [Google Scholar]
  • 10. Maxwell P. H., Wiesener M. S., Chang G. W., Clifford S. C., Vaux E. C., Cockman M. E., Wykoff C. C., Pugh C. W., Maher E. R., Ratcliffe P. J. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 [DOI] [PubMed] [Google Scholar]
  • 11. Lonser R. R., Glenn G. M., Walther M., Chew E. Y., Libutti S. K., Linehan W. M., Oldfield E. H. (2003) von Hippel-Lindau disease. Lancet 361, 2059–2067 [DOI] [PubMed] [Google Scholar]
  • 12. Brahimi-Horn M. C., Pouyssegur J. (2009) HIF at a glance. J. Cell Sci. 122, 1055–1057 [DOI] [PubMed] [Google Scholar]
  • 13. Walmsley S. R., Print C., Farahi N., Peyssonnaux C., Johnson R. S., Cramer T., Sobolewski A., Condliffe A. M., Cowburn A. S., Johnson N., Chilvers E. R. (2005) Hypoxia-induced neutrophil survival is mediated by HIF-1α-dependent NF-κB activity. J. Exp. Med. 201, 105–115 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Boutin A. T., Weidemann A., Fu Z., Mesropian L., Gradin K., Jamora C., Wiesener M., Eckardt K. U., Koch C. J., Ellies L. G., Haddad G., Haase V. H., Simon M. C., Poellinger L., Powell F. L., Johnson R. S. (2008) Epidermal sensing of oxygen is essential for systemic hypoxic response. Cell 133, 223–234 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Rius J., Guma M., Schachtrup C., Akassoglou K., Zinkernagel A. S., Nizet V., Johnson R. S., Haddad G. G., Karin M. (2008) NF-κB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1α. Nature 453, 807–811 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Heinis M., Simon M. T., Duvillie B. (2010) New insights into endocrine pancreatic development: the role of environmental factors. Horm. Res. Paediatr. 74, 77–82 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Ahlgren U., Jonsson J., Edlund H. (1996) The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development 122, 1409–1416 [DOI] [PubMed] [Google Scholar]
  • 18. Gradwohl G., Dierich A., LeMeur M., Guillemot F. (2000) neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc. Natl. Acad. Sci. U. S. A. 97, 1607–1611 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Scharfmann R. (2000) Control of early development of the pancreas in rodents and humans: implications of signals from the mesenchyme. Diabetologia 43, 1083–1092 [DOI] [PubMed] [Google Scholar]
  • 20. Cantley J., Selman C., Shukla D., Abramov A. Y., Forstreuter F., Esteban M. A., Claret M., Lingard S. J., Clements M., Harten S. K., Asare-Anane H., Batterham R. L., Herrera P. L., Persaud S. J., Duchen M. R., Maxwell P. H., Withers D. J. (2009) Deletion of the von Hippel-Lindau gene in pancreatic beta cells impairs glucose homeostasis in mice. J. Clin. Invest. 119, 125–135 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Puri S., Cano D. A., Hebrok M. (2009) A role for von Hippel-Lindau protein in pancreatic beta-cell function. Diabetes 58, 433–441 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Zehetner J., Danzer C., Collins S., Eckhardt K., Gerber P. A., Ballschmieter P., Galvanovskis J., Shimomura K., Ashcroft F. M., Thorens B., Rorsman P., Krek W. (2008) PVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta cells. Genes Dev. 22, 3135–3146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Cheng K., Ho K., Stokes R., Scott C., Lau S. M., Hawthorne W. J., O'Connell P. J., Loudovaris T., Kay T. W., Kulkarni R. N., Okada T., Wang X. L., Yim S. H., Shah Y., Grey S. T., Biankin A. V., Kench J. G., Laybutt D. R., Gonzalez F. J., Kahn C. R., Gunton J. E. (2010) Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets. J. Clin. Invest. 120, 2171–2183 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Rankin E. B., Tomaszewski J. E., Haase V. H. (2006) Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor. Cancer Res. 66, 2576–2583 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Haase V. H., Glickman J. N., Socolovsky M., Jaenisch R. (2001) Vascular tumors in livers with targeted inactivation of the von Hippel-Lindau tumor suppressor. Proc. Natl. Acad. Sci. U. S. A. 98, 1583–1588 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Biju M. P., Neumann A. K., Bensinger S. J., Johnson R. S., Turka L. A., Haase V. H. (2004) Vhlh gene deletion induces Hif-1-mediated cell death in thymocytes. Mol. Cell. Biol. 24, 9038–9047 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Miralles F., Czernichow P., Scharfmann R. (1998) Follistatin regulates the relative proportions of endocrine versus exocrine tissue during pancreatic development. Development 125, 1017–1024 [DOI] [PubMed] [Google Scholar]
  • 28. Miralles F., Serup P., Cluzeaud F., Vandewalle A., Czernichow P., Scharfmann R. (1999) Characterization of beta cells developed in vitro from rat embryonic pancreatic epithelium. Dev. Dyn. 214, 116–126 [DOI] [PubMed] [Google Scholar]
  • 29. Attali M., Stetsyuk V., Basmaciogullari A., Aiello V., Zanta-Boussif M. A., Duvillie B., Scharfmann R. (2007) Control of beta-cell differentiation by the pancreatic mesenchyme. Diabetes 56, 1248–1258 [DOI] [PubMed] [Google Scholar]
  • 30. Lee K., Lee J. H., Boovanahalli S. K., Jin Y., Lee M., Jin X., Kim J. H., Hong Y. S., Lee J. J. (2007) (Aryloxyacetylamino)benzoic acid analogues: A new class of hypoxia-inducible factor-1 inhibitors. J. Med. Chem. 50, 1675–1684 [DOI] [PubMed] [Google Scholar]
  • 31. Benit P., Goncalves S., Philippe Dassa E., Briere J. J., Martin G., Rustin P. (2006) Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples. Clin. Chim. Acta 374, 81–86 [DOI] [PubMed] [Google Scholar]
  • 32. Guillemain G., Filhoulaud G., Da Silva-Xavier G., Rutter G. A., Scharfmann R. (2007) Glucose is necessary for embryonic pancreatic endocrine cell differentiation. J. Biol. Chem. 282, 15228–15237 [DOI] [PubMed] [Google Scholar]
  • 33. Ginouves A., Ilc K., Macias N., Pouyssegur J., Berra E. (2008) PHDs overactivation during chronic hypoxia “desensitizes” HIFalpha and protects cells from necrosis. Proc. Natl. Acad. Sci. U. S. A. 105, 4745–4750 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Duvillie B., Attali M., Bounacer A., Ravassard P., Basmaciogullari A., Scharfmann R. (2006) The mesenchyme controls the timing of pancreatic beta-cell differentiation. Diabetes 55, 582–589 [DOI] [PubMed] [Google Scholar]
  • 35. Golosow N., Grobstein C. (1962) Epitheliomesenchymal interaction in pancreatic morphogenesis. Dev. Biol. 4, 242–255 [DOI] [PubMed] [Google Scholar]
  • 36. DeBerardinis R. J., Lum J. J., Hatzivassiliou G., Thompson C. B. (2008) The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab. 7, 11–20 [DOI] [PubMed] [Google Scholar]
  • 37. Kahn B. B., Alquier T., Carling D., Hardie D. G. (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 1, 15–25 [DOI] [PubMed] [Google Scholar]
  • 38. Gardner P. R., Nguyen D. D., White C. W. (1994) Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc. Natl. Acad. Sci. U. S. A. 91, 12248–12252 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Li C., Yin T., Dong N., Dong F., Fang X., Qu Y. L., Tan Y., Wu H., Liu Z., Li W. (2011) Oxygen tension affects terminal differentiation of corneal limbal epithelial cells. J. Cell. Physiol. 226, 2429–2437 [DOI] [PubMed] [Google Scholar]
  • 40. Lin Q., Lee Y. J., Yun Z. (2006) Differentiation arrest by hypoxia. J. Biol. Chem. 281, 30678–30683 [DOI] [PubMed] [Google Scholar]
  • 41. Gustafsson M. V., Zheng X., Pereira T., Gradin K., Jin S., Lundkvist J., Ruas J. L., Poellinger L., Lendahl U., Bondesson M. (2005) Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev. Cell 9, 617–628 [DOI] [PubMed] [Google Scholar]
  • 42. Rodesch F., Simon P., Donner C., Jauniaux E. (1992) Oxygen measurements in endometrial and trophoblastic tissues during early pregnancy. Obstet. Gynecol. 80, 283–285 [PubMed] [Google Scholar]
  • 43. Yochim J. M., Mitchell J. A. (1968) Intrauterine oxygen tension in the rat during progestation: its possible relation to carbohydrate metabolism and the regulation of nidation. Endocrinology 83, 706–713 [DOI] [PubMed] [Google Scholar]
  • 44. Cantley J., Grey S. T., Maxwell P. H., Withers D. J. The hypoxia response pathway and beta-cell function. Diabetes Obes. Metab. 12, 159–167 [DOI] [PubMed] [Google Scholar]
  • 45. Frew I. J., Krek W. (2008) pVHL: a multipurpose adaptor protein. Sci. Signal. 1, pe30. [DOI] [PubMed] [Google Scholar]
  • 46. Cejudo-Martin P., Johnson R. S. (2005) A new notch in the HIF belt: how hypoxia impacts differentiation. Dev. Cell 9, 575–576 [DOI] [PubMed] [Google Scholar]
  • 47. Ceradini D. J., Kulkarni A. R., Callaghan M. J., Tepper O. M., Bastidas N., Kleinman M. E., Capla J. M., Galiano R. D., Levine J. P., Gurtner G. C. (2004) Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat. Med. 10, 858–864 [DOI] [PubMed] [Google Scholar]

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