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1 CNRS UMR2501, Laboratoire Génétique Moléculaire, Signalisation et Cancer, Université Claude Bernard Lyon, Lyon F-69008, France2 International Agency for Research on Cancer, 69008 Lyon, France3 INSERM U845, Faculté de Médecine, Centre de Recherche Croissance et Signalisation, Université Paris Descartes, Hôpital Necker, 75015 Paris, France4 Leibniz Institute for Age Research, Fritz Lipmann Institute (FLI), 07745 Jena, Germany5 Faculty of Biology and Pharmacy, Friedlich-Schiller-University, 07743 Jena, Germany
(Correspondence should be addressed to C-X Zhang at Laboratoire Génétique Moléculaire, Signalisation et Cancer, CNRS, UMR5201, Faculté de Médecine, Université Claude Bernard Lyon 1, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France; Email: zhang{at}sante.univ-lyon1.fr)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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- and β-cells during the second transition, we show that loss of menin leads to the failure of endocrine cell development, altered pancreatic structure and a markedly decreased number of cells expressing neurogenin 3, indicating that menin is also required at this stage of the endocrine pancreas development. Taken together, our results suggest that menin plays an indispensable role in the development of the pancreatic endocrine cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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-, β- and pancreatic polypeptide (PP) cells, occur in 30–80% of MEN1 patients and account for the major cause of death in MEN1 patients due to malignancy (Thakker 1998). The loss of heterozygosity observed in MEN1 tumours (Larsson et al. 1988) supports the hypothesis that the MEN1 gene acts as a tumour suppressor in affected cells.
The protein product of the MEN1 gene, menin, has been shown to interact with a substantial number of well-known transcriptional factors and co-factors, including JUND (Agarwal et al. 1999), SMAD1, 3 and 5 (Kaji et al. 2001, Sowa et al. 2004), RELA, NF
B2 and NFB1 of NFB (Heppner et al. 2001), SIN3A (Kim et al. 2003), MLL1 and 2 (Hughes et al. 2004, Yokoyama et al. 2004, Milne et al. 2005), suggesting that menin is involved in transcriptional regulation of gene expression. Indeed, several genes have been identified as being regulated by menin, such as telomerase (Lin & Elledge 2003) and cyclin-dependent kinase inhibitors CDKN2C and CDKN1B (Karnik et al. 2007), as well as several hormones including insulin (Sayo et al. 2002), prolactin (Namihira et al. 2002) and parathyroid hormone (Sowa et al. 2004). However, little is known about the in vivo physiological function of menin, and the in vivo importance of the above-mentioned interactions between menin and its protein partners, especially in endocrine cells that are affected in MEN1 disease. It should be noted that Kaji et al. (2001) has shown that menin is required for TGFB1-induced ex vivo growth inhibition of anterior pituitary tumour cells and a similar observation has been made by Sowa et al. (2004) in primary parathyroid cells. By disrupting Men1 in mice, we have previously shown that menin was required for the development of several organs and tissues, such as neural tube and liver (Bertolino et al. 2003a). Consequently this elicits the question about the possible involvement of menin in the development of the pancreatic endocrine cells. Intriguingly, β-cell specific Men1 mutant mice showed normal islet structures at an early age (Bertolino et al. 2003c, Crabtree et al. 2003, Biondi et al. 2004). However, one can hardly exclude the possibility that this may be due to partial Cre-mediated gene disruptions among targeted cells during the embryonic stage. Alternatively, menin may play its role before Rip-Cre recombinase (insulin) expression, similar to the genes whose function in the pancreas development is needed only during a narrow period, as in the case of β-catenin (Heiser et al. 2006).
Substantial advances in the study of development of the pancreatic endocrine cells have been achieved in recent years. The current available data support the notion that sequential activation or repression (Edlund 2002, Wilson et al. 2003) of transcriptional factors and co-factors may play a central role in the control of pancreas development. Indeed, the endodermal region committed to pancreas development first expresses pancreatic–duodenal homeobox 1 (PDX1), detected starting from E8.5 in the mouse embryos in early progenitors and restricted to the β-cells at the late developmental stage (Guz et al. 1995). Targeted disruption of the Pdx1 gene in mice as well as homozygous mutation in human causes pancreas agenesis (Ahlgren et al. 1996, Stoffers et al. 1997), indicating its essential function in the morphogenesis and differentiation of the pancreas. The basic helix–loop–helix transcription factor neurogenin 3 (NEUROG3) is expressed transiently in the pancreatic endocrine progenitors (Apelqvist et al. 1999). Neurog3-deficient mice lack all pancreatic endocrine cell types (Gradwohl et al. 2000), indicating that it is required for differentiation of these cells. Further identification of the factors or co-factors involved in endocrine cell development would help to better understand the mechanisms implicated in this procedure. Considering the advance in the development of the pancreatic endocrine cells and available approaches allowing overcoming the lethality of Men1 null embryos, we decided to determine the possible involvement of menin in the development of the pancreatic endocrine cells. In the current study, we have combined different genetic approaches to investigate the role of menin in pancreas development of the mouse. Our data provide compelling evidence showing that menin is essential for the development of the pancreatic endocrine cells and its absence results in the deficient formation of NEUROG3-positive cells, a key factor in this process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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Mice carrying the targeted Men1 allele (Men1T/T) or the deleted Men1 allele (Men1
/) were used in the present study. Both genotypes are null mutation in mice as described previously (Bertolino et al. 2003a). All animal experiments were conducted in accordance with the standards of human animal care and were approved by IARC's Animal Care and Use Committee.
For establishing Men1 null ES cell lines, Men1+/T mice were inter-crossed. Blastocysts were flushed from the uteri of pregnant female mice at day 3.5 post coïtum and cultured individually on a murine fibroblastic cell layer for 5 days, in Dulbecco's modified Eagle's medium (15% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM β-mercaptoethanol and 10 ng/ml murine leukaemia inhibitory factor (Chemicon International, Temecula, CA, USA). The blastocysts were then dissociated with trypsin/EDTA, and put into culture for 7 days on a murine fibroblastic cell layer. Finally, the ES cells were amplified and karyotyped. To generate chimeric mice, two clones each of Men1 wild-type and Men1T/T ES cells were microinjected into blastocysts of C57BL/6 mice and transferred into pseudo-pregnant females.
Pancreas bud culture
The live embryos were harvested on E12.5. The pancreatic primordium was dissected and cultured on a filter (Millipore, Guyancourt, France) with RPMI 1640 medium (Invitrogen) containing penicillin (100 U/ml), streptomycin (100 µg/ml), HEPES (10 mmol/l), L-glutamine (2 mmol/l), non-essential amino acids (1x, Gibco), and 10% heat-inactivated serum (Hyclone, Logan, UT, USA) was added. Cultures were maintained at 37 °C in humidified 95% air, 5% CO2.
Mouse islet isolation
Pancreatic islets were isolated from mice according to the protocol previously described (Bertolino et al. 2003c). Briefly, 2 ml of 1 mg/ml collagenase (SERVA, Heidelberg, Germany) in Hank's buffered saline solution was injected into the pancreas through the bile duct. Pancreases were then removed and incubated for 20 min at 37 °C and dissociated by mechanical pipetting. Islets were hand-picked from dark-field dishes under a dissecting microscopy.
Tissue collection and fixation
Tissues of chimeric mice were collected between 3 and 4 weeks after birth. Embryos were collected at different embryonic stages between E11.5 and E18.5. These tissues were fixed in 4% buffered formaldehyde and embedded in paraffin. The pancreatic rudiments were fixed at the end of the culture period in 4% buffered formaldehyde, pre-embedded in agarose (4% of type VII low gelling temperature agarose (Sigma)), followed by embedding in paraffin.
Histological, immunohistochemical and immunofluorescence analyses
For embryos and the pancreas of the chimeras, 3 µm sections were used either for staining with haematoxylin and eosin for histological analyses or for immmunohistochemistry (IHC) and immunofluorescence (IF) staining as described previously (Bertolino et al. 2003b). Primary antibodies used were goat anti-menin C19 (1:500, Santa Cruz, CA, USA), rabbit anti-PDX1 (1:10 000 (Duvillie et al. 2003)) and mouse monoclonal anti-NEUROG3 (clone F25A1B3, 1:9000, DSHB). When using mouse antibodies as primary antibodies, Vector M.O.M. immunodetection kit was used (Vector Lab, Burlingame, CA, USA). For 5-bromodeoxyuridine (BrdU) analysis, bud cultures were incubated for 1 h with 10 µM BrdU (Sigma) before tissue fixation, then BrdU incorporation was evaluated by IHC using anti-BrdU antibody (Clone BU33 – Sigma). Primary antibodies used for IF experiments were guinea pig anti-insulin (1:500, Dako, Capintaria, CA, USA), rabbit anti-glucagon (1:600, NovoCastra, Milton Keynes, UK), anti-mucin-1 (1:200, Neomarkers, Fremont, CA, USA) and goat anti-menin (C19, 1:500).
For cultured pancreatic rudiments, 4 µm sections were processed for IF staining as previously described (Bhushan et al. 2001). The following antibodies were used: mouse anti-human insulin (1/2000, Sigma), rabbit anti-glucagon (1:1000, Diasorin, Stillwater, MN, USA), and rabbit anti-carboxypeptidase A (CPA1; 1:600, Biogenesis, Poole, UK). Quantification of the insulin- and glucagon-positive cells was performed with an IPLab 3.7 Eval software.
Apoptosis was examined on tissue sections by TUNEL staining using the ‘ in situ cell death detection ’ kit (Roche) and counterstained with DAPI (Vector Lab). Double TUNEL and glucagons analysis was done by combining the above-mentioned corresponding protocols.
In situ hybridization
For in situ hybridization, embryo tissues were fixed at 4 °C in 4% paraformaldehyde in PBS, cryoprotected in 15% sucrose–7.5% gelatin in PBS and frozen at –50 °C in isopentane. Cryosections (14 µm in sickness) were prepared. The Neurog3 probe (726 bp) was prepared as previously described (Ravassard et al. 1997). Plasmids were linearized and used as templates for synthesizing sense or antisense riboprobes using T7 or SP6 RNA polymerase (Roche), in the presence of digoxygenin-UTP (Roche). In situ hybridization was performed as described previously (Duvillie et al. 2006), and colorimetric revelation was performed with 5-bromo-4-chloro-3-indolyl phosphate (Promega) and nitroblue tetrazolium (Roche), to obtain a blue precipitate. Photographs were taken using a Hamamatsu C5810 cooled 3CCD camera. No signal was obtained when a sense riboprobe was used.
PCR and quantitative RT-PCR
For PCR analysis of chimerism, genomic DNA was isolated from chimeric tissues at age of 3 weeks. The following primers were used: NEO1-FW: 5'-GATGGATTGCACGCAGGTTC-3'; NEO1-RV: 5'-CAGGTCGGTCTTGACAAAAAG-3'; B2-FW: 5'-CACCGGAGAATGGGAAGCCGAA-3'; B2-RV: 5'-TCCACACAGATGGAGCGTCCAG-3'.
For RT-PCR analysis, total RNA was prepared from E12.5 wild-type and Men1/ pancreas buds using Tri-Reagent (Sigma) according to the protocol provided by the manufacturer. Total RNA (0.5 µg) was reverse transcribed with 0.5 µg oligo-dT using M-MLV (Invitrogen). First-strand cDNA was used for real-time PCR with the Light cycler instrument, except for Neurog3, and FastStart DNA Master SYBR Green I kit (Roche). Primers were designed with Primer3 software (primer3_www.cgi v 0.2 – Whitehead Institute/MIT, Cambridge, MA, USA) and were as follows: HES1-FW: 5'-GAAAGATAGCTCCCGGCATT-3'; HES1-RV: 5'-ACCTCGTTCATGCACTCGC-3'; PDX1-FW: 5'-CATCTCCCCATACGAAGTGC-3'; PDX1-RV: 5'-TGTAGGCAGTACGGGTCCTC-3'; ONECUT1-FW: 5'-AGACCTTCCGGAGGATGTG-3'; ONECUT1-RV: 5'-TTGGACGGACGCTTATTTTC-3'; FOXA2-FW: 5'-GACATACCGACGCAGCTACA-3'; FOXA2-RV: 5'-GAGAAAGCAGTCGTTGAAGG-3'; HPRT1-FW: 5'-TGTTGTTGGATATGCCCTTG-3'; HPRT1-RV: 5'-AACTTGCGCTCATCTTAGGC-3'. Serially diluted cDNA samples or PCR products were used to calculate the efficiency of each PCR with RealQuant software (Roche). After an initial Taq activation at 95 °C for 10 min, Light cycler PCR was performed using 45 cycles with the following cycling conditions: 95 °C for 15 s, 62 °C for 5 s, and 72 °C for 16 s. The Hprt1 transcript was used as internal standard to control the amplification variations.
For real-time PCR of Neurog3, we used the primers as followed: NEUROG3-FW 5'-TTCGCCCACAACTACATCTG-3'; NEUROG3-RV 5'-TTGGGAGACTGGGGAGTAGA-3', cyclophilin A-FW 5'-CAGGTCCTGGCATCTTGTTC-3'; cyclophilin A-RV 5'-TTGCTGGTCTTGCCATTCCT-3' using a SYBR Green PCR master Mix, Applied Biosystem (Foster City, CA, USA) according to the instructions of the manufacturer. For both PCR analyses, the mean of the control samples was set as 1 to normalize the results.
Statistical analysis
All values are expressed as means±S.D. Statistical significance was determined by using the two-tailed unpaired Student's t-test, and differences were considered to be statistically significant when P<0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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To investigate the role of menin in pancreas development in the mouse, we first analysed the menin expression pattern in embryonic pancreata of wild-type mice at different developmental stages respectively at E11.5, E12.5, E13.5, E15.5, E16.5 and E18.5. Menin protein was detected by immunostaining with anti-menin antibodies in the entire pancreatic epithelium and surrounding tissues throughout the developmental stages (Fig. 1A–F). To ensure that menin is expressed in both endocrine and ductal cells, which constitute minor cellular compartments in pancreata, we performed IF staining of menin in combination with the pancreatic cell-specific markers on E16.5 pancreata. The results showed that menin was detected in insulin- and glucagon-expressing endocrine cells as well as Mucin1-expressing ductal cells (Fig. 1G–I), indicating that menin is expressed in these cell populations in developing pancreas.
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We performed histological and IHC analyses on E12.5 pancreata, prior to the embryonic lethality of Men1 null embryos (Bertolino et al. 2003a, Crabtree et al. 2003). At this stage, pancreases undergo the first transition of endocrine cell differentiation and display glucagon-positive cells. The viability of all the analysed embryos was assessed by heart-beating. Both histological and IHC analyses were initially carried out at more than three different positions for each embryo. For those embryos where no glucagon staining was detected, the remaining tissues have been further subjected to the same analysis to confirm the result. The whole pancreata appeared histologically normal in E12.5 Men1 null embryos (Fig. 2B and C), the branching of pancreatic bud rudiment being not significantly different from that seen in control embryos (Fig. 2A). However, the glucagon-expressing cells were either absent, in four out of seven E12.5 Men1 null pancreata (Fig. 2E), or very few (Fig. 2F) in the remaining (three out of seven) compared with Men1 wild-type embryos. These observations suggest that menin is required to support the differentiation or the survival of glucagon-expressing cells at this stage. To investigate whether the reduced number of glucagon-positive cells is associated with apoptosis, TUNEL assay was applied to analyse these developing pancreata. Indeed, apoptotic cells were detected in three out of four E12.5 Men1 null embryos devoid of glucagon-positive cells, whereas they were completely absent in control embryos (Fig. 2G–L, Table 1). To further determine whether the absence of menin may trigger apoptosis in glucagon-positive cells, we carried out glucagon and TUNEL double staining. Indeed, a substantial proportion of glucagons-positive cells (70%) were found apoptotic in five Men1 null pancreata having glucagon-positive cells (Fig. 2M–P). Such double-positive cells were not seen in four Men1 wild-type control. These results suggest that menin prevents apoptosis in glucagon-secreting cells at this stage.
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Men1 null mutant ES cells failed to contribute normally to the formation of the pancreatic endocrine cells in chimeric mice
To determine whether the role of menin in the development of the pancreatic endocrine cells was due to the cell autonomous effects of menin, we next examined the capacity of Men1 null ES cells to differentiate into the pancreatic endocrine cells by chimerism assay. To this end, two Men1 wild-type and two Men1 null ES cell lines were independently established from blastocysts derived from inter-crosses of heterozygous Men1 mutant mice. After ascertaining their karyotype and ensuring that their gene expression profile corresponded to totipotent ES cells (Supplemental Fig. 1, see Supplementary data in the online of version of the Journal of Endocrinology at http://joe.endocrinology-journals.org/content/vol199/issue2/), they were injected into Men1 wild-type C57BL/6 blastocysts to generate chimeric mice. The proportion of Men1 null ES cell derivatives in the pancreatic islets of 3-week-old chimeric mice was then assessed by IHC (Fig. 3B–F and K) and DNA genotyping respectively (Fig. 3L). IHC analysis of six chimeric mice generated with Men1 null ES cells using anti-menin antibodies revealed that, while mixed populations of menin-positive and -negative cells could be detected in the pancreatic exocrine compartment and in adjacent tissues (Fig. 3B and C), the islets were mainly composed of menin positively stained cells (Fig. 3D–F). Quantification of the chimerism by IHC analysis showed that more than 85% of islets contain more than 80% of menin-positive cells (Fig. 3K). In these islets, the lack of Men1 null ES cell derivatives was seen both in - and β-cells, as demonstrated by double IF staining (Fig. 3G–J). The data indicate that the majority of Men1 null ES cells failed to contribute to the formation of pancreatic islet cells.
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Menin is required for the normal development of the pancreatic endocrine, but not exocrine cells in pancreatic bud cultures
The embryonic lethality of Men1 null embryos after E12.5 hampers the investigation of menin's role in the second transition of the endocrine pancreas. Therefore, to address this issue, we performed the pancreatic bud culture, which allows recapitulating of the second transition of endocrine cell differentiation (Duvillie et al. 2003, 2006, Attali et al. 2007). It was previously established that the cultured pancreases can develop exocrine and endocrine cells in the defined conditions, and the in vitro expression pattern of the major transcription factors found in the cultured pancreases, including NKX6-2, NEUROG3, PAX4, NEUROD1 and the markers of the mature β-cells PCSK1, PCSK2 and ABCC8, were similar to that found in vivo (Attali et al. 2007). Two independent series of pancreatic bud cultures using E12.5 Men1 wild-type (n=7) and Men1 null (n=6) heart-beating embryos were carried out. The development of endocrine and exocrine cells was evaluated after 7 days culture. Insulin/CPA1 double immunostaining revealed that, compared with the controls, Men1 null pancreatic buds exhibited a significant reduction in the number of insulin-positive cells, while CPA1 staining for exocrine cells in the same sample was normal, indicating that the absence of menin resulted in failure of endocrine cell development without affecting that of exocrine pancreas (Fig. 4A–H and Q). Importantly, by insulin/glucagon double immunostaining, we found that the development of both - and β-cells was clearly affected in Men1 null cultured pancreatic buds (Fig. 4I–P, and R), indicating that the need of menin is therefore not restricted to a specific type of pancreatic endocrine cells. These findings are consistent with the observation made by IHC and IF analysis in chimeric mice.
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We noticed that a substantial proportion (seven out of nine) of the Men1 null pancreatic buds cultured at day 7 formed cystic structures that varied in size in different cultured buds (Fig. 5A–C). Similar cysts were not seen in Men1 wild-type control (Fig. 5A). The formation of cystic structures in pancreas is considered to be a consequence of abnormal cellular differentiation of either ductal or endocrine cells (Cano et al. 2004, Pierreux et al. 2006). Indeed, we found that cells lining the cysts in Men1 null pancreatic bud culture were negatively stained with the antibodies against PDX1 and Mucin-1 respectively the mature pancreatic β-cell and ductal cell markers (Fig. 5D–F and G–L). Further analysis showed the lack of both insulin- and glucagon-secreting cells within large cystic structures in cultured Men1 null pancreatic buds at day 7 (Supplemental Fig. 4, see Supplementary data in the online of version of the Journal of Endocrinology at http://joe.endocrinology-journals.org/content/vol199/issue2/). All these data indicate that the cysts are delineated by an epithelium that follows neither mature - and β-cell nor ductal cell fate. Our work demonstrates that the lack of menin altered the cellular differentiation of the pancreatic cells.
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To investigate the possible molecular and cellular events leading to the aberrant pancreatic endocrine cell development in the absence of menin in bud culture, we analysed the number of cells expressing Neurog3, a key factor for the pancreatic endocrine cell development (Gradwohl et al. 2000) after 2 days of culture, where its expression reaches the highest level in normal cultured buds. We found that the number of Neurog3-expressing cells was markedly decreased in day 2 Men1 null pancreatic bud cultures (n=5) compared with Men1 wild-type controls (n=4; Fig. 6A–E). On the other hand, neither the cell proliferation analysed by BrdU staining (Fig. 6F and G), nor the cell death determined by TUNEL assay (Fig. 6H–M) showed any difference between Men1 null and wild-type cultured pancreatic buds at this time point. We also noticed that there was no obvious morphological alteration in day 2 Men1 null cultured pancreatic buds (data not shown). The abnormal pancreatic endocrine cell development and the decreased number of Neurog3-expressing cells in cultured Men1 null pancreatic buds are reminiscent of the phenotype of Neurog3 mutant mice (Gradwohl et al. 2000). The data suggest that the reduced Neurog3-expressing cell number in the Men1 null pancreatic bud cells could be the primary event leading to the observed defects.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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-, β- and PP cells, whereas its in vivo physiological function in these cells remains to be elucidated. Using different genetic approaches, we show that menin is a novel factor essential for the development of the pancreatic endocrine cells, including both - and β-cells. We also demonstrate that menin is required neither for the formation of the pancreatic buds nor for development of the exocrine pancreas, indicating clearly that the effect of menin in the pancreas is endocrine cell specific. More importantly, chimerism analysis demonstrates that the presence of surrounding menin-expressing cells is not sufficient to allow the Men1 null ES derivatives to develop into the islet cells, indicating an intrinsic need of menin in the pancreatic endocrine cells. Finally, we found that the inactivation of the Men1 gene affects the development of the pancreatic endocrine cells before the second transition of cellular differentiation of these cells. Our data thus indicate the importance of menin in both the early glucagon-expressing endocrine cells and the initial steps of the major pancreatic endocrine cell development. However, the contribution of menin to the later stage of the major endocrine pancreas development could not be assessed in the current work. This would require either the application of siRNA in late wild-type pancreatic bud explants or temporally inducible disruption of a floxed Men1 allele. Also, we noticed that a small number of islets from chimeric mice injected with Men1 null ES cells, less than 2% of examined islets, show a near-equal proportion of menin-positive and -negative cells. This, along with the observed presence of diminished, but not entirely absent, glucagon-secreting cells in some of the E12.5 Men1–/– embryonic pancreas, may suggest that the requirement of menin is either not absolute or can be biased by the occurrence of altered activities of other factors upon menin inactivation. The reduced number of glucagon-expressing cells and the apoptotic glucagon positive cells were seen in E12.5 Men1 null pancreata (under the first endocrine cell differentiation transition), suggesting that menin could play a role in cell survival in these cells at this early developmental stage. However, we did not observe any alteration of cell death in Men1 null pancreata cultured for 2 days, indicating that menin's role may imply different cellular functions in the second endocrine cell differentiation transition. Indeed, our data strongly suggest that menin is needed for the formation of NEUROG3-expressing cells during the crucial step of the second pancreatic transition where the progenitor cells commit to endocrine differentiation. We thus attribute the default of endocrine development in the cultured Men1 null embryos to the lack of activated NEUROG3 expression rather than cell death.
Transgenic lineage tracing studies demonstrated that the early glucagon-expressing cells are unlikely to be the progenitors of later mature endocrine cells (Herrera 2000). Therefore, the developmental failure of the pancreatic endocrine cells at the second transition in the absence of menin cannot be simply explained by the lack of early glucagon-expressing cells. Conversely, we noticed that cystic structures appeared in cultured Men1 null pancreatic buds, which are delineated with aberrantly differentiated cells. Considering the occurrence of deficient endocrine pancreas development, the formation of cystic structures in Men1 null pancreatic bud cultures could be the consequence of abnormal cellular differentiation of cells lacking menin expression.
We did not detect any altered expression of major genes involved in the control of early pancreas development in E12.5 Men1 null pancreata, including Pdx1 (Jonsson et al. 1994), Foxa2 (Lee et al. 2001), Onecut1 (Jacquemin et al. 2000) and Hes1 (Jensen et al. 2000), as well as Neurog3. However, the number of Neurog3-expressing cells, fated to become endocrine cells during the second endocrine cell differentiation transition (Edlund 2002, Wilson et al. 2003), was severely decreased in day 2 Men1 null pancreatic bud cultures. This discrepancy may be due to the different molecular mechanisms involved respectively in the first and the second pancreatic endocrine cell differentiation transitions. As menin is closely related to transcriptional regulation, it is plausible that menin is needed, directly or indirectly, for normal NEUROG3 expression at this second stage of endocrine pancreas development, by acting upstream of NEUROG3. Further studies are needed to determine in detail the role played by menin in regulating NEUROG3 expression and the fate of NEUROG3-expressing cells. We noticed that none of the currently known protein partners of menin has been reported to be involved in the regulation of the Neurog3 gene. Recently, Smart et al. (2006) has reported that the TGFB1 pathway plays an important biological role in pancreas development, but mainly in maturation of differentiating β-cells. Obviously, the interaction of menin with SMAD proteins alone is not sufficient to explain its role found in this study, although we cannot exclude the possibility that it may participate in the TGFB1 pathway at the later stage of pancreas development.
Among numerous factors implicated in the development of the pancreatic endocrine cells, MEN1 is perhaps the first gene that is identified to be involved both in development and tumorigenesis of pancreatic endocrine cells. The seemingly contradictory biological effects of menin during embryonic development and the adult life of pancreatic endocrine cells indicate that menin plays fundamental but different roles during these two periods. This is reminiscent of another tumour suppressor, the Rb gene, which is not only responsible for the occurrence of retinoblastoma when inactivated, but also critically required for normal retinal development during the embryogenesis (MacPherson et al. 2004). Our data are also in consistency with the recent findings revealing multifaceted biological functions of menin in different cells and tissues reported by other groups (Sowa et al. 2003, Yokoyama et al. 2005). In particular, Sowa et al. (2003) have demonstrated that menin is required for the commitment of mesenchymal stem cells into the osteoblast lineage. By addressing the mechanisms of menin's essential function in the development of the pancreatic endocrine cells, one may know better physiological functions of menin, especially those implicated in the control of cellular differentiation and proliferation in endocrine cells. On the other hand, albeit identification of numerous factors responsible for the development of the pancreatic endocrine cells, many biological aspects of these cells remain to be elucidated. The identification of menin's role in this procedure should be of help not only for a better understanding of these cells, but also for the development of new therapeutic strategies for the treatment of the diseases affecting the pancreatic endocrine cells, including both tumours and metabolic disorders.
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Declaration of interest |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of interestReferences |
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Funding |
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Acknowledgements |
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References |
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Received in final form 28 August 2008
Accepted 1 September 2008
Made available online as an Accepted Preprint 3 September 2008
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