Article |
Address correspondence to Harry Heimberg, Diabetes Research Center, Brussels Free University (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium. Tel.: 32-2-477-4548. Fax: 32-2-477-4545. E-mail: hheimber{at}vub.vub.ac.be
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: neurogenin 3; islets of langerhans; transdifferentiation; insulin; diabetes mellitus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments with transgenic mice have indicated key factors in the embryonic development of their endocrine pancreas. Analysis of null mutants for Pdx1/Ipf1, ngn3, NeuroD/ß2, Pax4, Nkx2.2, Nkx6.1, or Pax6 has identified a hierarchy of transcription factors that control embryonic formation of pancreatic islets (for review see Sander and German, 1997; Jensen et al., 2000a; Edlund, 2001). It is unknown whether these factors play a role in the postnatal growth of the pancreatic ß-cell mass and whether they can be used to induce formation of human ß-cells from postnatal nonendocrine cells. In this perspective, we examined the endocrinogenic potential of the class B basic helix-loop-helix (bHLH)* transcription factor neurogenin 3 (ngn3), which seems to function as a major and timely switch in the rodent embryonic pancreas (Gradwohl et al., 2000). When expression of ngn3 is directed ectopically into the embryonic epithelium, pancreas precursor cells develop prematurely and exclusively into glucagon-producing cells (Apelqvist et al., 1999; Schwitzgebel et al., 2000). Similarly, ngn3 induced premature differentiation into glucagon- and somatostatin-producing cells when introduced into early chicken endoderm (Grapin-Botton et al., 2001). The failure to induce insulin-producing cells might indicate that these immature cells lack the competence to drive ß-cell differentiation. The differentiating activity of ngn3 is under control of Notch signaling. Indeed, null mutant mice for the Notch ligand Dll1, for an intracellular mediator of Notch signaling RBP-J (Apelqvist et al., 1999), or for a downstream bHLH repressor HES1 (Jensen et al., 2000b), which possibly controls ngn3 (Tanabe and Jessell, 1996), all show premature endocrine differentiation. Therefore, we ectopically expressed ngn3 in adult duct cells to assess its role as a switch activating the expression of other developmental transcription factors and Delta-Notch proteins and consequently resulting in the appearance of endocrine differentiation markers, in particular insulin.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Ngn3 induces expression of neuroendocrine markers, in particular insulin, in postnatal human pancreatic duct cells
Control duct cell preparations were negative by RT-PCR analysis for the endocrine cell markers synaptophysin, chromogranin A, and prohormone convertases (PC1/3 and PC2), and for the islet cell markers insulin, somatostatin, glucagon, glucose transporter type II, and glucokinase. Transcripts encoding these proteins were clearly present in the human islet cell fraction (Fig. 5 A; not depicted). 10 d after infection with Adngn3 or AdNeuroD/ß2, duct cell preparations exhibited a strong activation of synaptophysin, chromogranin A, and PC1/3 gene expression, and a weaker increase for the glucokinase and insulin genes (Fig. 5 A). No signals were detected for Glut2, somatostatin, or glucagon transcripts. These effects of ectopic ngn3 expression were also reflected at the protein level (Fig. 5 B). The fraction of cells that were immunopositive for insulin and synaptophysin increased from 1% in control preparations to, respectively, 13 and 22% 10 d after Adngn3 infection. The percentage of glucagon- or somatostatin-positive cells remained under 2% (unpublished data). However, although ectopic ngn3 did not affect the number of glucagon-containing cells, it increased the percentage of somatostatin-positive cells 3.5-fold (0.2 ± 0.1% versus 0.7 ± 0.1%, P < 0.05; n = 4). A similar effect was observed after infection with AdNeuroD/ß2. Interestingly, most cells that expressed these endocrine markers were negative for ngn3 (Fig. 5 B, b, c, and g, arrowheads point to single positive cells). At this time point, the percentage of ngn3-positive cells was markedly lower than at day 3. Furthermore, the rare ngn3-positive cells that coexpressed endocrine markers exhibited a much lower ngn3 fluorescence intensity than those that did not (Fig. 5 B, b and g, arrows).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is unknown presently whether this forced transdifferentiation is restricted to a subpopulation of duct cells. A major proportion of ngn3-infected cells expressed the (neuro)endocrine markers, but only a fraction was insulin positive. The sequential activation of a comprehensive set of (neuro)endocrine-specific genes rather than the existence of (sub)populations of cells expressing only few individual markers is characteristic for the induction of a coordinated differentiation program. Based on its rapid nature and independence of cell proliferation (unpublished data), this process likely represents immediate transdifferentiation of the duct cells without the need for an intermediate cellular state (Shen et al., 2000; Slack and Tosh, 2001). Transdifferentiated cells are characterized by coexpression of the duct cell marker CK19 and the neuroendocrine marker synaptophysin. Such double positive cells are virtually absent in noninfected duct cell preparations. The expression levels of both markers changed gradually and reciprocally with time after infection. Only little cytokeratin positivity was left in the cells that finally expressed insulin. A combination of the transient nature of the adenoviral expression system and the elimination of fluorescence by GFP after extended fixation allowed simple tracing of cell fate. A direct relation between the ngn3-infected duct cells and the cells that became positive for endocrine markers was observed. Rare cells coexpressed endocrine marker proteins and traces of ngn3, suggesting a causal relationship between expression of ngn3 and the endocrine protein. Furthermore, several endocrine cells still contained the stable green fluorescent protein that remains present for days after its transcript, and consequently the mRNA encoding the less stable ngn3 protein, have disappeared (Corish and Tyler-Smith, 1999).
The mechanism whereby ngn3 induces duct cell differentiation into endocrine ß-cells seems to involve activation of the Pax4 and the NeuroD/ß2 promotor. Ngn3 is known to activate NeuroD/ß2 expression in chicken embryos (Grapin-Botton et al., 2001), Xenopus embryos, and (neuro)endocrine cell lines (Huang et al., 2000). This is also the case in HeLa cells that had been transfected with E47, a class A bHLH heterodimerization partner of ngn3 (Huang et al., 2000). Adult human duct cells contain high endogenous levels of E47 (unpublished data), allowing E-box binding of the ectopically expressed ngn3 and activation of the NeuroD/ß2 promotor. The delayed induction of NeuroD/ß2 by ngn3 suggests the existence of intermediate transcription factors. Moreover, the delay in NeuroD/ß2-induced Pax4 activation compared with ngn3 uncovers that both Pax4 and NeuroD/ß2 are ngn3 targets, instead of Pax4 being downstream of NeuroD/ß2. The present study thus supplements the hierarchy model of transcription factors involved in the formation of embryonic ß-cells (Schwitzgebel et al., 2000). It also demonstrates that the embryological program in mice can be recapitulated in postnatal human duct cells, leading to formation of insulin, and to a minor extent somatostatin-expressing cells. In experimental terms, adult duct cells infected with adenoviruses expressing recombinant transcription factors are a simple in vitro model for studying the molecular biology of endocrine transdifferentiation.
Ectopic expression of ngn3 or NeuroD/ß2 in isolated adult duct cells activates several (neuro)endocrine-specific genes, such as insulin and somatostatin, but not glucagon. In mouse or chicken embryonic endoderm cells in vivo, ectopic ngn3 induces glucagon but not insulin (Grapin-Botton et al., 2001). Activation of ß-cellspecific Pax4 in duct cells might be responsible for the absence of glucagon expression (Smith et al., 1999; Petersen et al., 2000). The role of Nkx2.2 and Nkx6.1 in this transdifferentiation process is unclear: Nkx2.2 was stimulated by ngn3 at the transcriptional level, and Nkx6.1 was stimulated at the posttranscriptional level. Both transcription factors appear essential during embryonic development of ß-cells, with Nkx2.2 acting upstream of Nkx6.1 (Sander et al., 2000). Adngn3-infected duct cells failed to generate glucose-induced insulin release within the limits of the present study, i.e., 10 d after infection (unpublished data). Our study can thus not be taken as evidence for the ability to produce functionally mature ß-cells from pancreatic duct cells. Nevertheless, the data are indicative for a selective ability of adult duct cells to differentiate toward (neuro)endocrine and islet cells. Ngn3 induced moderate to high expression of synaptophysin, chromogranin A, PC1/3, and glucokinase, but the degree of insulin gene activation is low and so is the cellular insulin content. Transdifferentiated pancreatic duct cells exhibit the ultrastructural characteristics of immature endocrine cells, which is consistent with the absence of a glucose-regulated secretory activity. The absence of Glut2 induction is compatible with the earlier report that Glut2 is poorly expressed in human ß-cells compared with human liver cells or rodent ß-cells (De Vos et al., 1995).
The incomplete differentiation of this particular phenotype might be caused by, or related to, a variety of factors. (a) Although Pax4 is subject to autoregulation in mouse islet cell lines (Smith et al., 2000), ngn3-induced activation of Pax4 in human duct cells was not. This allows the relatively high levels of Pax4 to exert their inhibitory action on insulin gene expression (Qiu et al., 1998; Smith et al., 1999). Low levels of Pax4 also occurred in normal adult human islet cells in contrast to its restriction to the embryonic mice pancreas (Sosa-Pineda et al., 1997; Smith et al., 1999, 2000). It is likely that in mature islet ß-cells a yet unknown factor overrules the Pax4 repressor activity. Given the transient presence of ngn3 and Pax4 in the embryonic mouse pancreas, it needs to be investigated whether a closer simulation of the embryonic situation by conditional expression of ngn3 in adult human duct cells would augment the insulin levels. (b) The state of the Delta-Notch pathway probably also has its influence on the degree of (neuro)endocrinogenesis. In uninfected duct cells, high levels of Notch1, 2, and 3 but not of Dll1, 3, and 4 were found. Ngn3 infection activated transcription of Dll1 and Dll4 and thus increased Notch signaling in neighboring pairs, which is expected to limit overall endocrine differentiation. It may thus be useful to add antimorphic forms of Delta that antagonize Notch signaling (Jen et al., 1997) or to introduce ngn3 targets that bypass stimulation of Delta genes in order to specifically drive the formation of endocrine cells. (c) The transdifferentiated cells likely are dependent on extracellular factors to promote their maturation. (d) Finally, repression of specific proneural regulators induced by ngn3 may be necessary to allow full endocrine differentiation. Although the transdifferentiated duct cells did not express genes that label differentiated neuronal cells (N-Cam, neurofilament, peripherin, and class III ß-tubulin [unpublished data]), major overlaps exist between the expression profile of endocrine pancreas and neurons (Atouf et al., 1997; unpublished data). These similarities were key to design conditions that drive embryonic stem cells to insulin production (Lumelsky et al., 2001). Moreover, insulin-producing cells are present in primary cell cultures from mammalian fetal brain (Clarke et al., 1986), and insulin-producing neurons in Drosophila brain show remarkable similarities with ß-cells in mammalian islets of Langerhans (Rulifson et al., 2002). All these data are highly suggestive for a common ancestral insulin-producing cell of neural origin, which complicates the identification of specific proneural factors that are nonessential for islet cell differentiation.
In more general terms, the present findings extend the role of lateral inhibition in cell differentiation from embryonic, poorly differentiated tissues (Artavanis-Tsakonas et al., 1999) to postnatal differentiated cell populations. Adult human duct cells were shown to transdifferentiate into neuroendocrine and into insulin-producing cells after ectopic expression of ngn3. Pancreatic duct and islet cells have a common embryonic progenitor, but according to a recent lineage tracing study ngn3 is never expressed in duct cells or their progenitors (Gu et al., 2002). However, our current study shows that ngn3 might serve as a master switch that drives transdifferentiation to a (neuro)endocrine phenotype when misexpressed in adult duct cells. It is still unknown whether ngn3 can be activated by environmental factors in normal duct cells of regenerating postnatal pancreas and thus result in the formation of new ß-cells. Current efforts focus on finding ways to differentiate embryonic stem cells into insulin-producing cells with the purpose of producing sufficient cells for transplantation in diabetes (Assady et al., 2001; Lumelsky et al., 2001). Our observations have indicated mechanisms through which adult duct cells could be forced to differentiate into insulin-producing cells. The abundance of duct cells and the ease of their in vitro manipulation support further attempts to explore their potential as a source for new ß-cells.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell isolation and cell culture
The duct cells in this study were obtained from heart-beating cadaveric nondiabetic donors as the discarded fraction of an islet cell isolation to prepare grafts for transplantation in type I diabetes patients. Human donor pancreases were procured at European hospitals and made available to the ß-Cell Bank in Brussels through the intermediate of Eurotransplant Foundation (Leiden, The Netherlands). The endocrine preparations contained >60% hormone-positive cells (Keymeulen et al., 1998). In the nonendocrine fraction, <2% expressed islet cell markers and >90% expressed the duct cellspecific phenotypic markers cytokeratin 19 and carbohydrate antigen 19.9 when cultured for at least 4 d (Bouwens et al., 1994). The nonendocrine cell preparation was cultured in suspension in Ham's F10 (Bio-Whittaker), 0.5% BSA (Boehringer Mannheim), 7.5 mM glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM L-glutamine at 37°C in a humidified atmosphere of 5% CO2. 24 h after isolation, the cells were washed, and the medium was renewed. On day 4 of culture, cells were counted and were infected and further cultured in suspension. Alternatively, day 4 cells were plated to form monolayers in the presence of 5% FBS (Life Technologies) and infected 6 d later. Under all conditions, cell culture medium was renewed every other day.
PC12 cells were cultured in suspension in RPMI 1640 with Glutamax (Life Technologies), 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. Rat ß-cells were prepared as described before (Heimberg et al., 1996).
Viral infection of isolated exocrine cells and PC12 cells
Cells were either infected with control virus (AdGFP) or with Adngn3 or AdNeuroD/ß2 as described previously (Heimberg et al., 2001). Adult human duct cells were infected at an MOI of 50 for 4 h at 37°C. PC12 cells were infected at an MOI 20 for 4 h at 37°C.
Protein analysis
Immunohistochemical analysis was performed on 4-µm-thick paraffin sections by indirect immunofluorescence as described (Heimberg et al., 2000). Similar methods were applied to cells from suspension cultures after fixation for 1 h in 4% paraformaldehyde and pelleting in 2% agarose before paraffin embedding. Before incubation with the first antibody, sections for CK19 staining were trypsin treated. Rabbit polyclonal PC1/3 antiserum was from I. Lindberg (Louisiana State University Health Sciences Center, New Orleans, LA) (Vindrola and Lindberg, 1992), rabbit polyclonal anti-ngn3 was from M. German (University of California San Francisco, San Francisco, CA), rabbit polyclonal Pax6 was from S. Saule (Institut Curie, Paris, France), and guinea pig polyclonal insulin was from C. Van Schravendijck (Brussels Free University). The mouse monoclonal anti-Nkx2.2 was developed by T. Jessell (Columbia University, New York, NY) and obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Rabbit polyclonal anti-Nkx6.1 has been described before (Oster et al., 1998). Rabbit polyclonal antisomatostatin was from J. DeMey (Brussels Free University), mouse monoclonal antichromogranin A was from Biogenex, rabbit polyclonal antisynaptophysin was from Novocastra, mouse monoclonal anticytokeratin 19 was from Dako, mouse monoclonal antiCA199 from Biomed, and mouse monoclonal anti-HA was from Boehringer. Secondary antibodies were Cy3- or FITC-labeled antirabbit, antimouse, and antiguinea pig (Jackson ImmunoResearch Laboratories). Immunoblot analysis was done as described before (Heimberg et al., 2000).
mRNA analysis
Total RNA was isolated using RNEasy columns (QIAGEN) and reverse transcription followed by PCR were performed and analyzed as described (Heimberg et al., 1996). Primers specifically amplified human Pdx1/Ipf1 (277 bp), 5'-CTGCCTTTCCCATGGATGAA-3' (forward) and 5'-CGCTTCTTGTCCTCCTCCTTT-3' (reverse), human ngn3 (286 bp), 5'-AGACGACGCGAAGCTCACC-3' (forward) and 5'-AAGCCAGACTGCCTGGGCT-3' (reverse), human NeuroD/ß2 (439 bp), 5'-ATCCCAACCCACCACCAACC-3' (forward) and 5'-CAGCGGTGCCTGAGAAGATT-3' (reverse), human Pax4 (496 bp), 5'-AGGAGGACCAGGGACTACCGT-3' (forward) and 5'-TTTAGGTGGGGTGTCACTCAG-3' (reverse), human Pax6 (301 bp), 5'-CAAAAGTCCAAGTGCTGGACAA-3' (forward) and 5'-CCCATCTGTTGCTTTTCGCT-3' (reverse), human Nkx2.2 (329 bp), 5'-TGCAGCACATGCAGTACAACG-3' (forward) and 5'-TCCCAAGGTTCAGAAGGAGAGG-3' (reverse), human Nkx6.1 (284 bp), 5'-TCTTCTGGCCCGGGGTGATG-3' (forward) and 5'-AGCCGCGTGCTTCTTCCTCC-3' (reverse), human Notch1 (160 bp), 5'-GAATCCAACCCTTGTGTCAAC-3' (forward) and 5'-GCAACGTCGTCAATACACGTG-3' (reverse), human Notch2 (231 bp), 5'-CGCTGCATTGACCTGGTCAAT-3' (forward) and 5'-TACATGTTGCACCCTTGCGA-3' (reverse), mouse Notch3 (240 bp), 5'-GGCATTGCTAGCTTCTCGTGT-3' (forward) and 5'-CATAACGGTTGATGCCATCAC-3' (reverse), human Jagged1 (417 bp), 5'-ATCTGTCCACCTGGCTATGCAG-3' (forward) and 5'-ATTTGCCTCCCGACTGACTCTT-3' (reverse), human Jagged2 (338 bp), 5'-GGAAGCCATGCCTTAACGCTT-3' (forward) and 5'-GCTCACAAAGGTCGACATCCA-3' (reverse), human Delta1 (309 bp), 5'-CCTGATGACCTCGCAACAGAA-3' (forward) and 5'-CATGCTGCTCATCACATCCAG-3' (reverse), human Delta4 (273 bp), 5'-ACCACTTCGGCCACTATGTGT-3' (forward) and 5'-TCTTGGTCACAAAACAGGCCT-3' (reverse), human synaptophysin (214 bp), 5'-GCCACATGCGGCAGCTACAG-3' (forward) and 5'-ACACGGCCACGGTGACAAAG-3' (reverse), human chromogranin A (286 bp), 5'-CCGCTGTCCTGGCTCTTCT-3' (forward) and 5'-CCGCTGTGTTTCTTCTGCTG-3' (reverse), human/rat insulin (438 bp), 5'-GCAGCCTTTGTGAACCAACA-3' (forward) and 5'-TCTGCGGTCATCAAATGAGG-3' (reverse), human glucagon (221 bp), 5'-CCCAAGATTTTGTGCAGTGGTT-3' (forward) and 5'-GCGGCCAAGTTCTTCAACAAT-3' (reverse), human glucokinase (607 bp), 5'-CTGGACGACAGAGCCAGGAT-3' (forward) and 5'-TCACCATTGCCACCACATCCAT-3' (reverse), human PC1/3 (355 bp), 5'-CAAGATACCAGGATGACGGCA-3' (forward) and 5'-GCCTCAATAGCATCCGTCACA-3' (reverse), mouse ngn3 (288 bp), 5'-CCGGATGACGCCAAACTTACA-3' (forward) and 5'-ACACCAGTGCTCCCGGGAG-3' (reverse), rat NeuroD/Beta2 (300 bp), 5'-GGACTTTCTTGCCTGAGCAGA-3' (forward) and 5'-AACTCGGTGGATGGTTCGTGT-3' (reverse), rat Pax4 (224 bp), 5'-ATGCGACCCTGTGACATCTCA-3' (forward) and 5'-AAGCCCTTCAGCACAAAGCTG-3' (reverse), rat Nkx2.2 (209 bp), 5'-CATGTCGCTGACCAACACAAAG-3' (forward) and 5'-TCGCTGCTGTCGTAGAAAGGA-3' (reverse), and rat/human ß-actin (361 bp), 5'-AGAGCTATGAGCTGCCTGAC-3' (forward) and 5'-CTGATCCACATCTGCTGGAA-3' (reverse). For in situ hybridization, human-specific NeuroD/ß2, Pax4, and ngn3 PCR products were subcloned into the pGEM-T Easy vector (Promega) and SalI linearized (Life Technologies). Digoxigenin-labeled transcripts were produced according to manufacturer's instructions (MAXI Script; Ambion). RNA in situ hybridization was performed as described (Gradwohl et al., 2000; Petersen et al., 2000). Detection of hybridized probes made use of an alkaline phosphatase-labeled antidigoxigenin antibody (Boehringer) and the substrate BM-Purple (Boehringer).
Electron microscopy
Cell preparations were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), postfixed in 1% osmium tetroxide, stained with 2% uranyl acetate, embedded in Spurr's resin, and ultra thin plastic sections were examined with a ZEISS EM 9S2 electron microscope.
Data analysis
Results obtained from infected cells were compared with uninfected and/or control virus-infected cells and statistically analyzed using the paired Student's t test.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
The authors are members of the Juvenile Diabetes Research Foundation Center for ß-Cell Therapy in Europe. Harry Heimberg is recipient of a Career Development Award from the Juvenile Diabetes Research Foundation and a Post-Doctoral Research Fellowship from the Fund for Scientific Research (Flanders, Belgium).
Submitted: 15 March 2002
Revised: 28 August 2002
Accepted: 28 August 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Artavanis-Tsakonas, S., M.D. Rand, and R.J. Lake. 1999. Notch signaling: cell fate control and signal integration in development. Science. 284:770776.
Assady, S., G. Maor, M. Amit, J. Itskovitz-Eldor, K.L. Skorecki, and M. Tzukerman. 2001. Insulin production by human embryonic stem cells. Diabetes. 50:16911697.
Atouf, F., P. Czernichow, and R. Scharfmann. 1997. Expression of neuronal traits in pancreatic beta cells. Implication of neuron-restrictive silencing factor/repressor element silencing transcription factor, a neuron-restrictive silencer. J. Biol. Chem. 272:19291934.
Bonner-Weir, S., M. Taneja, G.C. Weir, K. Tatarkiewicz, K.H. Song, A. Sharma, and J.J. O'Neil. 2000. In vitro cultivation of human islets from expanded ductal tissue. Proc. Natl. Acad. Sci. USA. 97:79998004.
Bouwens, L., R.N. Wang, E. De Blay, D.G. Pipeleers, and G. Kloppel. 1994. Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas. Diabetes. 43:12791283.[Abstract]
Corish, P., and C. Tyler-Smith. 1999. Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 12:10351040.
Edlund, H. 1999. Pancreas: how to get there from the gut? Curr. Opin. Cell Biol. 11:663668.[CrossRef][Medline]
Gradwohl, G., A. Dierich, M. LeMeur, and F. Guillemot. 2000. Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc. Natl. Acad. Sci. USA. 97:16071611.
Grapin-Botton, A., A.R. Majithia, and D.A. Melton. 2001. Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev. 15:444454.
Gu, G., J. Dubauskaite, and D.A. Melton. 2002. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development. 129:24472457.
He, T.C., S. Zhou, L.T. da Costa, J. Yu, K.W. Kinzler, and B. Vogelstein. 1998. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA. 95:25092514.
Heimberg, H., A. De Vos, K. Moens, E. Quartier, L. Bouwens, D. Pipeleers, E. Van Schaftingen, O. Madsen, and F. Schuit. 1996. The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells. Proc. Natl. Acad. Sci. USA. 93:70367041.
Heimberg, H., L. Bouwens, Y. Heremans, M. Van De Casteele, V. Lefebvre, and D. Pipeleers. 2000. Adult human pancreatic duct and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf-1. Diabetes. 49:571579.[Abstract]
Heimberg, H., Y. Heremans, C. Jobin, R. Leemans, A.K. Cardozo, M. Darville, and D.L. Eizirik. 2001. Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis. Diabetes. 50:22192224.
Huang, H.P., M. Liu, H.M. El-Hodiri, K. Chu, M. Jamrich, and M.J. Tsai. 2000. Regulation of the pancreatic islet-specific gene BETA2 (neuroD) by neurogenin 3. Mol. Cell. Biol. 20:32923307.
Jen, W.C., D. Wettstein, D. Turner, A. Chitnis, and C. Kintner. 1997. The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos. Development. 124:11691178.
Jensen, J., R.S. Heller, T. Funder-Nielsen, E.E. Pedersen, C. Lindsell, G. Weinmaster, O.D. Madsen, and P. Serup. 2000a. Independent development of pancreatic alpha- and beta-cells from neurogenin3-expressing precursors: a role for the notch pathway in repression of premature differentiation. Diabetes. 49:163176.[Abstract]
Keymeulen, B., Z. Ling, F.K. Gorus, G. Delvaux, L. Bouwens, A. Grupping, C. Hendrieckx, M. Pipeleers-Marichal, C. Van Schravendijk, K. Salmela, and D.G. Pipeleers. 1998. Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipients characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft. Diabetologia. 41:452459.[CrossRef][Medline]
Lumelsky, N., O. Blondel, P. Laeng, I. Velasco, R. Ravin, and R. McKay. 2001. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science. 292:13891394.
Oster, A., J. Jensen, H. Edlund, and L.I. Larsson. 1998. Homeobox gene product Nkx 6.1 immunoreactivity in nuclei of endocrine cells of rat and mouse stomach. J. Histochem. Cytochem. 46:717721.
Qiu, C., M.B. De Young, A. Finn, and D.A. Dichek. 1998. Cationic liposomes enhance adenovirus entry via a pathway independent of the fiber receptor and alpha(v)-integrins. Hum. Gene Ther. 9:507520.[Medline]
Rulifson, E.J., S.K. Kim, and R. Nusse. 2002. Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science. 296:11181120.
Sander, M., L. Sussel, J. Conners, D. Scheel, J. Kalamaras, F. Dela Cruz, V. Schwitzgebel, A. Hayes-Jordan, and M. German. 2000. Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development. 127:55335540.
Schwitzgebel, V.M., D.W. Scheel, J.R. Conners, J. Kalamaras, J.E. Lee, D.J. Anderson, L. Sussel, J.D. Johnson, and M.S. German. 2000. Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development. 127:35333542.
Shapiro, A.M., J.R. Lakey, E.A. Ryan, G.S. Korbutt, E. Toth, G.L. Warnock, N.M. Kneteman, and R.V. Rajotte. 2000. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N. Engl. J. Med. 343:230238.
Slack, J.M. 1995. Developmental biology of the pancreas. Development. 121:15691580.
Smith, S.B., H.C. Ee, J.R. Conners, and M.S. German. 1999. Paired-homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development. Mol. Cell. Biol. 19:82728280.
Smith, S.B., H. Watada, D.W. Scheel, C. Mrejen, and M.S. German. 2000. Autoregulation and maturity onset diabetes of the young transcription factors control the human PAX4 promoter. J. Biol. Chem. 275:3691036919.
Tanabe, Y., and T.M. Jessell. 1996. Diversity and pattern in the developing spinal cord. Science. 274:11151123.
Vindrola, O., and I. Lindberg. 1992. Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells. Mol. Endocrinol. 6:10881094.[Abstract]