Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function
Abstract
Neurog3 (Neurogenin 3 or Ngn3) is both necessary and sufficient to induce endocrine islet cell differentiation from embryonic pancreatic progenitors. Since robust Neurog3 expression has not been detected in hormone-expressing cells, Neurog3 is used as an endocrine progenitor marker and regarded as dispensable for the function of differentiated islet cells. Here we used 3 independent lines of Neurog3 knock-in reporter mice and mRNA/protein-based assays to examine Neurog3 expression in hormone-expressing islet cells. Neurog3 mRNA and protein are detected in hormone-producing cells at both embryonic and adult stages. Significantly, inactivating Neurog3 in insulin-expressing β cells at embryonic stages or in Pdx1-expressing islet cells in adults impairs endocrine function, a phenotype that is accompanied by reduced expression of several Neurog3 target genes that are essential for islet cell differentiation, maturation, and function. These findings demonstrate that Neurog3 is required not only for initiating endocrine cell differentiation, but also for promoting islet cell maturation and maintaining islet function.
Acknowledgments.
We thank Susan B. Hipkens, Kathy D. Shelton, Yanwen Xu, and Aizhen Zhao for technical assistance.; Chris Wright for help with writing the manuscript; and the Vanderbilt Transgenic/ES Cell Shared Resource for expertly performing blastocyst microinjections. We are also grateful to John Hutton for the use of his laboratory during establishment of the Neurog3tTA line. This research was supported by National Institutes of Health Grants DK065949 (to G.G.), DK072473 (to M.A.M.), DK072495 and DK68471 (to M.S.), and DK072495 (to P.S.); and Juvenile Diabetes Research Foundation Grant 2007-712 (to G.G.). P.S. was supported by the Juvenile Diabetes Research Foundation and the European Union 6th Framework Program. P.A.S. was supported by a Juvenile Diabetes Research Foundation postdoctoral fellowship (3-2004-608). J.N.J was supported by the Danish Research Counsel (271-05-0667), the Carlsberg Foundation, and a Julie von Müllens fund.
Supporting Information
Supporting Information (PDF)
Supporting Information
- Download
- 2.00 MB
References
1
SE Schonhoff, M Giel-Moloney, AB Leiter, Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types. Dev Biol 270, 443–454 (2004).
2
G Gu, J Dubauskaite, DA Melton, Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129, 2447–2457 (2002).
3
G Gradwohl, A Dierich, M LeMeur, F Guillemot, Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci USA 97, 1607–1611 (2000).
4
S Wang, et al., Myt1 and Ngn3 form a feed-forward expression loop to promote endocrine islet cell differentiation. Dev Biol 317, 531–540 (2008).
5
VM Schwitzgebel, et al., Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 127, 3533–3542 (2000).
6
KA Johansson, et al., Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types. Dev Cell 12, 457–465 (2007).
7
A Grapin-Botton, AR Majithia, DA Melton, Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev 15, 444–454 (2001).
8
A Apelqvist, et al., Notch signalling controls pancreatic cell differentiation. Nature 400, 877–881 (1999).
9
A Petri, et al., The effect of neurogenin3 deficiency on pancreatic gene expression in embryonic mice. J Mol Endocrinol 37, 301–316 (2006).
10
P White, CL May, RN Lamounier, JE Brestelli, KH Kaestner, Defining pancreatic endocrine precursors and their descendants. Diabetes 57, 654–668 (2008).
11
X Xu, et al., Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell 132, 197–207 (2008).
12
S Kodama, et al., Enhanced expression of PDX-1 and Ngn3 by exendin-4 during beta cell regeneration in STZ-treated mice. Biochem Biophys Res Commun 327, 1170–1178 (2005).
13
MV Joglekar, VS Parekh, S Mehta, RR Bhonde, AA Hardikar, MicroRNA profiling of developing and regenerating pancreas reveal post-transcriptional regulation of neurogenin3. Dev Biol 311, 603–612 (2007).
14
V Dror, et al., Notch signalling suppresses apoptosis in adult human and mouse pancreatic islet cells. Diabetologia 50, 2504–2515 (2007).
15
HM Yu, B Liu, F Costantini, W Hsu, Impaired neural development caused by inducible expression of Axin in transgenic mice. Mech Dev 124, 146–156 (2007).
16
HM Yu, B Liu, SY Chiu, F Costantini, W Hsu, Development of a unique system for spatiotemporal and lineage-specific gene expression in mice. Proc Natl Acad Sci USA 102, 8615–8620 (2005).
17
DK Gonda, et al., Universality and structure of the N-end rule. J Bio Chem 264, 16700–16712 (1989).
18
S Srinivas, et al., Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus BMC. Dev Biol 1, 4 (2001).
19
CS Lee, N Perreault, JE Brestelli, KH Kaestner, Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity. Genes Dev 16, 1488–1497 (2002).
20
CS Lee, DD De Leon, KH Kaestner, DA Stoffers, Regeneration of pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3. Diabetes 55, 269–272 (2006).
21
PA Seymour, et al., SOX9 is required for maintenance of the pancreatic progenitor cell pool. Proc Natl Acad Sci USA 104, 1865–1870 (2007).
22
S Zahn, J Hecksher-Sorensen, IL Pedersen, P Serup, O Madsen, Generation of monoclonal antibodies against mouse neurogenin 3: A new immunocytochemical tool to study the pancreatic endocrine progenitor cell. Hybrid Hybridomics 23, 385–388 (2004).
23
P Corish, C Tyler-Smith, Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng 12, 1035–1040 (1999).
24
M Gannon, C Shiota, C Postic, CV Wright, M Magnuson, Analysis of the Cre-mediated recombination driven by rat insulin promoter in embryonic and adult mouse pancreas. Genesis 26, 139–142 (2000).
25
S Wang, et al., Loss of Myt1 function partially compromises endocrine islet cell differentiation and pancreatic physiological function in the mouse. Mech Dev 124, 898–910 (2007).
26
W Nishimura, et al., A switch from MafB to MafA expression accompanies differentiation to pancreatic beta-cells. Dev Biol 293, 526–539 (2006).
27
J Jensen, et al., 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, 163–176 (2000).
28
A Villasenor, DC Chong, O Cleaver, Biphasic Ngn3 expression in the developing pancreas. Dev Dyn 237, 3270–3279 (2008).
29
J Jensen, et al., Control of endodermal endocrine development by Hes-1. Nat Genet 24, 36–44 (2000).
30
JY Lee, et al., RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function. J Biol Chem 281, 2649–2653 (2006).
31
TA Matsuoka, et al., The MafA transcription factor appears to be responsible for tissue-specific expression of insulin. Proc Natl Acad Sci USA 101, 2930–2933 (2004).
Information & Authors
Information
Published in
Classifications
Copyright
© 2009.
Submission history
Received: August 19, 2008
Published online: June 16, 2009
Published in issue: June 16, 2009
Keywords
Acknowledgments
We thank Susan B. Hipkens, Kathy D. Shelton, Yanwen Xu, and Aizhen Zhao for technical assistance.; Chris Wright for help with writing the manuscript; and the Vanderbilt Transgenic/ES Cell Shared Resource for expertly performing blastocyst microinjections. We are also grateful to John Hutton for the use of his laboratory during establishment of the Neurog3tTA line. This research was supported by National Institutes of Health Grants DK065949 (to G.G.), DK072473 (to M.A.M.), DK072495 and DK68471 (to M.S.), and DK072495 (to P.S.); and Juvenile Diabetes Research Foundation Grant 2007-712 (to G.G.). P.S. was supported by the Juvenile Diabetes Research Foundation and the European Union 6th Framework Program. P.A.S. was supported by a Juvenile Diabetes Research Foundation postdoctoral fellowship (3-2004-608). J.N.J was supported by the Danish Research Counsel (271-05-0667), the Carlsberg Foundation, and a Julie von Müllens fund.
Notes
This article contains supporting information online at www.pnas.org/cgi/content/full/0904247106/DCSupplemental.
Authors
Competing Interests
The authors declare no conflict of interest.
Metrics & Citations
Metrics
Citation statements
Altmetrics
Citations
If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.