Neurogenin3 inhibits proliferation in endocrine
progenitors by inducing Cdkn1a
Takeshi Miyatsuka, Yasuhiro Kosaka, Hail Kim, and Michael S. German1
Diabetes Center, Hormone Research Institute and Department of Medicine, University of California, San Francisco, CA 94143
Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 16, 2010 (received for review April 13, 2010)
During organogenesis, the final size of mature cell populations de-
pends on their rates of differentiation and expansion. Because
the developing pancreas initiates their differentiation to mature
islet cells, we examined the role of Neurog3 in cell cycle control
during this process. We found that mitotically active pancreatic
progenitor cells in mouse embryos exited the cell cycle after the
initiation of Neurog3 expression. Transcriptome analysis demon-
the mRNA encoding cyclin-dependent kinase inhibitor 1a (Cdkn1a).
In Neurog3 null mice, the islet progenitor cells failed to activate
Cdkn1a expression and continued to proliferate, showing that their
exit from the cell cycle requires Neurog3. Furthermore, induced
transgenic expression of Neurog3 in mouse β-cells in vivo markedly
decreased their proliferation, increased Cdkn1a levels, and eventu-
ally caused profound hyperglycemia. In contrast, in Cdkn1a null
mice, proliferation was incompletely suppressed in the Neurog3-
expressing cells. These studies reveal a crucial role for Neurog3 in
regulating the cell cycle during the differentiation of islet cells and
demonstrate that the subsequent down-regulation of Neurog3
allows the mature islet cell population to expand.
ganize as the organ grows. Within the pancreas, the size of the
islets of Langerhans and especially how many insulin-producing
β-cells they contain is a critical determinant of pancreaticfunction
and the risk of developing diabetes. The number of islet cells
dependson therateat whichnew endocrine cells [α-,β-,δ-,ε-,and
pancreatic polypeptide (PP) cells] differentiate from progenitors,
of the progenitors and mature endocrine cells. Understanding the
mechanisms that control these rates will help explain how distinct
cell populations assemble into functional organs.
The coordinated activity of numerous transcription factors
regulates the differentiation of the islet cells (1, 2). Among these
factors, Neurogenin3 (Neurog3/Neurog3), a member of the basic
helix–loop–helix (bHLH) transcription factor family, transiently
marks the progenitor cells that will become islet cells and ini-
tiates endocrine differentiation during embryonic development,
regeneration, and transdifferentiation (3–10).
Although we know that most descendants of Neurog3-
expressing cells exit the cell cycle (6, 11), we do not know whether
or how Neurog3 might drive cell cycle exit. To address these
questions, we used several mouse models with loss- and gain-of-
function mutations of Neurog3 and demonstrated that Neurog3 is
both necessary and sufficient to promote cellular quiescence in
pancreatic progenitors. Furthermore, transcriptome analysis with
high time resolution using the Neurog3-Timer mouse model
identified the cell cycle inhibitor Cdkn1a (p21/CIP1) as a down-
stream target of Neurog3 in the endocrine progenitors.
he mature structure of an organ depends on its constituent
cell populations and how those populations expand and or-
Cell Cycle Exit Follows Neurog3 Induction. To quantify cell division
during endocrine differentiation, we performed triple-labeled
immunohistochemistry with antibodies against Neurog3, eGFP,
and BrdU at embryonic day (E)15.5 in pancreases from hetero-
was replaced with eGFP (Neurog3eGFP/+mice; ref. 12) (Fig. 1 A–
D). Cells labeled by the anti-Neurog3 antibody but not anti-GFP
antibody (Neurog3+/eGFP-cells) represent the newest endocrine
progenitors that will shortly become Neurog3+/eGFP+double-
positive cells (13). Subsequently, the Neurog3+/eGFP+cells lose
Neurog3 immunoreactivity but temporarily retain eGFP immu-
noreactivity (Neurog3-/eGFP+cells) because of the long half-life
of green fluorescent protein (24 h or more; ref. 14) (Fig. 1E).
As outlined at the bottom of Fig. 1E, when Neurog3 expres-
sion starts, the first cells are Neurog3+, but eGFP-, because
maturation of the eGFP fluorophore takes time. These Neu-
rog3+/eGFP-cells progress to Neurog3+/eGFP+double-positive
cells after eGFP maturation. Then a few hours later when these
Neurog3+/eGFP+cells lose Neurog3 expression, the long half-
life eGFP leads to Neurog3-/eGFP+cells. Three hours after
BrdU labeling, 22.0% of Neurog3+/eGFP-cells, 7.2% of Neu-
rog3+/eGFP+cells, and 1.2% of Neurog3-/eGFP+cells were
labeled with BrdU (Fig. 1E). The differences in BrdU labeling
rates in these three populations indicate that cells stop entering
S-phase shortly after the initiation of Neurog3 expression.
To confirm the changes in cell cycle during endocrine matu-
ration, embryonic pancreases were dissected from Neurog3-Timer
embryos at E15.5 and E17.5, stained with the DNA dye Hoechst
33342, and analyzed by flow cytometry. The Timer (DsRed-E5)
fluorescent protein shifts its fluorescence emission peak from
green to red over time (15) and, thereby, overcomes the problems
caused by the long half-life of GFP (16, 17) and allows the de-
termination of the time because activation of Neurog3 in in-
shown in Fig. 1 F and G, at both E15.5 and E17.5, >98% of en-
docrine progenitors (gates A and B) were in G0/G1phase and,
thus, quiescent. In the mature endocrine cells in gate C, however,
a significant increase in cells in S/G2/M demonstrates the reentry
of cells into the cell cycle at E17.5, but not at E15.5 (Fig. 1G).
Neurog3 Is Required for Cell Cycle Exit. The studies in Fig. 1 cor-
relate cell cycle exit with Neurog3 expression, but do not indicate
whether Neurog3 drives the cells to exit the cell cycle, or whether,
alternatively, cells already programmed to exit the cell cycle
may preferentially activate Neurog3 expression. To address this
question, we analyzed Neurog3-deficient mice by histology and
flow cytometry. In Neurog3 null embryos (Neurog3eGFP/eGFP),
eGFP expression identifies those pancreatic progenitor cells in
which Neurog3 expression would have been activated. Immu-
nostaining for eGFP and BrdU demonstrated higher rates of
BrdU labeling in the eGFP-expressing cells of Neurog3eGFP/eGFP
Author contributions: T.M., Y.K., H.K., and M.S.G. designed research; T.M., Y.K., and H.K.
performed research; T.M. and M.S.G. analyzed data; and T.M. and M.S.G. wrote the
Conflict of interest statement: M.S.G. is an inventor on patents held by the University of
California covering Neurog3 and its use.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 4, 2011
| vol. 108
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11. Desgraz R, Herrera PL (2009) Pancreatic neurogenin 3-expressing cells are unipotent
islet precursors. Development 136:3567–3574.
12. Lee CS, Perreault N, Brestelli JE, Kaestner KH (2002) 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.
13. Miyatsuka T, Li Z, German MS (2009) Chronology of islet differentiation revealed by
temporal cell labeling. Diabetes 58:1863–1868.
14. Corish P, Tyler-Smith C (1999) Attenuation of green fluorescent protein half-life in
mammalian cells. Protein Eng 12:1035–1040.
15. Terskikh A, et al. (2000) “Fluorescent timer”: Protein that changes color with time.
16. Davis I, Girdham CH, O’Farrell PH (1995) A nuclear GFP that marks nuclei in living
Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic
fluorescence. Dev Biol 170:726–729.
17. Wu YL, et al. (2008) Development of a heat shock inducible gfp transgenic zebrafish
line by using the zebrafish hsp27 promoter. Gene 408:85–94.
18. Wang S, et al. (2010) Neurog3 gene dosage regulates allocation of endocrine and
exocrine cell fates in the developing mouse pancreas. Dev Biol 339:26–37.
19. Sander M, et al. (2000) Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the
major pathway of beta-cell formation in the pancreas. Development 127:5533–5540.
20. Teta M, Long SY, Wartschow LM, Rankin MM, Kushner JA (2005) Very slow turnover
of beta-cells in aged adult mice. Diabetes 54:2557–2567.
21. Meier JJ, et al. (2008) Beta-cell replication is the primary mechanism subserving the
postnatal expansion of beta-cell mass in humans. Diabetes 57:1584–1594.
22. Hara M, et al. (2003) Transgenic mice with green fluorescent protein-labeled
pancreatic beta -cells. Am J Physiol Endocrinol Metab 284:E177–E183.
23. el-Deiry WS, et al. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell
24. Wang Y, et al. (2008) Embryonic stem cell-specific microRNAs regulate the G1-S
transition and promote rapid proliferation. Nat Genet 40:1478–1483.
25. Abbas T, Dutta A (2009) p21 in cancer: Intricate networks and multiple activities. Nat
Rev Cancer 9:400–414.
26. Smith SB, et al. (2010) Rfx6 directs islet formation and insulin production in mice and
humans. Nature 463:775–780.
27. Smith SB, et al. (2003) Neurogenin3 and hepatic nuclear factor 1 cooperate in
activating pancreatic expression of Pax4. J Biol Chem 278:38254–38259.
28. Watada H, Scheel DW, Leung J, German MS (2003) Distinct gene expression programs
function in progenitor and mature islet cells. J Biol Chem 278:17130–17140.
29. Finegood DT, Scaglia L, Bonner-Weir S (1995) Dynamics of beta-cell mass in the growing
rat pancreas. Estimation with a simple mathematical model. Diabetes 44:249–256.
30. Huang HP, et al. (2000) Regulation of the pancreatic islet-specific gene BETA2
(neuroD) by neurogenin 3. Mol Cell Biol 20:3292–3307.
31. Dror V, et al. (2007) Notch signalling suppresses apoptosis in adult human and mouse
pancreatic islet cells. Diabetologia 50:2504–2515.
32. Wilson ME, et al. (2005) The HMG box transcription factor Sox4 contributes to the
development of the endocrine pancreas. Diabetes 54:3402–3409.
33. Wang S, et al. (2009) Sustained Neurog3 expression in hormone-expressing islet cells is
required for endocrine maturation and function. Proc Natl Acad Sci USA 106:9715–9720.
34. Lynn FC, et al. (2007) Sox9 coordinates a transcriptional network in pancreatic
progenitor cells. Proc Natl Acad Sci USA 104:10500–10505.
35. Milo-Landesman D, et al. (2001) Correction of hyperglycemia in diabetic mice
transplanted with reversibly immortalized pancreatic beta cells controlled by the tet-
on regulatory system. Cell Transplant 10:645–650.
| www.pnas.org/cgi/doi/10.1073/pnas.1004842108Miyatsuka et al.