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Retinoblastoma tumor suppressor protein in pancreatic progenitors controls α- and β-cell fate Erica P. Caia,b, Xiaohong Wuc, Stephanie A. Schroera, Andrew J. Eliad,e, M. Cristina Nostroa,f, Eldad Zacksenhausa, and Minna Wooa,b,e,g,1 aToronto General Research Institute and fMcEwen Centre for Regenerative Medicine, University Health Network, Toronto, ON, Canada M5G 1L7; bInstitute of Medical Science, University of Toronto, Toronto, ON, Canada M5S 1A8; cThe First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, China; dCampbell Family Institute for Breast Cancer Research, University Health Network, Toronto, ON, Canada M5G 2M9; eDepartment of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 2M9; and gDivision of Endocrinology, Department of Medicine, Toronto General Hospital, University Health Network, University of Toronto, Toronto, ON, Canada M5G 2C4 Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved July 16, 2013 (received for review February 20, 2013) Pancreatic endocrine cells expand rapidly during embryogenesis apoptosis (15), in a highly cell-specific and context-dependent by neogenesis and proliferation, but during adulthood, islet cells manner (16, 17). Although Rb is critical in regulating cell cycle have a very slow turnover. Disruption of murine retinoblastoma entry in proliferating cells, its role in postmitotic cells is more tumor suppressor protein (Rb) in mature pancreatic β-cells has limited. For example, Rb deficiency in proliferating myoblasts a limited effect on cell proliferation. Here we show that deletion induces increased proliferation and apoptosis (18), whereas fi fi of Rb during embryogenesis in islet progenitors leads to an in- Rb de ciency in postmitotic muscle bers does not lead to any crease in the neurogenin 3-expressing precursor cell population, defects (19). Rb has also been shown to control cell lineage which persists in the postnatal period and is associated with in- commitment in several cell types, including mesenchymal stem cells (20) and preadipocytes (21). In the pancreas, Rb has been creased β-cell mass in adults. In contrast, Rb-deficient islet precur- shown to play a minor role in well-differentiated β-cells, as evi- sors, through repression of the cell fate factor aristaless related denced by mice with rat insulin promoter-Cre-driven Rb deletion homeobox, result in decreased α-cell mass. The opposing effect on fi α β (22). However, E2f1 is required for normal pancreatic islet func- survival of Rb-de cient - and -cells was a result of opposing tion and homeostasis (23, 24). E2f1 directly regulates insulin se- effects on p53 in these cell types. As a consequence, loss of Rb cretion through modulating inward rectifier potassium channel in islet precursors led to a reduced α-toβ-cell ratio, leading to Kir6.2 expression (25). Although these results suggest a role for improved glucose homeostasis and protection against diabetes. Rb in islet development and function, the effect of Rb disruption in proliferating islet precursors is unknown. diabetes mellitus | insulin | glucagon | cell cycle | E2f To address this issue, we deleted Rb in Pdx1-expressing pan- creatic progenitors. Remarkably, as opposed to the minor effect β ancreatic α- and β-cells control glucose homeostasis through of Rb deletion in postmitotic -cells, disruption of Rb in pan- glucagon and insulin, which have opposing effects (1). The creatic progenitors had a profound effect on both pancreatic P α β fi pancreatic islet cell mass is achieved through a rapid surge of -and -cell fates. Rb de ciency led to increased Ngn3 and neu- neogenesis and proliferation that occurs during the perinatal rogenic differentiation 1 (NeuroD1) expression in islets, which fi period (2). However, this plasticity declines significantly in the represent multipotent endocrine islet cells. These Rb-de cient β postnatal period (3), resulting in a limited ability of islets to re- precursors showed enhanced -cell differentiation during em- generate in adulthood (4). However, molecular mechanisms that bryogenesis. Furthermore, Ngn3 expression persisted postnatally fi β govern both neogenesis and proliferation in islet cells remain in Rb-de cient islets, which were associated with increased -cell fi elusive. Better understanding of these processes may allow for mass. In contrast, Rb-de cient islet precursors failed to differ- α α the development of therapeutic strategies to treat diabetes. entiate into mature -cells because of the repression of an -cell During embryogenesis, the pancreas develops from foregut developmental gene, aristaless related homeobox (Arx), resulting α α β endoderm progenitors, which start expressing pancreatic and in a reduced -cell mass. These opposing effects on - and -cell duodenal homeobox 1 (Pdx1) at embryonic day 8.5 (E8.5) (5). differentiation and survival resulting from Rb loss led to improved Islet endocrine cells derive from neurogenin 3 (Ngn3)-expressing glucose homeostasis and protection against diabetes. precursors located in the pancreatic epithelium at E8.75 (6). α Results Once islet precursors adopt a cell identity by producing -or fi β β-cell hormones, these cells become postmitotic, after which only Rb De ciency in Islet Precursors Promotes -Cell Fate Through a limited number (<10%) of differentiated cells can reenter the Differentiation and Neogenesis. To investigate the physiologic cell cycle and contribute to prenatal islet expansion (7, 8). Neo- role of Rb in regulating islet cell fate, we deleted Rb in Pdx1- expressing pancreatic progenitor cells, using the Cre-loxP re- genesis, a proliferation process of undifferentiated precursors, is + fl fl combination system (26), yielding Pdx1-Cre Rb / mice, denoted the main mode through which rapid islet expansion is achieved fi in the late phase of fetal development (2). However, neogenesis herein as pancreas-speci c Rb knockout (p-RbKO) mice (Fig. S1A). To elucidate whether Rb is involved in regulating islet cell declines rapidly in the postnatal period, and Ngn3-expressing fi islet precursor cells become virtually absent by age 1 wk in mice differentiation, we rst examined the islet cell population in fetal (9, 10). Thereafter, the majority of islets are maintained in a pancreas at E16.5, when multipotent islet precursors are developing noncycling state without the ability to readily self-replicate. into single-hormone-expressing differentiated endocrine cells. The retinoblastoma tumor suppressor protein (Rb) functions MEDICAL SCIENCES as a major gatekeeper of cell cycle progression from G1 to S phase and is required for maintaining cells in the noncycling, Author contributions: E.P.C., M.C.N., E.Z., and M.W. designed research; E.P.C., X.W., S.A.S., quiescent stage (11). Rb inhibits cell cycle progression by binding and A.J.E. performed research; A.J.E., M.C.N., E.Z., and M.W. contributed new reagents/ to transcription factors such as E2f transcription factor 1 (E2f1) analytic tools; E.P.C. analyzed data; and E.P.C., M.C.N., E.Z., and M.W. wrote the paper. and inhibiting their transactivation (11). Rb phosphorylation The authors declare no conflict of interest. leads to its dissociation from E2f and the transcription of E2f- This article is a PNAS Direct Submission. regulated genes that are required for S phase entry (12). In ad- 1To whom correspondence should be addressed. E-mail: [email protected]. dition to cell cycle regulation, Rb controls multiple other cellular This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. processes, including differentiation (13), senescence (14), and 1073/pnas.1303386110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1303386110 PNAS | September 3, 2013 | vol. 110 | no. 36 | 14723–14728 Downloaded by guest on September 27, 2021 Intriguingly, we observed an increase in Ngn3 expression in islets Expression of Ngn3 persisted postnatally up to 4 wk of age in of p-RbKO pancreata at E16.5 compared with littermate con- p-RbKO mice compared with littermate controls (Fig. 1 B and C trols (Fig. 1A and Fig. S1B). The increase in this key determinant and Table S1). Consistent with this gene expression profile, – of endocrine lineage was observed along with another critical Ngn3-expressing islet cells were present in 4 8-wk-old p-RbKO downstream islet differentiation factor, NeuroD1 (Fig. 1A and mice, and these cells coexpressed insulin (Fig. 1D and Fig. S1C). These results suggest that Rb in islet precursors plays a critical Fig. S1B). To examine development into β-cell lineage, we next + role in regulating Ngn3 expression, and the preserved Ngn3 cell assessed for β-cell differentiation markers, NK6 homeobox 1 β fi population is capable of differentiating into mature -cells in the (Nkx6.1) and musculoaponeurotic brosarcoma oncogene family absence of Rb. proteins A (MafA), which were also increased (Fig. 1A and Fig. Ngn3 can activate downstream homeodomain-containing tran- S1B). All of these developmental markers also had a high pro- scription factor, paired box 4 (Pax4) (27), which can act as a portion of cells that coexpressed insulin (Fig. 1A and Fig. S1B), repressor of Arx; together, they mediate proper endocrine islet which together indicated that Rb deficiency in pancreatic pro- cell specification (28). Consistent with the findings in fetal p-RbKO genitors led to an increase in the number of endocrine precursors pancreas, the increased Ngn3 level was associated with a significant and enhanced differentiation into β-cells. induction of Pax4 expression in neonatal p-RbKO pancreas (Fig. 1E). In addition, expression of Pdx1 and MafA, which are also re- quired for β-cell specification, were significantly elevated (Fig. 1E). In keeping with these increases in β-cell neogenesis and differ- A BC entiation markers, p-RbKO pancreata exhibited an increase in Glu Ins Merge Control single or small clusters of insulin-expressing cells in close proximity p-RbKO to pancreatic ducts (Fig.

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