Disease Models & Mechanisms 3, 567-580 (2010) doi:10.1242/dmm.003210 © 2010. Published by The Company of Biologists Ltd RESEARCH ARTICLE

Rgs16 and Rgs8 in embryonic endocrine pancreas and mouse models of diabetes

Alethia Villasenor1, Zhao V. Wang2, Lee B. Rivera3, Ozhan Ocal4, Ingrid Wernstedt Asterholm2, Philipp E. Scherer2,5, Rolf A. Brekken3,4, Ondine Cleaver1,*,‡ and Thomas M. Wilkie4,*,‡

SUMMARY Diabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic -cells. Although -cells can replicate in younger adults, the available diabetes therapies do not specifically target -cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and -cell regeneration during islet expansion. Here, we show that the regulators of G protein signaling Rgs16 and Rgs8 are expressed in pancreatic progenitor and endocrine cells during development, then extinguished in adults, but reactivated in models of both type 1 and type 2 diabetes. Exendin-4, a glucagon-like peptide 1 (Glp-1)/incretin mimetic that stimulates -cell expansion, insulin secretion and normalization of blood glucose levels in diabetics, also promoted re-expression of Rgs16::GFP within a few days in pancreatic ductal- associated cells and islet -cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated -cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and -cell expansion in embryos and metabolically stressed adults. DMM INTRODUCTION ‘progenitor cells’ within the pre-pancreatic endoderm at around Diabetes affects over 246 million people worldwide and accounts embryonic day (E)8.75-9.0 (Golosow and Grobstein, 1962; Gittes for about 6% of annual global mortality (www.idf.org). This disease and Rutter, 1992; Kim and MacDonald, 2002; Yoshitomi and Zaret, is characterized by defective glucose metabolism and hyperglycemia 2004). By E12.5-14.5, endocrine progenitor cells proliferate, resulting from the destruction of insulin-producing -cells within delaminate and begin coalescing into small islet-like clusters. the pancreas (type 1), or defects in insulin signaling (type 2). During postnatal development, these clusters acquire recognizable Diabetes has no cure, although there are palliative treatments to islet anatomy; in mice, this consists of a core of -cells (that produce control its symptoms. There is a great need to understand the insulin) surrounded by a mantle of mostly -cells (that produce cellular and molecular basis for islet cell proliferation and glucagon), but also -cells (somatostatin), -cells (ghrelin) and PP differentiation in an effort to generate -cell regenerative therapies (pancreatic polypeptide) cells (Kim and MacDonald, 2002; Cleaver for diabetic patients. Although groundbreaking work has advanced and Melton, 2003; Collombat et al., 2006). In adulthood, there is our ability to drive stem cells towards the pancreatic endocrine cell little endocrine cell proliferation unless animals experience fate in culture (D’Amour et al., 2005; D’Amour et al., 2006; Kroon metabolic stresses that challenge their glucose homeostasis. et al., 2008), much remains unknown about the molecular pathways The cellular origin of the new endocrine cells remains regulating the differentiation of islet cell lineages (Lammert et al., controversial. Studies from Melton and others demonstrate that Disease Models & Mechanisms 2001; Cleaver and Melton, 2003; Lammert et al., 2003; Collombat new -cells derive from replication of pre-existing -cells rather et al., 2006; Oliver-Krasinski and Stoffers, 2008) and the than through proliferation of endogenous specialized progenitors mechanisms underlying islet regeneration (Dor et al., 2004; Bonner- (Dor et al., 2004; Teta et al., 2007). Work from Bonner-Weir, by Weir et al., 2008; Xu et al., 2008). contrast, supports the existence of ‘foci of regeneration’ or pools New tools required for the development of diabetes therapies of endocrine progenitors within the pancreatic ducts (Bonner-Weir can be fashioned using embryonic genes that are expressed during et al., 2004). Recent work by Heimberg and colleagues has shown pancreas development and later reactivated during pancreatic - that the adult pancreatic ducts have the ability to generate new - cell regeneration in models of diabetes (Inada et al., 2008; Xu et al., cell formation in response to extreme pancreatic injury (Gradwohl 2008). The earliest candidate genes are expressed in pancreatic et al., 2000; Xu et al., 2008). It is therefore plausible that both mechanisms occur, but depend on unspecified signals within the microenvironment. New biomarkers are therefore needed to 1Department of Molecular Biology and 5Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9148, further identify and examine expanding islets in different injury or USA disease models. These biomarkers will provide direct and rapid in 2 Department of Internal Medicine and Touchstone Diabetes Center, University of vivo validation of conditions that stimulate -cell replication and Texas Southwestern Medical Center, 75390 TX, USA 3Hamon Center for Therapeutic Oncology Research, University of Texas expansion. Southwestern Medical Center, 75390 TX, USA G protein-coupled receptor (GPCR) signaling pathways have 4Department of Pharmacology, University of Texas Southwestern Medical Center, been associated with -cell neogenesis. Glucagon-like peptide 1 6001 Forest Park Rd, Dallas, TX 75390-9041, USA (Glp-1) and exendin-4 (Byetta) are GPCR agonists that stimulate *These authors contributed equally to this work ‡Authors for correspondence ([email protected]; -cell replication and neogenesis and improve glucose tolerance in [email protected]) mouse models of type 1 diabetes (Xu et al., 1999; Tourrel et al.,

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2001; Kodama et al., 2005; Chu et al., 2007; Sherry et al., 2007; Wang et al., 2008). Glucose homeostasis is also improved in human type 2 diabetics (Fineman et al., 2003; Kendall et al., 2005). GPCRs activate G protein pathways, such as Gs- and Gq/11-stimulated cAMP and calcium signaling, respectively (Gilman, 1987; Simon et al., 1991). The regulators of G protein signaling (RGS) proteins are GTPase activating proteins (GAPs) for Gq/11 and Gi/o class  subunits, and thereby regulate the frequency and duration of GPCR signaling (Berman et al., 1996; Koelle and Horvitz, 1996; Ross and Wilkie, 2000). Interestingly, the expression of some Rgs genes is induced during GPCR signaling and can thereby serve as an indicator of active GPCR signaling (Dohlman et al., 1996). Furthermore, Rgs genes can be co-incidence detectors, induced by cross-talk pathways, to regulate Gi/q signaling in the same cell. Thus, Rgs gene expression can be used to identify where and when GPCR signaling is active during development and in tissues of healthy or diseased adults. The Rgs genes were first implicated in glucose homeostasis when we discovered that hepatic glucose production elevates the expression of Rgs16 in hepatocytes during fasting (Huang et al., Fig. 1. Rgs8::GFP and Rgs16::GFP are expressed in the pancreatic bud. 2006). To identify Rgs8/16 in other tissues that regulate glucose Rgs8 and Rgs16 are tandemly duplicated genes that are separated by homeostasis, we used two lines of bacterial artificial chromosome 42,682 bp (43 kb) of intragenic DNA (Sierra et al., 2002). (A,B)Transgenic mice containing a BAC with enhanced GFP (eGFP) inserted at the translation

DMM (BAC) transgenic mice that express either Rgs16::GFP or Rgs8::GFP. initiation site of either Rgs8::GFP (GENSAT ID: BX478, constructed from RP23- These reporter genes faithfully reproduce endogenous Rgs16 and 184B11) (A) or Rgs16::GFP (GENSAT ID: BX843, constructed from RP23-101N8) Rgs8 mRNA expression in mice (Gong et al., 2003; Su et al., 2004; (B) (Gong et al., 2003). (C,D)Expression of Rgs8::GFP (C) and Rgs16::GFP (D) in Huang et al., 2006; Morales and Hatten, 2006). Identifying the the dorsal pancreas during initial budding (arrows). Embryos were collected at ligands, conditions and cell types that induce the expression of these the indicated stages. dp, dorsal pancreatic bud; nt, neural tube. Bar, 250mm. Rgs genes is likely to further the development of novel therapies for metabolic diseases. Here, we show that Rgs16::GFP and Rgs8::GFP are expressed during pancreatic endocrine cell development and proliferation. Rgs16::GFP was expressed throughout the early gut tube Pancreatic progenitor cells and differentiating -cells express Rgs16 endoderm (E8.5) from the foregut to the tip of the open hindgut and Rgs8, beginning in the dorsal pancreatic anlagen and continuing (Fig. 2A and data not shown) and became restricted to the early throughout embryogenesis. In the perinatal pancreas, Rgs- liver and dorsal pancreatic bud epithelium after embryonic turning expressing cells aggregate into islets in tight association with (E9.5-10.5) (Fig. 2B,C). During dorsal bud outgrowth, Rgs16 was pancreatic blood vessels. In adults, pancreatic expression of Rgs16 expressed in a punctate pattern within the pancreatic epithelium and Rgs8 becomes quiescent. However, under conditions of chronic in a subpopulation of cells known to contain mostly epithelial and glucose stress, Rgs16 and Rgs8 are re-expressed in adult pancreatic endocrine cell types (Fig. 2C-E). In postnatal stages, Rgs16 Disease Models & Mechanisms islets. Here, we demonstrate Rgs16 expression in four different expression became restricted to aggregates of endocrine cells models of adult -cell expansion, including in (1) PANIC-ATTAC forming the islets of Langerhans (Fig. 2F). mice, a model of type 1 diabetes (Wang et al., 2008); and, together In contrast to the broad distribution of early Rgs16::GFP with Rgs8 expression, in (2) obese, hyperglycemic ob/ob mice, a expression in the endoderm, Rgs8::GFP expression was initially model of type 2 diabetes (Chua et al., 2002; Prentki and Nolan, localized to a distinct dorsal patch in a region fated to give rise to 2006); (3) midgestation pregnant females; and (4) following the pancreatic endoderm (E8.5) (Fig. 2G) (Wells and Melton, 1999). treatment with the GPCR agonist exendin-4. Rgs16 and Rgs8 are This striking expression was initiated prior to any cellular or sensitive reporters of conditions that stimulate the early stages of molecular evidence of pancreas specification, such as expression islet progenitor cell differentiation and -cell expansion in of Pdx1/Ipf1 or Ngn3 (Villasenor et al., 2008). By E9.5, Rgs8 development and disease. expression, like Rgs16, became largely restricted to the forming pancreatic bud (Fig. 2H,I). Later, Rgs8 was expressed in a pattern RESULTS that was almost identical to that of Rgs16, in scattered clusters of Rgs8/16 gene expression in endocrine progenitors during cells in the central region of the developing bud (Fig. 2J), but was development also weakly present in the exocrine pancreas (Fig. 2K,L). Overall, Two separate lines of GFP-expressing BAC transgenic mice (Fig. both patterns of expression were consistent with endocrine cell 1A,B) (Gong et al., 2003) were used to determine the expression distribution until postnatal stages (Fig. 2K,L). of the tandemly duplicated Rgs16 and Rgs8 genes during pancreatic Because the spatiotemporal distribution of Rgs16- and Rgs8- development. Both Rgs16::GFP and Rgs8::GFP were expressed expressing cells was reminiscent of endocrine precursors, their throughout embryonic and neonatal pancreas development (Fig. expression was compared with that of Ngn3, a well-characterized 1C,D; Fig. 2 and Table 1). endocrine progenitor marker (Gradwohl et al., 2000). Rgs16 and

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Fig. 2. Rgs16::GFP, Rgs8::GFP and Ngn3::GFP expression in the developing pancreatic bud. (A)Rgs16::GFP is broadly expressed in the endoderm at E8.5 but resolves (B) to the dorsal pancreas and liver bud by E9.5. (C)E10.5, expression in pancreatic endoderm, but not in mesoderm. (D,E)Expression in groups of cells along the central axis of the pancreatic tree, as branching elaborates. (F)In the postnatal pancreas, expression becomes restricted to forming islets (arrows). By contrast, (G) Rgs8::GFP is first expressed in a more localized domain within the foregut (arrowhead). (H-L)Similar to Rgs16, Rgs8 expression continues in scattered cells within the pancreatic endoderm between E9.5-10.5 (H,I), in the central region of the branching pancreatic bud between E12.5-E16.5 (J,K), and strongly in forming islets after birth (L). (G,M)Note that Rgs8::GFP is expressed in the dorsal pancreas prior to Ngn3::GFP (arrowheads), which is first detected in the dorsal pancreas at about E9.5 (compare G,H with M,N). (C-F,I-L,O-R) All three genes are expressed in the endocrine pancreas from E9.5 in a very similar pattern. Disease Models & Mechanisms (F,L,R) Arrows mark coalescing islets during postnatal development. The expression of Ngn3-eGFP was lower, but detectable, up to a few weeks post-birth. (D,E,J,K,P,Q) Dotted lines define the limit of the developing pancreas at each stage. Top row: schematics of the embryonic structures displayed in the rows below. aip, anterior intestinal portal; d, duodenum; dp, dorsal pancreatic bud; gte, gut tube endoderm; h, heart; l, liver; m, mesoderm; nf, neural folds; nt, neural tube; s, somite; vp, ventral pancreatic bud; ys, yolk sac. Bars, 100mm (A-E,G-K,M-Q); 50mm (F,L,R).

Rgs8 were expressed in a very similar pattern to Ngn3 (Fig. 2) on average about twofold longer than that of Rgs16::GFP). throughout development and until approximately 2 weeks Endogenous Rgs16 mRNA was also about twofold more abundant postnatal, when all three genes were extinguished in islets. However, than Rgs8 mRNA in E14.5 pancreas [quantitative PCR (qPCR); data the Rgs::GFP genes were expressed earlier than Ngn3::GFP (Fig. not shown]. Therefore, further investigation was focused on 2A,G,M) (see also Villasenor et al., 2008). Outside the pancreatic Rgs16::GFP expression during development and in adult models bud, endodermal Rgs8/16 and Ngn3 expression diverged, with of islet regeneration/expansion. Rgs16::GFP in the budding liver (Fig. 2B), Rgs8::GFP in the adjacent dorsal endoderm (Fig. 2H) and Ngn3::GFP faint in the posterior Rgs16::GFP in embryonic pancreatic endocrine cells duodenal endoderm (Fig. 2N,O). To determine whether endocrine cells within the pancreas In contrast to Rgs8::GFP and Ngn3::GFP, both of which were expressed Rgs16, the co-expression of Rgs16::GFP and endocrine extinguished shortly after birth, Rgs16::GFP remained strong in cell markers was analyzed (Fig. 3). We examined Rgs16 expression scattered cells along veins, ducts and arteries for the first 3-4 weeks at E8.75 through to E10.5, as the bud first evaginates and the first of postnatal development (see below). Rgs16::GFP expression was transition endocrine cells emerge, and at E15.5 during the rapid stronger than Rgs8::GFP throughout embryonic pancreatic wave of endocrine expansion and differentiation called the development (the photographic exposure time of Rgs8::GFP was ‘secondary transition’. Rgs16::GFP was expressed initially in most

Disease Models & Mechanisms 569 RESEARCH ARTICLE Rgs8/16 reactivated in expanding islets

+ Table 1. Expression of Rgs16::GFP in embryonic and postnatal patterns, greater than 98% of Rgs16::GFP cells were found to be pancreas Ki67 negative at E15.5, indicating that, at this stage, the vast majority Islet (Rgs16::GFP VDACs (Rgs16::GFP of Rgs16::GFP-expressing cells were non-replicating (only 1.45% Stage expression) expression) were Rgs16+/Ki67+) (supplementary material Fig. S3 and Table S1, E8.5 +/– NA and data not shown). As expected, Rgs16 expression did not co- E8.75-9.0 ++ NA localize with amylase-positive cells in acinar tissue (Fig. 3N). E9.5-16.5 +++ NA Rgs16-expressing cells were near blood vessels [marked by platelet P0 +++ NA endothelial cell adhesion molecule (PECAM)] (Fig. 3O; P1 +++ NA supplementary material Fig. S2J and data not shown), reflecting the known close association of endocrine and vascular tissues. At P3 +++ NA midgestation, Rgs16::GFP is predominately expressed in transient, P7 +++ NA post-delaminating endocrine cells that are aggregating into pre-islet P10 +++ + clusters. Together, these co-labeling data establish Rgs16 as a P11 + ++ marker for both early pancreatic progenitors and later P14 – ++ differentiating cells of the endocrine lineage. P15 – ++ P16-30 – + Rgs16::GFP in postnatal pancreas Adult (2 months) – – From E18.5 until a few weeks after birth, endocrine cells aggregate Adult (3 months) – – along the axes of pancreatic branches to form islets (Cleaver, 2004). During the early stages of this process, strong Rgs16 expression Arbitrary expression levels were assigned by independent visual analysis of relative intensity of fluorescence (two observers). Live dissected pancreatic tissue was could be detected within the forming islets (Fig. 4A) and always in examined in PBS, under fluorescence microscopy. P, pancreas-stage postnatal day. close association with blood vessels (Fig. 4B). Expression in islet

DMM Expression could be detected either in forming islets (middle column) or within endocrine cells declined rapidly after postnatal day (P)11 and was scattered cells along ducts and blood vessels (VDACs, right column). NA, not absent by P15 (compare Fig. 4A,B with 4C-E; supplementary applicable because strong expression in islets at these stages masks potential VDAC material Fig. S9). By contrast, Rgs16::GFP remained in discrete cells expression; (–) absent; (+) present; (++) medium; (+++) strong. and small cell clusters (two to ten cells) located along the central axis of distinct pancreatic branches (Fig. 4D-F). We termed these cells of the pancreatic bud epithelium and overlapped with the vessel- and ductal-associated Rgs16::GFP-positive cells (VDACs) pancreatic progenitor markers Sox9 and Pdx1 (Fig. 3A,B; (Fig. 4H). VDACs were frequently clustered around triad branch supplementary material Fig. S1A,B and Table S1). At about E9.5, points, where vessels and ducts branch coordinately (Fig. 4F). Rgs16::GFP expression overlapped with differentiating glucagon- Relatively strong VDAC expression could be observed throughout expressing cells and began to be excluded from Sox9-expressing early postnatal stages, up to and during the first 10 days following cells (supplementary material Fig. S1C and Table S1). Shortly weaning (pups were weaned at P16) (Table 1). VDACs were not thereafter, Rgs16::GFP expression became largely restricted to observed by 12 days post-weaning (Fig. 4G) or in the normal differentiating glucagon-expressing endocrine cells and to scattered glycemic adult pancreas (data not shown). E-cadherin-positive epithelial cells that expressed low levels of To identify the types of vessels associated with VDACs, Pdx1 but not Sox9 (Fig. 3C-E and supplementary material Fig. endothelial-lined blood vessels were distinguished from S1D-F). pancreatic ducts by immunofluorescence (Fig. 5A,B). Rgs16::GFP+ Disease Models & Mechanisms Following the secondary transition at E15.5, Rgs16::GFP VDACs were located along ducts (DBA) (Fig. 5B) closely expression was largely excluded from the tubular epithelium. associated with blood vessels (PECAM) (Fig. 5A) and lymphatics Most Rgs16::GFP-positive (Rgs16::GFP+) cells co-expressed (LYVE-1) (Fig. 5E). In addition, VDACs were often in close synaptophysin and islet hormones in delaminating endocrine cells proximity to developing islets expressing synaptophysin (Fig. 5D) (Fig. 3K-M). Only a small number of Rgs16::GFP+ cells co-expressed and, later, differentiation markers such as insulin (Fig. 5E). Indeed, either E-cadherin (3.5%) or Sox9 (5%) in the epithelium (Fig. 3F,G; Rgs16::GFP+ cells were often observed around the periphery of supplementary material Fig. S2A,B and Table S1 and data not islets or along duct/vessels triads in proximity to islets shown). Interestingly, Rgs16::GFP was never co-expressed with (supplementary material Fig. S10). Ngn3 (Fig. 3H; supplementary material Fig. S2C and Table S1). To elucidate the identity of Rgs16-expressing VDACs, a range In contrast to the lack of overlap with Ngn3, we found that 53% of endocrine markers of early versus late endocrine differentiation of Rgs16::GFP co-localized with Pdx1 (Fig. 3I; supplementary were examined. A small proportion of VDACs (~5%) expressed the material Fig. S2H and Table S1), a pancreatic progenitor marker earliest endocrine progenitor marker Sox9 (Fig. 5F), but none that also partially overlaps with insulin expression at E15.5 (Offield expressed later markers of endocrine differentiation such as et al., 1996). Rgs16::GFP co-localized with other markers of synaptophysin, insulin, glucagon, Glut2, somatostatin, PP or ghrelin endocrine cell fate, such as 52% with Nkx6.1 and 97% with (supplementary material Fig. S4 and data not shown). Furthermore, synaptophysin (Fig. 3J,K; supplementary material Fig. S2D,I and VDACs did not express markers of vascular endothelial, smooth Table S1). Rgs16::GFP was co-expressed with terminal endocrine muscle or macrophage lineages (supplementary material Fig. S4). differentiation markers such as glucagon and insulin (Fig. 3L,M and Some Rgs16+ VDACs are therefore similar to early progenitor cells supplementary material Fig. S2E-G), indicating that Rgs16 is in the epithelium in their expression of Sox9 and all are distinct expressed in islet lineages. Consistent with these expression from delaminating embryonic Rgs16+ endocrine cells (Fig. 3C-F).

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Fig. 3. Rgs16::GFP is expressed in early pancreatic progenitors and in delaminated cells of the endocrine lineage at E15.5. Rgs16::GFP is initially expressed in a subset of cells within the early pancreatic epithelium, at E8.75, and is co-expressed with Sox9 (A) and Pdx1(B). (C)A subset of Rgs16::GFP cells can be seen within the pancreatic bud epithelium, as shown by E-cadherin staining, at E10.0. (D)Later, Rgs16::GFP expression becomes mutually exclusive with Sox9 and (E) overlaps with the endocrine marker glucagon. By E15.5, Rgs16::GFP is primarily not co-expressed with E-cadherin (F) or Sox9 (G) in the pre-ductal epithelium, or with Ngn3 in endocrine cells prior to their delamination (H) (note that, in panel F, E-cadherin is expressed at low levels in budding endocrine cells that express high levels of Rgs16::GFP, green arrow). A few (2%) Rgs16::GFP-expressing cells, however, still reside in the epithelium (see inset in F). At E15.5, Rgs16::GFP is co- expressed (white arrows) with markers of differentiating endocrine cells: Pdx1(I), nkx6.1(J), synaptophysin (K), insulin (L) and glucagon (M). It is almost completely co-expressed with pooled antibodies to all differentiated endocrine cells (anti-ins, -gluc, -somatostatin, -ghrelin). Rgs16::GFP is not co-expressed with amylase (N) Disease Models & Mechanisms in exocrine cells or with PECAM (O) in vascular cells. (G-O)Nuclei are identified by DAPI staining of DNA (blue). The insets in F-M display either co-expression (F,I- M) or lack of co-expression (G,H) of markers, as indicated. Confocal micrographs, 50ϫ (A,B); 40ϫ (C-O).

Rgs16::GFP re-activated in the pancreas of pregnant females faded between E16.5 and E18.5 (supplementary material Fig. S5F; To determine whether Rgs16-expressing cells re-emerge in the data not shown). Between these stages, all pregnant females pancreas of metabolically stressed mice, the pancreata of pregnant expressed Rgs16::GFP in VDACs but only one expressed females were examined at different times during gestation. Islet Rgs16::GFP in the islets. By contrast, lactating females did not mass in females expands during pregnancy (Van Assche, 1978). express Rgs16::GFP in either VDACs or islets (data not shown). Although Rgs16::GFP expression was never observed in normal Rgs16::GFP expression along the ducts in pregnant females suggests adult pancreas, either within islets or as VDACs, Rgs16 was re- that increased maternal metabolic demands might be conveyed by expressed along vessel and duct tracts in mid- to late-gestation GPCR signaling and regulated by RGS proteins. female pancreas (supplementary material Fig. S5). Indeed, starting at approximately 8 days of gestation, rare GFP+ cells could be readily Rgs16::GFP re-activated in regenerating -cells of type 1 diabetic observed along the central axes of lateral pancreatic branches mice (supplementary material Fig. S5A), and increasing numbers of Given the correlation of Rgs16 expression with -cell proliferation, VDACs were present between E10.5-15.5 along many different both in the embryo and pregnant adults, we asked whether branches (supplementary material Fig. S5B-E). Rgs16::GFP Rgs16::GFP might also be re-expressed in islets during -cell expression was coincident with the initiation of a known phase of regeneration. Recently, islet regeneration was shown to occur -cell expansion in pregnant females (Gupta et al., 2007), but then following ablation in PANIC-ATTAC (pancreatic islet -cell

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Fig. 4. Rgs16::GFP+ cells are associated with blood vessels during neonatal islet formation. Neonatal Rgs16::GFP expression is observed in coalescing islets throughout the postnatal pancreas (at postnatal day indicated). (A)Rgs expression is evident in endocrine cells as they aggregate into clusters along blood vessels at P0. (B)A forming islet (white arrow) can be seen overlying a blood vessel (white arrowhead). (C)Expression continues in islets until approximately P11 (short red arrows), but expression remains in scattered cells, or VDACs, along the axes of lateral branches (short white arrows). (D-F)At weaning (P16), the expression of Rgs16::GFP is no longer detectable in islets (D); however, expression continues in VDACs lining axial blood vessels at the center of lateral pancreatic branches (short white arrows) (E,F). (F)GFP+ cells (arrow) are tightly associated with tracts of blood vessels and associated pancreatic ducts, and are especially enriched at vascular branch points (red arrowhead; the red dotted line delineates a mature islet). (G)Expression vanishes by P27 (12 days past weaning). (C-E)The dotted white lines depict margins of lateral pancreatic branches in postnatal pancreas. (H)Schematic of VDACs location along the ‘triad’ composed of artery (red), vein (blue) and duct

DMM (yellow). The cross section shows the approximate location of VDACs (green) at the interface between ducts and blood vessels. Scale bars, 100mm.

apoptosis through targeted activation of caspase 8) mice (Wang et al., 2008). In this model of type 1 diabetes, pancreatic -cells were targeted for cell death by the regulated expression of an FKBP- caspase 8 fusion protein, resulting in hyperglycemia. To assess the expression of Rgs16 during islet regeneration, the Rgs16::GFP transgene was crossed into the background of the PANIC-ATTAC mice. Following -cell apoptosis, Rgs16::GFP was co-expressed with insulin in a subset of pancreatic -cells during the first two weeks of islet regeneration (Fig. 6). The percentage of Rgs16::GFP+ cells was higher in hyperglycemic mice with more severe hyperglycemia and islet destruction than in mice with moderate glucose levels (Fig. 6A,B; supplementary material Fig. S6). Rgs16 was specifically expressed in -cells, as assessed by either insulin (Fig. Disease Models & Mechanisms 6A,B) or Glut2 co-staining (Fig. 6F), and was not observed in other endocrine cell types, such as -cells (Fig. 6C). Rgs16::GFP was also not expressed in the pancreas of either parental PANIC-ATTAC (Fig. 6D) or normoglycemic Rgs16::GFP;PANIC-ATTAC mice (Fig. 6E; supplementary material Fig. S6A,B,E). A time course study showed that hyperglycemia (>300 mg/dl) appeared in about half the males during the first three to five days of FKBP-ligand injection (supplementary material Fig. S6A,B). Rgs16::GFP was induced by chronic, but not acute, hyperglycemia. For example, Rgs16::GFP was not visible in day 5 hyperglycemic mice (n2) and only first appeared by day 7 in VDACs and a few cells within one or a small cluster of neighboring islets Fig. 5. Rgs16::GFP+ cells are associated with ducts and blood vessels (supplementary material Fig. S6E, panel a). Rgs16::GFP expression during postnatal islet formation. (A-E)Whole-mount staining for GFP in P15 expanded to more cells in more islets throughout the pancreas over BAC transgenic neonates demonstrates the close association of Rgs16- a 50-day interval of -cell regeneration (supplementary material expressing VDACs (arrows) with blood vessels (PECAM) (A), ducts (DBA lectin) Fig. S6D,E). Of note, if the blood insulin was at least 0.5 ng/ml, the (B), endocrine cells (synaptophysin) (C), late endocrine cells (insulin) (D) and day 5 blood glucose levels were good predictors of later Rgs16::GFP lymphatic vessels (Lyve-1) (E). Note the rare Rgs16::GFP+ cells observed within islets (yellow arrow in D). At higher resolution, a portion of VDACs (5%) can be expression (supplementary material Fig. S6B-D). + seen to express Sox9 (F). (A-E)Confocal micrographs of whole-mount Interestingly, the Rgs16::GFP cells in this model of -cell preparations, 30ϫ (A-E). Confocal micrographs of sectioned tissue, 100ϫ (F). regeneration displayed a higher proliferative capacity than the

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Fig. 6. Rgs16::GFP is expressed in regenerating islets of hyperglycemic PANIC-ATTAC mice. Rgs16::GFP and insulin are co-expressed in a few cells within islets of hyperglycemic PANIC-ATTAC;Rgs16::GFP transgenic mice during pancreatic -cell proliferation (recovery) following ablation. (A,B)Expression is detected in severely hyperglycemic (A) or moderate glycemic (B) mice after 2 weeks of islet recovery (arrows mark Rgs16::GFP+;insulin+ double-positive cells). (C)Rgs16::GFP is not found in glucagon-expressing -cells in the recovering islet (6 weeks of islet recovery). (D)No background GFP expression is detected in hyperglycemic PANIC-ATTAC mice. (E)Rgs16::GFP is never detected in normoglycemic mice PANIC-ATTAC;Rgs16::GFP mice. (F)Rgs16::GFP is expressed in Glut2+ -cells in the recovering islet (6 weeks of -cell recovery). Dotted white lines depict islet margin. Bars, 50mm.

Rgs16::GFP+ cells at E15.5 during embryogenesis (supplementary ob/ob mice correlated with chronic increases in both blood glucose +

DMM material Table S2). Over 5% of Rgs16::GFP cells in the PANIC- and insulin (Fig. 7A-C; supplementary material Fig. S7). Consistent ATTAC mice incorporated bromodeoxyuridine (BrdU), compared with this observation, Rgs16::GFP+ cells were never found in the with the surrounding Rgs16-negative endocrine cells, which showed pancreas of 4–5-week-old Rgs16::GFP;ob/ob mice, which had a rate of 0.98%. These data indicate that Rgs16 expression is found near-normal or recently elevated levels of glucose and insulin in adult regenerating islets within the proliferative -cell (supplementary material Fig. S7D, panel a; and data not shown). compartment. In the early phase of induction, especially in mice with high levels of either glucose or insulin and a modest elevation of the other, Rgs16::GFP re-activated in pancreas of ob/ob hyperglycemic mice Rgs16::GFP was expressed in VDACs (supplementary material Fig. -Cell proliferation also occurs in obese, diabetic ob/ob mice, a S10) and in a few cells in a small cluster of neighboring islets model of type 2 diabetes (Chua et al., 2002; Prentki and Nolan, (supplementary material Fig. S7D, panels b,c,g). The expression 2006). Therefore, Rgs16::GFP expression was assessed in normal expanded in islets throughout the pancreas as mice became both and hyperglycemic ob/ob mice (Fig. 7). Rgs16::GFP expression in hyperglycemic and hyperinsulinemic (supplementary material Fig.

Fig. 7. Rgs16::GFP is expressed in expanding islets of hyperglycemic ob/ob mice. (A)Rgs16::GFP is expressed in expanding islets of hyperglycemic ob/ob adult mice, but not Disease Models & Mechanisms in compound heterozygous mice (A), or in normoglycemic Rgs16::GFP;ob/ob or non-GFP ob/ob adults (supplementary material Fig. S7). (B,C)The number of islets expressing Rgs16::GFP generally increases with co-ordinate increases in glucose and insulin levels in Rgs16::GFP;ob/ob mice (see supplementary material Fig. S7). (C)Note the close association of Rgs16::GFP+ islets with large blood vessels (vessel, red arrowhead). (D-H)Double immunostaining for Rgs16::GFP and insulin (D), Glut-2 (E) and glucagon (F) demonstrates that Rgs16+ cells are found within islet -cells but not -cells, and are closely associated with blood vessels (PECAM) (G) and ducts (DBA) (H). (I)Cells with proliferative capacity (Ki67+, red arrows) are rare and equally distributed between cells that express Rgs16::GFP (white arrows) and those that do not express Rgs16::GFP. Dotted white lines depict islet margin. Bars, 100mm (A-C,G,H); 50mm (D-F,I).

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was similar to that observed in the islets of Rgs16::GFP;PANIC- ATTAC mice. As anticipated, Rgs16::GFP+ islet -cells were found in close association with islet capillaries (Fig. 7G) and ducts (Fig. 7H). Additionally, Rgs8::GFP was similarly induced specifically in the islets of hyperglycemic and hyperinsulinemic ob/ob mice (supplementary material Fig. S8). In older ob/ob mice with high glucose and insulin levels (as in Fig. 7C and supplementary material Fig. S7C,D, panel k), Rgs16::GFP expression was observed in up to half of the pancreatic islets (supplementary material Fig. S9), each containing 1-5% Rgs16::GFP+ -cells (in the three animals counted, 78% of ob/ob islets expressed Rgs16 in 0.5-5% of total endocrine cells, 17% expressed Rgs16 in 6-20% of total cells and 5% expressed Rgs16 in 21-50% of total cells). The majority of Rgs16::GFP+ islets were found along the central region of the main axis of the pancreas (Fig. 7C) but Rgs16::GFP expression appeared in islets of all sizes throughout the pancreas (supplementary material Fig. S7D). Although -cells in islets of ob/ob mice are known to proliferate (Bock et al., 2003), relatively modest numbers of Ki67+ cells were observed in hyperglycemic animals (36% of the ob/ob islets had 0.5-1% Ki67+ cells, 59% had 1-3% Ki67+ cells and about 5% had 3- 5% Ki67+ cells). In addition, Rgs16::GFP+ -cells in the islets of older

DMM ob/ob mice did not display a more highly proliferative state than Rgs16::GFP– -cells (supplementary material Table S3). Indeed, Rgs16-expressing cells co-stained for Ki67+ at the same rate as the surrounding -cells, at slightly over 1% (supplementary material Table S3). The total number of -cells expressing Rgs16::GFP declined in the pancreas of ob/ob mice as glycemia was lowered following initial -cell expansion, then increased with hyperinsulinemia in older ob/ob mice (supplementary material Fig. S7).

Exendin-4 induces Rgs16::GFP in adult pancreas Finally, to assess Rgs16::GFP expression using another model of - cell proliferation, we stimulated -cell neogenesis and islet expansion by injection of Rgs16::GFP transgenic mice with exendin- 4, a known GPCR agonist (Tourrel et al., 2001). Within 3 days of twice-daily injections of exendin-4 plus glucose, Rgs16 expression Disease Models & Mechanisms was induced in some VDACs but rarely, if at all, in islets (Fig. 8A,B). Later, by the sixth day of injection, Rgs16::GFP expression was observed in more VDACs and also in -cells within a few islets + Fig. 8. Exendin-4 induces Rgs16::GFP expression in adult pancreas. (Fig. 8C,D,H). Rgs16::GFP islets were also observed in mice Normoglycemic Rgs16::GFP BAC transgenic male mice (8-12 weeks) were injected daily with exendin-4 alone (three of five mice). As seen injected (intraperitoneally) twice daily for 6 days with PBS, glucose, exendin-4 previously in PANIC and ob/ob mice, Rgs16 expression first plus glucose (Ex+Glc), or exendin-4 alone (Ex). (A,B)After 3 days, Rgs16::GFP+ appeared in a small cluster of neighboring islets (Fig. 8E,F). By VDACs were observed but only in Ex+Glu-treated mice; (B) bright field of panel contrast, expression was not observed in islets in mice injected with + A (n3/group). (C-F)After 6 days, Rgs16::GFP VDACs and/or islets were glucose alone or PBS (Fig. 8G and supplementary material Fig. S11). observed in Ex+Glu (C,D) or Ex-treated (E,F) mice; (D,F) bright field of panels C These results confirmed that Rgs16::GFP can be used as a reporter and E. (G)An islet from a PBS-injected mouse at day 3; GFP expression was not observed in any PBS-treated mice (n2). (H)An islet of an Ex+Glc-injected of GPCR-stimulated -cell neogenesis in the pancreas. mouse at day 5; GFP expression was observed in the islets of Ex+Glu-injected mice (n2) (see supplementary material Fig. S11C). Dotted white lines depict DISCUSSION islet margin. Bars, 100mm (A-F); 50mm (G-H). In this study, we introduce regulators of G protein signaling as genetic beacons of -cell expansion during development and disease. Rgs16 and Rgs8 are expressed in pancreatic progenitor and S7D, panels d-f,j,k). Rgs16 expression occurred in -cells, but not endocrine cells during development, then extinguished in adults, -cells of the ob/ob islets, as Rgs16::GFP+ cells expressed insulin but reactivated in models of both type 1 and type 2 diabetes. and Glut2 (Fig. 7D,E) but not glucagon (Fig. 7F). This -cell Furthermore, Rgs16 expression is activated in response to exendin- specificity within islets and gradual expansion of Rgs16 expression 4, a GPCR agonist that is used to stimulate -cell expansion in type

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2 diabetes (Xu et al., 1999; Tourrel et al., 2001; Fineman et al., 2003; For example, Pdx1 expression is first observed throughout the Kendall et al., 2005; Kodama et al., 2005; Xu et al., 2006; Chu et al., pancreatic epithelium and its derivatives, but after birth it becomes 2007; Sherry et al., 2007; Wang et al., 2008). Based on their restricted to differentiating islets (Offield et al., 1996). The endocrine expression and known biochemical activities, Rgs16 and qualitatively different expression domains for Rgs16 may signify Rgs8 are likely to have redundant functions in the endocrine temporal regulation within a single cell lineage or expression in pancreas and may promote -cell differentiation and maturation, two separate lineages, the first appearing in the early epithelium although definitive functional characterization awaits simultaneous of the pancreatic bud and the other following transient expression deletion of both genes in -cells. Rgs16::GFP and Rgs8::GFP of Ngn3. transgenic mice are unique tools for comparative analysis of the endocrine pancreas during -cell expansion/regeneration in Rgs8/16::GFP expression in models of diabetes development, disease and response to therapy. Despite the strong expression of Rgs16 and Rgs8 throughout embryonic and neonatal isletogenesis, their expression ceases after RGS regulation of GPCR signaling 4 weeks of age in endocrine pancreas under normal conditions. RGS proteins are essential regulators of GPCR signaling, controlling However, both genes were re-expressed in models of -cell the timing of both signal activation and termination in diverse expansion or regeneration, suggesting that these genes are likely processes such as chemotaxis, cell cycle exit or re-entry, and neural to play a role in either endocrine proliferation, differentiation, or processing (Berman et al., 1996; Doupnik et al., 1997; Zeng et al., in response to chronic changes in glucose metabolism. The absence 1998; Zerangue and Jan, 1998; Luo et al., 2001). An intriguing aspect of RGS::GFP expression in normal adult pancreatic endocrine cells of mammalian Rgs genes related to Rgs16, but first noted in yeast suggests that, if Rgs16 and Rgs8 have a physiological role in adult Sst2/Rgs1, is that their expression is induced by ligands that are islets, low basal levels are sufficient to regulate daily postprandial feedback regulated by the RGS protein (Dohlman et al., 1996; Ross insulin release and glucose homeostasis. Dietary approaches to alter and Wilkie, 2000). Thus, the expression of Rgs16 and Rgs8 can be blood glucose and metabolism, such as high-fat or high-

DMM used to identify where and when GPCR signaling is active during disaccharide diets, or twice-daily intraperitoneal injections of development and in adult tissues of healthy or diseased individuals. glucose, failed to induce Rgs8/16 expression in otherwise-normal pancreatic islets (data not shown). However, the pancreatic Rgs8/16::GFP expression in embryonic pancreas expression of Rgs16 in pregnant females during midgestation, in Using Rgs8/16 BAC transgenic reporter lines, we assessed exendin-4-treated mice, and in the islets of hyperglycemic type 1 expression during key time points of pancreas development. Our and type 2 diabetic mice suggests that Rgs16 is a biomarker for results showed that, similar to Pdx1 and Ngn3 (Kim and cells that have reactivated an aspect of the embryonic -cell MacDonald, 2002; Villasenor et al., 2008; Cleaver and MacDonald, program during adult -cell proliferation/regeneration in response 2009), Rgs16::GFP and Rgs8::GFP are first observed throughout the to glucose stress. pancreatic epithelium, but later become restricted to -cells. Both Rgs16::GFP becomes re-expressed in hyperglycemic PANIC- Rgs16::GFP and Rgs8::GFP are first expressed in the ‘proto- ATTAC and ob/ob mice during the early phase of -cell differentiated’ early pancreatic epithelium at E8.75, when the regeneration, indicating that Rgs16 may regulate -cell replication essential progenitor cells are set aside for later pancreas during compensatory islet expansion. In these two models, the development (Offield et al., 1996; Zhou et al., 2007; Cleaver and pattern of expression is strikingly similar during disease progression MacDonald, 2009). At this early developmental stage, Rgs16::GFP and recovery. Hyperglycemia precedes Rgs16 induction in both is co-expressed with the pancreatic progenitor marker Sox9 in most PANIC and ob/ob mice (supplementary material Figs S6 and S7, Disease Models & Mechanisms multipotent progenitors. Interestingly, Rgs16::GFP and Rgs8::GFP respectively). This lag in expression suggests that Rgs16 is not are induced in the pancreatic bud in this early epithelium prior to directly induced in the pancreas by elevated blood glucose, but by Ngn3::GFP, a well-characterized marker of pancreatic endocrine physiologic responses to chronic glucose stress. Elevated insulin progenitors (Villasenor et al., 2008). may contribute to increased Rgs16::GFP expression but is not With the onset of endocrine differentiation, Rgs16::GFP sufficient, as some young hyperinsulinemic ob/ob mice express little expression becomes restricted to endocrine cells. By E15.5, or no Rgs16::GFP (supplementary material Fig. S7). However, Rgs16::GFP expression is predominantly restricted to delaminated combined hyperglycemia and hyperinsulinemia (and severe obesity) and/or differentiated endocrine cells that co-express synaptophysin, in older ob/ob mice are strongly correlated with Rgs16::GFP but not Ngn3. These Rgs16::GFP+ endocrine cells are primarily non- expression (supplementary material Fig. S7). replicating, as less than 1.5% of Rgs16::GFP+ cells are Ki67+. Given Another striking similarity during disease progression is Rgs16 these data, Rgs16 may promote -cell differentiation and/or inhibit distribution. In both mouse models of diabetes, Rgs16 expression cell cycle re-entry in delaminating endocrine cells. is first observed in the -cells of one or a small cluster of neighboring During development, Rgs16 expression identifies transient cell islets. As the disease progresses, Rgs16::GFP expression expands to states, an earlier progenitor state, and a later state that anticipates more cells, within more islets, throughout the pancreas and initiates hormone expression. The two ‘waves’ of Rgs16 (supplementary material Figs S6, S7). Rgs16::GFP expression persists expression, in the early pancreatic epithelium and later endocrine in non-proliferative -cells in both models of diabetes lineages, are similar to the ‘biphasic’ expression of pancreatic genes, (supplementary material Tables S2 and S3), reflecting expression such as Pdx1, Hlxb9 and Ngn3, before and after the secondary during embryogenesis and neonatal isletogenesis (supplementary transition (Pictet and Rutter, 1972; Offield et al., 1996; Li and material Table S1). This progressive and heterogeneous expression Edlund, 2001; Villasenor et al., 2008; Cleaver and MacDonald, 2009). of Rgs in islets during -cell expansion in PANIC and ob/ob mice

Disease Models & Mechanisms 575 RESEARCH ARTICLE Rgs8/16 reactivated in expanding islets

reflects the known heterogeneous response to hyperglycemia and of -cell differentiation during development and -cell regeneration early diabetes (Atkinson and Gianani, 2009). in models of metabolic stress and disease. Many GPCRs are Rgs16 has been suggested to inhibit -cell expansion during expressed in mouse islets but their functions are unknown (Regard postnatal development and in ob/ob mice (Poy et al., 2009). Indeed, et al., 2007). Furthermore, GPCRs that are expressed in embryonic Rgs16 and Rgs8 feedback may inhibit the GPCR signaling that endocrine progenitors, such as the Gq-coupled receptor Gpr56 (Gu initiates -cell expansion. Endogenous Rgs16 and Rgs8 genes are et al., 2004) (www.genepaint.org), may be induced during -cell similarly induced in the islets of 10-week-old ob/ob mice in both expansion in diabetes. A subset of these GPCRs may be regulated C57BL/6 and BTBR genetic backgrounds (Keller et al., 2008), where by Rgs16 and/or Rgs8 during pancreas development and islet -cell expansion occurs in the C57BL/6 mice but not in BTBR mice. regeneration. A challenge is to identify additional mitogens that Blood insulin levels are elevated in both backgrounds of mice, but stimulate -cell expansion. A practical approach would be to modestly in ob/ob BTBR mice (to about 10 ng/ml) in a constrained identify molecules that induce Rgs16 expression in surrogates of effort to manage insulin resistance and hyperglycemia. This pancreatic ducts and/or endocrine progenitors, such as primary indicates that BTBR islets perceive chronic glucose stress and cultured cells from pancreatic ductal adenocarcinoma (Aguirre et induce Rgs16 and Rgs8, even though they fail to undergo -cell al., 2003), ‘endocrine-differentiated’ embryonic stem (ES) cells expansion. These signals may be transmitted within the local (Kroon et al., 2008; Serafimidis et al., 2008), or induced pluripotent environment of responding islets and augment cellular responses stem (iPS) cells (Takahashi and Yamanaka, 2006; Nakamura et al., to elevated glucose and insulin (Llona, 1995). Feedback inhibition 2009). Superlative candidate molecules could be identified by in by Rgs16 (and Rgs8) could promote the cell cycle exit or prevent vivo validation in Rgs16::GFP transgenic mice, as demonstrated the cell cycle re-entry of maturing -cells that are still exposed to here for exendin-4. mitogens during diabetic islet expansion. We propose that Rgs16 and Rgs8 are fetal pancreatic endocrine genes that have redundant functions during embryonic pancreas Rgs16::GFP a biomarker of GPCR stimulation and -cell development and, later in adults, during islet regeneration

DMM Exendin-4 is an incretin mimetic prescribed for type 2 diabetics in type 1 diabetes (PANIC-ATTAC) and islet expansion in type 2 to improve glucose homeostasis by suppressing glucagon diabetes (ob/ob) (Herrera et al., 1994; Nir et al., 2007; Cano et al., secretion, stimulating insulin release and promoting -cell 2008; Wang et al., 2008). Future work will identify the ligands and expansion (Xu et al., 1999; Tourrel et al., 2001; Kodama et al., receptor-signaling pathways that are regulated by Rgs16 and Rgs8 2005; Bond, 2006; Lee et al., 2006; Sherry et al., 2007). Exendin- in hyperglycemic type 1 and type 2 diabetic mice. These ligands, 4 is a protease-resistant homolog of mammalian Glp-1, and both GPCRs and regulatory RGS proteins are likely to be important are agonists of the Glp-1 receptor (Glp-1R). Agonist binding signaling molecules in the progression towards diabetes, and key evokes Gs-mediated increases in intracellular cAMP levels, targets for diabetes therapy. promotes mitogenic and anti-apoptotic signals, and activates c- fos and other early response genes (Lee and Nielsen, 2009). METHODS However, RGS proteins are not Gs-GAPs (Berman1996). Green fluorescent protein (GFP) visualization in the pancreas from Therefore, Glp-1R either activates Gi or Gq proteins as well, or embryos, pups and adults stimulates the synthesis or release of other agonists of Gi/q- Rgs16::GFP and Rgs8::GFP BAC transgenic mice were generated coupled GPCRs in the pancreas, which would be regulated by by the Gene Expression Nervous System Atlas (GENSAT) project Rgs16 and Rgs8 (Wang et al., 2006). (Gong et al., 2003; Morales and Hatten, 2006). Pancreata were Daily injection of exendin-4 induced Rgs16::GFP expression in collected from Rgs16::GFP and Rgs8::GFP embryos (E8.5 through Disease Models & Mechanisms the pancreas. Rgs16::GFP expression was initiated in VDACs, to E16.5) and from postnatal stages P0 (birth) to P28. Pups were whereas -cell expression was observed in islets by day 6. weaned at P16 for time course studies of Rgs16::eGFP expression Interestingly, Glp-1R is specifically expressed in pancreatic ducts post-weaning. Tissues were dissected and transferred into ice-cold and islet -cells, and exendin-4 was reported to promote the 1ϫ PBS buffer. GFP visualization was accomplished by removing emergence of insulin-positive cells in cultured pancreatic ducts (Xu the embryonic gut tube and isolating the midgut, including the et al., 2006; Tornehave et al., 2008). The pattern of Rgs16::GFP pancreas, stomach and spleen. Tissue fragments were equilibrated induction by exendin-4 is strikingly similar to the early phase in in 40% glycerol for viewing. The pancreas was visualized using a PANIC and ob/ob mice, in which small numbers of VDACs appear Zeiss NeoLumar fluorescent microscope and photographed using transiently, and islet expression is first seen in one or a few closely an Olympus DP70 camera. Ngn3::eGFP embryos were generated clustered islets (supplementary material Fig. S10). Although by mating Ngn3::eGFP heterozygous males (generously provided speculative, the location of VDACs along pancreatic ducts, and their by Klaus Kaestner) with CD1 females. Embryos were dissected at appearance early in the time course of the hyperglycemic response different developmental stages, and pancreata were dissected and in mouse models of diabetes or in exendin-4 treatment, raises the visualized as described above. Pancreata from adult pregnant alluring possibility that these represent an endocrine progenitor females and ob/ob;Rgs16::eGFP transgenic mice were treated cell type. The contribution of endocrine progenitors within adult similarly to those from embryos, as described above. ducts to -cell regeneration is controversial. Future lineage-tracing experiments will be required to conclusively determine the Immunofluorescence staining of frozen sections endocrine potential of Rgs16+ VDACs. E15.5 dorsal pancreata were dissected from Rgs16::eGFP pregnant The induction of Rgs genes in VDACs and -cells implies females and were fixed overnight in 4% PFA in 1ϫ PBS at 4°C. The regulation of endogenous GPCRs that are important in the process next day the tissues were washed several times with 1ϫ PBS,

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equilibrated in 30% sucrose overnight and embedded in optimal Ki67 and Rgs16 were all determined, and rates of replication were cutting temperature (OCT) medium (Tissue-Tek). Cryosections calculated. (10 mm) of complete pancreata were mounted on SuperfrostPlus slides (Fisher) and immunofluorescence was carried out using: Immunofluorescence staining of frozen sections for VDACs chicken anti-GFP (1:500; Aves Labs), guinea pig anti-insulin (1:300; P15 pancreata from Rgs16::GFP mice were harvested and DakoCy), rabbit anti-glucagon (1:1000; LINCO), rat anti-CD31 immediately embedded in OCT medium and frozen. Sections (1:300; BD Pharmingen), rabbit anti-synaptophysin (1:200; (10 mm) were cut and allowed to dry overnight at room temperature. DakoCy), 5 mg/ml DBA (1:200; Vector Labs), rabbit anti-Pdx1 (1:600) Sections were then fixed for 5 minutes in ice-cold acetone and and mouse anti-ngn3 (1:4000) (Beta Cell Biology Consortium, rinsed several times in 1ϫ TBST (1ϫ TBS supplemented with 0.2% kindly provided by Dr Raymond MacDonald). TRITC secondary Tween). Sections were then blocked in 20% Aquablock in 1ϫ TBST, antibodies were from Jackson ImmunoResearch Laboratories and and stained using chicken anti-GFP (1:100) and one of the following anti-chicken Alexa 488 was from Invitrogen. Slides were primary antibodies: rabbit anti-sox9 (1:1000; Chemicon), guinea counterstained with DAPI and mounted with ProLong Gold pig anti-insulin (1:200; DakoCy), rabbit anti-glucagon (1:300; antifade reagent (Invitrogen). Images were acquired on a LSM 510 LINCO), goat anti-somatostatin (1:300; Santa Cruz), rabbit anti- META Zeiss confocal microscope. For PANIC-ATTAC samples, synaptophysin (1:300; DakoCy), rabbit anti-glut2 (1:200; Millipore), histology and immunofluorescence were performed as described 25 mg/ml rat anti-MECA32 (Rolf Brekken), 20 mg/ml rabbit anti- previously (Wang et al., 2008). Briefly, the pancreas was fixed in NG2 (Millipore), 20 mg/ml rat-anti Mac1(AbD Serotec). Sections 10% buffered formalin overnight. Paraffin sections (5 mm) were were stained overnight at 4°C then washed three times in 1ϫ TBST. incubated with guinea pig anti-insulin (1:500; DakoCy) and rabbit Bound primary antibody was visualized using appropriate Cy3- or anti-GFP (1:100; Invitrogen). The secondary antibodies used were FITC-conjugated secondary antibodies. Nuclei were visualized with donkey anti-guinea pig FITC (1:250; Jackson ImmunoResearch) and DAPI. Images were taken using a Nikon Eclipse E600 microscope. donkey anti-rabbit Cy3 (1:500; Jackson ImmunoResearch). Images

DMM were taken on a Leica TCS SP5 confocal microscope (Leica). Whole-mount immunofluorescence Pancreata were fixed for 1 hour in 4% PFA in PBS, washed and Immunofluorescence on paraffin sections dehydrated to 70% ethanol. Embryos were then washed in 50% E15.5 dorsal pancreata (Rgs16::eGFP) or adult pancreata from methanol for 1 hour and rinsed twice in 1ϫ PBS. The tissue was ob/ob;Rgs16::eGFP or PANIC-ATTAC;Rgs16::eGFP mice were permeabilized for 1 hour in 1% TritonX 100 in 1ϫ PBS, blocked dissected and fixed overnight in 4% PFA in 1ϫ PBS at 4°C. The in Cas-Block (Zymed), and immunofluorescence was then carried next day, the tissues were washed several times with 1ϫ PBS, out using: chicken anti-GFP (1:250; Aves Labs), guinea-pig anti- dehydrated and paraffin embedded in Paraplast Plus tissue insulin (1:200; DakoCy), rabbit anti-glucagon (LINCO), rat anti- embedding medium (McCormick). Sections (10 mm) of complete CD31 (1:200; BD Pharmingen), rabbit anti-synaptophysin (1:200; pancreata were mounted on SuperfrostPlus slides. Sections were DakoCy), 5 mg/ml DBA (1:200; Vector Labs), and rabbit anti-LYVE de-waxed in xylene; rehydrated through an ethanol series; washed (1:1000; Ambion). TRITC secondary antibodies were from Jackson several times in 1ϫ PBS; treated with R-Buffer A or R-Buffer B ImmunoResearch and anti-chicken Alexa 488 was from Invitrogen. (Electron Microscopy Sciences) in the 2100 Retriever; blocked for Tissues were dehydrated, cleared with BABB (1:2 mix of benzyl 1-2 hours with CAS-Block (Invitrogen); and incubated with chicken alcohol:benzyl benzoate), and visualized using a Zeiss NeoLumar anti-GFP (1:200), rabbit anti-sox9 (1:400; Chemicon), mouse anti- fluorescent microscope and photographed using an Olympus DP70 e-cadherin (1:200; Invitrogen), rabbit anti-glut2 (1:100; Abcam) and camera or LSM 510 META Zeiss confocal microscope. Disease Models & Mechanisms the four-hormones cocktail: guinea pig anti-glucagon (1:600; kindly provided by Raymond MacDonald), guinea pig anti-insulin (1:600; ob/ob;Rgs16::eGFP and ob/ob;Rgs8::eGFP mice DakoCy), goat anti-ghrelin (1:300; Beta Cell Consortium) and goat ob/+ breeder mice were obtained from Jackson Labs. Double anti-somatostatin (1:300; Santa Cruz). TRITC secondary antibodies heterozygous Rgs16::eGFP;ob/+ mice were intercrossed to obtain were from Jackson ImmunoResearch and anti-chicken Alexa 488 ob/ob, ob/ob;Rgs16::eGFP and ob/+;Rgs16::eGFP mice. Animals was from Invitrogen. Slides were counterstained with DAPI and were fed a standard rodent chow diet (Teklad) ad libitum. Blood mounted with ProLong Gold antifade reagent (Invitrogen). Images was acquired through tail clipping. Glucose levels were measured were acquired on a LSM 510 META Zeiss confocal microscope. by using a glucometer (AscensiaElite), and insulin levels were measured by using a rat/mouse insulin ELISA kit (Millipore) and Quantification of rates of replication read with a Sunrise microplater reader (Tecan). Immunofluorescence in paraffin sections of E15.5 dorsal pancreata (Rgs16::eGFP) or adult pancreata from ob/ob;Rgs16::GFP and PANIC-ATTAC;Rgs16::eGFP transgenic mice PANIC-ATTAC;Rgs16::GFP transgenic mice were performed PANIC-ATTAC transgenic mice were generated as described utilizing chicken anti-GFP (1:200) and either rabbit anti-Ki67 previously (Wang et al., 2008). Briefly, the rat insulin promoter was (1:200; Invitrogen) or rat anti-Brdu (1:50; Serotec). A total of eight used to drive the expression of an FKBP-caspase 8 fusion protein. fields of three different E15.5 embryonic pancreata, 70 islets of three Homozygous PANIC-ATTAC animals were crossed to Rgs16::eGFP different ob/ob;Rgs16::GFP pancreata, and 13 islets for PANIC- transgenic mice. All progeny were hemizygous for the PANIC- ATTAC;Rgs16::GFP transgenic pancreata were studied. The total ATTAC transgene. At about 12 weeks of age, animals were grouped number of endocrine cells per islet/field, the total number of Ki67+ into hemizygous PANIC-ATTAC;Rgs16::GFP transgenic mice cells, the total number of Rgs16+ cells, and the total overlap between (n2), hemizygous PANIC-ATTAC;Rgs16::eGFP-negative mice

Disease Models & Mechanisms 577 RESEARCH ARTICLE Rgs8/16 reactivated in expanding islets

(n4) and FVB control mice (n3). The dimerizer AP20187 was administered to animals according to the manufacturer’s TRANSLATIONAL IMPACT recommendations (Ariad Pharmaceuticals). For hemizygous Clinical issue PANIC-ATTAC mice, dimerizer (0.2 mg/g body weight) was Diabetes exacts one of the highest annual costs for treatment of any disease in injected either twice per day, every other day for eight days, or once the world and is the seventh leading cause of death in the USA. Most cases of per day at 12.00 h for five consecutive days. Fed glucose levels were both type 1 and type 2 diabetes involve the actual or functional loss of insulin- monitored using a glucometer and strips (Abbott Diabetes Care). producing pancreatic -cells. Although -cells can replicate, little is known After five days treatment, hemizygous PANIC-ATTAC males about how the process is regulated and available therapies for diabetes cannot showed either moderate (glucose <300 mg/dl) or severe (glucose specifically stimulate -cell regeneration. There is hope that understanding the signals that control islet cell proliferation and differentiation will help generate >300 mg/dl) hyperglycemia. Animals were sacrificed two weeks targeted -cell regenerative therapies for diabetic patients. later, with the pancreas processed for immunofluorescent staining (Fig. 6), or at the times indicated for analysis of GFP expression Results (supplementary material Fig. S6). Insulin levels were assessed as This study shows that two members of the gene family regulators of G protein signaling (RGS), Rgs8 and Rgs16, coordinate -cell expansion with metabolic per ob/ob mice.  need. Rgs8 and Rgs16 are expressed during islet development and in mouse models of type 1 and type 2 diabetes. Exendin-4, a Glp-1/incretin mimetic that +/– Exendin-4 , glucose or PBS treatment stimulates -cell expansion in diabetics, also promotes the expression of Male mice aged 8-12 weeks (30-40 g) were injected, Rgs16::GFP in pancreatic ductal-associated cells and islet -cells in mice. As the intraperitoneally, twice daily at zeitgeber time ZT6 (6 hours after name implies, RGS members regulate the frequency and duration of G protein- lights on) and ZT11. Exendin-4 was injected at a concentration of coupled receptor (GPCR) signaling. Since GPCR pathways are also associated 200 ng/mouse time point in a volume of 100 ml, glucose at 4.5 g/kg, with -cell expansion, this suggests that RGS proteins serve as sensitive and early beacons of G protein signaling in -cell progenitor expansion and and PBS at a volume of 100 ml. regeneration during development and in metabolically stressed adults.

DMM GFP quantification of exendin-4/glucose images Implications and future directions Original JPEG RGB images, at a resolution of 1360ϫ1024 pixels, This study indicates that the RGS proteins Rgs8 and Rgs16 coordinate GPCR ϫ signaling with islet development and, later in life, with metabolic stress. The of mouse pancreas taken with a 48 objective magnification were early response of these proteins to metabolic changes suggests that they may converted into 8-bit gray-scale format without rescaling in ImageJ be useful screening indicators. Furthermore, understanding the mechanisms (NIH). Background levels with a rolling ball radius of 50 pixels were that regulate GPCR induction of pancreatic -cell proliferation may provide a subtracted from images. Varying lower threshold adjustments crucial inroad to diabetes therapy. Future work should determine the potential were selected based on the contours of GFP+ regions. Integrated of RGS proteins to promote -cell development and islet regeneration. densities as the sum of 8-bit gray values, on a 0-255 scale per pixel, doi:10.1242/dmm.005850 were obtained from each particle with a size of three pixels or greater. The sum of analyzed integrated densities as a total GFP REFERENCES value was calculated using MS Excel. Three-dimensional bar graphs Aguirre, A. J., Bardeesy, N., Sinha, M., Lopez, L., Tuveson, D. A., Horner, J., correlating the levels of blood glucose, insulin, Rgs16::GFP Redston, M. S. and DePinho, R. A. (2003). Activated Kras and Ink4a/Arf deficiency expression in islets, and the age of ob/ob mice or the time after cooperate to produce metastatic pancreatic ductal adenocarcinoma. Genes Dev. 17, 3112-3126. treatment in PANIC mice, were created by Matlab. Atkinson, M. A. and Gianani, R. (2009). The pancreas in human type 1 diabetes: ACKNOWLEDGEMENTS providing new answers to age-old questions. Curr. Opin. Endocrinol. Diabetes Obes.

Disease Models & Mechanisms We thank M. E. Hatten (Rockefeller University) for kindly providing the Rgs16::GFP 16, 279-285. and Rgs8::GFP BAC transgenic mice; Klaus Kaestner (University of Pennsylvania Berman, D. M., Wilkie, T. M. and Gilman, A. G. (1996). GAIP and RGS4 are GTPase- School of Medicine) for providing the Ngn3-eGFP mice; Diana Chong for technical activating proteins for the Gi subfamily of G protein alpha subunits. Cell 86, 445-452. assistance; Raymond MacDonald for providing antibodies; Galvin Swift for E14.5 Bock, T., Pakkenberg, B. and Buschard, K. (2003). Increased islet volume but mRNA; Ariad Pharmaceuticals for providing the dimerizer for the PANIC-ATTAC unchanged islet number in ob/ob mice. Diabetes 52, 1716-1722. mice; Victor Pashkov for genotyping, qPCR and discussions; Alex Artyukhin for Bond, A. (2006). Exenatide (Byetta) as a novel treatment option for type 2 diabetes assistance with image analysis; and Raymond MacDonald, Galvin Swift, Nils mellitus. Proc. (Bayl. Univ. Med. Cent.) 19, 281-284. Bonner-Weir, S., Toschi, E., Inada, A., Reitz, P., Fonseca, S. Y., Aye, T. and Sharma, A. Halberg, Michael Dellinger and Deborah Clegg for helpful discussions and/or (2004). The pancreatic ductal epithelium serves as a potential pool of progenitor reading the manuscript. This work was supported by NIH R0161395, cells. 5 Suppl. 2, 16-22. P50MH066172, Welch I-1382 (to T.M.W.); NIH R01CA118240 (to R.A.B.); JDRF 1- Pediatr. Diabetes Bonner-Weir, S., Inada, A., Yatoh, S., Li, W. C., Aye, T., Toschi, E. and Sharma, A. 2008-16 (to P.E.S.); and NIH DK079862 and JDRF 99-2007-472 (to O.C.). Deposited (2008). Transdifferentiation of pancreatic ductal cells to endocrine -cells. in PMC for release after 12 months.  Biochem. Soc. Trans. 36, 353-356. COMPETING INTERESTS Cano, D. A., Rulifson, I. C., Heiser, P. W., Swigart, L. B., Pelengaris, S., German, M., The authors declare no competing financial interests. Evan, G. I., Bluestone, J. A. and Hebrok, M. (2008). Regulated -cell regeneration in the adult mouse pancreas. Diabetes 57, 958-966. AUTHOR CONTRIBUTIONS Chu, Z. L., Jones, R. M., He, H., Carroll, C., Gutierrez, V., Lucman, A., Moloney, M., O.C. and T.M.W. conceived the project and, with A.V., Z.V.W., R.A.B. and P.E.S., Gao, H., Mondala, H., Bagnol, D. et al. (2007). A role for -cell-expressed G protein- designed the experiments. A.V., Z.V.W., L.B.R., O.O., I.W.A., O.C. and T.M.W. coupled receptor 119 in glycemic control by enhancing glucose-dependent insulin performed the experiments. All authors analyzed data and edited the manuscript. release. Endocrinology 148, 2601-2609. A.V., O.C. and T.M.W. wrote the manuscript. Chua, S., Jr, Liu, S. M., Li, Q., Yang, L., Thassanapaff, V. T. and Fisher, P. (2002). SUPPLEMENTARY MATERIAL Differential beta cell responses to hyperglycaemia and insulin resistance in two Supplementary material for this article is available at novel congenic strains of diabetes (FVB-Lepr (db)) and obese (DBA-Lep (ob)) mice. http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.003210/-/DC1 Diabetologia 45, 976-990. Cleaver, O. (2004). Blood vessel signals during development and beyond. Curr. Top. Received 17 March 2009; Accepted 26 March 2010. Dev. Biol. 62, 1-36.

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Cleaver, O. and Melton, D. A. (2003). Endothelial signaling during development. Nat. Koelle, M. R. and Horvitz, H. R. (1996). EGL-10 regulates G protein signaling in the C. Med. 9, 661-668. elegans nervous system and shares a conserved domain with many mammalian Cleaver, O. and MacDonald, R. J. (2009). Developmental molecular biology of the proteins. Cell 84, 115-125. pancreas. In Handbook of Pancreatic Cancer (ed. J. A. J. Neoptolemos, M. Buchler and Kroon, E., Martinson, L. A., Kadoya, K., Bang, A. G., Kelly, O. G., Eliazer, S., Young, R. Urrutia), pp. 71-118. New York, NY: Springer. H., Richardson, M., Smart, N. G., Cunningham, J. et al. (2008). Pancreatic Collombat, P., Hecksher-Sorensen, J., Serup, P. and Mansouri, A. (2006). Specifying endoderm derived from human embryonic stem cells generates glucose-responsive pancreatic endocrine cell fates. Mech. Dev. 123, 501-512. insulin-secreting cells in vivo. Nat. Biotechnol. 26, 443-452. D’Amour, K. A., Agulnick, A. D., Eliazer, S., Kelly, O. G., Kroon, E. and Baetge, E. E. Lammert, E., Cleaver, O. and Melton, D. (2001). Induction of pancreatic (2005). Efficient differentiation of human embryonic stem cells to definitive differentiation by signals from blood vessels. Science 294, 564-567. endoderm. Nat. Biotechnol. 23, 1534-1541. Lammert, E., Cleaver, O. and Melton, D. (2003). Role of endothelial cells in early D’Amour, K. A., Bang, A. G., Eliazer, S., Kelly, O. G., Agulnick, A. D., Smart, N. G., pancreas and liver development. Mech. Dev. 120, 59-64. Moorman, M. A., Kroon, E., Carpenter, M. K. and Baetge, E. E. (2006). Production Lee, C. S., De Leon, D. D., Kaestner, K. H. and Stoffers, D. A. (2006). Regeneration of of pancreatic hormone-expressing endocrine cells from human embryonic stem pancreatic islets after partial pancreatectomy in mice does not involve the cells. Nat. Biotechnol. 24, 1392-1401. reactivation of neurogenin-3. Diabetes 55, 269-272. Dohlman, H. G., Song, J., Ma, D., Courchesne, W. E. and Thorner, J. (1996). Sst2, a Lee, Y. C. and Nielsen, J. H. (2009). Regulation of beta cell replication. Mol. Cell negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: Endocrinol. 297, 18-27. expression, localization, and genetic interaction and physical association with Gpa1 Li, H. and Edlund, H. (2001). Persistent expression of Hlxb9 in the pancreatic (the G-protein alpha subunit). Mol. Cell. Biol. 16, 5194-5209. epithelium impairs pancreatic development. Dev. Biol. 240, 247-253. Dor, Y., Brown, J., Martinez, O. I. and Melton, D. A. (2004). Adult pancreatic -cells Llona, I. (1995). Synaptic like microvesicles: do they participate in regulated are formed by self-duplication rather than stem-cell differentiation. Nature 429, 41- exocytosis? Neurochem. Int. 27, 219-226. 46. Luo, X., Popov, S., Bera, A. K., Wilkie, T. M. and Muallem, S. (2001). RGS proteins Doupnik, C. A., Davidson, N., Lester, H. A. and Kofuji, P. (1997). RGS proteins provide biochemical control of agonist-evoked [Ca2+]i oscillations. Mol. Cell 7, 651- reconstitute the rapid gating kinetics of G-activated inwardly rectifying K+ 660. channels. Proc. Natl. Acad. Sci. USA 94, 10461-10466. Morales, D. and Hatten, M. E. (2006). Molecular markers of neuronal progenitors in Fineman, M. S., Bicsak, T. A., Shen, L. Z., Taylor, K., Gaines, E., Varns, A., Kim, D. the embryonic cerebellar anlage. J. Neurosci. 26, 12226-12236. and Baron, A. D. (2003). Effect on glycemic control of exenatide (synthetic exendin- Nakamura, K., Salomonis, N., Tomoda, K., Yamanaka, S. and Conklin, B. R. (2009). 4) additive to existing metformin and/or sulfonylurea treatment in patients with type G(i)-coupled GPCR signaling controls the formation and organization of human 2 diabetes. Diabetes Care 26, 2370-2377. pluripotent colonies. PLoS One 4, e7780.

DMM Gilman, A. G. (1987). G proteins: transducers of receptor-generated signals. Annu. Rev. Nir, T., Melton, D. A. and Dor, Y. (2007). Recovery from diabetes in mice by beta cell Biochem. 56, 615-649. regeneration. J. Clin. Invest. 117, 2553-2561. Gittes, G. K. and Rutter, W. J. (1992). Onset of cell-specific gene expression in the Offield, M. F., Jetton, T. L., Labosky, P. A., Ray, M., Stein, R. W., Magnuson, M. A., developing mouse pancreas. Proc. Natl. Acad. Sci. USA 89, 1128-1132. Hogan, B. L. and Wright, C. V. (1996). PDX-1 is required for pancreatic outgrowth Golosow, N. and Grobstein, C. (1962). Epitheliomesenchymal interaction in pancreatic and differentiation of the rostral duodenum. Development 122, 983-995. morphogenesis. Dev. Biol. 4, 242-255. Oliver-Krasinski, J. M. and Stoffers, D. A. (2008). On the origin of the beta cell. Genes Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., Dev. 22, 1998-2021. Nowak, N. J., Joyner, A., Leblanc, G., Hatten, M. E. et al. (2003). A gene expression Pictet, R. L. and Rutter, W. J. (1972). Development of the embryonic pancreas. In atlas of the central nervous system based on bacterial artificial chromosomes. Nature Endocrine Pancreas: Handbook of Physiology (ed. D. F. Steiner and N. Frienkel), pp. 25- 425, 917-925. 66. Baltimore: Williams and Wilkins. Gradwohl, G., Dierich, A., LeMeur, M. and Guillemot, F. (2000). neurogenin3 is Poy, M. N., Hausser, J., Trajkovski, M., Braun, M., Collins, S., Rorsman, P., Zavolan, required for the development of the four endocrine cell lineages of the pancreas. M. and Stoffel, M. (2009). miR-375 maintains normal pancreatic - and -cell mass. Proc. Natl. Acad. Sci. USA 97, 1607-1611. Proc. Natl. Acad. Sci. USA 106, 5813-5818. Gu, G., Wells, J. M., Dombkowski, D., Preffer, F., Aronow, B. and Melton, D. A. Prentki, M. and Nolan, C. J. (2006). Islet beta cell failure in type 2 diabetes. J. Clin. (2004). Global expression analysis of gene regulatory pathways during endocrine Invest. 116, 1802-1812. pancreatic development. Development 131, 165-179. Regard, J. B., Kataoka, H., Cano, D. A., Camerer, E., Yin, L., Zheng, Y. W., Scanlan, T. Gupta, R. K., Gao, N., Gorski, R. K., White, P., Hardy, O. T., Rafiq, K., Brestelli, J. E., S., Hebrok, M. and Coughlin, S. R. (2007). Probing cell type-specific functions of Gi Chen, G., Stoeckert, C. J., Jr and Kaestner, K. H. (2007). Expansion of adult -cell in vivo identifies GPCR regulators of insulin secretion. J. Clin. Invest. 117, 4034-4043. mass in response to increased metabolic demand is dependent on HNF-4. Genes Ross, E. M. and Wilkie, T. M. (2000). GTPase-activating proteins for heterotrimeric G Dev. 21, 756-769. proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Disease Models & Mechanisms Herrera, P. L., Huarte, J., Zufferey, R., Nichols, A., Mermillod, B., Philippe, J., Biochem. 69, 795-827. Muniesa, P., Sanvito, F., Orci, L. and Vassalli, J. D. (1994). Ablation of islet Serafimidis, I., Rakatzi, I., Episkopou, V., Gouti, M. and Gavalas, A. (2008). Novel endocrine cells by targeted expression of hormone-promoter-driven toxigenes. Proc. effectors of directed and Ngn3-mediated differentiation of mouse embryonic stem Natl. Acad. Sci. USA 91, 12999-13003. cells into endocrine pancreas progenitors. Stem Cells 26, 3-16. Huang, J., Pashkov, V., Kurrasch, D. M., Yu, K., Gold, S. J. and Wilkie, T. M. (2006). Sherry, N. A., Chen, W., Kushner, J. A., Glandt, M., Tang, Q., Tsai, S., Santamaria, P., Feeding and fasting controls liver expression of a regulator of G protein signaling Bluestone, J. A., Brillantes, A. M. and Herold, K. C. (2007). Exendin-4 improves (Rgs16) in periportal hepatocytes. Comp. Hepatol. 5, 8. reversal of diabetes in NOD mice treated with anti-CD3 monoclonal antibody by Inada, A., Nienaber, C., Katsuta, H., Fujitani, Y., Levine, J., Morita, R., Sharma, A. enhancing recovery of -cells. Endocrinology 148, 5136-5144. and Bonner-Weir, S. (2008). Carbonic anhydrase II-positive pancreatic cells are Sierra, D. A., Gilbert, D. J., Householder, D., Grishin, N. V., Yu, K., Wensel, T. G., progenitors for both endocrine and exocrine pancreas after birth. Proc. Natl. Acad. Otero, G., Copeland, N. G., Jenkins, N. A., Wilkie, T. M. et al. (2002). Evolution of Sci. USA 105, 19915-19919. the regulators of G-protein signaling multigene family in mouse and human. Keller, M. P., Choi, Y., Wang, P., Davis, D. B., Rabaglia, M. E., Oler, A. T., Stapleton, D. Genomics 79, 177-185. S., Argmann, C., Schueler, K. L., Edwards, S. et al. (2008). A gene expression Simon, M. I., Strathmann, M. P. and Gautam, N. (1991). Diversity of G proteins in network model of type 2 diabetes links cell cycle regulation in islets with diabetes signal transduction. Science 252, 802-808. susceptibility. Genome Res. 18, 706-716. Su, A. I., Wiltshire, T., Batalov, S., Lapp, H., Ching, K. A., Block, D., Zhang, J., Soden, Kendall, D. M., Riddle, M. C., Rosenstock, J., Zhuang, D., Kim, D. D., Fineman, M. S. R., Hayakawa, M., Kreiman, G. et al. (2004). A gene atlas of the mouse and human and Baron, A. D. (2005). Effects of exenatide (exendin-4) on glycemic control over protein-encoding transcriptomes. Proc. Natl. Acad. Sci. USA 101, 6062-6067. 30 weeks in patients with type 2 diabetes treated with metformin and a Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from sulfonylurea. Diabetes Care 28, 1083-1091. mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676. Kim, S. K. and MacDonald, R. J. (2002). Signaling and transcriptional control of Teta, M., Rankin, M. M., Long, S. Y., Stein, G. M. and Kushner, J. A. (2007). Growth pancreatic organogenesis. Curr. Opin. Genet. Dev. 12, 540-547. and regeneration of adult beta cells does not involve specialized progenitors. Dev. Kodama, S., Toyonaga, T., Kondo, T., Matsumoto, K., Tsuruzoe, K., Kawashima, J., Cell 12, 817-826. Goto, H., Kume, K., Kume, S., Sakakida, M. et al. (2005). Enhanced expression of Tornehave, D., Kristensen, P., Romer, J., Knudsen, L. B. and Heller, R. S. (2008). PDX-1 and Ngn3 by exendin-4 during beta cell regeneration in STZ-treated mice. Expression of the GLP-1 receptor in mouse, rat, and human pancreas. J. Histochem. Biochem. Biophys. Res. Commun. 327, 1170-1178. Cytochem. 56, 841-851.

Disease Models & Mechanisms 579 RESEARCH ARTICLE Rgs8/16 reactivated in expanding islets

Tourrel, C., Bailbe, D., Meile, M. J., Kergoat, M. and Portha, B. (2001). Glucagon-like Xu, G., Kaneto, H., Lopez-Avalos, M. D., Weir, G. C. and Bonner-Weir, S. (2006). GLP- peptide-1 and exendin-4 stimulate -cell neogenesis in streptozotocin-treated 1/exendin-4 facilitates -cell neogenesis in rat and human pancreatic ducts. Diabetes newborn rats resulting in persistently improved glucose homeostasis at adult age. Res. Clin. Pract. 73, 107-110. Diabetes 50, 1562-1570. Xu, X., D’Hoker, J., Stange, G., Bonne, S., De Leu, N., Xiao, X., Van de Casteele, Van Assche, F. A., Aerts, L. and De Prins, F. (1978). A morphological study of the M., Mellitzer, G., Ling, Z., Pipeleers, D. et al. (2008). Beta cells can be endocrine pancreas in human pregnancy. Br. J. Obstet. Gynaecol. 85, 818-820. generated from endogenous progenitors in injured adult mouse pancreas. Cell Villasenor, A., Chong, D. C. and Cleaver, O. (2008). Biphasic Ngn3 expression in the 132, 197-207. developing pancreas. Dev. Dyn. 237, 3270-3279. Yoshitomi, H. and Zaret, K. S. (2004). Endothelial cell interactions initiate dorsal Wang, C., Kerckhofs, K., Van de Casteele, M., Smolders, I., Pipeleers, D. and Ling, Z. pancreas development by selectively inducing the transcription factor Ptf1a. (2006). Glucose inhibits GABA release by pancreatic -cells through an increase in Development 131, 807-817. GABA shunt activity. Am J. Physiol. Endocrinol. Metab. 290, E494-E499. Zeng, W., Xu, X., Popov, S., Mukhopadhyay, S., Chidiac, P., Yagaloff, K. A., Fisher, S. Wang, Z. V., Mu, J., Schraw, T. D., Gautron, L., Elmquist, J. K., Zhang, B. B., L., Ross, E. M., Muallem, S., Wilkie, T. M. et al. (1998). The N-terminal domain of Brownlee, M. and Scherer, P. E. (2008). PANIC-ATTAC, a mouse model for inducible RGS4 confers receptor-selective inhibition of G protein signaling. J. Biol. Chem. 273, and reversible -cell ablation. Diabetes 57, 2137-2148. 34687-34690. Wells, J. M. and Melton, D. A. (1999). Vertebrate endoderm development. Annu. Rev. Zerangue, N. and Jan, L. Y. (1998). G-protein signaling: fine-tuning signaling kinetics. Cell Dev. Biol. 15, 393-410. Curr. Biol. 8, R313-R316. Xu, G., Stoffers, D. A., Habener, J. F. and Bonner-Weir, S. (1999). Exendin-4 stimulates Zhou, Q., Law, A. C., Rajagopal, J., Anderson, W. J., Gray, P. A. and Melton, D. A. both -cell replication and neogenesis, resulting in increased -cell mass and (2007). A multipotent progenitor domain guides pancreatic organogenesis. Dev. Cell improved glucose tolerance in diabetic rats. Diabetes 48, 2270-2276. 13, 103-114. DMM Disease Models & Mechanisms

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