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1 ~ 5 % ltion > 99 % ltion Gly yemi (mmol/l) Time fter -ell ltion (dys) Survivl (%) & ~ 5 % ltion 75 > 99% ltion (dys) 7.4 d 1.25 lood ph d ody weight h nge mo e insulin o ontent (ng/mg of p nres) ~ 5% > 99% β-ell ltion f Fold Chnge Fold Chnge insulin 1 mrna insulin 2 mrna 7d 15d 1mo Supplementry Figure S1: Aute -ell ltion., Fsting glyemi in RIP-DTR mie either untreted or fter ltion (n=13-41 per group)., Mortlity urves. Mie treted with DT died within 2 months. n=3-4 mie/group., Blood ph dropped to 7.15 rpidly fter DT dministrtion (, 7.35±.2, n=6; 7 dys fter DT tretment: 7.14±.6, n=6). P<.1. d, Body weight vrition during 1 month fter DT (1 indites no hnge etween dy nd dy 3). DT-treted mie hve 15% ody weight derese during this period (, 1.7±.4, n=5; 1 month fter DT:.84±.2, n=6). P<.1. e, Dorsl pnreti insulin ontent dropped 15 dys fter DT from 175.9±11.9 ng/mg (untreted, n=9) to 4.4±14.2 ng/mg in hemizygous femles (n=9) nd.44±.9 ng/mg in mles nd homozygous femles (n=19). This represents β-ell destrution of 41% nd 99.8%, respetively. P<.5. f, Rel-time quntittive PCR of the 2 insulin trnsripts efore nd fter β-ell ltion. Reovery of insulin trnsription is signifint 3 dys fter DT, inditing tht there is erly regenertion fter -ell removl. n=5 mie/group. P<
2 insulin glugon H&E; DT, 3d 99% ltion o ontrol d DT, 3d Supplementry Figure S2: DT seletively triggers -ell loss y poptosis. -, β-ells re lmost undetetle 2 weeks fter DT. DAPI (lue). Brs: 5 µm., Pnreti setion stined with hemtoxilin & eosin (H&E). Mssive islet ell poptosis is oserved 3 dys fter one DT injetion (pyknoti nulei in inserts). Sle rs: 5µm nd 2 µm (inserts). d, Eletron mirosopy on pnret 3 dys fter DT. Left nd middle pnels: poptoti -ells with highly ondensed hromtin. Right pnel: n endophgosome in n djent endorine ell ontins -ell poptoti ody filled with insulin grnules. Sle rs re 5nm. 2
3 DT Glyemi (mmol/l) insulin tretment no insulin tretment dieti reovered (months) insulin glugon - -less islets (% of totl islets) d mo; re. 15d mo; reovered Supplementry Figure S3: -ell regenertion., Fsting glyemi in 8 DT-treted mles. Mie reeived insulin during 5 months. Four nimls rehed ner norml vlues ( reovered )., Proportion of islets ontining up to 2 β-ells/setion (n=3-4 mie/group; 232, 225 nd 117 islets sored in s nd 15 dys or months fter DT, respetively). One-wy ANOVA (p=.241) nd Mnn-Whitney test ( p<.5)., Pnret.5 nd months fter DT. Brs: 5 m. 3
4 RIP Cre-ERT Ros26 STOP YFP RIP DTR -ell trgeting induile YFP leling -ell ltion regenertion from esping -ells new -ells (YFP+) -ell leling (YFP+) -ell ltion DT other origins new -ells (YFP-) TAM NO TAM -ell regenertion (7 dys) (1 mo) Glugon rtta TetO Cre Ros26 STOP YFP RIP DTR -ell trgeting induile YFP leling -ell ltion - to - new -ells s (YFP+) induile leling of -ells (YFP+) remining -ells (<1%) other origins new -ells (YFP-) Birth DOX No DOX -ell regenertion (1 mo) (15 dys) (15 dys) DT (1 mo) Supplementry Figure S4: Conditionl ell linege tring nlyses., Experimentl design of -ell linege tring., Experimentl design of onditionl -ell linege tring. 4
5 Glugon ontent (ng g/mg of pnres) Glug gonemi (pg/ml) 2 15 lugon mrna (fold hnge) d 15d 7d 15d 1mo G d e Ki67+ ells (% of -ells) ell mss ( g) d 7d 15d 1mo mo 15d 1mo mo f Glugon + e ells (% of -ells s) g 1mo insulin glugon merged 7d 15d 1mo mo Supplementry Figure S5: -ell ltion influenes -ells.,, Inrese in pnreti () nd irulting () glugon fter DT tretment, determined y RIA. (n= 5- mie/group). P<.5; P<.1., Rel time PCR of glugon mrna revels the inresed glugon gene expression 1 month fter -ell ltion (n=5 mie/group). d, -ell prolifertion determined with nti-glugon nd nti-ki67 ntiodies. n=3-7 mie per time group; 119 to 82 -ells sored/mouse, from -2 islets/individul. e, Histogrms of -ell mss (n=3-4 mie per time group). f, g, Cololiztion of glugon nd insulin fter β-ell ltion. f, Proportion of glugon+/insulin+ ells fter DT (n=4-11 mie/group; 7 dys post- DT: 4-2 -ells sored/mouse, from 2- islets/individul; 15 dys: ells/mouse, from 4-16 islets/mouse; 1 month: ells/mouse, from islets/mouse; months: ells/mouse, from 8-18 islets/mouse). Groups were ompred using one-wy ANOVA (p=.69) nd Mnn-Whitney tests ( p<.5, 5 for eh omprison with s). g, Pnret from DT-treted mie, with nti-insulin insulin (green) nd ntiglugon (red) ntiodies. Arrow: ell ontining insulin nd glugon simultneously. Brs re 2 m. 5
6 Insulin pdx1 glugon pdx1 Insulin glugon Merged ells on+ ells) PDX1+ (% of glugo mo mo DT, 1mo Supplementry Figure S6: Pdx1 expression in -ells fter -ell ltion., Pdx1 is expressed in frtion of -ells fter -ell ltion. Proportions of -ells stined with nti-pdx1 ntiody 1 nd month fter DT (n=3-4 mie/group; ells sored/mouse, from 9-14 islets/individul). P<.5. 5, Pnreti setions immunostined with nti-insulin insulin (green), nti-glugon (red) nd nti-pdx1 (white) ntiodies, from RIP-DTR mie efore () nd 1 month fter DT. Arrows show ells ontining Pdx1, Insulin nd Glugon; rrowheds, ells ontining Glugon nd Pdx1, ut not Insulin. Brs re 2 m. 6
7 No DOX DOX Glugo on YFP DAPI.2% 88% Insulin Glugon d YFP DAPI.2% Somtosttin e f Glugon.2% Supplementry Figure S7: Induile -ell leling in Glugon-rtTA; Tet-Cre; R26-YFP; RIP-DTR trnsgeni mie., DOX-untreted mie (negtive s): less thn.3% of islet ells re YFP+ (.24±.11% for β-ells; n=4 mie;,44 islet ells sored). -f, DOX tretment (2 weeks fter wening). The glugon-rtta g system is effiient nd speifi: Most -ells re leled (88.9%±4.42%; n=3 mie; 2,258 -ells sored from 8 islets) wheres lmost no β- nd -ells re YFP-tgged with DOX (.28±.3% for β-ells; 624 β-ells sored from8 islets; n=3, nd.27±.27 for -ells; 315 -ells sored from 82 islets; n=3). 7
8 Glugon Cre Ros26 STOP YFP RIP DTR -ell trgeting onstitutive YFP leling -ell ltion -ells (YFP+) DT -ell differentition from glugon-expressing ells other origins -ell regenertion (1mo) new -ells (YFP+) new -ells (YFP-) lls) YFP+ ells (% of insulin+ el 3 2 insulin YFP glugon merged d e f g 1mo h i j k 1mo l m n o Supplementry Figure S8: Glugon-expressing ells give rise to new -ells., Cell linege tring nlysis with Glugon-Cre; R26-YFP; RIP-DTR trnsgeni mie., Experimentl design. Mie hving most of their -ells leled with YFP were given DT. A month lter, insulinontining ells derived from tgged -ells, or from ells trnsiently expressing the glugon gene fter ltion, should express the reporter moleule., Proportions of YFP-leled insulin-ontining ells. Before DT injetion, no -ell expressed YFP. One month fter DT tretment, 25.1±3.5% of the -ells ws YFP+ (n=3-4 mie/group; s:.36±.18%; ells/mouse, from to 14 islets/mouse; DT-treted mie: 45-1 β-ells/mouse, from 11-2 islets/mouse). d-o, Pnret from untreted (, d-g) or DT-treted mie (h-o) nd srified 1 month lter. In s, lmost no insulin+ ells express the YFP reporter. One month fter DT, insulin+ ells re frequently YFP+ (h-o; white rrowhed in h-k), inditing their origin from ells hving expressed glugon. Insulin (red), glugon (purple) nd YFP (green). Dshed lines delinete one islet (h-k). Sle rs: 2µm. 8
9 e ) Glugon surf (% of totl re Control (DT, 7d) 1 insulin glugon 1 Glu gon ontent (pg/m mg of pnres) Glu-DTR Control (DT, 7d) Glu-DTR DT, 7d d Glu-DTR e insulin 4 glugon Glugon + ells (% of insulin+ells) 3 2 RIP-DTR (DT, 15d) RIP-DTR Glu-DTR RIP-DTR DT, 15d RIP-DTR; Glu-D DTR Supplementry Figure S9: Simultneous o-ltion of - nd -ells. -. -ell ltion in Glugon-DTR mie ( Glu-DTR )., -ell re in s nd 7 dys fter DT. -ell ltion represents redution of 96.6% 6% of vlue (.6±.2% 2%vs 2±.2±.4%, 4% n=3 mie per group). p<.5., Pnreti glugon ontent 7 dys fter DT. The mount of glugon drops from 1,685±284 pg/mg in s to 2.4±.3 pg/mg 7 dys fter DT (n=5 nd 6, respetively). This represents derese of 99.8%. p<.1., Pnreti setions from Glugon-DTR mie efore ( ) nd 7 dys fter DT ( DT, 7d ), stined for insulin (green) nd glugon (red). DT triggers the destrution of lmost ll -ells. Sle r: µm (left pnels) nd µm (right pnels, 1-4). d, Proportion of insulin+ ells lso expressing glugon fter ltion of β-ells ( RIP- DTR ) or fter the o-ltion of β- nd -ells ( RIP-DTR; Glu-DTR ). In RIP-DTR; Glugon-DTR mie, glugon/insulin o-expressing ells do not pper fter the o-ltion of - nd -ells (1 glugon+/insulin+ ell ws oserved in 265 islets nlyzed; in RIP-DTR only mie, 43 glugon+/insulin+ ells were found in totl of 225 islets studied; n=3 mie per group). p<.1. e, Pnreti setions from RIP-DTR nd from RIP-DTR; Glugon-DTR mie, 15 dys fter DT tretment. At low mgnifition with nti-insulin (green) nd nti-glugon (red) stining, islets n e esily depited in RIP-DTR euse of the presene of glugon+ ells (upper left pnel), wheres islets re undetetle in RIP-DTR; Glugon-DTR (lower left pnel, dshed lines). One glugon+/insulin+ ell is shown in RIP-DTR mie (upper right), nd few ells ontining insulin or glugon re shown in RIP-DTR; Glugon-DTR islet (lower right; dshed line). Sle rs re 5 m (left pnels) nd 2 m (right pnels). 9
10 RIP-DTR/Glugon-rtTA/TetO-CRE/R26-YFP dithizone YFP DO OX- terted Control d DT-treted isolted islets insulin glugon -174x D7 DT Supplementry Figure S: Vritions of islet gene expression fter ner-totl β-ell ltion. The regenertive regultion of -ell-speifi genes is estlished within islets., -ell linege gene expression fter DT (totl pnres Q-PCR). All genes re down-regulted t dy 15 ( D-D15 period), ut upregulted during reovery initition ( D15-D3 ). Vlues re rtios etween one regenertive time-point (dy D15 or D3) nd the previous ( seline )., Islet isoltion in RIP-DTR; Glugon-rtTA; TetO-Cre; R26-YFP trnsgeni mie. Isolted islets from n untreted mouse (Top) nd DT-treted mouse (ottom), s seen either with right-field mirosopy (left pnels; dithizone) or epifluoresene (right pnels). Note the islet size differene etween s nd DT-treted, whih is due to the loss of -ells ( -ells re YFP+; sme mgnifition in ll pnels). Islets were isolted 7 dys fter -ell ltion with DT., Semi-quntittive RT-PCR using RNA extrted of islets isolted from nd DT-treted mie (see Methods online). There is 174-fold derese for insulin2 trnsripts fter -ell ltion, wheres the glugon trnsripts remin like in isolted DT-untreted islets. d, Q-PCR for -ell-speifi genes using RNA extrted of islets isolted from nd DT-treted mie t different time-points following DT (7 nd 15 dys). Note tht the initil downregultion ( D-D7 period, i.e. 7 dys fter ltion) t is reversed e t lter time-points ts when the trnsription of these genes es grdully resumes ( D7-D15 period; 15 dys fter ltion). Vlues represent the rtio etween ertin regenertive time-point (dy D7 or D15) nd the previous one ( seline, i.e. dy D or D7, respetively).
11 Ins YFP Glut2 Ins Glut2 Glut2 YFP Merged+DAPI d e Control f g h i j DT, 1mo Glu Ins Glut2 Ins Glut2 Glut2 Glu Merged k l m n o Control p q r s t DT, 1 mo u vq w x y Supplementry Figure S11: Reprogrmmed -ells express the β-ell mturity mrker Gluose trnsporter type2 (Glut2). -y, Confol imges of Glugon-rtTA; TetO-Cre; R26-YFP; RIP-DTR trnsgeni mie treted with DOX during 2 weeks fter wening. -e, DT-untreted nimls (). Glut2 is expressed on the surfe of funtionl β-ells (sterisk). f-i, DT-treted mie. A month fter β-ell ltion, more thn 1/3 of YFP+/insulin+ ells express Glut2, like funtionl -ells (38.2±2.9%; 51 YFP+/insulin+ ellsfrom 62 islets; rrowhed). k-o, DT-untreted nimls (). Glugon-expressing -ells re Glut2-negtive (rrowhed). p-y, DT-treted mie. One month fter DT, ¾ of insulin+ ells er Glut2 on their surfe (74.4±.4%; 262 insulin+ ells from 62 islets). Interestingly, some of these re insulin+/glugon+ o-expressing ells (reprogrmmed -ells; p-t); while others hve lost glugon expression (u-y). Brs: m. pnels). 11
12 % norml gluose homeostsis 5% fint or no regenertion (hemizygous RIP-DTR femles; not shown) 3% to % -ell mss gluose intolerne regenertion y -ell replition (Nir, 7; Wng, 8; Cno, 8) dietes 1% regenertion from heterologous origins (this work) Supplementry Figure S12: -ell regenertion from heterologous origins. Contriution of -ells to the emergene of new -ells is proportionl to the degree of -ell ltion, suh tht the more -ells remin (f. i. with lower DT doses not shown-, nd in other pulished models), the less importnt is the diret involvement of -ells. Only profound derese of the -ell mss triggers -ell regenertion; the heterologous formtion of new -ells is oserved only if lmost no -ells remin. 12
13 Supplementry Methods Aville mie: RIP-CreER mie were generted nd kindly provided y D. Melton 1. Glugon-Cre, TetO- Cre, nd R26-EYFP mie were previously desried 2-4. Physiologil studies: Gluose tolerne tests, pnreti glugon nd insulin dosges (immunossys), gene expression nlyses y rel time PCR s well s histologil, ultrstruturl nd morphometri nlyses were performed s desried 5,6. Immunofluoresene: Prffin nd ryostt setions were 5 m- or m-thik, respetively. The ntiodies used were: rit nti-glut2 (kind gift of B. Thorens, 1/2), rit nti-pdx1 (kind gift of C. Wright, 1/5,), guine pig nti-porine insulin (Dko, 1/4), mouse ntiporine glugon (Sigm, 1/1,), rit nti-humn somtosttin (Sigm, 1/2), mouse nti-humn Ki67 (BD Trnsdution Lortories, 1/2), rit nti-gfp (Moleulr Proes, 1/2), rit nti-nkx6.1 (BCBC, AB69) nd mouse nti-ki67 (BD Trnsdution Lortories, 1/2). Seondry ntiodies were oupled to either Alex 45, 488, 647 (Moleulr Proes), Cy3, Cy5 (Jkson Immunoreserh), or TRITC (Southern Biotheh). We were unle to find optiml onditions llowing the immunodetetion of severl -ellspeifi mturity mrkers suh s MfA, GCK or PC1/3 simultneously with insulin nd glugon while preserving the endogenous YFP. Setions were exmined with Lei TCS SPE or SP2 AOBS, or Zeiss LSM 5 onfol mirosopes, Nikon Coolsope or Teni G212 eletron mirosope, where pproprite. Some of the pitures were proessed with Voloity progrm (Remove Noise medium filter, Brightness nd Contrst Enhnement). Totl RNA extrtion, DNA synthesis nd Q-PCR: Adult pnret (n=3 mie/time-point) were hrvested t 3 time-points during regenertion (, 15 dys nd 3 dys) nd proessed for RNA extrtion s desried 7. Adult islets were isolted s desried 6 nd pooled from 4 mie for eh time-point (, 7 dys nd 15 dys post ltion). Totl-RNA extrtion from isolted islets ws done with the Qigen RNesy Miro kit, inluding the DNse-I step (Qigen #744). Qulity for RNA integrity ws performed y pillry eletrophoresis on Agilent 2 Bionlyzer. For oth totl pnres nd isolted islets, 2ng totl RNA/smple ws proessed with the DNAfree TM kit (Amion #196) for removl of ontminting gdna, following the rigorous DNse tretment steps desried in the kit. Next, 5ng of totl RNA/smple ws reverse-trnsried with the Qigen Sensisript RT Kit (Qigen #25211) using Oligo-dT primers (Invitrogen # ). The resulting DNA ws sumitted to Q-PCR retion using the pproprite primer mixes for eh gene s well s the Universl qpcr SuperMix omponent provided in the Express SyBr GreenER kit (Invitrogen #1652). We used the CorettRootis4 root nd the PCR retion ws ompleted in the CorettReserh6 series yler using 4 yles progrm. Normliztion nd nlysis of dt were done with the RT- PCRnlysis_mro v1.1 (ourtesy of the NCCR Genomi Pltform, University of Genev) using 3 different normliztion genes ( -tin, 18S, GAPDH). 13
14 Gene -tin 18S GAPDH Glut2 Ins2 MfA Ngn3 Nkx6.1 Px4 Pdx1 Primer sequene F 5 AAG GCC AAC CGT GAA AAG AT 3 R 5 GTG GTA CGA CCA GAG GGA TAC 3 F 5 CAG ATT GAT GGC TCT TTC TCG 3 R 5 AGA CAA ATC GCT CCA CCA AC 3 F 5 TCC ATG ACA ACT TTG GCA TTG 3 R 5 CAG TCT TCT GGG TGG CAG TGA 3 F 5 TCT TCA CGG CTG TCT CTG TG 3 R 5 AAT CAT CCC GGT TAG GAA CA 3 F 5 TCA ACA TGG CCC TGT GGA T 3 R 5 AAA GGT GCT GCT TGA AAA AGC 3 F 5 CAG CAG CGG CAC ATT CTG 3 R 5 GCC CGC CAA CTT CTC GTA T 3 F 5 GTC GGG AGA ACT AGG ATG GC 3 R 5 GGA GCA GTC CCT AGG TAT G 3 F 5 AGA GAG CAG GCT TGG CCT ATT C 3 R 5 GTC GTC AGA GTT CGG GTC CAG 3 F 5 GCA AGC CTC TGG TCT TCC TGA A 3 R 5 GGA CAA GGC TCC CAG TGT GT 3 F 5 CAG TGG GCA GGA GGT GCT TA 3 R 5 GCC CGG GTG TAG GCA GTA C 3 Sttistil nlyses: Mnn-Whitney, Fisher s test, Dunn s multiple omprison, nd ANOVA were pplied where pproprite, using GrphPd s Prism 4.. -ell mss ws lulted s the reltive -ell re reported to pnreti weight. -ell replition: sine -ells re so sre fter ltion, no men vlue per individul ws otined; insted, the ounts were pooled nd glol verge vlue from t lest 5 nimls per group ws lulted. All error rs represent stndrd error of the men (SEM). Supplementry Notes Dor, Y., Brown, J., Mrtinez, O.I., & Melton, D.A., Adult pnreti et-ells re formed y self-duplition rther thn stem-ell differentition. Nture 429 (6987), (24). Herrer, P.L., Adult insulin- nd glugon-produing ells differentite from two independent ell lineges. Development 127 (11), (2). Perl, A.K., Wert, S.E., Ngy, A., Loe, C.G., & Whitsett, J.A., Erly restrition of peripherl nd proximl ell lineges during formtion of the lung. Pro Ntl Ad Si U S A 99 (16), (22). Srinivs, S. et l., Cre reporter strins produed y trgeted insertion of EYFP nd ECFP into the ROSA26 lous. BMC Dev Biol 1 (1), 4 (21). Herrer, P.L. et l., Emryogenesis of the murine endorine pnres; erly expression of pnreti polypeptide gene. Development 113 (4), (1991). Strom, A. et l., Unique mehnisms of growth regultion nd tumor suppression upon Ap intivtion in the pnres. Development 134 (15), (27). Bonl, C. et l., Pnreti Intivtion of -My Dereses Ainr Mss nd Trnsdifferentites Ainr Cells Into Adipoytes in Mie. Gstroenterology 136, (29). 14
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Supplementry Figure S Tissue weights (g).... Liver Hert Brin Pncres Len mss (g) 8 6 -% +% 8 6 Len mss Len mss (g) (% ody weight) Len mss (% ody weight) c Tiilis nterior weight (g).6...... Qudriceps weight
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