a ) evolution of glycemia in -cell - ablated adults 
 ( middle - aged ) and aged mice . area under the curve  ( auc ) in 
 middle - aged ( 2-month - old , n=4 ) and aged ( 1- and 1.5-year - old , n=5 and n=3 ) mice before 
 and after stopping insulin administration revealed no statistical difference between 
 groups ( welch s test [ p[0 - 45mpa]=0.1029 , 0.3321 ; 
 p[4.5 - 7mpa]=0.1748 , 0.5007 ] , one - way anova [ p=0.1161 ; p=0.2681 ] , and mann 
 whitney [ p=0.1640 , 0.4519 ] ) . b ) evolution of glycemia in 14 aged mice 
 during 14 months post - ablation (  mpa  ) . mice were treated with insulin for 
 4.5 months ; most of them ( 5/7 in each group ) subsequently recovered from diabetes . c - e ) pancreatic islets before ( c ) and after ( d , 
 e ) -cell ablation in 1.5-year - old mice ; -cell mass increases 
 3.5-fold between 0.5 and 1 mpa , 12-fold at 7 mpa and 32-fold at 14 mpa , in all age 
 groups . table s1 , 
 2-month - old : n(0.5mpa)=4 , n(1mpa)=4 , 
 n(7mpa)=4 ; 1-year - old : n(0.5mpa)=5 , 
 n(1mpa)=5 , n(7mpa)=5 , n(14mpa)=8 ; 
 1.5-year - old : n(0.5mpa)=3 , 
 n(1mpa)=3 , n(7mpa)=3 , n(14mpa)=8 ) . f ) 
 -cell proliferation is very low in aged mice , whether control ( 1.5% , n=8 , 39,790 
 insulin - cells scored ) or ablated ( 0.2% , n=6 , 938 790 
 insulin - cells scored ) ( supp . g ) proportion of insulin cells also 
 containing glucagon after dt is not different between groups ( supp . table s3 ; 
 control : n[2-month - old]=3 , n[1-year - old]=3 , 
 n[1.5-year - old]=3 ; 0.5mpa : n[2-month - old]=5 , 
 n[1-year - old]=5 , n[1.5-year - old]=6 ; 1mpa : 
 n[2-month - old]=4 , n[1-year - old]=6 , n[1.5-year - old]=4 ; 
 7mpa : n[2-month - old]=5 , n[1-year - old]=5 , 
 n[1.5-year - old]=6 ; one - way anova [ p=0.6796 , 0.4297 , 0.9266 , 0.2411 ] ) ; note 
 that 40% of the cells containing insulin at 1 mpa also contained glucagon . the 
 proportion of glucagon / insulin cells remains constant between 
 0.5 and 7 mpa , while the number of insulin cells increases with time 
 ( e , 
 supp . table s1 ) , suggesting 
 that there is acumulative recruitment of -cells into insulin production . h ) islet with yfp / glucagon / insulin 
 cells in 1-year - old glucagon - rtta ; teto - cre ; r26-yfp ; rip - dtr mice , 7 
 mpa ; rtta expression allows the selective irreversible yfp - labeling of adult 
 -cells upon administration of doxycycline ( dox ) before -cell ablation . i ) proportion of yfp - labeled insulin - expressing cells in dox - treated 
 mice . 80% of insulin cells are yfp after 7 mpa , in all age 
 groups ( supp . table s4 ; 
 control : n[2-month - old]=3 , n[1-year - old]=3 , 
 n[1.5-year - old]=3 ; 1mpa : n[2-month - old]=5 , 
 n[1-year - old]=3 , n[1.5-year - old]=3 ; 7mpa : 
 n[2-month - old]=5 , n[1-year - old]=5 , n[1.5-year - old]=5 ; 
 one - way anova [ p=0.9417 , 0.8910 , 0.9641 ] ) . j , k ) 
 yfp / glucagon / insulin cells at 7 mpa , following a 
 dox pulse - labeling at 5.5 months after -cell loss ( supp . table s5 , control : 
 n[1-year - old]=5 , n[1.5-year - old]=5 ; 7mpa : 
 n[1-year - old]=5 , n[1.5-year - old]=5 ; welch s correction 
 [ p=0.8272,0.8926 ] , mann - whitney [ p=0.9444 ] . on average , 15% of the insulin 
 cells found were yfp - labeled , some of which no longer contained glucagon as in 
 ( j ) , lower row . note the decreased proportion of yfp - labeled 
 insulin cells when -cells are tagged late after ablation ( from 80% 
 to 15% ; compare ( i ) and ( k ) ) , and the presence of yfp - labeled 
 insulin / glucagon - negative cells in the latter situation ( j ) , 
 suggesting that bihormonal -cells slowly but gradually lose glucagon gene 
 activity . a ) evolution of glycemia (  auc  ) between 2.5 and 4 
 mpa , in pups and adults ( see fig 1b , welch s 
 test [ p=0.0188 ] ) . b ) qpcr of insulin2 mrna after -cell ablation ; 
 insulin2 transcripts are 25-fold more abundant in pups than in adults at 2 mpa ( n=3 
 mice / group , each individual sample was run in triplicate in each reaction , for a total 
 of 3 independent reactions ) ; built - in welch s test [ p=0.0134 , 0.0049 ] , error 
 bars : s.d . ) . c ) glucose tolerance tests ( ipgtt ) for dt - treated ( 4.5 mpa , 
 n=4 ) and age - matched controls ( n=4 ) ; note the fold - increase between glucose injection 
 and the glycemic peak during ipgtt for each animal , and fold - decrease between glycemic 
 peak and t120 ( two - tailed unpaired t - test , [ p(i)=0.5836 , 
 p(ii)=0.4937 ] ) . d ) plasma insulin at t0 , t15 and t30 during the 
 ipgtt ( control : n=4 ; dt : n=4 ; two - tailed paired t - test 
 [ p=0.0008 ] ) . e ) insulin tolerance tests ( itt ) performed 1,5 year after 
 -cell ablation at 2 weeks of age ( controls : n=7 ; 
 dt : n=10 ) . f ) 4.5 months after -cell ablation 
 ( at 2 weeks ) , 3 mice became normoglycemic and received a second treatment with dt . ablation of regenerated insulin - cells in recovered mice leads to the 
 appearance of glucagon / insulin cells , corresponding to the type 
 of  -cell - dependent  regeneration observed in adults ( 31% of 
 insulin+ cells also contained glucagon , supp . table 
 s9 , control : n[1-mo - old]=3 , 6,006 
 insulin - cells scored , n[2-mo - old]=3 , 6,358 
 insulin - cells scored ; dt : n[0.5mpa]=5 , 412 
 insulin - cells scored ; n[1.5mpa]=3 , 675 
 insulin - cells scored ; welch s test [ p=0.1197 , p=0.0688 ] , error bars : 
 s.e.m . ) . h ) islet cell proliferation is increased ( 3.5-fold ; 
 ki67 cells ) in islets of dt - treated pups at 0.5 mpa 
 ( control : n(1-month - old)=3 , 95 islets scored ; 
 n(1.5-month - old)=3 , 94 islets scored ; n(2-month - old)=3 , 90 
 islets scored ; n(2.5-month - old)=3 , 89 islets scored ; n(3-month - old 
 ctrl)=3 , 91 islets scored ; n(3.5-month - old)=3 , 93islets scored ; 
 n(18.5-month - old)=3 , 83 islets scored ; 19(19-month - old ctrl)=3 , 
 83 islets scored ; n(19.5-month - old)=3 , 88 islets scored ; 
 dt(2-week - old ) : n(0.5mpa)=6 , 333 islets scored ; 
 n(1mpa)=3 , 91 islets scored ; n(1.5mpa)=3 , 90 islets scored ; 
 dt(2-month - old ) : n(0.5mpa)=3 , 76 islets scored ; 
 n(1mpa)=3 , 77 islets scored ; n(1.5mpa)=3 , 81 islets scored ; 
 dt(1.5-year - old ) : n(0.5mpa)=3 , 74 islets scored ; 
 n(1mpa)=3 , 81 islets scored ; n(1.5mpa)=3 , 77 islets scored ; 
 error bars : s.d . welch s test , one - way anova [ p<0.001 ] mann - whitney 
 [ p=0.0238 ] ) . i ) ki67 cells are hormone , chromogranin 
 a - negative ; lineage - traced - and dt - spared -cells are ki67-negative . a ) the number of somatostatin cells transiently 
 decreases by 80% during the 2 week after ablation 
 ( n[control]=255 islets , 7 mice ; n[3dpa]=240 islets , 5 mice ; 
 n[5dpa]=228 islets , 5 mice ; n[7dpa]=251 islets , 5 mice ; 
 n[0.5mpa]=267 islets , 6 mice ; n[1mpa]=266 islets , 5 mice ; 
 n[1.5mpa]=206 islets , 5 mice ; error bars : s.d . ; welch s test 
 [ p=0.0008 , 0.0229 , 0.006 , 0.035 ] , one - way anova [ p<0.0001 ] , mann - whitney 
 [ p=0.0043 ] ) . b ) relative somatostatin gene expression sharply decreases 2 
 weeks after -cell ablation in 2-week - old mice ( n=3 mice / group , each individual 
 sample of each experimental group was run in triplicate , in 3 independent reactions ) ; 
 built - in welch s test [ p=0.0002 ] , error bars : s.d . ) . d ) quantitative values of reporter gene expression in islet cells ( n=4 , 
 1,263 yfp - cells scored ) . observed and expected numbers of somatostatin and 
 insulin cells per islet section , before and after -cell ablation . 3a ) 
 correspond (  test ) with estimates made assuming that 
 dedifferentiated proliferating -cells yield 2 types of progeny ( as deduced from 
 fig . dashed arrows , phenotypic stability ; 
 plain arrows , dynamic behavior ( dedifferentiation and replication ) . a ) immunofluorescence showing yfp - labeled insulin 
 cells at 1.5 month following streptozotocin ( stz)-induced ablation of -cells in 
 2-week - old mice . arrows : yfp / insulin cells ; arrowhead : 
 yfp / somatostatin cell ; asterisks : escaping -cells . b ) number of remaining -cells per islet section at 2 weeks after 
 streptozotocin or dt treatment in pups , reflecting difference in ablation efficiency of 
 the 2 methods ( supp . table 
 s18)(n[stz]=87 islets , 3 mice ; n[dt]= 361 islets , 4 mice ; 
 welch s test [ inter - islet p<0.0001 ; inter - individual p=0.0109 ] , 
 mann - whitney [ p<0.001 ] ) . c ) the number of 
 yfp / insulin cells per islet section at 1.5 mpa is not 
 significantly different between the two -cell ablation methods ( supp . table 
 s19)(n[stz]=88 islets , 3 mice ; n[dt]=193 islets , 7 mice ; 
 welch s test [ p=0.4786 ] ) . d ) -cell numbers per islet section 
 in controls ( n=3 , 174 islets scored ) , 0.5 mpa ( n=4 , 140 islets scored ) and 1 mpa ( n=3 , 
 86 islets scored ) ( unpaired t - test , two - tailed , [ p=0.6386 ; p=0.5406 ] ) . e ) 
 immunofluorescence for yfp and ki67 2 weeks ( 0.5 mpa ) after dt , in 
 somatostatin - cre ; r26-yfp ; rip - dtr mice . f ) experimental 
 design for -cell tracing in -cell - ablated somatostatin - cre ; 
 r26-yfp ; rip - dtr mice at 2 months of age , and immunofluorescence for 
 somatostatin , yfp and insulin at 1.5 mpa . arrow : 
 yfp / insulin / somatostatin cell . g ) at 1.5 mpa , 17% of insulin cells coexpress yfp 
 vs. almost 100% in ablated prepubescent mice 
 ( control : n=4 ; dt : n=8 ; unpaired t - test , two - tailed 
 [ p=0.0462 ] ) . h ) at 1.5 mpa , 98% of the yfp cells are 
 somatostatin , and 1% are insulin cells ( vs. 
 44% in mice ablated before puberty ; n=8 , unpaired a ) islet transplantation design : 400 - 600 islets isolated from 
 2-week - old somatostatin - cre ; r26-yfp ; rip - dtr transgenics were 
 transferred under the kidney capsule of 2-month - old immunodeficient ( scid ) mice ( n=3 ) . b ) experimental design : after one week of engraftment , adult host mice 
 were dt - treated and left to regenerate for 6 weeks . c ) - 
 to- conversion was observed in -cell - ablated engrafted islets , like in 
 the pancreas of juvenile mice . scale bars : 20 m once differentiated from -cells ( yfp ) , the newly formed 
 -cells re - enter the cell cycle ( ki67 cells ) . two waves of massive 
 replication occur , at 3 and 4 months after injury , respectively ( supp . b ) 
 qpcr for -cell - specific genes using rna extracted from islets isolated from 
 control and dt - treated mice , either 2 weeks or 4 months following dt administration 
 (  0.5 mpa  and  4 mpa  ) . note that after an initial extreme 
 downregulation of all the -cell - specific markers explored , their levels 
 significantly recover after 4 months , which correlates with the observed robust 
 regeneration and diabetes recovery . values represent the ratio between each regeneration 
 time - point and its age - matched control . d ) qpcr comparison between regenerated 
 cherry / insulin cells isolated from mice 4 months after 
 -cell ablation , and cherry -cells obtained from age - matched 
 controls ( 4.5-month - old ) . all markers tested are expressed at identical levels in both 
 groups ; non--cell markers are expressed at extremely reduced levels ( ct ranging 
 from 28 to 31 ) , showing the same degree of purity in both types of cell preparations . e , f ) interestingly , in contrast to bona fide 
 -cells isolated from 4.5-month - old controls , regenerated insulin 
 cells have lower levels of cyclin - dependent kinase inhibitors , foxo1 and smad3 . this 
 correlates with their increased proliferative capacity at this specific time - point . scale bars : 20 m ; qpcrs : n=3 mice / group , each individual sample of each 
 experimental group was run in triplicate , in 3 independent reactions ) ; built - in 
 welch s test ; error bars : s.d . a ) qpcr for ngn3 mrna after -cell ablation reveals a 
 transitory 5-fold upregulation of ngn3 transcripts 6 weeks after -cell ablation 
 when -cell ablation is performed before puberty , but not in adult mice . ( controls : n[1-month - old]=3 , 
 n[1.5-month - old]=3 , n[2-month - old]=6 , 
 n[2.5-month - old]=3 , n[2.5-month - old]=3 , 
 n[3-month - old]=3 , n[3.5-month - old]=3 , n[4-month - old]=3 ; 
 dt(2-week - old ) : n[0.5mpa]=3 , n[1mpa]=3 , 
 n[1.5mpa]=6 , n[2mpa]=3 ; dt(2-month - old ) : 
 n[0.5mpa]=3 , n[1mpa]=3 , n[1.5mpa]=3 , 
 n[2mpa]=3 ; each individual sample ( mouse ) was run in triplicate , in each 
 one of 3 independent reactions ; built - in welch s test [ p=0.0112 , 0.0178 ] ) . b ) ngn3 transcriptional activity can be monitored in 
 ngn3-yfp knock - add - on mice because ngn3 promoter activity results in 
 yfp expression . in non - ablated age - matched control pups , or in ablated adults , no islet 
 yfp cells were found ( not shown ) , yet when -cells are ablated at 2 
 weeks of age , 86% of insulin cells also express yfp at 1.5 mpa 
 ( control : n=3 , 6,358 insulin - cells scored 
 dt : n=3 , 675 insulin - cells scored ; welch s test 
 [ p=0.0010 ] ) ; c ) at 1.5 mpa , 81% of yfp cells co - express 
 insulin , but no glucagon , somatostatin or pp ( not shown ) . two weeks later , 
 yfp cells are almost absent , reflecting the downregulation of ngn3 
 expression reported in a ) , and suggesting that insulin cells 
 originate from cells transiently activating ngn3 expression after ablation 
 ( control : n[1-month - old]=3 ; n[1.5-month - old]=3 ; 
 n[2-month - old]=3 ; n[2.5-month - old]=3 ; absent 
 yfp - cells in all control conditions ; dt : 
 n[0.5mpa]=3 , 31 yfp - cells ; n[1mpa]=3 , 123 
 yfp - cells ; n[1.5mpa]=3 , 729 yfp - cells ; 
 n[2mpa]=3 ; 47 yfp - cells ; welch s test and anova 
 [ p<0.0001 ] ) . d ) irreversible lineage - tracing of ngn3-expressing 
 cells at 1 and 1.5 mpa upon tamoxifen ( tam ) administration in ngn3-creert ; 
 r26-yfp ; rip - dtr mice ; immunofluorescence analyses reveal that in absence of 
 -cell ablation , there is no yfp induction ( controls ) . in ablated mice , nearly 
 all insulin cells are yfp with time ( arrows ) . at early 
 time - points ( 1 mpa ) , yfp / hormone - negative cells e , f ) in 
 -cell - ablated ngn3-creert ; r26-yfp ; rip - dtr pups , 91% of 
 insulin cells coexpress yfp ( control : n=3 , 
 3,472 insulin - cells scored , dt : n=3 , 489 
 insulin - cells scored ) ( e ) and inversely , 93% of the 
 yfp cells are insulin ( f ) 
 ( control : n=3 ; absent yfp - cells in all control 
 conditions , dt : n=3 , 478 yfp - cells scored ) . g ) 
 experimental design to block ngn3 upregulation in -cell - ablated prepubescent 
 mice , by administrating dox to mice bearing 5 mutant alleles : 
 ngn3-tta ; tre - ngn3 ; rip - dtr . in these 
 mice the ngn3 coding region is replaced by a dox - sensitive transactivator gene ( tta ) ; 
 the endocrine pancreas develops normally because ngn3 expression is allowed in absence 
 of dox by the binding of tta to the promoter of tre - ngn3 transgene . pups were given dt at 2 weeks of age and then dox 2 weeks later , to block ngn3 
 upregulation . they were euthanized when ngn3 peaks after ablation 
 ( 2-month - old).h ) islets from non - ablated (  no dt  ) and 
 ablated (  dt  ) mice , exposed ( ngn3 inhibition ) or not ( normal ngn3 
 expression ) to dox treatment from 4 weeks of age . -cell regeneration is 
 efficient in absence of dox ( as previously shown ) , but decreases after ngn3 blockade , 
 resulting in the appearance of glucagon / insulin bihormonal cells . i ) 
 sharply decreased regeneration by blocking ngn3 expression in dox - treated mice reveals 
 the requirement of ngn3 for efficient -cell regeneration in pups 
 ( dt : n=266 islets scored , 3 mice ; dt+dox : n=167 , 4 
 mice ; welch s test [ inter - islet p<0.0001 ; inter - animal p=0.0352 ] , 
 mann - whitney [ p<0.0001 ] ) . j ) glucagon+/insulin+ bihormonal cells 
 appear in dox - treated -cell - ablated pups ( ngn3 inhibition ) , suggesting a switch 
 to an  adult - like  , less efficient , mechanism of regeneration 
 ( control+dox : n=3 , 9233 insulin - cells scored ; 
 dt : n=3 1385 insulin - cells scored ; 
 dt+dox : n=4 , 141 insulin - cells scored ; welch s 
 test [ p=0.0081 ] , anova [ p<0.0001 ] ) . k ) combined double lineage 
 tracing of -cells ( tomato ) and ngn3-expressing cells 
 ( yfp ) show by immunofluorescence that nearly all insulin cells 
 express both reporters , but no somatostatin ( arrows ) . a ) cartoon depicting the foxo1 network involved in the regulation 
 of cell cycle progression and cellular senescence : foxo1 arrests the cell cycle by 
 repressing activators ( cyclind1 , cyclind2 ) and inducing inhibitors ( cdkn1a / p21 , 
 cdkn1b / p27 , cdkn2b / p15ink4b , cdkn1c / p57 ) [ pmid : 10102273 ; pmid : 17873901 ] . cdkn1a / p21 
 and cdkn2b / p15ink4b activation , a sign of cellular senescence [ pmid : 17667954 ] , is 
 regulated by foxo1 through direct interaction with skp2 protein . in turn , skp2 blocks 
 foxo1 and , together with cks1b , cdk1 and cdk2 , triggers the direct degradation of 
 cdkn1a / p21 and cdkn1b / p27 , thus promoting proliferation [ pmid : 15668399 ] . foxo proteins 
 are inhibited mainly through pi3k / akt - mediated phosphorylation [ pmid : 10102273 , pmid : 
 12621150 , pmid : 21708191 , pmid : 10217147 , pmid : 17604717 ] : pdk1 , the master kinase of 
 the pathway , stimulates cell proliferation and survival by directly activating akt , 
 which phosphorylates ( inhibits ) the foxos [ pmid : 10698680 , pmid : 19635472 ] . pi3k / akt / foxo1 circuit requires active tgf/smad signaling [ pmid : 24238962 , pmid : 
 15084259 ] in order to co - regulate cdkn1a / p21-dependent cell senescence . active 
 tgf signaling downregulates the bmp pathway downstream effectors id1 and id2 , 
 known to promote dedifferentiation and proliferation during embryogenesis and cancer 
 progression , probably through cdkn2b / p15ink4b regulation [ pmid : 11840321 , pmid : 
 16034366 ] . b ) -cell ablation in adults triggers foxo1 upregulation 
 and the subsequent cell cycle arrest in -cells . a - d ) the 1 week foxo1 inhibition with the compound as1842856 in 
 control unablated adult mice ( a ) results in dedifferentiation of one - fourth 
 of the -cell population ( b ; 
 supp . table s30 , 
 treated : n=3 , 1,347 yfp - cells scored ; 
 untreated : n=4 , 1,224 , yfp - cells scored ; error bars : 
 s.d . ) , without leading to insulin ( c ; 
 supp . table s31 , 
 treated : n=3 , 3,249 insulin - cells scored ; 
 untreated : n=4 , 9,562 insulin - cells scored ; error bars : 
 s.d . ; welch s test [ p=0.1590 ] ) or glucagon ( d ; 
 supp . table s32 , 
 treated : n=2 , 728 yfp - cells scored ; error bars : s.e.m . ) e ) one month following foxo1 transient inhibition in 
 -cell - ablated adults , dedifferentiated -cells do not express glucagon 
 ( supp . table s36 , 
 treated : n=2 , 986 yfp - cells scored ; error bars : s.e.m . ) . f ) transient foxo1 inhibition long - time ( 1 month ) after -cell 
 ablation also leads to the appearance of lineage - traced dedifferentiated -cells 
 that express insulin ( supp . table 
 s37 - 39 , 
 treated : n=3 , 71 islets scored ; 300 insulin - cells scored ; 
 1216 yfp - cells scored ; error bars : s.d . ) .
total or near - total loss of insulin - producing -cells is a situation found 
 in diabetes ( type 1 , t1d ) 1,2 . 
 restoration of insulin production in t1d is 
 thus a major medical challenge . 
 we previously observed in mice in which -cells are 
 completely ablated that the pancreas reconstitutes new insulin - producing cells in absence 
 of autoimmunity 3 . 
 the process involves 
 the contribution of islet non--cells ; specifically , glucagon - producing 
 -cells begin producing insulin by a process of reprogramming 
 ( transdifferentiation ) without proliferation 3 . here 
 we studied the influence of age on -cell reconstitution 
 from heterologous islet cells after near - total -cell loss . 
 we found that 
 senescence does not alter -cell plasticity : -cells can reprogram to 
 produce insulin from puberty through adulthood , and also in aged individuals , even a 
 long - time after -cell loss . 
 in contrast , prior to puberty there is no detectable 
 -cell conversion , although -cell reconstitution after injury is more 
 efficient , always leading to diabetes recovery ; it occurs through a newly discovered 
 mechanism : the spontaneous en masse reprogramming of somatostatin - producing 
 -cells . the younglings display  somatostatin - to - insulin  
 -cell conversion , involving de - differentiation , proliferation and re - expression of 
 islet developmental regulators . 
 this juvenile adaptability relies , at least in part , upon 
 combined action of foxo1 and downstream effectors . 
 restoration of insulin producing - cells 
 from non--cell origins is thus enabled throughout life via - or 
 -cell spontaneous reprogramming . 
 a landscape with multiple intra - islet cell 
 interconversion events is emerging , thus offering new perspectives .