A FoxA2+ long-term stem cell population is necessary for growth plate cartilage regeneration after injury

ARTICLE A FoxA2+long- e m s em cell popula ion is necessa y o g ow h pla e ca ilage egene a ion a e inju y Shanmugam Mu uganandan 1, Rachel Pie ce1, Dian As a i Teguh 2, Rocio Fuen e Pe ez3, Nicole Bell4, B andon Nguyen5, Ka he ine Hohl 2,6, B ian D. Snyde 7, Ma k W. G ins a 6, Hannah Albe ico1, Do i Woods1, Yiwei Kong1, Co neliu Sima 8, Sanke Bhaga 9, Kailing Ho10, Vicki Rosen 10, Lau a Game 10 & And eia M. Ionescu 1✉ Longi udinal bone g ow h, achie ed h ough endochond al ossifica ion, is accomplished by a ca ilaginous s uc u e, he physis o g ow h pla e, comp ised o mo phologically dis inc zones ela ed o chond ocy e unc ion: es ing, p oli e a ing and hype ophic zones. The es ing zone is a s em cell- ich egion ha gi es ise o he g ow h pla e, and exhibi s egene a i e capabili ies in esponse o inju y. We disco e ed a FoxA2+g oup o long- e m skele al s em cells, si ua ed a he op o es ing zone, adjacen he seconda y ossifica ion cen e , dis inc om he p e iously cha ac e ized PTH P+s em cells. Compa ed o PTH P+ cells, FoxA2+cells exhibi highe clonogenici y and longe i y. FoxA2+cells exhibi dual os eo-chond o-p ogeni o ac i i y du ing ea ly pos na al de elopmen (P0-P28) and chon- d ogenic po en ial beyond P28. When he g ow h pla e is inju ed, FoxA2+cells expand in esponse o auma, and p oduce physeal ca ilage o g ow h pla e issue egene a ion. h ps://doi.o g/10.1038/s41467-022-30247-1 OPEN 1Depa men o Biology, 134 Muga Li e Sciences Building, No heas e n Uni e si y, 360 Hun ing on A e, Bos on, MA 02115, USA. 2Cen e o Ad anced O hopedic S udies, Be h Is ael Deaconess Medical Cen e , 330 B ookline A enue, Bos on, MA 02215, USA. 3Di ision o Pedia ics, Uni e si y o O iedo, O iedo 33206, Spain. 4New Yo k Uni e si y College o Den is y, 345 E.24 h S , New Yo k, NY 10010, USA. 5Mode na The apeu ics, One Upland Rd, No wood, Ohio, MA 02062, USA. 6Depa men s o Biomedical Enginee ing, Chemis y, and Medicine, Bos on Uni e si y, 590 Commonweal h A e, SCI 518, Bos on, MA 02215, USA. 7Depa men o O hopedic Su ge y, Bos on Child en’s Hospi al, 300 Longwood A e, Bos on, MA 02115, USA. 8Depa men o O al Medicine, In ec ion, and Immuni y, Ha a d School o Den al Medicine, 188 Longwood A enue, Bos on, MA 02115, USA. 9Ul agenyx Pha maceu ical, 840 Memo ial D i e, Camb idge, MA 02139, USA. 10 Depa men o De elopmen al Biology, Ha a d School o Den al Medicine, 188 Longwood A enue, Bos on, MA 02115, USA. ✉email: a.ionescu@no heas e n.edu NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions 1 1234567890():,; Longi udinal bone g ow h is achie ed by endochond al ossi- fica ion, implemen ed by he physis o g ow h pla e (GP), a ca ilaginous s uc u e loca ed be ween he epiphysis and me aphysis a he ends o long bones o ming he appendicula skele on. Human GP is comp ised o fi e mo phologically dis inc zones: es ing (RZ), p oli e a ion (PZ), hype ophy (HZ), calci- fica ion (CZ), and ossifica ion (OZ)1. Physeal inju ies a e a majo cause o skele al mo bidi y in g owing child en, mani es ing as angula o sho ened limb de o mi ies, due o pa ial o comple e a es o GP unc ion2. Clinically, GP inju ies a e classified acco ding o he Sal e –Ha is (SH) sys em, p edica ed on ac- u e mo phology in ol ing he physis ± epiphysis and/o me a- physis: SH1) physeal shea sepa a ion; SH2) physis +me aphysis; SH3) physis +epiphysis; SH 4) physis +epiphysis +me aphysis; SH 5) c ush inju y o physis. SH1 ac u es ha e he bes p og- nosis, healing wi hou dis up ed g ow h. The clea age plane is p ima ily h ough he HZ, s uc u ally he weakes zone com- p ising he GP, owing o lack o calcifica ion and limi ed col- lagenous ma ix. S i ened by a collagen ma ix, he adjacen RZ and PZ a e una ec ed3,4. In con as , SH 3–5 inju ies dis u b all zones, esul ing in dis u bed bone g ow h3,4. These clinical sce- na ios sugges ha in ol emen o he RZ a ec s he abili y o he GP o egene a e in esponse o inju y. In abbi s, e en when he PZ and HZ om ulna GP we e excised, he es o a ion o he GP was s ill possible, as long as he RZ emained in ac 5,6. As he name implies, he “ es ing zone”houses a quiescen p ogeni o cell popula ion1. Se e al s udies ha e iden ified in mouse7, a 8, and abbi 9slow-cycling cells p esen in he RZ immedia ely adjacen o he seconda y ossifica ion cen e (SOC). In pulse- chase expe imen s, animals we e adminis e ed [3H] hymidine o 5-b omo-2-deoxyu idine (B dU) o a p olonged pe iod su ficien o allow slow-cycling s em cells o comple e one cycle. A he end o he chase, he label diminishes in p oli e a ing cells, bu emains undilu ed in slow-cycling cells. While hese epo s demons a e he p esence o slow-cycling, po en ial p ogeni o cells a he RZ, hei gene ic signa u e emains undefined, as he e is no bioma ke o isola e and cha ac e ize hese cells. Recen ly New on e al., using clonal gene ic acing wi h a col2CRE;ERT;R26-Con e i mouse line and unc ional pe u ba ions, es ablished ha o ma ion o he SOC igge ed GP chon- d op ogeni o s o unde go a adical shi in clonali y and o acqui e sel - enewal capabili ies10. Howe e , since “Con e i” labeling is based on collagen ype II exp ession, an ube -ma ke o all GP cells, he e was no unique bioma ke o isola e and/o cha ac e ize indi idual s em cell subg oups. Nex , Mizuhashi e al. employed a PTH PC e.ERT;Toma o /+mouse s ain o e eal ha PTH P ma ked a subse o s em cells loca ed a he bo om o he RZ11. Labeling, acing and isola ing RZ cells based on hei PTH P exp ession bo h in i o and in i o, demons a ed ha a subg oup PTH P+(posi i e) s em cell e ol ed p edominan ly in o columna chond ocy es11. Toge he wi h ano he ye uni- den ified PTH P−(nega i e) subg oup loca ed a he op o he RZ, adjacen o he SOC, PTH P+cells, loca ed p ima ily a he bo om o he RZ, con ibu ed o long- e m GP g ow h11. In he p esen wo k, we p o e ha a popula ion o Fox- A2+col10−cells, loca ed a he op o he RZ, a e he PTH P- (nega i e) s em cell popula ion, and ep esen a subg oup o long- e m skele al s em cells (LTSSC) capable o dual os eo-chond o- p ogeni o ac i i y du ing ea ly pos na al de elopmen (P0-P28) and chond ogenic po en ial beyond P28, undamen al o GP u no e and egene a ion ollowing inju y. Resul s FoxA2 exp ession p efigu es SOC o ma ion. We p e iously disco e ed ha FoxA ansc ip ion ac o s a e key egula o s o chond ocy e hype ophy12. FoxA1-3 a e highly exp essed in he hype ophic zone (HZ) o newbo n mice GP (Fig. 1a, , k). Howe e , unlike FoxA3, which is exp essed in a b oade domain h oughou he GP (Fig. 1 ), o FoxA1, which is highly exp essed in he HZ bu e y li le elsewhe e (Fig. 1k), FoxA2 is exp essed in wo sepa a e domains: he HZ and a disc e e pe ia icula domain loca ed a he ends o he long bones (Fig. 1a). Th oughou pos na al de elopmen (P0-P14), his dis inc FoxA2+popula ion is con inuously enla ging, possibly p efigu ing he o ma ion o he SOC (Fig. 1a–e). FoxA1 and FoxA3 also co ela e wi h SOC de elopmen , bu eme ge in he epiphyseal ca ilage a ound P5 (Fig. 1 –o), much la e han FoxA2, which is exp essed om bi h (P0) (Fig. 1a–e). In he p esump i e SOC, he fi s col10+cells appea by P7, as shown in Tg.col10mChe y mice, bu FoxA2+cells appea as ea ly as P0 (Fig. 1p– ). Compa ison be ween FoxA2+ cells in he pe ia icula egion wi h he columna GP cells, did no e eal any his ological o mo phological di e ences and no no iceable change in Ki67 exp ession and p oli e a ion, p io o P5 (Fig. 1u–y). Howe e , a e P5 FoxA2+cells down egula e he exp ession o Ki67, in p epa a ion o hype ophic di e en ia- ion (Fig. 1w, x). Al oge he , hese expe imen s sugges ha FoxA2 exp ession ma ks he epiphyseal ca ilage, mos o which will o m he seconda y ossifica ion cen e , a soone han col.10 exp ession. FoxA2+cells gi e ise o he GP RZ op compa men .To de e mine whe he FoxA2 ma ks he a e o he cells des ined o become SOC, we labeled and aced FoxA2+cells, using a Fox- A2C eERT/+mouse line consis ing o a amoxi en-inducible C eERT2 d i e knocked-in he FoxA2 3’UTR13. Two amox- i en pulses, adminis e ed P3-4 o P7-8, o FoxA2C eERT/+; ZsG eenfl/+;Tg.col10mche y mice ga e ise, by P18, o ZsG een+ cells p esen p ima ily in he SOC, wi h a ew ZsG een+cells p esen a he op o he GP RZ (Fig. 2A(a, b, e)). I was p e- iously published ha hype ophic chond ocy es become bone cells and con ibu e o he os eogenic lineage14–16. FoxA2 is no exp essed in he epiphyseal bone o he SOC, bu ins ead i is Ki67 P0 P3 P5 P7 FoxA2 Tg.Col10 mche y abcd p s u wx y s P14 e FoxA3 FoxA1 gij Immunohis ochemis y FoxA2 / Hoesch h klmn o q Fig. 1 FoxA1-3 exp ession domain p edic s he o ma ion o he SOC. Immunohis ochemis y o FoxA2 (a–e), FoxA3 ( –j), FoxA1 (k–o), Ki67 (u–y), and fluo escence mic oscopy on Tg.col10a1mche y mice (p– )on pos na al day P0, P3, P5, P7, P14 ibia sec ions. FoxA1-3 (yellow), Ki67 (whi e), Hoechs (blue), mche y fluo escence ( ed). Scale ba s, 100 µm. ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 2NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions es ic ed o he GP hype ophic ca ilage and a small disc e e domain a he end o he long bones, as shown by immunohis- ochemis y o FoxA2 (Fig. 1a–e). Thus, he p esence o Fox- A2+p ogeny in he epiphyseal SOC i is likely he esul o FoxA2+hype ophic chond ocy es con ibu ing o he os eo- genic lineage. To confi m his, we adminis e ed 2x amoxi en injec ions o FoxA2C e.ERT/+;Toma o / ;Tg.col1GFP mice a P3-4, P7-8, o P13-14 and we ollowed hem 3 mon hs la e , demon- s a ing ha Toma o+p ogeny (de i ed om FoxA2+cells) a e col.1+exp essing bone cells (Fig. 2B(a–c)). By P13, du ing SOC enla gemen , we see a significan build-up o ZsG een+cells in he GP ca ilage. In FoxA2C eERT2/+;ZsG eenfl/+; Tg.col10mche y mice injec ed wi h amoxi en P13-14 and ha es ed a P18, we coun ed 126 ± 40 cells/hindlimb. Fi e amoxi en injec ions, adminis e ed P13-17 o FoxA2C eERT2/+;ZsG eenfl/+;Tg.col10 mche y mice, led o a u he inc ease in ZsG een+cells (387 ± 109 cells/ hindllimb) (Fig. 2A(c, d, e)). As FoxA2+cells a e loca ed on he bo de be ween GP and SOC, we sough o delinea e he ca ilage/bone in e ace by coun e - s aining wi h 5-DTAF(4,6-Dichlo o iazinyl Aminofluo escein) dye, which labels e ficien ly he bone ma ix bu no he ca ilage ma ix in Agg ecanC eERT/+;Toma ofl/+mice (Supplemen a y Fig. 1A(a)). A P13-17 amoxi en pulse adminis e ed o FoxA2C eERT2/+; Toma ofl/+mice, e ealed ha 75% o FoxA2+cells a e in he GP ca ilage, ou side he 5-DTAF labeled bone domain, and 25% a e in he g een labeled SOC (Supplemen a y Fig. 1A(b, c)). This FoxA2+popula ion loca ed a he op o he GP RZ, a he ca ilage/bone in e ace, i is no only p esen in mice, bu in abbi s as well (Supplemen a y Fig. 1B). To in es iga e whe he he FoxA2+cells p esen in he RZ in ea ly pos na al de elopmen (P0-P14) s ill pe sis in he GP ca ilage 3 mon hs la e , we pe o med a long- e m pulse chase o he FoxA2C eERT/+;Toma ofl/+mice. Tamoxi en pulses a P3-4 (du ing SOC p efigu a ion), a P7-8 (SOC hype ophy), and P13-14 (SOC enla gemen ) o FoxA2C eERT/+;Toma ofl/+mice ga e ise o Toma o+labeled cells which, 3 mon hs la e , we e s ill p esen in he RZ ei he as single cells, clus e s o columns o p ogeny (Fig. 2B(d– )), o became col.1+bone cells in he SOC (Fig. 2B(a–c)). Al oge he , hese expe imen s sugges ha a FoxA2+popula ion, le behind om epiphyseal ca ilage A a*** *** Tam P3-4 P7-8 P13-14 P13-17 Quan i ica ion o GP RZ FoxA2+ cells ( ) . SOC FoxA2+ cells ( ) using FoxA2C e.ERT/+; ZsG een /+Tg.col10mche y mice a1 a1 b1 b1 c1 c1 d1 d1 FoxA2+ cells om GP FoxA2+ cells om SOC bcd e * Tam Ha es P3-4 P18 Tam Ha es P7-8 P18 Tam Ha es P13-14 P18 Tam Ha es P13-17 P18 # FoxA2+ cells Tam Ha es P3-4 P90 Tam P7-8 P90 Ha es P13-14 P90 BDual con ibu ion o FoxA2+ cells o os eogenic and chond ogenic lineages FoxA2C e.ERT/+Toma o / Tg.Col1GFP mice FoxA2C e.ERT/+Toma o / 5-DTAF mice Ha es P3-4 P90 Ha es P7-8 P90 Ha es P13-14 P90 abcde 1 a1 2 a2 b1 b2 211 2 c1 c2 d1 d2 2 1 e1 e2 1 2 1 2 2 1 0 200 400 600 800 1000 *** *** Tam Tam Tam Tam Ha es Fig. 2 A popula ion o FoxA2+col10−cells p efigu es SOC o ma ion and gi es ise o he op compa men o he GP RZ. A Tibia sec ions ha es ed om FoxA2C eERT2/+;ZsG eenfl/+; Tg.Col10mche y mice ea ed wi h 2x amoxi en (Tam) injec ions a pos na al days P3-4 (a), P7-8 (b), P13-14 (c), o 5x amoxi en injec ions P13-P17(d), and ha es ed a P18. Hoechs (blue), ZsG een fluo escence mic oscopy (g een), mche y fluo escence mic oscopy ( ed). Scale ba s, 100 µm. Rep esen a i e de ails, om he in e ace be ween GP and SOC, a e shown in numbe ed inse s (a1–d1). Quan ifica ion o he numbe o FoxA2+(ZsG een+) cells in GP is achie ed by coun ing ZsG een+cells loca ed in GP (ex ending 100 µm away om he GP/SOC in e ace, owa ds he GP). The numbe o ZsG een+cells ep esen s a sum o n=8 sec ions pe mouse hindlimb. Simila quan ifica ion is pe o med o FoxA2+cells in SOC. Da a p esen ed as mean ± SD, n=3 mice. The as e isks indica e significan di e ence: One-way ANOVA, Tukey es ; ***p=0.000003 (P3-4 GP s P3-4 SOC), 0.000005 (P7-8 GP s P7-8 SOC), 0.000569 (P3-4 GP s P13-17 GP), 0.000828 (P7-8 GP s P13-17 GP), and *p=0.011031(P13-17 GP s P13-17 SOC). Comple e s a is ical in o ma ion is p o ided in Supplemen a y Table 1 (e). BTibia sec ions om FoxA2C eERT2/+;Toma ofl/+;Tg.Col1GFP mice ea ed wi h 2x amoxi en injec ions P3-P4 (a), P7-P8 (b), P13-P14 (c), and ha es ed a P90. Tibia sec ions om FoxA2C eERT2/+;Toma ofl/+mice ea ed wi h 2x amoxi en injec ions P3-P4 (d), P7-P8 (e), P13-P14 ( ), ha es ed a P90. Toma o ( ed), Hoechs (blue), GFP (g een) (a–c), 5-DTAF (g een) (d– ). Scale ba s, 100 µm. Rep esen a i e de ails a e shown in numbe ed inse s (a1– 1). NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions 3 pos na al de elopmen (du ing SOC p efigu a ion, hype ophy and enla gemen ), con ibu es o he GP RZ op compa men . FoxA2+long- e m s em cells . PTH P+sho - e m s em cells. Gi en ha FoxA2+cells eside in he RZ, a known loca ion o s em cells5,11, and ha FoxA2+cells a e loca ed in close p oxi- mi y o he SOC, which is iewed as a signaling cen e o RZ s emness10, we asked whe he FoxA2+cells a e di e en han he p e iously cha ac e ized PTH P+s em cell subse 11. Using a PTH Pmche y mouse line, Mizuhashi e al. ha e ecen ly shown ha PTH P+cells a e es ic ed o he bo om o he RZ, and hey a e dedica ed, a leas o some deg ee, o gi ing ise o col- umns o p ogeny11. He e we show ha FoxA2+cells a e loca ed a he op o he RZ and hey a e geog aphically sepa a ed om he p e iously cha ac e ized PTH P+cells11, in bo h ibia and ulna o FoxA2C eERT/+;ZsG eenfl/+;PTH Pmche y mice (Fig. 3A(a, b)). To confi m ou his ological findings, we pe - o med FACS analysis on cells isola ed om GP issue o FoxA2C eERT/+;ZsG eenfl/+;PTH Pmche y mice injec ed wi h amoxi en P13 o P17, and sac ificed a P18. We so ed he FoxA2+(ZsG een+) cells, he PTH P+(mChe y+) cells and he double posi i e (DP) cells. Consis en wi h he his ological da a, we obse ed no o e lap be ween FoxA2+cells and PTH P+cells (Fig. 3A(d)). The pe cen age o double posi i e (DP) cells (0.017 ± 0.004%) is he same as in insic (backg ound) fluo es- cence o double nega i e (DN) con ol cells (0.014%). To in es iga e whe he FoxA2+cells can become PTH P+cells o e ime, we pulse-chased FoxA2C eERT/+;ZsG eenfl/+;PTH Pmche y mice, by injec ing hem wi h amoxi en P13 o P17, and sac ificing hem a P40 (Fig. 3A(c, e)). His ological da a e eals he p esence o yellow FoxA2+PTH P+cells, while FACS analysis confi ms an 0.1% o double posi i e (DP) cells (Fig. 3A(c, e)), confi ming ha FoxA2+cells can become PTH P+cells o e ime. To inqui e abou FoxA2+cells sel - enewabili y and mul ipo ency, we used iple ansgenic mice FoxA2C eERT2/+;ZsG eenfl/+; Tg.col10mChe y, ea ed wi h amoxi en P13-P17 and ha es ed a P18, ha allowed ei he g een labeling o RZ FoxA2+col10−cells, o yellow labeling o HZ FoxA2+col10+cells (Supplemen a y Fig. 2A). To compa e FoxA2+cells wi h PTH P+cells, we isola ed PTH P+ cells om P18 GP issue o PTH Pmche y mice. Bo h FoxA2+cells and PTH P+cells isola ed om GP ca ilage ha e high le els o ca ilage specificgenes(collagen ype 2,agg ecan) and low le els o bone ma ke s (collagen ype 1, alkaline phospha ase) when compa ed wi h bone ma ow mesenchymal cells (BMSC) (Supplemen a y Fig. 2B). In a c u (colony o ming uni ) assay, RZ FoxA2+col.10-(g een) cells o med dis inc colonies (Supplemen a y Fig. 3a–c), while HZ FoxA2+col.10+(yellow) cells ailed (Supplemen a y Fig. 3d– ). This indica es ha FoxA2+cells, om RZ no HZ, ha e he capaci y o o m clones when cul u ed ex i o. Nex we compa ed he sel - enewabili y o FoxA2+cells (isola ed om FoxA2C eERT2/+;ZsG eenfl/+;Tg.col10mChe y mice), wi h ha o PTH P+cells (isola ed om PTH Pmche y mice) (Fig. 3B, Supplemen a y Fig. 4A). A pla ing, PTH P+cells ga e ise o 40% mo e colonies han FoxA2+cells (51 s. 37), bu only 11% (6/51) PTH P+p ima y colonies could o m seconda y colonies, whe eas 38% (14/37) FoxA2+p ima y colonies could be u he passaged (Fig. 3B, Supplemen a y Fig. 4B). FoxA2+colonies ha e highe clonogenici y and longe i y han colonies es ablished om PTH P+cells. Abou 9% (10/112) FoxA2+colonies each Passage 9 and beyond, whe eas only 1.4% (2/143) PTH P+ colonies could each Passage 5 (Fig. 3B, Supplemen a y Fig. 4B). To inqui e whe he FoxA2+clones a e mul ipo en , indi idual FoxA2+clones, om Passage 1 and 9, we e u he expanded in i o, and gene a ed Alcian-blue ma ix, Aliza in ed mine alized ma ix and Oil Red d ople s du ing cul u e in chond ogenic, os eogenic, o adipogenic di e en ia ion media (4/4 clones) (Fig. 3C). Al oge he , hese expe imen s demons a e ha , in i o, FoxA2+cells ha e mul ipo ency and highe sel - enewabili y and longe i y han PTH P+cells. Dual os eo-chond o-po en ial o FoxA2+cells p io o P28. To in es iga e he dynamics o FoxA2+cells clonali y in i o, we aimed o label su ficien FoxA2+cells o demons a e significan con ibu ion o he GP issue, bu spa se enough o allow assessmen o single clones (cells, clus e s and columns o p o- geny) o e ime. Two amoxi en injec ions, adminis e ed P14-P15 o FoxA2C eERT/+;Toma ofl/+mice, labeled 7.92 ± 0.9 FoxA2+ cells, which is 8% om he o al numbe o FoxA2+cells de ec ed by immunohis ochemis y a P14 (97.67 ± 9 cells) (Supplemen- a y Fig. 5A(a, b, e)). Fi e amoxi en injec ions (adminis e ed P14-P18 o FoxA2C eERT/+;Toma ofl/+mice) labeled 22.6 ± 4 FoxA2+cells, inc easing he labeling e ficiency ela i e o he numbe o FoxA2+cells iden ified ia immunohis ochemis y, om 8 o 23% (Supplemen a y Fig. 5A(a, c, e)). Fi e amoxi en injec ions (adminis e ed P14-P18 o FoxA2C eERT2/+;Toma oflfl mice), labeled 36.9 ± 2.3 FoxA2+cells, inc easing he e ficiency o labeling om 23% (one Toma o floxed allele) o 38% ( wo Toma o floxed alleles) (Supplemen a y Fig. 5A(a, d, e)). This inc ease in labeling e ficiency was eflec ed in a 2- old inc ease in he numbe o columns in FoxA2C eERT/+;Toma oflfl mice as compa ed wi h FoxA2C eERT/+;Toma ofl/+mice ha es ed 9 mon hs a e he las amoxi en injec ion (Supplemen a y Fig. 5B). O e all, inc eased labeling o he FoxA2+cells demons a ed inc eased con ibu ion o he FoxA2+cells o he GP ca ilage, bu i also inc eased densi y o he Toma o+cells hinde ing single clone analysis. As such, in lineage acing expe imen s, we labeled a mode a e amoun o FoxA2+cells by gi ing 5x amoxi en injec ions (P14 o P18) o FoxA2C eERT/+; Toma ofl/+mice (wi h one Toma o floxed allele) (Fig. 4A). When FoxA2C eERT2/+;Toma ofl/+mice we e pulsed wi h amoxi en P14-P18 and chased o 1 day o 1, 3, 9 mon hs we obse ed inc easing numbe s o double s, clus e s, and columns o p ogeny (Fig. 4A). Al hough he ini ial pe cen age o columns is 1% a 1 mon h, he e was subs an ial inc ease in column o ma ion o e ime, and by 9 mon hs, he columns o p ogeny accoun o 26% o he o al combined s uc u es (single cells, clus e s, and columns) (Fig. 4C1, Supplemen a y Fig. 6C). This is in con as wi h PTH P+cells, which ga e ise o a la ge numbe o columns ini ially, bu he numbe dec eased significan ly o e ime11. FoxA2+cells beha e as LTSSC, fi s gi ing ise o p ogeny, which hen p oduce columns o chond ocy es. Conse- quen ly, he pe cen age o FoxA2+clus e s peaks ea ly (43% by 1 mon h), bu d ops o 31% by 9 mon hs, when FoxA2+column o ma ion is high (Fig. 4C1). Las ly, despi e o ming clus e s and columns, a significan numbe o FoxA2+cells emains as single cells. The pe cen age o single cells dec eases apidly o e ime, om 82% (1 day), o 55% (1 mon h), bu he e is s ill a high ac ion o single cells as la e as 9 mon hs a e labeling (Fig. 4C1). Al oge he , hese findings sugges ha FoxA2+cells cons i u e a mo e quiescen , LTSSC popula ion loca ed nea he SOC, which gi es ise o an inc easing numbe o columns o p ogeny o e ime. Nex , we compa ed he clonali y o FoxA2+cells, labeled du ing SOC enla gemen (P14-P18), wi h he clonali y o FoxA2+cells, labeled du ing SOC os eogenic ma u a ion (P28- P37). Quan ifica ion o he pe cen age o FoxA2+clones (single cells, clus e s o columns) o he o al numbe o Toma o+uni s, shows a simila dynamic. Bo h FoxA2+cells labeled a a younge age ( amoxi en P14-18), o a an olde age ( amoxi en P28-37), ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 4NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions exhibi he same LTSSC cha ac e is ics in i o, as shown by an inc eased pe cen age o FoxA2+columns o e ime (Fig. 4A–C). Howe e , quan ifica ion o he numbe o FoxA2+clones high- ligh s highe he e ogenei y in he mice labeled a a younge age. A high numbe (138 FoxA2+clones/hindlimb) a e ini ially labeled in FoxA2C eERT/+;Toma ofl/+mice ( ea ed wi h amoxi en P14-P18), bu his numbe dec eases significan ly ( o 41 FoxA2+clones/hindlimb) by 3 mon hs a e he ini ial labeling (Fig. 4(C3), Supplemen a y Fig. 6C). In con as , in FoxA2C eERT/+; Toma ofl/+mice ( ea ed wi h amoxi en P28-P37) he numbe o FoxA2+clones is no significan ly changed be ween 1-day (55 FoxA2+clones/hindlimb) and 3 mon hs (57 FoxA2+clones/ hindlimb) a e he ini ial labeling (Fig. 4C4, Supplemen a y Fig. 6C). These esul s aise he possibili y ha FoxA2+cells labeled a e P28 emain mos ly in GP, and hei con ibu ion o he SOC slows down significan ly o e ime. 0 20 40 60 Colonies om FoxA2+ (Zsg een+) s em cells Colonies om PTHRP+ (mche y+) s em cells c u assay o FoxA2+ cells s PTH P+ cells B FoxA2 C e.ERT/+ ZsG een /+ PTH P mche y mice PTH P+ FoxA2+ a P oximal ibia a Dis al ulna PTH P+ FoxA2+ ab a1 b1 a1 b1 A P18 Tam Ha es P13-17 a Dis al ulna P40 Tam Ha es P13-17 PTH P+ FoxA2+ c1 c1 FoxA2+ PTH P+ Passage 1 Passage 9 G ow h media Adipogenic media Os eogenic media Chond ogenic media T ilineage di e en ia ion assay o FoxA2+ cells abc d e gh C c d. Ha es a P18 P1 P2 P3 P4 P5 P6 P7 P8 P9 Passage numbe Colonies / 1000 cells 10 102 103104 105 106 102 103104 105 106 ZsG een mChe y 102 103104 105 106 mChe y 10 10 10 102 103104 105 106 ZsG een e. Ha es a P40 1.579% 0.015% 0.681% 2.445% 0.108% 0.396% Fig. 3 FoxA2+cells a e long- e m s em cells, and hey a e dis inc om he sho - e m PTH P+s em cells. A Tibia (a) and ulna (b) omFoxA2C eERT2/+; ZsG eenfl/+;PTH Pmche y mice ea ed wi h amoxi en (Tam) P13-P17 and ha es ed a P18, and om FoxA2C eERT2/+;ZsG eenfl/+;PTH Pmche y mice ea ed wi h amoxi en P13-P17 and ha es ed a P40 (c). Flo escence mic oscopy o ZsG een (g een), mche y ( ed), Hoechs (blue). Scale ba s, 50 µm. Sepa a ion o FoxA2+(g een) cells om PTH P+( ed) cells and double posi i e FoxA2+PTH P+(yellow) cells by Fluo escence-ac i a ed cell so ing (FACS) o GP cells isola ed om FoxA2C eERT2/+;ZsG eenfl/+;PTH P mche y mice, injec ed wi h amoxi en om P13 o P17, and ha es ed a P18 (d)o P40(e). BColony o ming uni (c u) assay o FoxA2+(ZsG een+) cells ( om FoxA2C eERT2/+;ZsG eenfl/+;col.10mche y mice ea ed wi h amoxi en P13-P17 and ha es ed a P18) and PTH P+ (mche y+)cells ( om PTH Pmche y mice ha es ed a P18). Quan ifica ion o he numbe o colonies (pe 103cells) p esen ed as mean ± SD om n=3 expe imen s. CT ilineage di e en ia ion o FoxA2+(ZsG een+) clones, om passage P1 (a–d) and passage P9 (e–h). Fou independen clones, o each passage, we e es ed in ei he g ow h media (a,e), o adipogenic (b, ), os eogenic (c,g) and chond ogenic (d,h) media. Scale ba s, 100 µm. NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions 5 Indeed, o FoxA2C eERT2/+;Toma ofl/+mice ea ed wi h amoxi en P14 –P18, and ha es ed a P19, a 1-day chase e eals ha 137 cells o he FoxA2+cells a e in he GP and 43 in he SOC (Supplemen a y Fig. 6A1). A 1-mon h chase inc eases he SOC popula ion, om 43 o 253, sugges ing ha FoxA2+cells can con ibu e o he epiphyseal bone, du ing SOC os eogenic ma u a ion (Supplemen a y Fig. 6A1). This con ibu ion is no longe endochond al, as RZ FoxA2+cells no longe exp ess col.10 by P21 (Supplemen a y Fig. 6B). Ins ead, FoxA2+cells a e os eo-p ogeni o s ha suppo di ec ly he os eogenic lineage. This is consis en wi h ou p e ious findings demons a ing ha FoxA2+cells labeled du ing ea ly pos na al de elopmen ga e ise o col.1+p ogeny in he SOC (Fig. 2B). In addi ion, as p e iously shown, he FoxA2+cells labeled P14 o P18 gi e ise o clus e s and inc easing columns o p ogeny o e ime, sugges ing chond o-p ogeni o quali y (Fig. 4A). Al oge he , hese findings highligh dual os eo-chond o-p ogeni o a e o FoxA2+cells labeled p io o P28. In con as , FoxA2+cells labeled a e P28 emain mos ly in GP, and hei con ibu ion o he SOC slows down (Supplemen- a y Fig. 6A2). Fo olde mice, 1-day chase e eals ha 55 FoxA2+cells a e in he GP and 10 FoxA2+cells in he SOC abo e (Supplemen a y Fig. 6A2). A 1-mon h chase shows a simila numbe o FoxA2+cells (23) in he SOC, sugges ing ha Ha es : 1day ab dgjc e hi kl Lineage acing FoxA2C e.ERT/+Toma o /+ mice, Tamoxi en s a ing P28 Quan i ica ion Toma o+ cells, clus e s, columns Ha es : 1 mon h Ha es : 3 mon hs Ha es : 9 mon hs j g d ba B1 B2 B3 B4 B k ab b a c ce d ig gi ed h A1 A2 A3 jkl lj k A4 Ha es : 1day Lineage acing FoxA2C e.ERT/+ Toma o /+ mice, Tamoxi en a P14 Ha es : 1 mon h Ha es : 3 mon hs Ha es : 9 mon hs A C FoxA2C e.ERT2; Toma o /+ Tamoxi en a P14 FoxA2C e.ERT2; Toma o /+ Tamoxi en a P28 # FoxA2+clones / HL # FoxA2+clones / HL C3 Numbe o FoxA2+ clones single cells clus e s columns 0 40 80 120 160 1d 1mo 3mo 9mo C4 Pe cen age (%) o FoxA2+ clones FoxA2C e.ERT2; Toma o /+ Tamoxi en a P14 FoxA2C e.ERT2; Toma o /+ Tamoxi en a P28 % FoxA2+clones / HL % FoxA2+clones / HL C1 single cells clus e s columns C2 0 40 80 120 1d 1mo 3mo 9mo 0 40 80 120 1d 1mo 3mo 9mo 0 40 80 120 160 1d 1mo 3mo 9mo Tam Ha es P14-18 P19 Tam Ha es P14-18 P48 Tam Ha es P14-18 P108 Tam Ha es P14-18 P288 Tam Ha es P28-37 P38 Tam Ha es P28-37 P67 Tam Ha es P28-37 P127 Tam Ha es P28-37 P307 Fig. 4 Dual os eo-chond o-po en ial o FoxA2+cells in he ea ly weeks o pos na al de elopmen (P14) and s onge chond ogenic po en ial beyond P28. A Tibia sec ions ha es ed om FoxA2C eERT2/+;Toma ofl/+mice ea ed wi h 5x amoxi en (Tam) injec ions s a ing a P14, and ha es ed a 1 day (A1), 1 mon h (A2), 3 mon hs (A3), and 9 mon hs (A4) a e he las injec ion. Hoechs (g ay), 5-DTAF (g een). Ba s, 100 µm. Rep esen a i e de ails om GP sec ions, in numbe ed inse s (a–l). BTibia sec ions ha es ed om FoxA2C eERT2/+;Toma ofl/+mice ea ed wi h 10x amoxi en injec ions s a ing a P28, and ha es ed a 1 day (B1), 1 mon h (B2), 3 mon hs (B3), and 9 mon hs (B4) a e he las injec ion. Hoechs (g ay), 5-DTAF (g een). Ba s, 100 µm. Rep esen a i e de ails om GP sec ions, in numbe ed inse s (a–l). CQuan ifica ion o he numbe (C3,C4) and he pe cen age (C1,C2) o single cells, clus e s, columns o med a 1 day, and 1, 3, 9 mon hs a e he las injec ion. Pe cen age (%) single cells is calcula ed by he numbe o single Toma o+ cells in he GP (ou side he g een 5-DTAF domain) o he o al numbe o Toma o+uni s (single cells, clus e s and columns). Each poin ep esen s he sum o n=8 sec ions pe mouse hindlimb. Simila quan ifica ion o pe cen age (%) clus e s and columns. One Toma o+uni , is defined by 1 single cell, o 1 clus e comp ised o wo o mo e cells g ouped oge he , o by 1 column o cells. ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 6NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions FoxA2+cells con ibu ion o he epiphyseal bone slows down significan ly a e P28 (Supplemen a y Fig. 6A2). Sel - enewabili y o FoxA2+cells in a econs i u ion assay.To demons a e sel - enewal and mul ipo en capabili ies o FoxA2+ cells in i o, we pe o med a se ial ansplan a ion assay. We isola ed FoxA2+(ZsG een+) cells om FoxA2C eERT2/+; ZsG eenfl/+;Tg.col10mChe y mice, as p e iously desc ibed, labeled hem wi h DiD fluo escen dye, and ansplan ed hem subcu aneously in o ZsG een- li e ma es (Fig. 5A, Supplemen- a y Fig. 7A). A e 1 mon h, FoxA2+(ZsG een+) cells om he g a s exhibi ed sel - enewabili y and e ained colony- o ming abili y (Fig. 5B(a)). In addi ion, he g a ed cells ha e unde gone mul iple ounds o cell di ision shown by a log- old educed fluo escence, obse ed as a shi in he in ensi y his og am plo s (Fig. 5B(c)). Peak DiD in ensi y a day 0 (D0), immedia ely be o e ansplan a ion, was 1.2 × 104, and i was educed o 1.1 × 103on day 30 (D30) pos - ansplan ion (Fig. 5B(c)). The pe cen age o ZsG een+cells wi h high in ensi y DiD fluo escence (DiDhigh) Se ial ansplan a ion o FoxA2+ cells in i o A P ima y T ansplan a1 a2 a3 1 2 3 1 23 b2 b3 b1 Isola ion and assessmen o sel - enewabili y po en ial o FoxA2+ cells B ab Seconda y T ansplan FACS sepa a ion Label wi h DiD Fi s T ansplan Explan a e 1mo. In i o colony o ma ion and expansion Label again wi h DiD dye Second T ansplan In i o ZsG +colony o ma ion (a e i s ansplan ) Chond ocy e isola ion Take cells o DiD analysis by FACS (Day 30 i s ansplan ) Take cells o DiD analysis by FACS (Day 0 second ansplan ) Explan a e 1 mo. Take cells o DiD analysis by FACS (Day 30 second ansplan ) In i o ZsG +colony o ma ion (a e second ansplan ) T ansplan a ion o p imed p ogeni o s 105 cells Take cells o DiD analysis by FACS (Day 0 i s ansplan ) ZsG –li e ma e Seconda y ecipien ZsG –li e ma e Te ia y ecipien FoxA2+Col10- His ological analysis o explan s a 1mo. Lineage p iming FoxA2C e.ERT/+ ZsG een /+ Tg.col10mche y mice P ima y hos Chond ogenic Adipogenic Os eogenic *** 0 20 40 60 80 100 *** DiDhigh D0 D30 % ZsG een+ cells 0 20 40 60 80 100 DiDlow D0 D30 % ZsG een+ cells 0 20 40 60 80 100 0 20 40 60 80 100 *** *** D0 D30 % ZsG een+ cells D0 D30 ZsG een+ cells coun DiDhigh DiDlow de g h ZsG een+ cells coun c 100 50 0 100 50 0 100 50 0 100 50 0 DiD in ensi y 101 103105107 DiD in ensi y 101 103105107 100 50 0 % ZsG een+ cells 100 50 0 Fig. 5 Se ial ansplan a ion o FoxA2+cells in i o. A Expe imen al s a egy o he isola ion and assessmen o sel - enewabili y and di e en ia ion po en ial o FoxA2+cells in i o. BRep esen a i e images o colonies de i ed om FoxA2+cells isola ed om p ima y (a) and seconda y ansplan s (b). Scale ba s, 1 cm. DiD fluo escence in ensi y his og am p ofiles a Day 0 ( ed), Day 30 (blue), and nega i e con ol (black) om p ima y (c) and seconda y ( ) ansplan s. Pe cen age o DiDhigh cells in he FoxA2+cells om p ima y (d) and seconda y (g) ansplan s. Pe cen age o DiDlow cells in he FoxA2+ cells om p ima y (e) and seconda y (h) ansplan s. Da a a e p esen ed as mean ± SD o each g oup, n=3 mice. The as e isks indica e significan di e ence: wo- ailed S uden ’s unpai ed samples es ; ***p=7.0148E−07 (DiDhigh p ima y ansplan D0 s D30), 7.84512E−06 (DiDlow p ima y ansplan D0 s D30), 3.32176E−06 (DiDhigh seconda y ansplan D0 s D30), and 3.46695E−06 (DiDlow seconda y ansplan D0 s D30). Comple e s a is ical in o ma ion is p o ided in Supplemen a y Table 1. NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions 7 was 75 ± 1% p io o ansplan a ion (D0), bu i was educed o 27.3 ± 1.1% in he cells de i ed om g a s a D30 pos - ansplan a ion (Fig. 5B(d)). Simul aneously, he pe cen age o cells exhibi ing low in ensi y DiD fluo escence (DiDlow) was significan ly inc eased om 20.3 ± 0.6% a D0 o 42.3 ± 1.1% a D30 (Fig. 5B(e)). This sugges ha FoxA2+(ZsG een+) cells p oli e a e in he p ima y ansplan s and dilu e he DiD fluo - escen dye. Nex , FoxA2+(ZsG een+)cells om he p ima y g a we e s ained wi h DiD dye and ansplan ed in o he e ia y ecipien mice (Fig. 5A, Supplemen a y Fig. 7A). A simila educ ion in DiDhigh cell popula ion ( om 75.3 ± 1.5% on D0 o 29.7 ± 1.5% on D30) and a p opo iona e inc ease in DiDlow cell popula ion ( om 19.7 ± 0.6% on D0 o 46.7 ± 1.1% on D30) we e consis en wi h he p ope ies obse ed wi h cells de i ed om p ima y g a s (Fig. 5B( , g, h)). In addi ion, FoxA2+(ZsG een+) cells om he seconda y g a s e ain colony o ming abili y (Fig. 5B(b)). The cells collec ed a he end o se ial ansplan a ion exhibi ed mul i-lineage di e en ia ion capabili ies, as indica ed by posi i e Alcian blue, LipidTox o Aliza in Red-S s aining in c oss-sec ions o e ia y g a s (Supplemen a y Fig. 7B(a–c)). These e ia y g a s we e p oduced by ansplan a ion o lineage-p imed (chond o- genic, adipogenic, os eogenic) second g a -de i ed FoxA2+(Zs- G een+) cells (Fig. 5A, Supplemen a y Fig. 7B(a–c)). In con as , second g a -de i ed FoxA2+(ZsG een+) cells, wi hou lineage p iming, p oduced e ia y g a s ha we e nega i e o cell di e en ia ion (Fig. 5A, Supplemen a y Fig. 7B(d– )). Al oge he , hese expe imen s indica e ha FoxA2+cells p ese e bo h hei sel - enewing abili y and hei mul ipo ency du ing in i o se ial ansplan a ion. GP ca ilage egene a ion a e SH1-like inju y. To in es iga e whe he FoxA2+cells expand and epai he GP ca ilage in esponse o auma, we de eloped a mu ine model o GP inju y. Sal e –Ha is ype1 ac u es (SH1), which in ol e sepa a ion ac oss he en i e physis, h ough he HZ, ha e he bes p ognosis o epai and a e he leas likely o cause g ow h a es 3,4. We ep oduced his clinical obse a ion and de eloped a SH1-like su gical model, o es i FoxA2+cells could epai he GP ca ilage. A 30 g needle is passed in he ans e se plane, h ough he HZ o he p oximal ibial physis, c ea ing a disc e e de ec , isualized by Con as -Enhanced Compu ed Tomog aphy (CECT)17,18 (Fig. 6A, B). Quan i a i e analysis o sequen ial sagi al planeimageses ima es ha hede ec acks, on a e age, 2216 ± 195.6 μm medial o la e al, 280 ± 60 μm an e io o pos e io , 165.6 ± 36.9 μm heigh , and 28.6° ± 5.5 angle ela i e o mid-co onal plane (Supplemen a y Fig. 8A, B). Colo maps o ep esen a i e CECT images o con ol and ope a ed ibias e eal ha inju y si e heals wi hin 7 days, i espec i e o mouse gende (Fig. 6C). The inju y gap olume dec eases significan ly om 0.29 ± 0.07 mm3a 1 day, o 0.04 ± 0.04 mm3, close o backg ound le el, a 7-day pos -op (Fig.6D).O e alllongi udinalg ow hwasno a ec ed a 7- and 21-day pos -op (Supplemen a y Fig. 8C). A 1-day pos -op, he inju ed GP displays significan damage in he HZ (Fig. 6E(a, b)). In addi ion, gi en ha he cen al egion is mo e cu ilinea , wi h a sho e GP in he middle, he inju y causes mo e damage in his a ea, des oying he HZ, he PZ, and mos o he RZ. A 1-day pos -op, he heal hy ca ilage a ea is 42% o he o al measu ed a ea (heal hy plus inju ed) (Fig. 6F). Wi hin days, he de ec shows healing and he ca ilage is egene a ing. By he 3 d day, he cen al gap is closing in, and he heal hy ca ilage a ea is up o 63% ((Fig. 6E(c, d), F). By he 7 h day, he GP is 96% egene a ed, displaying all h ee laye s (RZ, PZ, HZ) ((Fig. 6E(e, ), F). The inju ed GP healed wi h physeal ca ilage, no fib oca ilage o bone. FoxA2+cells expand and egene a e he GP a e SH1-like inju y. A e SH1-like su ge y on FoxA2C eERT/+;Toma ofl/+ mice, he e was subs an ial expansion o Toma o+cells in he inju ed GP (Fig. 7A, B, Supplemen a y Fig. 9). We quan ified he numbe o FoxA2+cells in GP ca ilage as well as in neighbo ing me aphyseal bone (MB) and epiphyseal bone (SOC) (Fig. 7B). A 1-day pos -op, he e was no di e ence be ween GP FoxA2+cells in con ol e sus ope a ed mice (Fig. 7A(a, d), Fig. 7B(b1)). Howe e , he e is a significan loss (94%) o FoxA2+cells a he HZ/MB bo de , which con inues o be sus ained 3-day pos -op (Fig. 7B(b3)). This indica es ha he e is no expansion o he MB Toma o+cells in esponse o inju y. Simila ly, he e is no expansion o he Toma o+cells in he SOC a 1 day o 3 days a e SH1-like su ge y (Fig. 7B(b2)). Howe e , he e is a 2.7- old expansion o Toma o+cells in he GP 3 days a e he SH1-like su ge y (Fig. 7B(b1)). These findings a e suppo ed by a p oli e a ion assay, which demons a es ha Toma o+cells a e B dU+(Supplemen a y Fig. 10A). This expansion o FoxA2+cells a 3 days pos -op is he esul o FoxA2+cells labeled p io o su ge y who a e mul iplying in esponse o inju y, and no esh labeling o FoxA2+cells due o de no o exp ession o FoxA2 in he inju ed ca ilage emnan s. Immunohis ochemis y o FoxA2 in he inju ed GP ca ilage shows he exp ession o FoxA2 in i s es ablished RZ domain, bu no in he ca ilage emnan s a d1, d2 o d3 pos -op (a–c) (Supplemen a y Fig. 10B). By 7-day pos -op, FoxA2+(Toma o+) cells a e significan ly educed in he GP issue, and in pa allel, we see a su ge in Toma o+cells in he MB (3- old inc ease) (Fig. 7B(b3)). This sugges s ha he Toma o+cells, ha ha e expanded in he GP issue a 3-day pos -op, ansi ed below in he bone spongiosa by 7-day pos -op (Fig. 7A( 2)). In he cen e , whe e he wide gap was now educed o a hin sli , he las Toma o+cells a e mig a ing down, aligned in sho (4–8 cell) s acks o clones (Fig. 7A( 1)). FoxA2+cells a e needed o ca ilage egene a ion. To de e - mine whe he FoxA2+cells pa icipa e in egene a ing he inju ed GP issue ia mul i-lineage di e en ia ion, we asked i FoxA2+cells di e en ia e in o col.10+cells (chond ogenic lineage) o col.1+cells (os eogenic lineage). SH1-like su ge y on FoxA2C e.ERT/+;ZsG een /+; Tg.col10mche y mice demons a es ha FoxA2+(ZsG een+)cells gene a e yellow hype ophic cells (col.10mche y+)(Fig.8A(b)), while SH1-like su ge y on FoxA2C e.ERT/+;Toma o / ;Tg.col1GFP mice shows ha FoxA2+(Toma o+) cells ansi in he me aphyseal bone o become yellow bone cells (col.1GFP+)(Fig.8A(a)). To inqui e whe he FoxA2+cells a e needed o ca ilage egene a ion, we gene a ed FoxA2C eERT/+;DTA / mice, in which FoxA2+cells we e abla ed by p oduc ion o diph he ia oxin (DTA). In FoxA2C eERT2/+;DTA /+;Toma ofl/+mice he e was a 36% dec ease in FoxA2+(Toma o+) cells compa ed wi h FoxA2C eERT2/+;Toma ofl/+li e ma es (Fig. 8B). Loss o FoxA2+ cells lead o a delay in ca ilage closu e in FoxA2C eERT2/+;DTA / mice 7-day pos -op (Fig. 8C(b1, d1)). Ca ilage egene a ion in FoxA2C eERT2/+;DTA / mice was significan ly educed (down o 72%) compa ed wi h con ol mice (Fig. 8C, D), sugges ing ha FoxA2+cells a e necessa y o GP epai . Summa y In summa y, we demons a e ha a popula ion o FoxA2+col10- cells, esidual om epiphyseal ca ilage pos na al de elopmen , con ibu es o he op sub-compa men o he RZ (Fig. 9). Based on hei loca ion, FoxA2+cells a e physically sepa a ed om he PTH P+cells, which a e loca ed a he bo om o he RZ, and exhibi highe clonogenici y and longe i y han PTH P+cells. Sho - e m pulse-chase o he FoxA2+cells gene a es dyads/clus e s ARTICLE NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 8NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions o cells, whe eas long- e m pulse-chase gene a es inc easing col- umns o p oli e a ing p ogeny, sugges ing ha FoxA2+cells may fi s gi e ise o p ogeny ha subsequen ly di e en ia e in o col- umns o chond ocy es. P io o P28, FoxA2+cells ha e a dual os eo-chond o-p ogeni o a e, and a e P28, FoxA2+LTSSC emain mos ly in GP, while hei con ibu ion o he SOC winds down significan ly. In a mu ine model o GP inju y, FoxA2+cells expand in esponse o auma and pa icipa e in p oduc ion o physeal ca ilage allowing o success ul egene a ion. In conclusion, FoxA2+cells a e a LTSSC popula ion necessa y o bo h GP u no e and ca ilage egene a ion ollowing inju y. Discussion While POC (p ima y ossifica ion cen e s) o he long bones o m a E15.5, du ing mouse emb yonic de elopmen , he SOCs appea some ime la e , p ima ily a e bi h, a ound pos na al day P719,20. Chond ocy e hype ophy (P7-P8) is ollowed by RZ PZ HZ SH1 – like GP inju y model CECT imaging o he inju y wi h CA4+ AB Con ol + CA4+Su ge y + CA4+ Con ol - CA4+ 1a 2a 3a 1b 2b 3b CECT imaging o GPC egene a ion . C ** Inju y gap olume (mm3) P=0.055 d1 d3 d7 d1 d3 d7 sopsyad-op Su ge y Con ol Quan i ica ion ca ilage olume D 0.0 0.1 0.2 0.3 0.4 0.5 Con ol Su ge y A B d1 d3 d7 d1 d3 d7 1 2 3 ab d c e aa. SO/FG in con ol s. ope a ed mice sopsyad-op % GPC egene a ion % SO+ a ea (A) s o al a ea (A+B) 1 2 3 E * Image J quan i ica ion F ** *** 0 20 40 60 80 100 -1000 Hounds ield Uni s (HU) 5000 100 80 60 40 20 0 Fig. 6 A mu ine model o GP inju y. A Schema ics o Sal e –Ha is ype 1 (SH1)-like inju y model. BCECT (con as -enhanced compu ed omog aphy) imaging o he SH1-like inju y, 1-day a e su ge y. Rep esen a i e sagi al (1a,2a,3a) and ans e se (1b,2b,3b)μCT slices o he p oximal ibia, a 1-day pos -su ge y, be o e (1a,1b) and a e (2a,2b,3a,3b) exposu e o CA4+. Scale ba s, 500 µm. CColo maps o ep esen a i e con as -enhanced μCT images o con ol and ope a ed ibias a 1, 3, and 7 days pos -ope a ion, iewed om he ans e se plane. The colo scale co esponds o Hounsfield Uni s (HU). Fo e e ence, ai a enua es a −1000 H and wa e a 0 HU. Scale ba s, 500 µm. DQuan ifica ion o he inju y gap olume using CECT imaging. Da a a e p esen ed as mean ± SD, n=5 mice o each ime poin (d1,d3,d7)). The as e isks indica e significan di e ence: Two- ailed S uden ’s unpai ed samples es ; **p=0.0079 (d1 s d7). ESa aninO/Fas G een s aining o con ol and ope a ed ibias, 1 day (a,b), 3 days (c,d), and 7 days (e, ) a e SH1-like su ge y. Inse , cen al a ea o he de ec (1–3). Scale ba s, 100 µm. FImageJ quan ifica ion o he inju y. A is heal hy ca ilage a ea (Sa aninO+), B is inju ed a ea (Sa aninO−), and A+B is he o al a ea. Pe cen age (%) ca ilage egene a ion is A/A+B. Da a a e p esen ed as mean ± SD, n=3 mice. The as e isks indica e significan di e ence: Two- ailed S uden ’s unpai ed samples es ; ***p=0.000839 (d1 s d7), **p=0.007121 (d3 s d7), *p=0.037861 (d1 s d3). Comple e s a is ical in o ma ion is p o ided in Supplemen a y Table 1. NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-022-30247-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:2515 | h ps://doi.o g/10.1038/s41467-022-30247-1 | www.na u e.com/na u ecommunica ions 9