Pathophysiology of aniridia-associated keratopathy: developmental aspects and unanswered questions

The Ocula Su ace 22 (2021) 245–266 A ailable online 11 Sep embe 2021 1542-0124/© 2021 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Pa hophysiology o ani idia-associa ed ke a opa hy: De elopmen al aspec s and unanswe ed ques ions L. La a a , b , * , F.C. Figuei edo c , R. Ashe y-Padan d , J.M. Collinson e , J. Daniels , S. Fe a i g , N. Szen m´ a y a , S. Sol´ a h , R. Shalom-Feue s ein i , M. Lako j , S. Xapelli k , l , D. Abe dam m , n , ** , N. Lagali o , p , *** a D . Rol . M. Schwie e Cen e o Limbal S em Cell and Ani idia Resea ch, Saa land Uni e si y, Hombu g, Saa , Ge many b Depa men o Oph halmology, Saa land Uni e si y Medical Cen e , Hombu g, Saa , Ge many c Depa men o Oph halmology, Royal Vic o ia In i ma y, Newcas le Upon Tyne, Uni ed Kingdom d Sackle Facul y o Medicine, Tel A i Uni e si y, Tel A i , 69978, Is ael e School o Medicine, Medical Sciences and Nu i ion, Uni e si y o Abe deen, Ins i u e o Medical Sciences, Fo es e hill, Abe deen, AB25 2ZD, Uni ed Kingdom Cells o Sigh , UCL Ins i u e o Oph halmology, Uni e si y College London, London, EC1V 9EL, UK g The Vene o Eye Bank Founda ion, Venice, I aly h Resea ch Ins i u e o Medicines (iMed.ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa, Lisbon, Po ugal i Depa men o Gene ics and De elopmen al Biology, The Rappapo Facul y o Medicine and Resea ch Ins i u e, Technion - Is ael Ins i u e o Technology, Hai a, Is ael j Biosciences Ins i u e, Facul y o Medical Sciences, Newcas le Uni e si y, Newcas le Upon Tyne, UK k Ins i u o Fa macologia e Neu ociˆ encias, Faculdade de Medicina, Uni e sidade de Lisboa, Lisboa, Po ugal l Ins i u o de Medicina Molecula Jo˜ ao Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, Lisboa, Po ugal m Cen e de Reche che des Co delie s, INSERM U1138, Team 17, F ance n Uni e si ´ e de Pa is, 75006, Pa is, F ance o Depa men o Biomedical and Clinical Sciences, Link¨ oping Uni e si y, Link¨ oping, Sweden p Depa men o Oph halmology, Sø lande Hospi al A endal, A endal, No way ARTICLE INFO Keywo ds: Ani idia-associa ed ke a opa hy (AAK) PAX6 An e io chambe De elopmen Limbal s em cells (LSC) Ani idia Ani idia animal and cellula models Clinical esea ch Resea ch s a egies Limbal niche ABSTRACT Ani idia, a a e congeni al disease, is o en cha ac e ized by a p og essi e, p onounced limbal insu iciency and ocula su ace pa hology e med ani idia-associa ed ke a opa hy (AAK). Due o he cha ac e is ics o AAK and i s bila e al na u e, clinical managemen is challenging and complica ed by he mul iple coexis ing ocula and sys emic mo bidi ies in ani idia. Al hough i is p ima ily assumed ha AAK o igina es om a congeni al limbal s em cell de iciency, in ecen yea s AAK and i s pa hogenesis has been ques ioned in he ligh o new e idence and a e ined unde s anding o ocula de elopmen and he biology o limbal s em cells (LSCs) and hei niche. He e, by consolida ing and compa ing he la es clinical and p eclinical e idence, we discuss key unanswe ed ques ions ega ding ocula de elopmen al aspec s c ucial o AAK. We also highligh hypo heses on he po en ial ole o LSCs and he ocula su ace mic oen i onmen in AAK. The insigh s hus gained lead o a g ea e app ecia ion o he ole o de elopmen al and cellula p ocesses in he eme gence o AAK. They also highligh a eas o u u e esea ch o enable a deepe unde s anding o ani idia, and he eby he po en ial o de elop new ea men s o his a e bu blinding ocula su ace disease. 1. In oduc ion Ani idia is a a e, pan-ocula , bila e al and congeni al disease a ec ing he no mal de elopmen and unc ion o almos all eye s uc- u es. Ani idia is inhe i ed in an au osomal dominan manne , wi h high pene ance and ye wi h a iable pheno ypic exp essi i y e en wi hin he same amily. A a iable deg ee o i is hypoplasia o o al absence o i is is he hallma k o he disease. O e 90% o cases o ani idia in ol e haploinsu iciency o he PAX6 gene, commonly due o he e ozygous non-sense mu a ions on one copy o he gene. Well o e 500 PAX6 * Co esponding au ho . D . Rol . M. Schwie e Cen e o Limbal S em Cell and Ani idia Resea ch, Saa land Uni e si y, Hombu g, Saa , Ge many. ** Co esponding au ho . Cen e de Reche che des Co delie s, INSERM U1138, Team 17. *** Co esponding au ho . Depa men o Biomedical and Clinical Sciences, Link¨ oping Uni e si y, Link¨ oping, Sweden. E-mail add esses: [email p o ec ed] (L. La a), [email p o ec ed] (D. Abe dam), [email p o ec ed] (N. Lagali). Con en s lis s a ailable a ScienceDi ec The Ocula Su ace jou nal homepage: www.else ie .com/loca e/j os h ps://doi.o g/10.1016/j.j os.2021.09.001 Recei ed 10 Ap il 2021; Recei ed in e ised o m 19 July 2021; Accep ed 8 Sep embe 2021 The Ocula Su ace 22 (2021) 245–266 246 unique mu a ions ha e been iden i ied in pa ien s wi h amilial o spo- adic ani idia (h p://LOVD.nl/PAX6) occu ing in all exons and in non- coding egions o he gene. They include poin mu a ions leading o amino acid subs i u ion, dele ions, inse ions, p ema u e e mina ion, splicing de ec s o loss o he en i e gene. Ani idia can occu ei he as an isola ed mal o ma ion o as pa o a synd ome such as WAGR (also known as WAGR complex, Wilms umou -ani idia synd ome, ani idia- Wilms umou synd ome) caused by la ge dele ions ha a ec bo h PAX6 and he adjacen WT1 gene. WAGR is cha ac e ized by Wilms umou , Ani idia, Geni ou ina y anomalies and de elopmen al delay ( o me ly ‘men al Re a da ion’) [1,2]. Ani idia may also occu in Gil- lespie synd ome, caused by mu a ion in a di e en gene, ITPR1, con- sis ing o ani idia, ce ebella a axia and in ellec ual impai men [3]. Al hough nea ly all PAX6-ani idia pa ien s de elop p og essi e opaci- ica ion o he co nea e med ani idia-associa ed-ke a opa hy (AAK), glaucoma and ca a ac , he unde lying molecula mechanisms and physiological causes o he many pa hological ea u es o he disease ha e no ye been elucida ed. A main eason o his is ha ocula de elopmen , main enance and egene a ion in ol e c oss alk be ween di e en issues, and complex in e ac ions wi h he immune, ne ous, and me abolic sys ems. AAK, also some imes e med ani idia- ela ed ke a opa hy (ARK) o ani idic ke a opa hy, is a highly p e alen condi ion in PAX6- he e ozygo es ha is po en ially pain ul and se e ely limi s unc ional ision [4]. As he co nea is eadily accessible and can be pa ly o ully eplaced, AAK is a p ime a ge o he apies o imp o e ision in ani idia. E en a small educ ion in he se e i y o AAK can ansla e in o signi ican bene i s in ision and ocula su ace symp oms. Mo eo e , AAK is p og essi e, so i is wo hwhile o concen a e e o s in o un- de s anding i s pa hogenesis and pa hophysiology, because he po en ial may exis o slowing, al e ing, o e en a es ing i s p og ession in younge subjec s. Symp oms o AAK include b eakdown o he co neal su ace, wi h epi helial hinning o loss, in lamma ion wi h immune cell in il a ion, ascula iza ion and ch onic p og essi e opac i ica ion. These symp oms o e lap pa ly o wholly wi h hose ha a ise when he s em cells a he pe iphe y o he co nea – limbal s em cells (LSCs) – a e de icien due o disease o inju y. T adi ionally, i has been widely belie ed ha AAK is a consequence o a p og essi e limbal s em cell de iciency (LSCD) [5,6], al hough o da e, he e is no de ini i e p oo o loss o deg ada ion o limbal s em cells (LSCs) o hei niche as he causal e en igge ing AAK. Gi en he complexi y o he pa hology p esen in AAK [7], i is mo e likely ha PAX6 con ols mul iple physiological and biological ac o s ha ac oge he , and ha hei dys egula ion in ani idia leads o AAK de elopmen . Iden i ying he possible unde lying pa hogenic mechanisms leading o AAK de elopmen and p og ession holds he po en ial o es ablishing new he apeu ic op ions o he single g ea es unme need o pa ien s se e ely a ec ed by ani idia. In his e iew, we highligh key ques ions o impo ance – many s ill un esol ed – conce ning de elopmen al as- pec s and he eme gence o AAK. We show ha o ega d AAK as pu ely a limbal s em cell de iciency is o igno e he mul iple de elopmen al and pa hogenic e en s in ani idic eyes, a ec ing mul iple issues, ha may con ibu e o he onse o p og ession o he disease. We highligh some po en ial he apeu ic s a egies ha may a ise om a ulle unde - s anding o he de elopmen al basis o AAK. The opics discussed a e also ele an mo e gene ally o di e se ypes o en i onmen al o congeni al co neal pa hologies ha in ol e LSCD, co neal neo- ascula iza ion and opaci ica ion. Finally, we p esen ecen indings on ansla ion o esul s om animal models o humans as a c i ical s ep in de eloping u u e he apies o ea AAK and unde s anding hei ele- ance o o he co neal diseases. 2. A e de elopmen al de iciencies in ol ed in he eme gence o AAK? PAX6 1 codes o a key ansc ip ion ac o ha is essen ial o eye de elopmen and main enance. PAX6 is exp essed a he ea lies s ages o eye de elopmen and in mul iple eye issues h oughou li e. Se e al Pax6-he e ozygous mu an mu ine s ains (collec i ely ‘small eye’ o ‘Sey’ mice) ha e been cha ac e ized and used as in i o models o ani idia, o s udy he oles o he gene and he de elopmen al and pa hological aspec s o he diso de [8]. The mouse models ep esen a spec um o mu a ions. Some such as he Ha well allele Pax6 Sey−H and ‘Dickies small eye’ Pax6 Sey−Dey a e la ge dele ions a ec ing Pax6 and su ounding genes, and show se e e pheno ypes ha a e pe haps no ideal o s udying human ani idia and AAK [9–12]. O he s, including Pax6 Sey (=Pax6 SeyMH ), Pax6 Sey−Neu (=Pax6 Sey−Neu1 ), Pax6 ADD4802 , Pax6 Coop , Pax6 Aey11 , Pax6 AEY18 and he allelic se ies Pax6 2Neu o Pax6 10Neu and Pax6Leca1 – 4 include poin mu a ions leading o p ema- u e e mina ion, ameshi s o splice de ec s [9,13–16]. The e a e also enginee ed dele ions (Pax6 LacZ ) and loxed alleles (Pax6 lox ) ha yield la ge dele ions upon ac ion o C e ecombinase [17–19]. Wi h he excep ion o some hypomo ph alleles (Pax6 4Neu , Pax6 7Neu and Pax6 Coop ) and a gain o unc ion (Pax6 ADD4802 ), mos alleles lis ed abo e a e hough o known on he basis o nonsense-media ed RNA decay, phe- no ypes, and/o allelic complemen a ion s udies, o be null o Pax6. Unless s a ed o he wise, all Pax6 mouse mu an s discussed below ep esen null alleles. Pax6-knockou esul s in ea ly ailu e in lens placode de elopmen and anoph halmia (eye absence) bu he e ozygo es display a pheno ype ha esembles human ani idia, including a p og essi e AAK [14,28]. Al hough he pheno ype o Pax6 mouse mu an s is a ec ed by gene ic backg ound and he e is indi idual a ia ion in se e i y e en wi hin li e s, o e all hey a e ema kably consis en models o AAK. Conco - dance wi h human ani idia, howe e , is no comple e. Fo example, Pax6 +/− mice exhibi mic oph halmia wi h a 10% educ ion in eye diame e and educed lens size; while mic oph halmia can also occu in human ani idia, mos pa ien s ha e a ela i ely no mal eye and lens size [20]. The ex en o which mic oph halmia in mice may modula e he an e io segmen dysgenesis associa ed wi h Pax6 is no known, bu i should be no ed ha he ansgenic ‘PAX77’ mouse ha o e exp esses 5–6 copies o human PAX6 exhibi s mic oph halmia (including mic o- co nea) wi hou an AAK pheno ype [21,22]. Typically, he Pax6 he e ozygous newbo n mice exhibi mo pho- logical al e a ions in he co neal epi helium. Sepa a ion o he lens om he co nea du ing de elopmen is delayed in Pax6-mu an mice and i idoco neal and/o lens-co nea adhesions simila o Pe e s’ anomaly may be obse ed a bi h, dependen on mouse s ain and he Pax6 allele [23–26]. The g oss abno mali ies hese mice can exhibi a bi h a e small eyes and/o an opaci y o he cen al co nea due o delayed o ailed de achmen o he lens om he co nea. In con as o he mu ine models, he ocula su ace o he majo i y o ani idia pa ien s does no exhibi any appa en g oss abno mali y a bi h. Excep ionally, a e cases o Pe e s anomaly a e caused by PAX6 mu a ions [27] bu in nea ly all cases o congeni al ani idia, a p ope sepa a ion o he co nea and lens is obse ed [28]. Despi e a seemingly no mal co nea obse ed ea ly in li e, howe e , close inspec ion o he cen al co nea in a 4-yea -old child en wi h congeni al ani idia indica ed educed sensi i i y o me- chanical ouch, educed sub-basal ne e densi y and ele a ed p esence o an igen-p esen ing dend i ic cells [29]. Mo eo e , he co neal hick- ness is known o be pa hologically inc eased in ani idia, e en in child- hood [29]. As ou lined below, PAX6 is exp essed in many eye s uc u es du ing de elopmen (sec ion 2.1). The e o e, i is p obable ha AAK is 1 Human nomencla u e is used whene e applicable. I indings a e species- ela ed he gene/p o ein nomencla u e o he species is used acco ding o he li e a u e sou ce. L. La a e al. The Ocula Su ace 22 (2021) 245–266 247 a leas pa ly in luenced by de elopmen al de ec s and no solely due o pos na al eme gence o pa hological co neal unc ion. AAK pene ance is ull, bu i s pheno ypic exp essi i y is highly a iable be ween in- di iduals, e en be ween siblings [30]. This sugges s ha s ong en i- onmen al and s ochas ic componen s, and/o modi ie genes ha ac in conce wi h PAX6 and a y be ween indi iduals can in luence he exp ession o he disease. This could also be ue o epigene ic di e - ences be ween indi iduals ha may modula e AAK se e i y. I would mo eo e be impo an o unde s and how he di e en eye s uc u es in luence each o he du ing co neal de elopmen , and he ole o PAX6 le els in in luencing he onse and se e i y o AAK. PAX6/Pax6, he mammalian o hologue o he Eyeless gene (Ey) in D osophila, is a pai ed and homeodomain ansc ip ion ac o which is essen ial o eye de elopmen . The ac i i y o PAX6 in eye de elopmen is e olu iona ily conse ed as he human o mouse Pax6 genes can induce ec opic o ma ion o he compound eye om D osophila imag- inal discs as well as in Xenopus emb yos [31,32]. PAX6, howe e is also impo an o b ain, gu and panc eas de elopmen [33–35]; see e iew [36]. This mul iple o gan in ol emen pa ially explains why hap- loinsu iciency o PAX6 causes in adul s, no only ani idia pheno ype, bu also se e al non-ocula condi ions such as obesi y, glucose in ole - ance and diabe es, and anosmia [37,38]. Sleep diso de s a e also e- po ed in pa ien s and PAX6 may impac b ain s uc u es such as he pineal gland [39]. The impo ance o PAX6 o di e en s uc u es o he eye is high- ligh ed by he ac ha ani idia pa ien s su e om mul iple eye ab- no mali ies in bo h an e io and pos e io segmen s o he eye. PAX6 is exp essed in he mul iple ocula cell ypes om he ea lies s ages o eye de elopmen and h oughou li e (Figs. 1-3). The exac ole o PAX6 in lens and op ic cup de i a i es has been sys ema ically in es iga ed using condi ional mu agenesis ( e iewed in Re . [40]). By con as , he impac o Pax6 on co neal de elopmen con inues o be u he explo ed, equi ing e icien gene ic dele ion in he a ious co neal cell ypes [41]. 2.1. How is he an e io segmen o he eye o med? In e eb a e eye de elopmen , Pax6 exp ession is de ec ed in he an e io neu al pla e in he eye ield egion [42] and subsequen ly in bo h he neu oec ode m and su ace ec ode m p ogeni o s o he eye [18,33,42,43]. The su ace ec ode m gi es ise o he lens and co neal epi helial laye s (lens, limbus and co neal epi helium), while he neu- oec ode m popula es he op ic esicles ha unde go mo phogenesis o o m he op ic cups. The ou e laye o he op ic cups is popula ed by he e inal pigmen ed epi helium p ogeni o s (RPE), while he inne laye s o he op ic cup di e en ia e o o m all o he e inal neu ons and he Mülle glia cells. The an e io op ic cup im gi es ise o he i is and cilia y body pigmen ed epi helia Fig. 1). The ocula mesenchyme su - ounding he op ic cup im e en ually con ibu es o he i is and co neal s oma. The high and con inuous exp ession o Pax6 in cells ha de i e om su ace ec ode m and op ic cup (lens, co neal epi helium, i is and cilia y epi helium) is equi ed o he exp ession o genes encoding ansc ip ion ac o s, s uc u al and signaling molecules, which a e c i ical o he mo phogenesis and di e en ia ion o he neu onal, pig- men ed and he anspa en co nea and lens eye lineages. The ole o Fig. 1. Eye de elopmen and PAX6 exp ession in he mouse. The g een box shows he de eloping mu ine eye a di e en s ages. The colo code ma ks he emb yonic o igin o hese issues. (Blue: su ace ec ode mal, G een: Neu al ec ode mal; G ey: Neu al c es ; Yellow: Mesode mal). The yellow Box below shows he ela i e PAX6 exp ession in hese di e en s uc u es. The neu al ec ode m o ms he op ic cup as a double-laye ed s uc u e and is impo an o lens placode de elopmen o he su ace ec ode m. The dis al ips o he op ic cup will de elop i is s uc u es, and his coincides wi h e y high PAX6 exp ession le els. The inne laye o he op ic cup will o m he neu al e ina he ou e laye de elops o e inal pigmen epi helium. Upon sepa a ion o lens and su ace ec ode m se e al wa es o neu al c es and mesode mal cells mig a e in o he an e io segmen con ibu ing o he co neal s oma, co neal endo helium, i is s oma and an e io chambe . Mos o he da a displayed de i e om s udies on mu ine de elopmen [19,33,43]. *No e ha endo helium and s oma di e en ia ion is displayed o mouse whe e endo helium and s oma di e en ia e om he same cell mass mig a ed in a i s wa e. Howe e , i is unknown i endo helial cells a e al eady speci ied p io mig a ion [111]. A second mig a ion wa e la e appea s in he angle be ween u u e co nea and op ic cup and di e en ia e in o s oma o i is and cilia y body [47]. In human and bi ds h ee mig a ion wa es a e obse ed. Fi s endo helium is speci ied hen mesenchyme mig a es be ween epi helium and endo helium o di e en ia e o s oma and he hi d wa e con ibu es o i is and cilia y body [46]. G aphics adap ed om di e en sou ces [44,47,112]. L. La a e al. The Ocula Su ace 22 (2021) 245–266 248 Pax6 in each o hese s uc u es and he signaling cues ha media e hei coo dina ed de elopmen du ing he o ma ion o he an e io segmen o he eye, as ele an o he onse o AAK, is b ie ly summa ized in he nex sec ion. The an e io segmen o he eye includes he co nea, conjunc i a an e io chambe , i is, lens and associa ed s uc u es. In de elopmen o hese s uc u es, he neu al and su ace ec ode mal cells in e ac wi h mesenchymal cells o neu al c es and mesode mal o igin. These a ious and complex in e ac ions a e b ie ly desc ibed below. Fo a de ailed desc ip ion o he associa ed p ocesses, see e iews [44–47]. The pu a- i e in luence o PAX6 dosage on hese di e en p ocesses in de elop- men o he co neal pheno ype is discussed in sepa a e sec ions. 2.1.1. Ea ly de elopmen – su ace ec ode m and op ic esicle Mo phologically, he de elopmen o he eye is e iden wi h he o ma ion o he op ic esicles. The op ic esicles a e PAX6-exp essing bila e al e agina ions om he diencephalon ha gi e ise o he op ic cup h ough mo phogenesis ( e iewed in Re s. [48,49]). The op ic cup in e ac s wi h su ace ec o- de m, lens and mig a ing mesenchymal cells. Dis up ion o de elop- men al p ocesses has been desc ibed by manipula ing signaling pa hways. A sauce -shaped op ic cup, en al coloboma, o a de iciency o pe iocula mesenchyme we e obse ed by manipula ing ei he Wn -, L p6-, o e inoic acid signaling [50]. The pe iphe al im o he op ic cup con ain p ogeni o s ha will gi e ise, du ing pos na al s ages o he pigmen ed and non-pigmen ed laye s o he i is and cilia y body ( e iewed in Re . [51]). Wn ligands and BMP signaling om he su ace ec ode m elici Wn /GSK3β- esponse in e inal pigmen epi helium p ogeni o s (RPE) and a e c ucial o p oduc ion o he co ec numbe o RPE cells and p ope cu a u e o he op ic cup [50,52]. 2.1.2. Lens As soon as he op ic esicle o ms, in mice a E8.5, Pax6 is exp essed in neu onal and su ace ec ode m p ogeni o s o he eye (See E8.5 Fig. 1). The lens de i es om Pax6-exp essing lens-compe en acial ec ode m which is con ac ed by he op ic esicle [53]. A e he lens placode has been induced, i in agina es o o m he lens esicle. The de ailed mechanisms o lens induc ion and gene ic and signaling ne wo ks a e e iewed in Re . [54]. In he lens and op ic cup, Pax6 exp ession is speci ied independen ly, by cis- egula o y elemen s [40,54]. Pax6 au o egula es i s own exp ession and he Pax6 su ace ec ode mal enhance elemen d i ing PAX6 au o egula ion in e ac s wi h ano he ansc ip ion ac o , SOX2 [55]. Sox2 exp ession is induced in he su ace ec ode m upon an induc i e signal om he op ic esicle and de e mines lens placode o ma ion (See E9.5 Fig. 1) [56]. The cells om he an e io pole o he lens esicle gi e ise o he lens epi helial cells, whe eas he pos e io cells di e en ia e in o lens ibe cells. PAX6 con inues o be exp essed du ing lens in agina ion and Fig. 2. Aligned de elopmen al s ages o c i ical e en s in an e io eye de elopmen in se e al species. * Emb yonic days o weeks o ges a ion (WG). A1,2,3). Ea lies exp ession o PAX6 in su ace ec ode m and neu oec ode m is u he obse ed and sus ained in se e al eme ging s uc u es such as op ic esicle, op ic cup de eloping lens and su ace epi helium [33,43,113,114]. No e ha gi en ime poin s o each species showed he ea lies obse ed exp ession in he ci ed s udies. B) Neu al c es mig a ion is impo an o con ibu ing o co neal s uc u es such as endo helium and s oma as well as i is s oma. In mice wo mig a ion wa es a e obse ed and in a ian and human he e a e h ee epo ed wa es, al hough he e a e inconsis encies in he li e a u e [44–47,111]. B1) In mice neu al c es cells inc ease a E13 [115]. In a second wa e, mesenchymal cells mig a e and con ibu e o s oma o he i is (E15.5) [47]. B2) In humans he i s wa e is in he 7 h WG as no endo helial cells can be obse ed be o e [116]. In bi ds, he s oma is buil up by mesenchymal cells mig a ing be ween epi helium and endo helium a ound he 8 h WG [117]. Al hough he e a e desc ip ions o an e io chambe angle de elopmen , no e ha he mig a ion pa h o hese mesenchymal cells has no been add essed in hose s udies [118,119]. B3) In chicken he e a e h ee wa es o NCC. The i s wa e leads o o ma ion o he endo helium [120]; he second wa e de elops in o co neal ke a ocy es [120] and he hi d wa e o NCC con ibu es o i is and mesode mal cells become dis inguishable om endo helium a he se en h day [121]. C) The iming o limbus o ma ion, speci ica ion and ma u a ion a he molecula le el is s ill enigma ic C1) Be o e ke a in su ace ma ke s become dis inguishable (See D), exp ession changes could lead o lineage seg ega ion o co neal and conjunc i al cells. In he a , CX43 exp ession is los a E12 and could explain a spa ial sepa a ion o limbus and co nea epi helium [74]. C2) In he human, single cell analysis iden i ied clus e s o co neal, conjunc i al and limbal cell lineages [70]. KRT15 s a s o become es ic ed o limbal epi helium i e weeks la e a WG17 in humans [71]. A a simila imepoin , single cell analysis also iden i ies di e en s em cell and p ogeni o ma ke s [70]. C3) In he chicken, a di usion ba ie is es ablished in he limbal egion be o e cell lines can be dis inguished by cy oke a in exp ession changes. In e es ingly, he limbal ba ie is es ablished p io o exp ession changes in CX43 [73]. D1,2) As appa en in he imeline, he changes in su ace exp ession o ke a ins o in eg ins become clea e wi h u he s a i ica ion. These p ocesses occu much la e han he i s e idence o lineage seg ega ion o conjunc i a, limbal and co neal epi helium (See Sec ion 2.1.3). (Time poin s depic ed in he g aphics a e ob ained om ci ed li e a u e. S ages a e aligned wi h Ca nie S age compa ison). Ges a ion Week (WG), E (Emb yonic), P (Pos na al) [308]. L. La a e al. The Ocula Su ace 22 (2021) 245–266 249 di e en ia ion [19,57] as e iewed by C ekl A e al. [58] and is main- ained in he lens epi helium h oughou li e. 2.1.3. Co neal s oma and endo helium A e he lens, op ic cup and ocula su ace ec ode m ha e been speci ied, neu al c es cells and mesode mal cells mig a e be ween hese s uc u es. The co neal s oma is o med by neu al c es -de i ed cells om he pe iocula mesenchyme, a popula ion o mesenchymal cells loca ed nea he op ic cup and p esump i e lens. Fa e mapping o mesode m-de i ed cells showed a con ibu ion o he co neal endo he- lium and s oma [59,60]. In humans (and bi ds), h ee wa es o neu al c es cells (NCCs) mig a ion a e epo ed in con as o wo wa es in mice (No e ha Fig. 1 e e s o mouse de elopmen ). A i s wa e o NCCs mig a e be ween he lens and he su ace ec ode m o o m he co neal endo helium. A sec- ond wa e o ms he s omal ke a ocy es. The hi d wa e o NCCs con- ibu es o he cilia y body and i is s uc u es (see Sec ion 2.1.4). In mice a single wa e o neu al c es mig a ion gi es ise o bo h endo helium and s oma. S udies isola ing p ima y s omal cells om ansgenic mouse co - neas e ealed simila i ies in mRNA exp ession p o iles (Twis , snail, Slug and Sox9) be ween neu al c es -de i ed p ecu so s and he isola ed co neal p ecu so cells. This inding was ega ded as e idence o a neu al c es o igin o hese cells, which a e impo an o he u no e o s omal issue [61]. The co neal endo helium p o ides an impo an pump unc ion, ac i ely main aining a luid and elec oly e balance be ween he an e- io chambe and co neal s oma o p e en co neal swelling, hus main aining co neal anspa ency. The cellula densi y o his single endo helial cell laye is c i ical o main aining an adequa e pump unc ion. F om a his ologic analysis o human e uses, i has been shown ha he cellula i y o he endo helium apidly dec eases in he p ena al pe iod om 16 weeks o ges a ion o e m, a he same ime he co nea g ows in size [62]. The educ ion in endo helial cell densi y du ing his pe iod is abou 50%, while he densi y educes u he by a hi d du ing he i s wo yea s o li e. A pu a i e e ec o PAX6 le els on s omal, limbal s omal and endo helial de elopmen and how his could in lu- ence AAK is discussed in Sec ion 2.6. 2.1.4. I is and an e io chambe angle The an e io chambe angle is he angle be ween he i is and he co neal endo helium in he limbal egion. The i idoco neal angle con- ains impo an aqueous humo d ainage s uc u es such as he abec- ula meshwo k and Schlemm’s canal. The con iguous i is and cilia y body epi helia de i e om he im o he op ic cup while he s omal laye s de i e om he ocula mesen- chyme [26]. A Pax6 exp ession g adien is obse ed in he op ic cup wi h he highes le el om he dis al (close o he lens) o p oximal side (close o he op ic ne e [33] (Fig. 1, E 15.5). Fo he i idoco neal angle, lineage acing expe imen s in mice e ealed he con ibu ion o NCCs o he cilia y muscles, cilia y blood essels, an e io i is, abecula meshwo k and Schlemm’s canal in he i idoco neal angle [59,63,64]. Mesode mal cells also con ibu e o s uc u es such as he lining o Schlemm’s canal and he i is s oma, bu no o he cilia y muscles [59]. Pax6 may pa icipa e in egula ion o he ac o s equi ed o he mig a ion o NCC in o he eye [65] (see also e iews on neu al c es o he eye [44,66]). In mouse emb yos (E 15.5), i is and cilia y body p ogeni o cells can be molecula ly dis inguished om cells which o m he p esump i e neu al e ina. Thus he p ope de elopmen o he i is elies on he co ec compa men aliza ion o he op ic cup (See e iew in Re . [26] o molecula de ails). Fig. 3. Pos na al PAX6 gene exp ession in humans. The g een Box (A–D) shows he di e en eye s uc u es in human issue. The colo code ma ks he emb yonic o igin o hese issues. (Blue: su ace ec ode mal; G een: Neu al ec ode mal; G ey: Neu al c es ; Yellow: Mesode mal). The yellow box below shows he same ocula issues which exhibi sus ained PAX6 exp ession du ing pos na al s ages, hus likely also playing a ole in adul hood. No e some Da a om o he model o ganisms a e also ans e ed o he human s uc u es shown he e (E– H). The e is e idence o Pax6 exp ession in lac imal and meibomian glands based on exp ession a ays [122] (A, E). Pax6 is exp essed in lens epi helium, cilia y body (B, F), limbal, co neal and conjunc i al epi helium (C, G). In he adul e ina, Pax6 is exp essed in he ganglion cell laye and he amac ine cells o he inne egion o inne nuclea laye (D, H). G aphics eely adop ed om di e en In e ne sou ces [306,307]. L. La a e al. The Ocula Su ace 22 (2021) 245–266 250 Al hough he molecula mechanism esponsible o he no mal de elopmen o he i idoco neal angle has no ye been elucida ed, a se ies o de elopmen al s eps a e desc ibed in he mouse whe e inal ma u a ion o Schlemm’s canal and he abecula meshwo k ex ends pos na ally o P42 [67]. In humans, he de elopmen al p ocesses a e simila , wi h all udimen a y s uc u es de eloped a bi h [68]. Fu he ma u a ion and eo ganiza ion ake place, likely due o mechanical s ess and aqueous humo low and his p ocess could las 1–8 yea s pos na ally [69]. The impac o changing PAX6 le els on an e io chambe de elop- men and possible impac on he ocula su ace is discussed in Sec ion 2.5. 2.1.5. Limbus and limbal s em cells A e he lens has o med, he PAX6-posi i e cells o he su ace ec ode m seg ega e and gi e ise o he lineages o he an e io ocula su ace epi helia, including conjunc i al and limbal/co neal lineages [19] (Fig. 1, E8.5-E15.5).). In humans (bu no mice), specialized ana omical s uc u es, he ‘palisades o Vog ’, de elop a he limbus – he bounda y ing a ound he pe iphe y o he co nea whe e he s em cells eside in adul li e. The de elopmen al aspec s o hei o ma ion is o u mos in e es , as hey a e impo an o s em cell homeos asis and a e a ec ed in AAK. To make he o e iew easie o unde s and, a compa ison o he key e en s o he an e io chambe de elopmen in di e en animal models is shown (Fig. 2). A 8.5 WG (week o ges a ion), he human e al co nea is s ill con inuous wi h he su ace ec ode m. A 12–22 WG, in u n, indi idual componen s such as he conjunc i a, co nea and limbus can be dis in- guished by gene exp ession [70]. In 12 WG e al co neas, he p e- sump i e limbus is obse ed as ‘ idge like’ ea u e o a ‘dimpling’ in he epi helium [71,72]. In human e al co neas, he palisades o Vog a e no ye de ec ed and a e p obably o med pos na ally. Du ing 8–22 WG, he limbal epi helium s a s o become hicke han in he su ounding co nea and conjunc i a. Single cell RNA-seq s udies o he de eloping human co nea indica e he p esence o a p oli e a ing epi helial p o- geni o clus e wi h highly exp essed co neal epi helial s em and p o- geni o ma ke s TP63, CLDN1, CLDN4 and TXNIP a 18 WG, indica ing he i s p esence o “a pe iphe al limbal-like egion” ha bo ing he limbal s em and/o p ogeni o cells [70]. Due o he lack o de ailed molecula and mechanis ic s udies o cell a e de e mina ion in he mammalian limbus, co neal epi helium and conjunc i a, he e we e e also o unc ional s udies done in chickens, o elucida e he ime poin s when he co nea and conjunc i a become spa ially sepa a ed by a limbus. In he chickens, a di usion ba ie be ween he co neal epi helium and conjunc i a is es ablished a emb yonic day 8 (See Fig. 2 o species compa ison) [73]. This obse a ion is consis en wi h ecen ansc ip- ional da a om he de eloping human co nea a 12 WG, whe e he e a e wo sepa a e clus e s o ocula su ace and conjunc i al epi helial cells [70]. This sepa a ion in chicken may be achie ed h ough he di e en ial exp ession o a connexin, CX43, o in esponse o o he e en s allowing di e en ial esponses o, as ye unknown, induc i e signals [73–75]. CX43 is p esen in he conjunc i a and is also s ongly exp essed in he co neal epi helium, bu is absen a he limbal bo de egion. This ea u e is hough o isola e limbal cells om signals in he su ounding issues and o play a ole in hei main enance in an un- di e en ia ed s a e [73]. The de ailed signaling pa hways unde lying hese p ocesses, howe e , emain o be u he elucida ed. In e es ingly, se e al ma ke s o adul LSCs a e ini ially exp essed h oughou he en i e co neal epi helium and i is only a e s a i ica ion ha hei labeling pa e n becomes es ic ed o he limbal epi helium. In common wi h o he adul s em cell sys ems, i hus appea s ha he adul s em cells a e a spa ially es ic ed subpopula ion o a p ogeni o popula ion ha is speci ied du ing emb yogenesis. Al hough he limbus is speci ied p ena ally, he e may be some o e lap in s a i ica ion o co neal epi helium and u he ma u a ion o limbal s uc u es. Thus, i could be hypo hesized ha signals om he s a i ying co neal epi helium a e also necessa y o u he ma u a ion o he limbus (See Sec ion 2.3), as could in luence unde lying co neal s omal cells (See Sec ion 2.6). 2.1.6. Speci ica ion o conjunc i al-, co neal and limbal epi helia Despi e hei p oximi y wi hin a con iguous ocula su ace epi he- lium and hei common and pe sis en exp ession o Pax6 du ing de elopmen and h oughou li e, he co neal epi helial cells and he conjunc i al epi helial cells belong o wo dis inc lineages [76,77] a ising om di e en popula ions [78]. These wo lineages a ise simul aneously om Pax6 posi i e ec ode mal cells ha emain on he emb yonic ec ode mal su ace o he de eloping eye once he lens esicle has o med [79,80]. In i o s udies wi h ocula epi helial cells isola ed om abbi s and ansplan ed in o mice ha e shown ha limbal and co neal epi helial cell-de i ed cys s con ained only s a i ied squamous- ype epi helial cells. In con as , conjunc i al epi helial cell-de i ed cys s con ained s a i ied columna - ype epi helial cells in e spe sed wi h Pe iodic Acid Schi (PAS) s aining-posi i e cells wi h goble -like s uc u e [78]. Despi e he ac ha such isola ed cells migh no con ain s em cells o may be in luenced by di e en ia ed co- ansplan ed cells, such indings would suppo he hypo hesis ha co neal and limbal epi helial cells o igina e om a di e en emb yonic lineage han conjunc i al epi helial cells, and ha goble cells o igina e om he conjunc i al compa men and no ollowing ex e nal modula ion, as o iginally p oposed [78]. Ne e heless, lineage acing o hese hypo hesized p ogeni o pop- ula ions will be needed o unequi ocally add ess his issue in wild ype mice and u he mo e, explo e he ocula lineage speci ica ion in PAX6-mu a ed mice. In spi e o hei impo ance, ela i ely li le is known abou he ac o s egula ing conjunc i al goble cell de elopmen . Conjunc i al epi helial cells and goble cells de i e om a common bipo en p o- geni o [81,82]. SAM Poin ed Domain-Con aining ETS T ansc ip ion Fac o (SPDEF) has been desc ibed as a c ucial ansc ip ion ac o o goble cell di e en ia ion [83]. Goble cell di e en ia ion and mucin sec e ion appea o be di ec ly ela ed o he eyelid opening. In humans, he eyelids a e used un il he 5 h-6 h mon h o in au e ine li e, and goble cells appea in he o nix ex ending owa d he palpeb al and bulba egions om he 8 h o 9 h week o ges a ional age [84,85]. S udies a e cu en ly unde way o e alua e when he neu al egula ion o goble cell sec e ion becomes unc ional [86] and he hie a chical ne wo k o ansc ip ion ac o s egula ing goble cell de elopmen in heal hy and pa hophysiological condi ions ( e iewed in Re . [87]). Some signals which p e en he conjunc i al pheno ype in he cen- al co nea epi helium a e known [88,89]. The ex en o PAX6 in ol emen in hese p ocesses will be discussed u he in Sec ion 2.2. In con as o he poo ly known mechanisms de e mining cell a es, he issue speci ic ma ke s (mos ly cy oke a ins) o co neal limbal and conjunc i al epi helia a e well desc ibed, al hough species-speci ic di - e ences in some o hese ma ke s exis . The conjunc i al, limbal and co neal epi helium a e composed o one o wo cell laye s be o e eyelid opening, a e which he hickness o hese cell laye s inc eases o 4–5 cell laye s (see Fig. 2 o animal model compa ison). Cells become s a i ied and dis inguishable by hei mo phology, wi h basal cells ha ing a cuboidal shape and being a ached o he epi helial basemen memb ane. The in e media e wing cells (an e io o he basal cells) a e p esen , and a e in u n co e ed by la ened supe icial squamous epi helial cells. In he e us, only mino ke a in exp ession is isible in he supe icial shedding cells [72,90]. These p ocesses a e simila in di e en model o ganisms and ha e been p e iously summa ized [91]. Ea ly mu ine co neal epi helial di e en ia ion akes place a em- b yonic day 15 (E15), as exp ession o he Pax6- a ge gene K 12 be- comes appa en and is a speci ic ma ke o he co neal epi helium h oughou li e [92,93]. This is likely also ue in humans, as he neu al coun e pa o KRT12, KRT3, becomes isible in human e al co neas L. La a e al. The Ocula Su ace 22 (2021) 245–266 251 om 12 WG o 17 WG in supe icial cells. By 20 WG, KRT3 can be de ec ed in he supe icial cells o limbus and co nea, simila o he adul co nea [71]. No ably, he sequen ial appea ance o he KRT3 and KRT12 pai is di e en in chicken and abbi animal models [94]. In mice he de eloping limbus ma u es upon co neal epi helial s a i ica ion ha is associa ed wi h eyelid opening a ound pos na al day 10–14 (P10-14). KRT12 is exp essed in he supe icial epi helial cells and ex ends o all sup abasal cells and in la e emb yonic s ages o all laye s o he epi helium upon ma u a ion [95,96]. The co neal epi he- lium con inues o ma u e up o 3–6 mon hs pos na ally [92]. T an- sc ip ional changes in he co neal epi helium upon eye opening in mice, which a e simila o humans, ha e been summa ized [91]. One week a e bi h, α 9 in eg in, which ma ks he limbal egion in humans and in mice [97], is equally dis ibu ed ac oss he en i e co neal epi helium and becomes es ic ed o he limbus in mice and a s a 8 weeks pos na ally [90,98]. Simila ly, ke a ins can be used o obse e limbal ma u a ion. KRT19 is ound o be exp essed h oughou he en i e mu ine co neal epi helium p io o eyelid opening, howe e a e s a i ica ion, he exp ession o his ke a in becomes es ic ed o he mouse limbal epi helium [99]. KRT19 is also epo ed o be concen- a ed in human limbal epi helium bu species di e ences du ing ma u a ion o co neal epi helium a e likely [100]. In ea ly human de elopmen (8–14 WG), KRT15 is exp essed ac oss he en i e ocula su ace including conjunc i a, limbal and co neal epi helia, bu becomes es ic ed o he limbal epi helium om 17 WG, p io o eye opening (See Fig. 2 C 2 ) [71]. These da a seem o sugges ha , be o e s a i ica ion o he ocula su ace epi helium, s em cells a e no es ic ed o he limbus bu a he dis ibu ed h oughou he en i e limbal/co neal epi helium. (This would also poin o he impo an ole o s omal s em cell niche in main aining s em cell capaci y in adul s as desc ibed in 3.1 Con o e sy on limbal s em cells). Addi ional suppo o his hypo hesis came om he use o a X-ch omosomal LacZ ansgene, which allows andom and i e e sible labeling o emb yonic blas ocys cells in emale mice based on X-ch omosome inac i a ion mosaicism a ea ly emb yonic blas ocys s age. In e es ingly, in he i s 3–4 weeks a e bi h, a diso ganized mosaic pa e n o LacZ + -labelled pa ches o cells appea ed dispe sed h oughou he en i e limbal/co neal epi helia. Howe e , om P30 onwa d, a ypical adial s ipe pa e n o cell mig a ion in o he co nea became e iden as he s em cells became es ic ed o he limbus [101]. These elegan s udies [101,102] oge he wi h o he s udies men ioned abo e, indica e ha he co neal epi helium is sel -sus ained by i s own pool o s em cells, hese s em cells p obably di e en ia e be o e P30 and om ha s age, he limbus becomes he unique s em cell loca ion. No ably, he o iginal ‘XLacZ’ mouse model has limi a ions due o he ac ha acing is o blas ocys -s age (E3-4) cells and because i does no allow cell ype-speci ic acing in a clonal, empo al manne by i al mic oscopy. Howe e , ad anced quan i a i e lineage acing s udies has u he shown ha his concep is co ec , e ealing addi ional aspec s o limbal s em cell biology (discussed in sec ion 3.1 below [103–108]). 2.1.7. Co neal ne es In mice, b anching ne e bundles co e he en i e co neal s oma by E16.5. The co neal epi helium is i s inne a ed a E16.5 and ne es subsequen ly o m a swi l pa e n in he subbasal ne e plexus a abou h ee weeks pos na ally [109]. The oph halmic ne es a ise om he igeminal ganglion which is bo h de i ed om neu al c es and ec o- de mal cells (see he e iew in Re . [45]. A leas in chickens, he co nea is inne a ed solely by he neu al c es de i ed neu ons o he igeminal ganglion [110]. 2.2. Could dis u bed seg ega ion and sepa a ion o co neal and conjunc i al cell lineages con ibu e o AAK? Lineage seg ega ion o co neal epi helium om he conjunc i a in humans appea s a e sus ained PAX6 induc ion a he ime o su ace ec ode m speci ica ion. In mice, PAX6 is ound in he ocula su ace ec ode m a E8, and in humans a emb yonic day 42 (Fig. 2). Al e ed PAX6 le els in he co neal and conjunc i al epi helium du ing de el- opmen could impac signals equi ed o de ine bo de s and sel - main enance o co neal and conjunc i al issue iden i y. Deepe un- de s anding o conjunc i al and co neal di e en ia ion is he e o e essen ial o dis inguish be ween he de elopmen al and pos na al ole o PAX6. In he mouse, he egula o y ne wo k o Pi x2 and downs eam Dkk2 exp essed in he mesenchyme inhibi s Wn /β-ca enin signaling esul ing in he inhibi ion o he conjunc i al a e in he cen al co nea [88,89,123]. PITX2 is also desc ibed o in eg a e e inoic acid (RA) signaling om he su ace ec ode m, op ic cup and lens. Since RA signaling is al e ed in Pax6 Sey/Sey (Pax6 −/− ) mouse eyes [124], his could a ec he c oss alk be ween he pe iocula mesenchyme and he su ace ec ode m. In addi ion, ansc ip ional analysis in human subjec s wi h ani idia indica es ha RA me abolism could be al e ed in he conjunc i al cells due o PAX6 mu a ion [125], bu he mechanism e- qui es u he con i ma ion. Whe he PAX6 p o ein le els could impac p ope seg ega ion o co neal and conjunc i al issue du ing de elopmen , and he e o e po en ially in luence he de elopmen o AAK, emains unknown; howe e , ecen s udies a e beginning o add ess his ques ion. A ecen single-cell RNA-seq analysis o human co neal de elopmen [70] encompassing 12–23 weeks o ges a ion indica ed low o e all PAX6 exp ession h oughou he de eloping co nea; howe e , in all cases he highes exp ession was obse ed in he epi helial laye . The same was also ue o he adul co nea wi h highes PAX6 exp ession obse ed in he co neal and conjunc i al epi helium. Since se e al cell popula ions can be now iden i ied by hei ansc ip ional p o ile, i is now possible o in es iga e he ex en o which PAX6 is exp essed in hese cell clus e s o i some cells in hese clus e s exhibi highe o ansien PAX6 exp ession. Ano he ecen s udy has epo ed pos na al modi ica ion o PAX6 dosage in he Pax6 Sey−Neu/+ (Pax6 +/− ) mouse model by inhibi ing he mi ogen-ac i a ed p o ein kinase (MEK) pa hway [126]. In ha s udy, pha macologic MEK inhibi ion by ocula o sys emic ou es inc eased PAX6 p o ein exp ession in he basal epi helial laye s o no mal le els as in wild- ype mice, esul ing in es o a ion o co neal ana omy and anspa ency o a no mal pheno ype. This esul p o ides e idence o a PAX6-dependen ole in epi helial cell speci ica ion, e en pos na ally. 2.3. Does he limbus o AAK pa ien s exhibi de elopmen al de ec s? Simila i ies be ween ani idia synd omes and LSCD may sugges ha epi helial de ec s in AAK a e he esul o LSC ailu e. Howe e , since pa hological changes in ani idia a e also obse ed in he ne es, in- lamma o y cells and co neal epi helium ea ly in li e, e en be o e he limbus becomes o e ly a ec ed [29], i sugges s ha mul iple pa ho- logical mechanisms may be in ol ed and may p ecede and/o p omo e LSC insu iciency. The exac loca ion and di e en ia ion cha ac e is ics o LSCs o hei p ecu so s du ing human de elopmen is no well s udied a he mo- lecula le el, especially he pos na al o ma ion o limbus niche s uc- u es which di e om hose obse ed in mice [127]. I has been p e iously hypo hesized ha he delayed o ma ion o he limbus could be a cause o he la e onse o AAK in ani idia [71]. The ΔN iso o m o Tp63 α (ΔNp63 α ) is conside ed as a LSC ma ke [128] and as a mas e induce needed o he p og ession om an em- b yonic ec ode mal monolaye epi helium in o a dynamic, s a i ied epi helium ound in he adul s age (e.g. in he co neal epi helium and epide mis) [129]. PAX6 exp ession occu s de elopmen ally be o e he exp ession o TP63 [130,131]. In con as o PAX6 ha is widely exp essed in eye issues, TP63 exp ession is limi ed o he ocula su ace epi helia (conjunc i a, limbus, co nea) and is associa ed wi h ocula glands (lac imal and meibomian) [132,133]. Mu a ions in he TP63 gene lead o Ec odac yly-Ec ode mal dysplasia-Cle lip/pala e (EEC) L. La a e al. The Ocula Su ace 22 (2021) 245–266 252 and Ankyloblepha on-Ec ode mal de ec s-Cle lip/pala e (AEC) syn- d omes, wo synd omes wi h nume ous issues a ec ed. These syn- d omes can also be associa ed wi h LSCD [134]. TP63 was desc ibed o be equally exp essed in he e al limbal and cen al co nea [71]. In adul humans, TP63 exp ession is es ic ed o he limbus, and posi i e s aining is only de ec ed in cen al co neal epi helium when he co neal epi helium is egene a ing a e wounding [71,135]. The occu ence o limbal s em cell dysgenesis shows ha s em cells a e equi ed o co neal main enance, howe e iden i ying he LSCs hemsel es is challenging [136]. Two ecen single-cell RNA-seq s udies led o he iden i ica ion o glycop o ein ho mone subuni alpha 2 (GPHA2) as a no el ma ke o an ou e popula ion o limbal s em cells. GPHA2, is a la gely unexplo ed gene whose unc ion appea s o be essen ial o LSC sel - enewal and di e en ia ion [70,107]. GPHA2-o e exp essing ansgenic animals showed no g oss pheno ype al e a ions [137], bu i would be in e es ing o examine he pheno ype o hese mice and gene a e a knockou mouse s ain. GPHA2 exp ession was d ama ically educed o ba ely de ec able le els ollowing cul i a- ion o human LSCs and in immunode icien mice. This sugges s ha GPHA2 may be egula ed by T cells c i ical o he adap i e immune esponse, and ha may se e as an impo an con ibu o o he LSC niche [70,107]. No iceably, like he pu a i e LSC ma ke KRT15, GPHA2 is no only exp essed by basal limbal epi helial cells (i.e. LSCs), bu i is also occasionally de ec ed in limbal sup a-basal cells [107], and he e o e was also p oposed o ma k limbal commi ed o di e en ia ed cells [138]. The p ocesses, howe e , esponsible o c ea ing he limbal niche s uc u e sec e ion o i s basemen memb ane, as well as associa ion and ec ui men o niche cells, a e no su icien ly unde s ood. I would be o in e es o de e mine whe he GPHA2 exp ession is al e ed in AAK pa- ien s and/o in animal models o ani idia, o yield u he e idence o he loss o LSC unc ion, as is widely belie ed. In addi ion, lineage acing in Pax6 +/− mice enabled he de ec ion o pa hogenic mecha- nisms associa ed wi h ani idia and he LSCs in ligh o de elopmen al p ocesses. Fo example, Pax6 +/− LSCs in Pax6 +/− ↔ Pax6 +/+ chime ic mice a e unc ional and p oduce s eams o epi helial cells ha mig a e no mally in o he co nea, al hough hese p ogeny a e less likely o each he cen e o he co nea han wild- ype cells [139]. This sugges s ha dosage de iciency o Pax6 does no p eclude no mal speci ica ion o LSCs in a cell-au onomous manne . I emains unknown, howe e , i he ela i ely no mal beha io o Pax6 +/− cells in he chime ic mouse limbus ep esen s a non-au onomous ‘ escue’ by sec e ed p o ein ac o s such as GPHA2 eleased om he wild- ype cells. 2.4. Does lens de elopmen impac AAK? In con as o ani idia pa ien s, Pax6 Sey/+ (Pax6 +/− ) mice ha e a mo e p ominen an e io segmen dysplasia and he lens o en emains a ached o he co nea. The mouse lens is la ge in p opo ion o he es o he eye, as compa ed o he human eye. This could esul in a mo e se e e lens and co neal pheno ype in he mouse compa ed o he human eye [11]. In he chicken, i was shown ha su gical emo al o he lens a ec s mul iple eye de elopmen al p ocesses including eye g ow h, and inhibi s no mal de elopmen o he pe iphe al e ina, cilia y body, i is, and mig a ion o NCC in o he co nea [140]. The co neal cell a e o su ace ec ode m is s abilized by NCC mig a ing in he lens pe iphe al ec ode m. The o med s oma p e en s PAX6 down egula ion in he co neal epi helium [141]. The de eloping lens is a key signaling cen e du ing eye de elop- men . Du ing o ma ion o he lens placode, ligands o Wn , BMP and e inoic acid sec e ed om he su ace ec ode m play a ole in pa e ning o he op ic cup [50,52]. Su gical emo al o he lens om de eloping chicken eyes leads o down egula ion o genes associa ed wi h e inoic acid, BMP and Wn signaling in he pe iphe al e ina, including he cilia y body, and some aspec s o he lens-de icien pheno ype (e.g. mic oph halmia) can be ecapi ula ed by inhibi ing e inoic acid signaling, o escued in lens- emo ed eyes by es o ing e inoic acid [142]. Pax6 is essen ial o ea ly s ages o lens induc ion, possibly h ough in luencing modi ica ion and emodeling o ch oma in [19,52] as e iewed in Re . [54]. In addi ion o i s ole wi hin he lens lineage, Pax6 also plays a ole in he adjacen op ic esicle o igge lens o ma ion. When Pax6 is knocked ou a an ea ly s age in he op ic esicle, he lens does no de elop [113]. Consis en wi h he impo ance o Pax6 in lens o ma ion, he e is e idence ha he lens is exquisi ely sensi i e o he co ec Pax6 gene dosage. In Mexican Ca e ish As yanax mexicana, i is he loss o Pax6 exp ession speci ically du ing lens de elopmen , ha p ecipi a es lens apop osis which in u n leads o ailu e o e inal g ow h and he loss o an e io segmen s uc u es [143]. In mice, less han 80% o mo e han 120% o no mal Pax6 ac i i y is hough o esul in lens de ec s ha a ec he es o eye de elopmen , e en hough o adul hood [21]. Mo eo e , esul s om expe imen al inac i a ion one allele o Pax6 in mice, speci ically in he lens o in he op ic cup, indica e ha Pax6 exp ession in he lens is necessa y o no mal de elopmen o he an e io chambe [112]. Fu he e idence o he sensi i i y o lens de elopmen o Pax6 le els was concluded based on mouse chime a expe imen s indica ing ha he e ozygous Pax6 Sey−Neu/+ (Pax6 −/+ ) cells do no con ibu e o he de eloping emb yonic lens, in con as o hei con ibu ion o he o he eye issues [144]. Addi ionally, in chime ic mice whe e he lens was wild- ype, i ually all o he aspec s o an e io segmen de elopmen we e es o ed, including no mal i is de elopmen , co neal epi helial mo phology and limbal unc ion [145,146]. A plausible wo king model o he de elopmen al de ec s unde lying he de elopmen o he ani idia pheno ype is ha lens signals egula ed by he co ec Pax6 dosage a e equi ed o no mal de elopmen o o he an e io segmen s uc u es. Iden i ica ion o hese lens signals o ches- a ing an e io segmen de elopmen should he e o e be a high p i- o i y in eye esea ch, i espec i e o hei oles in ani idia. An ea ly onse o ca a ac is p e alen in ani idia [147]. This is likely due o abno mali y o he lens epi helium and lens ibe cells, and a hinning o he lens capsule [148–150]. Non-cell-au onomous mecha- nisms o ca a ac in ani idia ha e been also p oposed as abno mal zonula ibe s a e epo ed o be associa ed wi h congeni al ca a ac in ani idia [151,152]. An e io opaci ies in he lens and i is emnan s in he an e io chambe a e some imes obse ed in ani idic eyes (Fig. 4), sugges ing ha he sepa a ion o he lens and i is om he co nea may some imes be a es ed in human emb yos [4]. Fu he de ailed s udies a e neces- sa y o in es iga e i he lens s a us a ec s he co neal endo helium. Since he ke a opa hy is mos ly p esen in he an e io laye s o he co nea, i is no ob ious whe he an incomple e sepa a ion o he lens o i is om he co nea could impac AAK. The pu a i e c oss alk be ween he lens and o he de eloping ocula issues has been discussed abo e. Simila o dys unc ional lens epi helial cells, an abno mally hin co neal epi helium in he he e ozygous Pax6 +/− mouse [28] may ep esen de elopmen ally imma u e epi helial cells (including limbal s em and p ogeni o cells) incapable o o m he ully s a i ied mul iple co neal epi helial laye s ha no mally a ise pos na ally in he mouse co nea [87]. Howe e , i mus be no ed ha any appa en undi e en ia ed s a e may be a seconda y consequence o he ch onic ab asion and wound-healing physiology o he ani idic co nea [153,154]. 2.5. Does an e io chambe and i is mal o ma ion impac AAK de elopmen ? The condi ional inac i a ion o a single Pax6 allele in mice om ei he he inne laye o he ou e pigmen ed epi helium o he dis al op ic cup esul s in a p o ound i is hypoplasia [51]. The esul ing educ ion in Pax6 dosage in e up s di e en s ages o i is de elopmen : om educ ion in he size o he p ogeni o s, o delayed onse o L. La a e al. The Ocula Su ace 22 (2021) 245–266 253 muscle-speci ic ma ke s and ab oga ed i is sphinc e mo phogenesis [51,155]. Indeed se e al key ac o s o i is de elopmen a e educed in he de eloping i is o he Pax6 Sey−1Neu/+ mice including: Pi x2, Ig 2, Foxc1, TGFb2, Zic2 and BMP4 [156]. These ansc ip ion ac o s and ligands could impac he di e en ia ion o he i is p ogeni o s as well as he mig a ion o NCC ha popula e he i is s oma and he co nea [157]. The majo i y (50%–75%) o ani idia pa ien s de elop glaucoma mos likely as a consequence o abno mal di e en ia ion o he abecula meshwo k and/o comple e absence o Schlemm’s canal [158,159]. No ably, condi ional haploinsu iciency o Pax6 in he mouse lens and co nea - bu no in he de eloping op ic cup laye s - dis up ed abecula meshwo k and Schlemm’s canal de elopmen and esul ed in glaucoma [145]. I is cu en ly unclea , howe e , i his esul is due o Pax6 ac- i i y in he lens and co nea egula ing ac o s equi ed o he de el- opmen o he d ainage s uc u es, o due o he abno mal mo phology o he eye due o adhesion be ween lens, co nea and i is epi helium in he model [145]. Ano he unsol ed opic is how pa ial o comple e loss o i is and he abno mal di e en ia ion o he d ainage s uc u es in ani idia impac AAK p og ession. I should be conside ed ha he posi ioning o he an e io chambe angle may be needed o signaling o LSC and hei niche o de elop co ec ly, as well as o ensu e he p ope low o aqueous humo impo an o main ain he co ec eye p essu e and nu i ion o he an e io segmen s uc u es. In a ecen s udy examining 87 eyes o ani idia pa ien s, 21 o which had a pa ial i is, i was shown ha he pa ial p esence o an i is was s ongly associa ed wi h a milde deg ee o AAK [29]. Al hough his could suppo a connec ion be ween i is o chambe angle de elopmen and AAK, he mild ke a opa hy could also be caused by he common causa i e mu a ion i sel . 2.6. A e mesenchymal s uc u es (co neal s oma and endo helium) a ec ed du ing de elopmen ? A numbe o clinical s udies ha e epo ed ha he co neal s oma is abno mally hick in almos all cases o ani idia [6,160,161]. Al hough he causes o a hick s oma in ani idia a e no ye cla i ied (as he co neal endo helium appea s o unc ion no mally), i has been epo ed ha , du ing no mal human de elopmen , he co neal s oma is hicke in u e o and p og essi ely hins wi h inc easing ges a ional age [162]. This has led o he hypo hesis ha he no mal hinning o he co neal s oma in la e de elopmen al s ages in u e o is dis up ed in ani idia [29]. A de elopmen al o igin o he hickened co neal s oma is sup- po ed by he lack o clinical signs o s omal edema and a su icien ly high endo helial cell densi y in ani idia o main aining p ope s omal hyd a ion. In he epi helia o he de eloping lens, e ina, cilia y body, i is and co nea, PAX6 is exp essed a high le els, and his is easily de ec able by in si u hyb idiza ion, Wes e n blo and immunohis ochemis y. In he mesenchymal componen o some o he ocula issues, such as he co neal endo helium, co neal s omal ke a ocy es and abecula meshwo k mesenchyme, low and ansien le els o PAX6 (a he limi s o de ec ion by he abo e echniques) ha e been ep oducibly demon- s a ed du ing mid-la e s ages o de elopmen [163,164]. These low le els o PAX6 ha e ne e heless been shown expe imen ally o ep e- sen a cell-au onomous equi emen o con ibu ion o cells o he co neal endo helium and s oma, and also play a ole in he di e en i- a ion o abecula meshwo k [47,163,164]. Al hough PAX6 is down- egula ed in he abecula meshwo k o no mal adul eyes upon di e en ia ion [163], ecen single cell analysis has e ealed he p es- ence o PAX6 ansc ip s in no mal limbal co neal ke a ocy es, co neal s omal ke a ocy es as well as in co neal s omal s em cells, in o adul - hood [70]. This co ela es wi h he p e ious obse a ion o PAX6 exp ession in a popula ion o s omal s em cells [165]. A he single-cell le el in humans, PAX6 mRNA is de ec ed h ough all de elopmen al s ages a a low le el [70]. (Fig. 5). Impai ed de elopmen o he an e io chambe angle, i is and endo helium due o PAX6 haploinsu iciency has been ca e ully s udied and e iewed in a numbe o epo s [28,40,47,155,163], bu he impac o PAX6-de icien co neal o limbal s omal cells on AAK emains elusi e [166]. PAX6 dosage is sel -e iden ly c ucial o no mal eye de elop- men , so he molecula mechanism by which di e en issues equi e ei he ‘high’ o ‘low’ le els o Pax6, and how dosage is con olled ia egula o y DNA elemen s, s ill equi es cla i ica ion. PAX6 di ec ly in- e ac s wi h mul iple o he p o eins [167,168] and he p esence and s oichiome y o di e en binding pa ne s in di e en cell ypes is ex- pec ed o modula e PAX6 ac i i y. Hund eds o genes a e egula ed, di ec ly o indi ec ly, by PAX6 du ing eye de elopmen [169] and a e a ec ed o di e en deg ees by changes in dosage. I is he e o e ex- pec ed ha mesenchymal cells wi h ‘low’ le els o PAX6 will exhibi a di e en p o ile o downs eam gene exp ession om epi helial cells wi h ‘high’ le els. The biological impac o PAX6-he e ozygosi y o low le els o exp ession in mesenchymal cells, i any, a e un esol ed. Taken oge he , he mos pa simonious scena io is ha PAX6, exp essed a high le els, is unc ioning cell-au onomously in he op ic cup, lens and co neal epi helium p ogeni o s, and ha hese issues ha e a non-au onomous in luence on he su ounding an e io segmen al neu- al c es and mesode mal lineages (See Fig. 5, blue a ows). As he Schlemm’s canal lining and i is s oma a e o med by mesode mal cells [59] and a e absen in Pax6 +/− mice [65,170], i may be possible ha ansien PAX6 exp ession in hese cells u he con ibu es di ec ly o hei o ma ion. 2.7. Do meibomian and lac imal gland o ma ion impac AAK de elopmen ? The unc ions o lac imal and meibomian glands a e essen ial o he p oduc ion, s abili y and unc ion o he ea ilm. Any ac o dis u bing he homeos asis o he ocula su ace uni may dis up he s abili y o he ea s, leading o damage o co neal and conjunc i al epi helia and possibly impac ing LSC unc ion [171]. These suppo i e glands a e de i ed om he ocula su ace ec ode m, bu o da e he e is no e i- dence ega ding he impac o hese s uc u es on o he an e io s uc- u es du ing eye de elopmen . The mo phogenic e en s necessa y o he lac imal gland de elopmen in Pax6 Sey/Sey mice a e de ec i e [172]. In Pax6 Sey/+ a E19.5, he lac imal bud becomes isible bu i s s uc u e emains es igial [173]. In lac imal gland o ganoid models, Pax6 is necessa y o exp ession o he genes encoding he sec e ion machine y (aquapo ins and neu o ansmi e s) bu hese analyses we e pe o med wi h o al Pax6 knockou model [174]. In addi ion, PAX6 is one o he ansc ip ion ac o s necessa y o d i e explan cul u es o induced plu ipo en s em cells (iPSCs) in o a lac imal gland cellula a e [173, 175]. The de elopmen o he meibomian glands equi es p ope eyelid closu e and eyelid usion du ing emb yonic de elopmen . PAX6 could in luence his p ocess since i is exp essed a low le els du ing eyelid de elopmen in a complex exp ession pa e n [176,177]. PAX6 is exp essed du ing de elopmen in he acina cells o meibomian glands, al hough i s con ibu ion o de elopmen o hese glands equi es u he in es iga ion [178]. P o ein composi ion o he ea ilm is al e ed in ani idia, and an ele a ion in in lamma o y cy okine le els has been obse ed [179]. Meibomian gland dys unc ion has also been documen ed in ani idia pa ien s [180–183]. S ill, i is unknown whe he ea ilm and meibomian gland abno mali ies a ise om de elopmen al de ec s o whe he he unc ion is impai ed pos na ally due o de icien ocula su ace epi helia. AAK may unde pin he in lamma o y p ocess and ac as a possible igge mechanism o d y eye and meibomian gland dys unc ion. Ele a ed in e leukins in he ea ilm could be also caused by he ch onic wound healing s a e o he co neal epi helium in ani idia o om in lamma ion in he limbus and co neal s oma [7]. I is also impo an o keep in mind ha he de elopmen al de ec s obse ed in mice may no necessa ily be mi o ed in humans. Longi udinally L. La a e al. The Ocula Su ace 22 (2021) 245–266 260 eyes, he basal side o he Palisades o Vog is densely popula ed by co puscula ne e endings, sugges ing ha neu o ophic ac o s may suppo s em cells no only di ec ly, bu indi ec ly by main enance o he limbal niche [287,288]. Human co neal-limbal o ganoids appea o main ain good niche unc ion wi hou inne a ion in i o [289], bu his does no p eclude an in i o ole in niche de elopmen o main e- nance. In mammals, hough no in bi ds, PAX6 is exp essed ansien ly du ing ea ly de elopmen o he igeminal ganglion, in cells o bo h he OpV and mmV-de i ed componen s, and his co ela es wi h delayed inne a ion o senso y s uc u es such as whiske ollicles in mu an s (unpublished da a). Pu a i e links be ween PAX6 mu a ion, dis up ion o limbal-co neal inne a ion, he deg ada ion o palisade s uc u e and [207,270] de iciency o he limbal niche equi e u he in es iga ion. As immune cell in il a ion in o he co nea acili a es (and is acili- a ed by) neo ascula iza ion, a de ici o co neal ne es may di ec ly o indi ec ly play an impo an ole in de elopmen o neo ascula iza ion in AAK. In expe imen al models o co neal neo ascula iza ion and i- geminal ne e abla ion in he mouse, blood essel in asion was shown o occu only in a eas de oid o ne es, whe e in lamma o y leukocy es and mac ophage-lineage cells also in il a ed he co nea [290]. Con e sely, whe e he ne e supply emained in ac , he co nea emained essel- ee. A loss o an iangiogenic ac o s was no ed in dene a ed co neas, in pa icula loss o ascula endo helial g ow h ac o ecep o -3 (VEGFR3) exp ession in he co neal epi helium and loss o pigmen epi helium-de i ed ac o (PEDF) exp ession in he co neal s oma. In a di e en mouse model o in lamma ion-induced d y eye disease, co neal ne es we e diminished and exp essed ele a ed le els o he p oin lamma o y neu opep ide SP [291]. Blockade o SP o i s neu okinin-1 ecep o e ec i ely p e en ed ascula endo helial cell ac i a ion and educed co neal neo ascula iza ion. In addi ion, VEGF has been shown o media e co neal epai in ab asion-induced co neal ne e damage models, whe e VEGF blockade e ec i ely supp essed ne e egene a ion [292,293]. The sou ce o VEGF in he co neal ab asion model is in il a ing T-cells and neu ophils [292]. Taken oge he , hese s udies indica e ha co neal ne es, whe e damaged o accompanied by in lamma ion o when los en i ely, lose hei abili y o exp ess angios a ic ac o s and ins ead exp ess p oin lamma o y and egene a i e ac o s, con ibu ing o enhanced co neal neo- ascula iza ion. These e ec s can be conside ed pa o he no mal wound healing esponse, and may hus explain why he co nea in AAK, which is in a ch onically in lamed wound-like s a e, is cha ac e ized by a de ici o ne es and an abundance o blood essels. Po en ial he a- peu ic app oaches supplying de icien ac o s such as VEGFR3 o PEDF, o blocking ac o s aiding neo ascula g ow h such as SP o VEGF, wa an u he in es iga ion in ele an in i o and in i o ani idia models. Gi en he ole o co neal ne es in main aining a ascula i y and epi helial in eg i y, es o a ion o a heal hy co neal ne e popula ion may ep esen a iable he apeu ic app oach o AAK. As desc ibed abo e, au ologous se um eye d ops ha e been shown o elie e symp- oms o neu opa hic co neal pain and inc ease he abundance o co neal ne es in non-ani idia subjec s [294]. In subjec s wi h oxic co neal epi heliopa hy induced by an i-glaucoma eye d ops, a cou se o au ol- ogous se um d ops signi ican ly imp o ed co neal sensi i i y [295]. In a coho o hi een subjec s wi h AAK, au ologous se um eye d ops ins illed o e an 8-week pe iod esul ed in subjec i e imp o emen in ke a opa hy symp oms and healed co neal epi helial de ec s in se e al cases; howe e , neo ascula iza ion and s omal sca ing did no signi - ican ly imp o e du ing he cou se o ea men [296]. Longe - e m ea men , ca e ul pa ien selec ion (e.g., based on AAK g ade) and/o use o molecules wi h speci ic ne e egene a i e capaci y may be o bene i in AAK. The ecen ly app o ed ecombinan human ne e g ow h ac o (NGF) ea men (Cenege min) may be o in e es in his ega d. Indica ed o ea men o mode a e o se e e neu o ophic ke a opa hy (a a e disease cha ac e ized by diminished co neal inne - a ion, non-healing epi helial wounds and co neal ulce a ion), NGF s imula es co neal epi helial cell g ow h and su i al, aids in he main enance o limbal s em cell unc ion, p omo es ea p oduc ion and suppo s co neal e-inne a ion [297]. In clinical s udies, 65–75% o pa ien s ecei ing an 8-week cou se o Cenege min eye d ops exhibi ed comple e co neal healing, al hough elapse occu ed in abou 20% while eye pain and educed isual acui y we e equen ly epo ed ad e se e ec s [297,298]. No ably, i has also been shown ha co neal subbasal ne e densi y signi ican ly inc eased ollowing an 8-week cou se o Cenege min [299]. Whe he hese p omising e ec s could be ex ended o AAK is unknown and would need o be explo ed; howe e , use o Cenege min o condi ions o he han neu o ophic ke a opa hy is cu en ly o -label. He e, animal models would be use ul o ini ial in es iga ions. 4. Molecula biology and gene ics pe spec i e Due o he many obse ed PAX6 mu a ions independen ly leading o ani idia and he he e ogeneous clinical pheno ype, i is di icul o associa e speci ic mu a ions o AAK se e i y [4]. Howe e , some classes o mu a ions (selec ed missense o non-coding mu a ions) a e associa ed wi h educed AAK p og ession and mild o absen LSCD [29]. Compa a i e OMICs o such pa ien samples could help elucida e co - ela ions be ween speci ic mu a ions and deg ee o AAK se e i y a he molecula le el [125,300]. Nonsense media ed decay (NMD) o mu an mRNA is he mos common mechanism o PAX6 p o ein de iciency, bu he e is no e idence his is modula ed by he posi ion o mu a ions in he ansc ip . The e ec o missense mu a ion is di icul o p edic bu some in i o s udies desc ibed he e ec o he mu a ion in di e en binding domains. I migh no be use ul o compa e di e en missense mu a ions wi h each o he since hey could ha e di e en p ope ies. Fo PAX6 un-on mu a ions (wi h a mu a ed s op codon), i is hough (bu no ye demons a ed) ha he mu a ed PAX6 p o ein is deg aded o no p oduced. Mu a ions in egula o y domains o PAX6 (also ound as 3′-cis- egula o y egion dele ions) a e in e es ing since PAX6 educ ion could be less p onounced, and hese mu a ions ha e been ela ed o milde pheno ypes [4,206,300]. In a e cases, genes o he han PAX6 can pu a i ely cause ani idia [301–303]. P ima y cul u e o pa ien cells (bo h epi helial and mesenchymal) should help o iden i y de egula ed genes impo an o AAK de elopmen especially in cases o ani idia wi h mild AAK, o disc imina e om PAX6-dependen exp ession changes ha a e no causa i e o AAK [125]. Recen comp ehensi e e iews summa ize in de ail he gene ics o congeni al ani idia [1,2, 304]. Al hough he impac o PAX6 le els and splice a ian s on i is and cilia y body de elopmen ha e been sys ema ically s udied [155], we lack such de ailed knowledge o LSCs and co neal epi helial cells. Also, PAX6 p o ein has been desc ibed o be loca ed in he nucleus, cy osol o e en in sec e ed o m. I mus be u he e alua ed how localiza ion o PAX6 is con olled by pos - ansla ional modi ica ions. PAX6 mRNA exp ession may be no as d ama ically al e ed as a he p o ein le el, and hus mRNA exp ession and ansla ion o PAX6 need o be s udied in de ail in cells such as LSCs and di e en ia ed epi helium. In e es ingly, i has ecen ly been shown ha Pax6 exp ession is nega i ely egula ed by he mic oRNAs miR-7 and miR-135, and ha p o ec ion o his inhibi o y mechanism was capable o es o ing PAX6 p o ein le els in isola ed panc ea ic isle s in an ani idia mouse model [305]. I emains o be de e mined whe he a simila egula o y mechanism also occu s in LSCs. Ne e heless, hese da a, in addi ion o he pu a i e a ge miR-204-5p discussed ea lie , appea o sugges ha RNA-based he a- pies could ep esen a po en ial inno a i e he apeu ic s a egy o AAK. 5. Conclusions o u he esea ch s a egies Nea ly all pa ien s wi h ani idia su e om AAK [4]. The onse o clinically appa en AAK di e s be ween indi iduals e en wi h he same mu a ions. Based on ou p esen le el o knowledge, i is no clea i AAK is caused by LSCD while i seems likely ha some loss o LSC unc ion L. La a e al. The Ocula Su ace 22 (2021) 245–266 261 occu s, i is no necessa y o in oke LSCD as he main cause o he pheno ype seen in AAK eyes. Clinical mic oscopy indings sugges ha a minimal deg ee o ke a opa hy is likely o exis in all cases o ani idia e en be o e ocula su ace changes become isible a he sli lamp. This ‘minimal ke a opa hy’ includes educed mechanical ouch sensi i i y, a de ici o co neal ne es, and inc eased in lamma o y cell p esence in he cen al co nea [4]. LSCD may in ac be a consequence o he ea ly minimal ke a opa hy in subjec s whe e he speci ic PAX6 mu a ion p edisposes he co nea o a p og essi e AAK pheno ype. The minimal ke a opa hy may in u n ha e a de elopmen al o igin. The ch onic wound healing pa hology o he co neal epi helium in AAK may o e - whelm he PAX6 +/− limbal egene a i e po en ial du ing no mal li e [153,154]. Essen ial knowledge, howe e , is s ill lacking conce ning he c i ical ac o s needed o speci y and main ain he limbal niche and how hese ela e o PAX6 exp ession bo h p ena ally and pos na ally; he e- o e, pa hogenic mechanisms a he molecula le el a e s ill specula i e. Ea ly indica ions a e ha PAX6 egula ion o and by o he genes and ac o s is complex, and mul iple signaling pa hways, molecula and cellula mechanisms and eedback loops appea o be ac i e, esul ing in he obse ed AAK pheno ypes. Deciphe ing some o he key pa hways and mechanisms in ol ed can p o ide insigh s ha will be impo an o u u e and new he apies a ge ing AAK, keeping in mind ha he complexi y i sel may p o ide mul iple po en ial he apeu ic a ge s. 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