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.
Decla a ion o compe ing in e es
No con lic ing ela ionship pe aining o his wo k exis s o any
au ho .
Acknowledgmen s
This e iew is based upon wo k om he Wo k G oup5 o he COST
Ac ion CA18116 (Ani idia: ne wo king o add ess an unme medical,
scien i ic, and socie al challenge), suppo ed by COST (Eu opean
Coope a ion in Science and Technology). The Eu opean Join P o-
g amme on Ra e Diseases (EJP RD 2020) is acknowledged o i s suppo
o NL, LL, DA, MJC, NS, RAP (AAK-INSIGHT). The wo k o LL and NS a
he D . Rol M. Schwie e Cen e o Limbal S em Cell and Congeni al
Ani idia Resea ch a Saa land Uni e si y, Ge many was suppo ed by
he D . Rol M. Schwie e Founda ion.
Re e ences
[1] Waw ocka A, K awczynski MR. The gene ics o ani idia - simple hings become
complica ed. J Appl Gene 2018;59:151–9.
[2] Lima Cunha D, A no G, Co on M, Moosajee M. The spec um o PAX6 mu a ions
and geno ype-pheno ype co ela ions in he eye. Genes 2019;10.
[3] Ge be S, Alzayady KJ, Bu glen L, B ´
emond-Gignac D, Ma chesin V, Roche O,
e al. Recessi e and dominan de no o ITPR1 mu a ions cause Gillespie
synd ome. Am J Hum Gene 2016;98:971–80.
[4] Lagali N, Wow a B, F ies FN, La a L, Moslemani K, U heim TP, e al.
em>PAX6</em>mu a ional s a us de e mines ani idia-associa ed ke a opa hy
pheno ype. Oph halmology 2020;127:273–5. ≤.
[5] Nishida K, Kinoshi a S, Ohashi Y, Kuwayama Y, Yamamo o S. Ocula su ace
abno mali ies in ani idia. Am J Oph halmol 1995;120:368–75.
[6] Lagali N, Eden U, U heim TP, Chen X, Riise R, Dellby A, e al. In i o mo phology
o he limbal palisades o og co ela es wi h p og essi e s em cell de iciency in
ani idia- ela ed ke a opa hy. In es Oph halmol Vis Sci 2013;54:5333–42.
[7] Lagali N, Wow a B, Dob owolski D, U heim TP, Fage holm P, Wylegala E. S age-
ela ed cen al co neal epi helial ans o ma ion in congeni al ani idia-associa ed
ke a opa hy. Ocul Su 2018;16:163–72.
[8] F eund C, Ho s o d DJ, McInnes RR. T ansc ip ion ac o genes and he
de eloping eye: a gene ic pe spec i e. Hum Mol Gene 1996:1471–88. 5 Spec No.
[9] Hill RE, Fa o J, Hogan BL, Ton CC, Saunde s GF, Hanson IM, e al. Mouse Small
eye esul s om mu a ions in a pai ed-like homeobox-con aining gene. Na u e
1992;355:750.
[10] Lyon MF, Phillips RJ, Fishe G. Dose- esponse cu es o adia ion-induced gene
mu a ions in mouse oocy es and hei in e p e a ion. Mu a Res 1979;63:161–73.
[11] Glase T, Lane J, Housman D. A mouse model o he ani idia-Wilms umo
dele ion synd ome. Science 1990;250:823–7.
[12] Theile K, Va num DS, S e ens LC. De elopmen o Dickie’s small eye: an ea ly
le hal mu a ion in he house mouse. Ana Emb yol 1980;161:115–20.
[13] Thaung C, Wes K, Cla k BJ, McKie L, Mo gan JE, A nold K, e al. No el ENU-
induced eye mu a ions in he mouse: models o human eye disease. Hum Mol
Gene 2002;11:755–67.
[14] Lyon MF, Bogani D, Boyd Y, Guillo P, Fa o J. Fu he gene ic analysis o wo
au osomal dominan mouse eye de ec s, Ccw and Pax6(coop). Mol Vis 2000;6:
199–203.
[15] Fa o J, Pe e s H, He mann T, Schmahl W, Cha e jee B, Neuh¨
ause -Klaus A, e al.
Molecula cha ac e iza ion o Pax6(2Neu) h ough Pax6(10Neu): an ex ension o
he Pax6 allelic se ies and he iden i ica ion o wo possible hypomo ph alleles in
he mouse Mus musculus. Gene ics 2001;159:1689–700.
[16] G aw J, L¨
os e J, Puk O, Müns e D, Haubs N, Soewa o D, e al. Th ee no el
Pax6 alleles in he mouse leading o he same small-eye pheno ype caused by
di e en consequences a a ge p omo e s. In es Oph halmol Vis Sci 2005;46:
4671–83.
[17] Simpson TI, P a T, Mason JO, P ice DJ. No mal en al elencephalic exp ession
o Pax6 is equi ed o no mal de elopmen o halamoco ical axons in
emb yonic mice. Neu al De 2009;4:19.
[18] S -Onge L, Sosa-Pineda B, Chowdhu y K, Mansou i A, G uss P. Pax6 is equi ed
o di e en ia ion o glucagon-p oducing alpha-cells in mouse panc eas. Na u e
1997;387:406–9.
[19] Ashe y-Padan R, Ma qua d T, Zhou X, G uss P. Pax6 ac i i y in he lens
p imo dium is equi ed o lens o ma ion and o co ec placemen o a single
e ina in he eye. Genes De 2000;14:2701–11.
[20] Vosk esenskaya A, Pozdeye a N, Ba ko Y, Vasilye a T, Ma akhono A, Wes RA,
e al. Mo phome ic analysis o he lens in human ani idia and mouse Small eye.
Exp Eye Res 2021;203:108371.
[21] Do `
a N, Ou J, Kuce o a R, Pa isi I, Wes JD, Collinson JM. PAX6 dosage e ec s on
co neal de elopmen , g ow h, and wound healing. De Dynam : an o icial
publica ion o he Ame ican Associa ion o Ana omis s 2008;237:1295–306.
[22] Schedl A, Ross A, Lee M, Engelkamp D, Rashbass P, an Heyningen V, e al.
In luence o PAX6 gene dosage on de elopmen : o e exp ession causes se e e eye
abno mali ies. Cell 1996;86:71–82.
[23] Ramaesh T, Ramaesh K, Ma in Collinson J, Chanas SA, Dhillon B, Wes JD.
De elopmen al and cellula ac o s unde lying co neal epi helial dysgenesis in
he Pax6+/−mouse model o ani idia. Exp Eye Res 2005;81:224–35.
[24] Da is J, Duncan MK, Robison J WG, Pia igo sky J. Requi emen o Pax6 in
co neal mo phogenesis: a ole in adhesion. J Cell Sci 2003;116:2157–67.
[25] Hickmo JW, Gunawa dane U, Jensen K, Ko ecki AJ, Simpson EM. Epis asis
be ween Pax6(Sey) and gene ic backg ound ein o ces he alue o de ined hyb id
mouse models o he apeu ic ials. Gene The 2018;25:524–37.
[26] Da is-Silbe man N, Ashe y-Padan R. I is de elopmen in e eb a es; gene ic and
molecula conside a ions. B ain Res 2008;1192:17–28.
[27] Weh E, Reis LM, Happ HC, Le in AV, Wheele PG, Da id KL, e al. Whole exome
sequence analysis o Pe e s anomaly. Hum Gene 2014;133:1497–511.
[28] Ramaesh T, Collinson JM, Ramaesh K, Kau man MH, Wes JD, Dhillon B. Co neal
abno mali ies in Pax6 +/−small eye mice mimic human ani idia- ela ed
ke a opa hy. In es Oph halmol Vis Sci 2003;44:1871–8.
[29] Lagali N, Wow a B, F ies FN, La a L, Moslemani K, U heim TP, e al. Ea ly
pheno ypic ea u es o ani idia-associa ed ke a opa hy and associa ion wi h PAX6
coding mu a ions. Ocul Su 2020;18:130–40.
[30] Wang GM, P aso L, Al-Hasani H, Ma s CER, Tolia S, Wiinikka-Buesse L, e al.
Pheno ypic a ia ion in a ou -gene a ion amily wi h ani idia ca ying a no el
PAX6 mu a ion. J Oph halmol 2018:5978293. 2018.
[31] Halde G, Callae s P, Geh ing WJ. New pe spec i es on eye e olu ion. Cu Opin
Gene De 1995;5:602–9.
[32] Chow RL, Al mann CR, Lang RA, Hemma i-B i anlou A. Pax6 induces ec opic
eyes in a e eb a e. De elopmen 1999;126:4213–22.
[33] Wal he C, G uss P. Pax-6, a mu ine pai ed box gene, is exp essed in he
de eloping CNS. De elopmen 1991;113:1435–49.
[34] Sande M, Neubüse A, Kalama as J, Ee HC, Ma in GR, Ge man MS. Gene ic
analysis e eals ha PAX6 is equi ed o no mal ansc ip ion o panc ea ic
ho mone genes and isle de elopmen . Genes De 1997;11:1662–73.
[35] Gosmain Y, Ma hine E, Cheyssac C, Gu´
e a del A, Mamin A, Ka z LS, e al. Pax6
con ols he exp ession o c i ical genes in ol ed in panc ea ic
α
cell
di e en ia ion and unc ion. J Biol Chem 2010;285:33381–93.
[36] C ekl A, Callae s P. PAX6: 25 h anni e sa y and mo e o lea n. Exp Eye Res
2017;156:10–21.
[37] Ding J, Gao Y, Zhao J, Yan H, Guo S-y, Zhang Q-x, e al. Pax6 haploinsu iciency
causes abno mal me abolic homeos asis by down- egula ing glucagon-like
pep ide 1 in mice. Endoc inology 2009;150:2136–44.
[38] Sisodiya SM, F ee SL, Williamson KA, Mi chell TN, Willis C, S e ens JM, e al.
PAX6 haploinsu iciency causes ce eb al mal o ma ion and ol ac o y dys unc ion
in humans. Na Gene 2001;28:214–6.
[39] Be n sson SG, K is o e sson A, Daniilidou M, Dahl N, Eks ¨
om C, Semnic R, e al.
Ani idia wi h PAX6 mu a ions and na colepsy. J Sleep Res 2020;29:e12982.
[40] Shaham O, Menuchin Y, Fa hy C, Ashe y-Padan R. Pax6: a mul i-le el egula o o
ocula de elopmen . P og Re in Eye Res 2012;31:351–76.
[41] Do `
a NJ, Manuel M, Kleinjan D-J, P ice DJ, Collinson JM, Hill RE, e al.
A condi ional Pax6 deple ion s udy wi h no mo phological e ec on he adul
mouse co neal epi helium. BMC Res No es 2018;11:705.
[42] Zube ME, Ges i G, Viczian AS, Ba sacchi G, Ha is WA. Speci ica ion o he
e eb a e eye by a ne wo k o eye ield ansc ip ion ac o s. De elopmen 2003;
130:5155–67.
[43] G indley JC, Da idson DR, Hill RE. The ole o Pax-6 in eye and nasal
de elopmen . De elopmen 1995;121:1433–42.
L. La a e al.
The Ocula Su ace 22 (2021) 245–266
262
[44] Williams AL, Bohnsack BL. Neu al c es de i a i es in ocula de elopmen :
disce ning he eye o he s o m. Bi h De ec s Res C Emb yo Today 2015;105:
87–95.
[45] Jas apu ia-Ag awal S, Lwigale PY. Chap e 10 - neu al c es cells in ocula
de elopmen . In: T aino PA, edi o . Neu al c es cells. Bos on: Academic P ess;
2014. p. 189–203.
[46] Lwigale PY. Chap e ou - co neal de elopmen : di e en cells om a common
p ogeni o . In: Hej mancik JF, Nicke son JM, edi o s. P og ess in molecula
biology and ansla ional science. Academic P ess; 2015. p. 43–59.
[47] C ekl A, Tamm ER. An e io eye de elopmen and ocula mesenchyme: new
insigh s om mouse models and human diseases. Bioessays 2004;26:374–86.
[48] Fuh mann S. Eye mo phogenesis and pa e ning o he op ic esicle. Cu Top De
Biol 2010;93:61–84.
[49] Adle R, Can o-Sole MV. Molecula mechanisms o op ic esicle de elopmen :
complexi ies, ambigui ies and con o e sies. De Biol 2007;305:1–13.
[50] Ca pen e AC, Smi h AN, Wagne H, Cohen-Taya Y, Rao S, Wallace V, e al. Wn
ligands om he emb yonic su ace ec ode m egula e ’bime allic s ip’ op ic cup
mo phogenesis in mouse. De elopmen 2015;142:972–82.
[51] Da is-Silbe man N, Kalich T, O on-Ka ni V, Ma qua d T, K oebe M, Tamm ER,
e al. Gene ic dissec ion o Pax6 dosage equi emen s in he de eloping mouse
eye. Hum Mol Gene 2005;14:2265–76.
[52] S ein eld J, S ein eld I, Co ona o N, Hampel M-L, Laye PG, A aki M, e al. RPE
speci ica ion in he chick is media ed by su ace ec ode m-de i ed BMP and Wn
signalling. De elopmen 2013;140:4959–69.
[53] Collinson JM, Hill RE, Wes JD. Di e en oles o Pax6 in he op ic esicle and
acial epi helium media e ea ly mo phogenesis o he mu ine eye. De elopmen
2000;127:945–56.
[54] C ekl A, Ashe y-Padan R. The cellula and molecula mechanisms o e eb a e
lens de elopmen . De elopmen 2014;141:4432–47.
[55] Ao a S-i, Nakajima N, Sakamo o R, Wa anabe S, Iba aki N, Okazaki K. Pax6
au o egula ion media ed by di ec in e ac ion o Pax6 p o ein wi h he head
su ace ec ode m-speci ic enhance o he mouse Pax6 gene. De Biol 2003;257:
1–13.
[56] Kamachi Y, Uchikawa M, Collignon J, Lo ell-Badge R, Kondoh H. In ol emen o
Sox1, 2 and 3 in he ea ly and subsequen molecula e en s o lens induc ion.
De elopmen 1998;125:2521–32.
[57] Shaham O, Smi h AN, Robinson ML, Take o MM, Lang RA, Ashe y-Padan R. Pax6
is essen ial o lens ibe cell di e en ia ion. De elopmen 2009;136:2567–78.
[58] C ekl A, Zhang X. Signaling and gene egula o y ne wo ks in mammalian lens
de elopmen . T ends Gene 2017;33:677–702.
[59] Gage PJ, Rhoades W, P ucka SK, Hjal T. Fa e maps o neu al c es and mesode m
in he mammalian eye. In es Oph halmol Vis Sci 2005;46:4200–8.
[60] T aino PA, Tam PP. C anial pa axial mesode m and neu al c es cells o he
mouse emb yo: co-dis ibu ion in he c anio acial mesenchyme bu dis inc
seg ega ion in b anchial a ches. De elopmen 1995;121:2569–82.
[61] Yoshida S, Shimmu a S, Nagoshi N, Fukuda K, Ma suzaki Y, Okano H, e al.
Isola ion o mul ipo en neu al c es -de i ed s em cells om he adul mouse
co nea. S em Cell 2006;24:2714–22.
[62] Mu phy C, Al a ado J, Jus e R, Maglio M. P ena al and pos na al cellula i y o
he human co neal endo helium. A quan i a i e his ologic s udy. In es
Oph halmol Vis Sci 1984;25:312–22.
[63] Johns on MC, Noden DM, Hazel on RD, Coulomb e JL, Coulomb e AJ. O igins o
a ian ocula and pe iocula issues. Exp Eye Res 1979;29:27–43.
[64] Noden D. Pe iocula mesenchyme: neu al c es and mesode mal in e ac ions.
Duane’s unda ions o clinical oph halmology 1993;1:1–23.
[65] Kanakubo S, Nomu a T, Yamamu a K-i, Miyazaki J-i, Tamai M, Osumi N.
Abno mal mig a ion and dis ibu ion o neu al c es cells in Pax6 he e ozygous
mu an eye, a model o human eye diseases. Gene Cell 2006;11:919–33.
[66] Akula M, Pa k JW, Wes -Mays JA. Rela ionship be ween neu al c es cell
speci ica ion and a e ocula diseases. J Neu osci Res 2019;97:7–15.
[67] Smi h RS, Zabale a A, Sa ino a OV, John SWM. The mouse an e io chambe
angle and abecula meshwo k de elop wi hou cell dea h. BMC De Biol 2001;
1:3.
[68] Rem´
e C, Lali e d’Epinay S. Pe iods o de elopmen o he no mal human chambe
angle. Doc Oph halmol 1981;51:241–68.
[69] McMenamin PG. A mo phological s udy o he inne su ace o he an e io
chambe angle in p e and pos na al human eyes. Cu Eye Res 1989;8:727–39.
[70] Collin J, Queen R, Ze i D, Bojic S, Do gau B, Moyse N, e al. A single cell a las o
human co nea ha de ines i s de elopmen , limbal p ogeni o cells and hei
in e ac ions wi h he immune cells Ocul Su . 2021.
[71] Da ies SB, Chui J, Madigan MC, P o is JM, Wake ield D, Di Gi olamo N. S em cell
ac i i y in he de eloping human co nea. S em Cell 2009;27:2781–92.
[72] Se el D, Isaacs R. A e-e alua ion o co neal de elopmen . T ans Am Oph halmol
Soc 1988;86:178–207.
[73] Kubilus JK, Zapa e i Mo ales C, Linsenmaye TF. The co neal epi helial ba ie
and i s de elopmen al ole in isola ing co neal epi helial and conjunc i al cells
om one Ano he De elopmen o he co neal-conjunc i al ba ie . In es
Oph halmol Vis Sci 2017;58:1665–72.
[74] Wolosin JM, Schü e M, Zieske JD, Budak MT. Changes in connexin43 in ea ly
ocula su ace de elopmen . Cu Eye Res 2002;24:430–8.
[75] Wolosin JM, Budak MT, Akinci MA. Ocula su ace epi helial and s em cell
de elopmen . In J De Biol 2004;48:981–91.
[76] K use FE, Chen JJ, Tsai RJ, Tseng SC. Conjunc i al ansdi e en ia ion is due o
he incomple e emo al o limbal basal epi helium. In es Oph halmol Vis Sci
1990;31:1903–13.
[77] Wei ZG, Wu RL, La ke RM, Sun TT. In i o g ow h and di e en ia ion o abbi
bulba , o nix, and palpeb al conjunc i al epi helia. Implica ions on conjunc i al
epi helial ansdi e en ia ion and s em cells. In es Oph halmol Vis Sci 1993;34:
1814–28.
[78] Wei ZG, Sun TT, La ke RM. Rabbi conjunc i al and co neal epi helial cells
belong o wo sepa a e lineages. In es Oph halmol Vis Sci 1996;37:523–33.
[79] Da is JA, Reed RR. Role o ol -1 and pax-6 ansc ip ion ac o s in
neu ode elopmen . J Neu osci 1996;16:5082–94.
[80] Ko oma BM, Yang JM, Sundin OH. The Pax-6 homeobox gene is exp essed
h oughou he co neal and conjunc i al epi helia. In es Oph halmol Vis Sci
1997;38:108–20.
[81] Pelleg ini G, Golisano O, Pa e na P, Lambiase A, Bonini S, Rama P, e al. Loca ion
and clonal analysis o s em cells and hei di e en ia ed p ogeny in he human
ocula su ace. J Cell Biol 1999;145:769–82.
[82] Wei ZG, Lin T, Sun TT, La ke RM. Clonal analysis o he in i o di e en ia ion
po en ial o ke a inocy es. In es Oph halmol Vis Sci 1997;38:753–61.
[83] Ma ko CK, Menon BB, Chen G, Whi se JA, Cle e s H, Gipson IK. Spde null mice
lack conjunc i al goble cells and p o ide a model o d y eye. Am J Pa hol 2013;
183:35–48.
[84] Sellheye K, Spi znas M. Ul as uc u al obse a ions on he de elopmen o he
human conjunc i al epi helium. G ae es A ch Clin Exp Oph halmol 1988;226:
489–99.
[85] Miyashi a K, Azuma N, Hida T. Mo phological and his ochemical s udies o goble
cells in de eloping human conjunc i a. Jpn J Oph halmol 1992;36:169–74.
[86] Ríos JD, Fo de K, Diebold Y, Ligh man J, Zieske JD, Da DA. De elopmen o
conjunc i al goble cells and hei neu o ecep o sub ype exp ession. In es
Oph halmol Vis Sci 2000;41:2127–37.
[87] Swamyna han SK. Ocula su ace de elopmen and gene exp ession. Jou nal o
Oph halmology 2013:22. 2013.
[88] Gage PJ, Kuang C, Zacha ias AL. The homeodomain ansc ip ion ac o PITX2 is
equi ed o speci ying co ec cell a es and es ablishing angiogenic p i ilege in
he de eloping co nea. De Dynam 2014;243:1391–400.
[89] Gage PJ, Qian M, Wu D, Rosenbe g KI. The canonical Wn signaling an agonis
DKK2 is an essen ial e ec o o PITX2 unc ion du ing no mal eye de elopmen .
De Biol 2008;317:310–24.
[90] Chung EH, Bukusoglu G, Zieske JD. Localiza ion o co neal epi helial s em cells in
he de eloping a . In es Oph halmol Vis Sci 1992;33:2199–206.
[91] Zieske JD. Co neal de elopmen associa ed wi h eyelid opening. In J De Biol
2004;48:903–11.
[92] Tani uji-Te ai N, Te ai K, Hayashi Y, Chikama T-i, Kao WW-Y. Exp ession o
ke a in 12 and ma u a ion o co neal epi helium du ing de elopmen and
pos na al g ow h. In es Oph halmol Vis Sci 2006;47:545–51.
[93] Liu JJ, Kao WW, Wilson SE. Co neal epi helium-speci ic mouse ke a in K12
p omo e . Exp Eye Res 1999;68:295–301.
[94] Chaloin-Du au C, Dhouailly D, Sun T-T. Appea ance o he ke a in pai K3/K12
du ing emb yonic and adul co neal epi helial di e en ia ion in he chick and in
he abbi . Cell Di e De 1990;32:97–108.
[95] Liu C-Y, Zhu G, Wes e hausen-la son A, Con e se R, Candace WCK, Sun T-T, e al.
Co nea-speci ic exp ession o K12 ke a in du ing mouse de elopmen . Cu Eye
Res 1993;12:963–74.
[96] Ku pakus MA, Maniaci MT, Esco M. Exp ession o ke a ins K12, K4 and K14
du ing de elopmen o ocula su ace epi helium. Cu Eye Res 1994;13:805–14.
[97] Chen Z, de Pai a CS, Luo L, K e ze FL, P lug elde SC, Li D-Q. Cha ac e iza ion o
pu a i e s em cell pheno ype in human limbal epi helia. S em Cell 2004;22:
355–66.
[98] Pajoohesh-Ganji A, Ghosh SP, S epp MA. Regional dis ibu ion o
α
9β1 in eg in
wi hin he limbus o he mouse ocula su ace. De Dynam 2004;230:518–28.
[99] Kalha S, Sh es ha B, Sanz Na a o M, Jones KB, Klein OD, Michon F. Bmi1+
p ogeni o cell dynamics in mu ine co nea du ing homeos asis and wound
healing. S em Cell 2018;36:562–73.
[100] Eche a ia TJ, Di Gi olamo N. Tissue- egene a ing, ision- es o ing co neal
epi helial s em cells. S em Cell Re iews and Repo s 2011;7:256–68.
[101] Collinson JM, Mo is L, Reid AI, Ramaesh T, Keigh en MA, Flockha JH, e al.
Clonal analysis o pa e ns o g ow h, s em cell ac i i y, and cell mo emen
du ing he de elopmen and main enance o he mu ine co neal epi helium. De
Dynam 2002;224:432–40.
[102] Dou a as P, Webb S, Whi ake DA, Do `
a N, Hill RE, Do in JR, e al. Ra e co neal
clones in mice sugges an age- ela ed dec ease o s em cell ac i i y and suppo
he limbal epi helial s em cell hypo hesis. S em Cell Res 2012;8:109–19.
[103] Ami ai-Lange A, Al shule A, Bubley J, Dbaya N, Tiosano B, Shalom-Feue s ein R.
Lineage acing o s em and p ogeni o cells o he mu ine co neal epi helium.
S em Cell 2015;33:230–9.
[104] Di Gi olamo N, Bobba S, Ra i aj V, Delic NC, Slape o a I, Nico ich PR, e al.
T acing he a e o limbal epi helial p ogeni o cells in he mu ine co nea. S em
Cell 2015;33:157–69.
[105] Richa dson A, Lobo EP, Delic NC, Mye scough MR, Lyons JG, Wake ield D, e al.
Ke a in-14-posi i e p ecu so cells spawn a popula ion o mig a o y co neal
epi helia ha main ain issue mass h oughou li e. S em cell epo s 2017;9:
1081–96.
[106] Nasse W, Ami ai-Lange A, So e iou D, Hanna R, Tiosano B, Fuchs Y, e al.
Co neal-commi ed cells es o e he s em cell pool and issue bounda y ollowing
inju y. Cell Rep 2018;22:323–31.
[107] Al shule A, Ami ai-Lange A, Ta azi N, Dey S, S inko sky L, Hadad-Po a S, e al.
Disc e e limbal epi helial s em cell popula ions media e co neal homeos asis and
wound healing. Cell S em Cell 2021;28:1248–61. e8.
L. La a e al.
The Ocula Su ace 22 (2021) 245–266
263
[108] Bha acha ya S, Mukhe jee A, Pisano S, Al shule A, Nasse W, Dey S, e al. So
limbal niche main ains s em cell compa men aliza ion and unc ion h ough
YAP. bioRxi 2021:2021. 05.25.445490.
[109] McKenna CC, Lwigale PY. Inne a ion o he mouse co nea du ing de elopmen .
In es Oph halmol Vis Sci 2011;52:30–5.
[110] Lwigale PY. Emb yonic o igin o a ian co neal senso y ne es. De Biol 2001;239:
323–37.
[111] Reneke LW, Sil e sides DW, Xu L, O e beek PA. Fo ma ion o co neal
endo helium is essen ial o an e io segmen de elopmen - a ansgenic mouse
model o an e io segmen dysgenesis. De elopmen 2000;127:533–42.
[112] Fujimu a N. WNT/β-Ca enin signaling in e eb a e eye de elopmen . F on ie s in
Cell and De elopmen al Biology 2016;4.
[113] Kawakami A, Kimu a-Kawakami M, Nomu a T, Fujisawa H. Dis ibu ions o PAX6
and PAX7 p o eins sugges hei in ol emen in bo h ea ly and la e phases o
chick b ain de elopmen . Mech De 1997;66:119–30.
[114] Nishina S, Kohsaka S, Yamaguchi Y, Handa H, Kawakami A, Fujisawa H, e al.
PAX6 exp ession in he de eloping human eye. B J Oph halmol 1999;83:723–7.
[115] Pei YF, Rhodin JAG. The p ena al de elopmen o he mouse eye. Ana Rec 1970;
168:105–25.
[116] Wulle KG, Le che W. Elec on mic oscopic obse a ions o he ea ly de elopmen
o he human co neal endo helium and desceme ’s memb ane. Oph halmologica
1969;157:451–61.
[117] Feneck EM, Lewis PN, Meek KM. Iden i ica ion o a p ima y s oma and no el
endo helial cell p ojec ions in he de eloping human co nea. In es Oph halmol
Vis Sci 2020;61:5.
[118] O’Rahilly R. The p ena al de elopmen o he human eye. Exp Eye Res 1975;21:
93–112.
[119] Peces-Pe˜
na MD, de la Cuad a-Blanco C, Vicen e A, M´
e ida-Velasco JR.
De elopmen o he cilia y body: mo phological changes in he dis al po ion o
he op ic cup in he human. Cells Tissues O gans 2013;198:149–59.
[120] Ba d JBL, Hay ED, Melle SM. Fo ma ion o he endo helium o he a ian co nea:
a s udy o cell mo emen in i o. De Biol 1975;42:334–61.
[121] Rena d G, Hi sch M, Sa oldelli M, Pouliquen Y. The de elopmen o he i ido-
co neal angle in he chick emb yo. Alb ech Von G ae es A ch Klin Exp
Oph halmol 1980;212:135–42.
[122] Tsai PS, E ans JE, G een KM, Sulli an RM, Schaumbe g DA, Richa ds SM, e al.
P o eomic analysis o human meibomian gland sec e ions. B J Oph halmol 2006;
90:372–7.
[123] Kuma S, Dues e G. Re inoic acid signaling in pe iop ic mesenchyme ep esses
Wn signaling ia induc ion o Pi x2 and Dkk2. De Biol 2010;340:67–74.
[124] Enw igh JF, G ainge RM. Al e ed e inoid signaling in he heads o small eye
mouse emb yos. De Biol 2000;221:10–22.
[125] La a L, Ludwig N, K ammes L, S achon T, F ies FN, Mukwaya A, e al. Abno mal
neo ascula and p oli e a i e conjunc i al pheno ype in limbal s em cell
de iciency is associa ed wi h al e ed mic oRNA and gene exp ession modula ed
by PAX6 mu a ional s a us in congeni al ani idia. Ocul Su 2020.
[126] Rabiee B, Anwa KN, Shen X, Pu a I, Liu M, Jung R, e al. Gene dosage
manipula ion alle ia es mani es a ions o he edi a y <em>PAX6</em>
haploinsu iciency in mice. Sci T ansl Med 2020;12:eaaz4894.
[127] Majo F, Rocha A, Nicolas M, Jaoude GA, Ba andon Y. Oligopo en s em cells a e
dis ibu ed h oughou he mammalian ocula su ace. Na u e 2008;456:250–4.
[128] Pelleg ini G, Dellamb a E, Golisano O, Ma inelli E, Fan ozzi I, Bondanza S, e al.
p63 iden i ies ke a inocy e s em cells. P oc Na l Acad Sci U S A 2001;98:3156–61.
[129] Shalom-Feue s ein R, Lena AM, Zhou H, De La Fo es Di onne S, Van
Bokho en H, Candi E, e al. ΔNp63 is an ec ode mal ga ekeepe o epide mal
mo phogenesis. Cell Dea h Di e 2011;18:887–96.
[130] Li G, Xu F, Zhu J, K awczyk M, Zhang Y, Yuan J, e al. T ansc ip ion Fac o PAX6
(Pai ed Box 6) con ols limbal s em cell lineage in de elopmen and disease.
J Biol Chem 2015;290:20448–54.
[131] Bha acha ya S, Se o L, Ni E, Dhi aj D, Al shule A, Kh eish M, e al. SOX2
egula es P63 and s em/p ogeni o cell s a e in he co neal epi helium. S em Cell
2019;37:417–29.
[132] Ga g A, Zhang X. Lac imal gland de elopmen : om signaling in e ac ions o
egene a i e medicine. De Dynam 2017;246:970–80.
[133] Ga za-Leon M, Le´
on-Cach´
on RBR, Villa ue e-de la C uz R, Ma ínez-T e i˜
no DA.
In a ed meibog aphy and molecula assessmen o p63 gene mu a ions in a
Mexican pa ien wi h EEC synd ome. A ch Soc Esp O almol 2018;93:562–6.
[134] Di Io io E, Kaye SB, Ponzin D, Ba ba o V, Fe a i S, B¨
ohm E, e al. Limbal s em cell
de iciency and ocula pheno ype in ec odac yly-ec ode mal dysplasia-cle ing
synd ome caused by p63 mu a ions. Oph halmology 2012;119:74–83.
[135] Di Io io E, Ba ba o V, Ruzza A, Ponzin D, Pelleg ini G, De Luca M. Iso o ms o
Del aNp63 and he mig a ion o ocula limbal cells in human co neal
egene a ion. P oc Na l Acad Sci U S A 2005;102:9523–8.
[136] Guo ZH, Zhang W, Jia YYS, Liu QX, Li ZF, Lin JS. An insigh in o he di icul ies in
he disco e y o speci ic bioma ke s o limbal s em cells. In J Mol Sci 2018;19:
1982.
[137] Okada SL, Ellswo h JL, Du nam DM, Haugen HS, Holloway JL, Kelley ML, e al.
A glycop o ein ho mone exp essed in co ico ophs exhibi s unique binding
p ope ies on hy oid-s imula ing ho mone ecep o . Mol Endoc inol 2006;20:
414–25.
[138] Dou S, Wang Q, Qi X, Zhang B, Jiang H, Chen S, e al. Molecula iden i y o
human limbal he e ogenei y in ol ed in co neal homeos asis and p i ilege. Ocul
Su 2021;21:206–20.
[139] Collinson JM, Chanas SA, Hill RE, Wes JD. Co neal de elopmen , limbal s em cell
unc ion, and co neal epi helial cell mig a ion in he Pax6 +/−mouse. In es
Oph halmol Vis Sci 2004;45:1101–8.
[140] Beebe DC, Coa s JM. The lens o ganizes he an e io segmen : speci ica ion o
neu al c es cell di e en ia ion in he a ian eye. De Biol 2000;220:424–31.
[141] Collomb E, Yang Y, Fo iel S, Cadau S, Pea on DJ, Dhouailly D. The co neal
epi helium and lens de elop independen ly om a common pool o p ecu so s.
De Dynam 2013;242:401–13.
[142] Smi h JN, Walke HM, Thompson H, Collinson JM, Va gesson N, E skine L. Lens-
egula ed e inoic acid signalling con ols expansion o he de eloping eye.
De elopmen 2018;145:de 167171.
[143] Yamamo o Y, Je e y WR. Cen al ole o he lens in ca e ish eye degene a ion.
Science 2000;289:631–3.
[144] Collinson JM, Quinn JC, Buchanan MA, Kau man MH, Wedden SE, Wes JD, e al.
P ima y de ec s in he lens unde lie complex an e io segmen abno mali ies o
he <em>Pax6</em>he e ozygous eye. P oc Na l Acad Sci Uni S a es Am
2001;98:9688–93.
[145] K oebe M, Da is N, Holzmann S, K i zenbe ge M, Shelah-Go aly M, O i R, e al.
Reduced exp ession o Pax6 in lens and co nea o mu an mice leads o ailu e o
chambe angle de elopmen and ju enile glaucoma. Hum Mol Gene 2010;19:
3332–42.
[146] Klimo a L, Kozmik Z. S age-dependen equi emen o neu o e inal Pax6 o lens
and e ina de elopmen . De elopmen 2014;141:1292–302.
[147] Shiple D, Finklea B, Laude dale JD, Ne land PA. Ke a opa hy, ca a ac , and d y
eye in a su ey o ani idia subjec s. Clin Oph halmol 2015;9:291–5.
[148] Schneide S, Oshe RH, Bu k SE, Lu z TB, Mon ione R. Thinning o he an e io
capsule associa ed wi h congeni al ani idia. J Ca a ac Re ac Su g 2003;29:
523–5.
[149] Ma inez G, de Iongh RU. The lens epi helium in ocula heal h and disease. In J
Biochem Cell Biol 2010;42:1945–63.
[150] Ed´
en U, Lagali N, Dellby A, U heim TP, Riise R, Chen X, e al. Ca a ac
de elopmen in No wegian pa ien s wi h congeni al ani idia. Ac a Oph halmol
2014;92:e165–7.
[151] Wol M, Zabel B, Lo enz B, Blankenagel A, Gho bani MB, Schwenn O, e al.
[Molecula gene ic s udy o he PAX6 gene in ani idia pa ien s]. Oph halmologe
1998;95:828–30.
[152] To nq is K. Ani idia: sigh - h ea ening and ha d o cu e. Ac a Oph halmol 2008;
86:704–5.
[153] Ou J, Lowes C, Collinson JM. Cy oskele al and cell adhesion de ec s in wounded
and Pax6+/- co neal epi helia. In es Oph halmol Vis Sci 2010;51:1415–23.
[154] Ou J, Walczysko P, Kuce o a R, Rajnicek AM, McCaig CD, Zhao M, e al. Ch onic
wound s a e exace ba ed by oxida i e s ess in Pax6+/- ani idia- ela ed
ke a opa hy. J Pa hol 2008;215:421–30.
[155] Da is N, Yo e C, Ra i S, An es R, Be ge J, Holzmann S, e al. Pax6 dosage
equi emen s in i is and cilia y body di e en ia ion. De Biol 2009;333:132–42.
[156] Wang X, Shan X, G ego y-E ans CY. A mouse model o ani idia e eals he in i o
downs eam a ge s o Pax6 d i ing i is and cilia y body de elopmen in he eye.
Biochim Biophys Ac a (BBA) - Mol Basis Dis 2017;1863:60–7.
[157] I ne LM, Wu dak H, Schwe d ege K, Kunz T, Ille F, Le een P, e al. Compound
de elopmen al eye diso de s ollowing inac i a ion o TGFβ signaling in neu al-
c es s em cells. J Biol 2005;4:11.
[158] Nelson LB, Spae h GL, Nowinski TS, Ma go CE, Jackson L. Ani idia. A e iew.
Su ey o Oph halmology. 1984;28:621–42.
[159] B ´
emond-Gignac D. Le glaucome dans l’ani idie. J F Oph almol 2007;30:196–9.
[160] B and JD, Casuso LA, Budenz DL. Ma kedly inc eased cen al co neal hickness:
an un ecognized inding in congeni al ani idia. Am J Oph halmol 2004;137:
348–50.
[161] Whi son JT, Liang C, God ey DG, Pe oll WM, Ca anagh HD, Pa el D, e al.
Cen al co neal hickness in pa ien s wi h congeni al ani idia. Eye Con ac Lens
2005;31:221–4.
[162] Ki wan C, O’Kee e M, Fi zsimon S. Cen al co neal hickness and co neal diame e
in p ema u e in an s. Ac a Oph halmol Scand 2005;83:751–3.
[163] Baulmann DC, Ohlmann A, Flügel-Koch C, Goswami S, C ekl A, Tamm ER. Pax6
he e ozygous eyes show de ec s in chambe angle di e en ia ion ha a e
associa ed wi h a wide spec um o o he an e io eye segmen abno mali ies.
Mech De 2002;118:3–17.
[164] Collinson JM, Quinn JC, Hill RE, Wes JD. The oles o Pax6 in he co nea, e ina,
and ol ac o y epi helium o he de eloping mouse emb yo. De Biol 2003;255:
303–12.
[165] Funde bu gh ML, Du Y, Mann MM, Sunda Raj N, Funde bu gh JL. PAX6
exp ession iden i ies p ogeni o cells o co neal ke a ocy es. Faseb J 2005;19:
1371–3.
[166] C ekl A, Tamm ER. An e io eye de elopmen and ocula mesenchyme: new
insigh s om mouse models and human diseases. Bioessays : news and e iews in
molecula , cellula and de elopmen al biology 2004;26:374–86.
[167] Kamachi Y, Uchikawa M, Tanouchi A, Sekido R, Kondoh H. Pax6 and SOX2 o m a
co-DNA-binding pa ne complex ha egula es ini ia ion o lens de elopmen .
Genes De 2001;15:1272–86.
[168] Coope ST, Hanson IM. A sc een o p o eins ha in e ac wi h PAX6: C- e minal
mu a ions dis up in e ac ion wi h HOMER3, DNCL1 and TRIM11. BMC Gene
2005;6:43.
[169] Chauhan BK, Reed NA, Zhang W, Duncan MK, Kilimann MW, C ekl A.
Iden i ica ion o genes downs eam o Pax6 in he mouse lens using cDNA
mic oa ays. J Biol Chem 2002;277:11539–48.
[170] Ma suo T, Osumi-Yamashi a N, Noji S, Ohuchi H, Koyama E, Myokai F, e al.
A mu a ion in he Pax-6 gene in a small eye is associa ed wi h impai ed
mig a ion o midb ain c es cells. Na Gene 1993;3:299–304.
[171] A agona P, Rolando M. Towa ds a dynamic cus omised he apy o ocula su ace
dys unc ions. B J Oph halmol 2013;97:955.
L. La a e al.
The Ocula Su ace 22 (2021) 245–266
264
[172] Hogan BLM, Ho sbu gh G, Cohen J, He he ing on CM, Fishe G, Lyon MF.
em>Small eyes</em>(<em>Sey</em>): a homozygous le hal mu a ion on
ch omosome 2 which a ec s he di e en ia ion o bo h lens and nasal placodes in
he mouse. J Emb yol Exp Mo phol 1986;97:95–110.
[173] Maka enko a HP, I o M, Go inda ajan V, Fabe SC, Sun L, McMahon G, e al.
FGF10 is an induce and Pax6 a compe ence ac o o lac imal gland
de elopmen . De elopmen 2000;127:2563–72.
[174] Bannie -H´
elaou¨
e M, Pos Y, Ko ing J, T ani Bus os M, Geha H, Beg hel H, e al.
Explo ing he human lac imal gland using o ganoids and single-cell sequencing.
Cell S em Cell 2021.
[175] Hi ayama M, Ko SBH, Kawaki a T, Akiyama T, Gopa aju SK, Soma A, e al.
Iden i ica ion o ansc ip ion ac o s ha p omo e he di e en ia ion o human
plu ipo en s em cells in o lac imal gland epi helium-like cells. npj Aging and
Mechanisms o Disease 2017;3:1.
[176] Wang J, Call M, Mongan M, Kao WW-Y, Xia Y. Meibomian gland mo phogenesis
equi es de elopmen al eyelid closu e and lid usion. Ocul Su 2017;15:704–12.
[177] Shi F, Fan Y, Zhang L, Meng L, Zhi H, Hu H, e al. The exp ession o Pax6 a ian s
is subjec o pos ansc ip ional egula ion in he de eloping mouse eyelid. PloS
One 2013;8:e53919.
[178] Call M, Fischesse K, Lunn MO, Kao WW. A unique lineage gi es ise o he
meibomian gland. Mol Vis 2016;22:168–76.
[179] Landsend ECS, U heim ØA, Pede sen HR, Aass HCD, Lagali N, Da DA, e al. The
le el o in lamma o y ea cy okines is ele a ed in congeni al ani idia and
associa ed wi h meibomian gland dys unc ion. In es Oph halmol Vis Sci 2018;
59:2197–204.
[180] Jas aneiah S, Al-Rajhi AA. Associa ion o ani idia and d y eyes. Oph halmology
2005;112:1535–40.
[181] Landsend ECS, Pede sen HR, U heim ØA, Xiao J, Adil MY, Tashbaye B, e al.
Meibomian gland dys unc ion and ke a opa hy a e associa ed wi h d y eye
disease in ani idia. B J Oph halmol 2019;103:119–24.
[182] Ihna ko R, Ed´
en U, Lagali N, Dellby A, Fage holm P. Analysis o p o ein
composi ion and p o ein exp ession in he ea luid o pa ien s wi h congeni al
ani idia. Jou nal o P o eomics 2013;94:78–88.
[183] Landsend ECS, U heim OA, Pede sen HR, Aass HCD, Lagali N, Da DA, e al. The
le el o in lamma o y ea cy okines is ele a ed in congeni al ani idia and
associa ed wi h meibomian gland dys unc ion. In es Oph halmol Vis Sci 2018;
59:2197–204.
[184] Bonna E, Logan P, Eus ace P. Absen meibomian glands: a ma ke o EEC
synd ome. Eye 1996;10:355–61.
[185] Townsend WM. The limbal palisades o Vog . T ans Am Oph halmol Soc 1991;89:
721–56.
[186] Sho AJ, Secke GA, Mun o PM, Khaw PT, Tu SJ, Daniels JT. Cha ac e iza ion
o he limbal epi helial s em cell niche: no el imaging echniques pe mi in i o
obse a ion and a ge ed biopsy o limbal epi helial s em cells. S em Cell 2007;
25:1402–9.
[187] Li W, Hayashida Y, Chen Y-T, Tseng SCG. Niche egula ion o co neal epi helial
s em cells a he limbus. Cell Res 2007;17:26–36.
[188] Shanmugana han VA, Fos e T, Kulka ni BB, Hopkinson A, G ay T, Powe DG,
e al. Mo phological cha ac e is ics o he limbal epi helial c yp . B J Oph halmol
2007;91:514–9.
[189] Co sa elis G, Cheng SZ, Dong G, Sun TT, La ke RM. Exis ence o slow-cycling
limbal epi helial basal cells ha can be p e e en ially s imula ed o p oli e a e:
implica ions on epi helial s em cells. Cell 1989;57:201–9.
[190] Sun TT, Tseng SC, La ke RM. Loca ion o co neal epi helial s em cells. Na u e
2010;463:E10–1. discussion E1.
[191] Daniels JT, Da JKG, Tu SJ, Khaw PT. Co neal s em cells in e iew. Wound
Repai Regen 2001;9:483–94.
[192] Fa elly O, Suzuki-Ho iuchi Y, B ews e M, Ku i P, Huang S, Rice G, e al. Two-
pho on li e imaging o single co neal s em cells e eals compa men alized
o ganiza ion o he limbal niche. Cell S em Cell 2021;28:1233–47. e4.
[193] Tseng SC. Regula ion and clinical implica ions o co neal epi helial s em cells.
Mol Biol Rep 1996;23:47–58.
[194] Dua HS, Azua a-Blanco A. Limbal s em cells o he co neal epi helium. Su
Oph halmol 2000;44:415–25.
[195] Puangs icha e n V, Tseng SC. Cy ologic e idence o co neal diseases wi h limbal
s em cell de iciency. Oph halmology 1995;102:1476–85.
[196] Li W, Chen YT, Hayashida Y, Blanco G, Khei kah A, He H, e al. Down- egula ion
o Pax6 is associa ed wi h abno mal di e en ia ion o co neal epi helial cells in
se e e ocula su ace diseases. J Pa hol 2008;214:114–22.
[197] Dziasko MA, Daniels JT. Ana omical ea u es and cell-cell in e ac ions in he
human limbal epi helial s em cell niche. Ocul Su 2016;14:322–30.
[198] Dua HS, Mi i A, Aloma T, Yeung AM, Said DG. The ole o limbal s em cells in
co neal epi helial main enance: es ing he dogma. Oph halmology 2009;116:
856–63.
[199] Huang AJ, Tseng SC. Co neal epi helial wound healing in he absence o limbal
epi helium. In es Oph halmol Vis Sci 1991;32:96–105.
[200] Blanpain C, Fuchs E. S em cell plas ici y. Plas ici y o epi helial s em cells in issue
egene a ion. Science 2014;344:1242281.
[201] Rama P, Ma uska S, Paganoni G, Spinelli A, De Luca M, Pelleg ini G. Limbal s em-
cell he apy and long- e m co neal egene a ion. N Engl J Med 2010;363:147–55.
[202] Kolli S, Ahmad S, Lako M, Figuei edo F. Success ul clinical implemen a ion o
co neal epi helial s em cell he apy o ea men o unila e al limbal s em cell
de iciency. S em Cell 2010;28:597–610.
[203] Ouyang H, Xue Y, Lin Y, Zhang X, Xi L, Pa el S, e al. WNT7A and PAX6 de ine
co neal epi helium homeos asis and pa hogenesis. Na u e 2014;511:358–61.
[204] Le Q, Deng SX, Xu J. In i o con ocal mic oscopy o congeni al ani idia-associa ed
ke a opa hy. Eye 2013;27:763–6.
[205] Schl¨
o ze -Sch eha d U, La a L, Gießl A, Zenkel M, F ies FN, K¨
asmann-Kellne B,
e al. Dys unc ion o he limbal epi helial s em cell niche in ani idia-associa ed
ke a opa hy. Ocul Su 2021;21:160–73.
[206] Vosk esenskaya A, Pozdeye a N, Vasilye a T, Ba ko Y, Shipuno A, Gagloe B,
e al. Clinical and mo phological mani es a ions o ani idia-associa ed
ke a opa hy on an e io segmen op ical cohe ence omog aphy and in i o
con ocal mic oscopy. Ocul Su 2017;15:759–69.
[207] Lagali N, Ed´
en U, U heim TP, Chen X, Riise R, Dellby A, e al. In i o mo phology
o he limbal palisades o og co ela es wi h p og essi e s em cell de iciency in
ani idia- ela ed ke a opa hy. In es Oph halmol Vis Sci 2013;54:5333–42.
[208] Chen YT, Chen FY, Vijmasi T, S ephens DN, Gallup M, McNama a NA. Pax6
down egula ion media es abno mal lineage commi men o he ocula su ace
epi helium in aqueous-de icien d y eye disease. PloS One 2013;8:e77286.
[209] McNama a NA, Gallup M, Po co TC. Es ablishing PAX6 as a bioma ke o de ec
ea ly loss o ocula pheno ype in human pa ien s wi h Sjog en’s synd ome. In es
Oph halmol Vis Sci 2014;55:7079–84.
[210] Ljubimo AV, Saghizadeh M. P og ess in co neal wound healing. P og Re in Eye
Res 2015;49:17–45.
[211] Swamyna han SK, Ka z JP, Kaes ne KH, Ashe y-Padan R, C aw o d MA,
Pia igo sky J. Condi ional dele ion o he mouse Kl 4 gene esul s in co neal
epi helial agili y, s omal edema, and loss o conjunc i al goble cells. Mol Cell
Biol 2007;27:182–94.
[212] Tiwa i A, Loughne CL, Swamyna han S, Swamyna han SK. KLF4 plays an
essen ial ole in co neal epi helial homeos asis by p omo ing epi helial cell a e
and supp essing epi helial-mesenchymal ansi ion. In es Oph halmol Vis Sci
2017;58:2785–95.
[213] Reis LM, Tyle RC, Volkmann Kloss BA, Schil e KF, Le in AV, Low y RB, e al.
PITX2 and FOXC1 spec um o mu a ions in ocula synd omes. Eu J Hum Gene
2012;20:1224–33.
[214] Saman M, Chauhan BK, La h op KL, Nischal KK. Congeni al ani idia: e iology,
mani es a ions and managemen . Expe Re Oph halmol 2016;11:135–44.
[215] Leipe LJ, Walczysko P, Kuce o a R, Ou J, Shanley LJ, Lawson D, e al. The oles
o calcium signaling and ERK1/2 phospho yla ion in a Pax6+/- mouse model o
epi helial wound-healing delay. BMC Biol 2006;4:27.
[216] Ha mon RM, Simpson CL, Johnson JL, Koe sie JL, Dubash AD, Najo NA, e al.
Desmoglein-1/E bin in e ac ion supp esses ERK ac i a ion o suppo epide mal
di e en ia ion. J Clin In es 2013;123:1556–70.
[217] Pajoohesh-Ganji A, Pal-Ghosh S, Tad alka G, S epp MA. Co neal goble cells and
hei niche: implica ions o co neal s em cell de iciency. S em Cell 2012;30:
2032–43.
[218] Ramos T, Sco D, Ahmad S. An upda e on ocula su ace epi helial s em cells:
co nea and conjunc i a. S em Cell In 2015;2015:601731.
[219] Be olin M, B eda C, Fe a i S, Van Acke SI, Zaka ia N, Di Io io E, e al.
Op imized p o ocol o egene a ion o he conjunc i al epi helium using he cell
suspension echnique. Co nea 2019;38:469–79.
[220] Roux Isabelle P, Romain D, Jean-Paul C, Jieqiong Q, Huiqing Z, e al. Modeling o
ani idia- ela ed ke a opa hy by CRISPR/Cas9 genome edi ing o human limbal
epi helial cells and escue by ecombinan PAX6 p o ein. S em Cell 2018.
[221] Dou a as P, Mo RL, Edwa ds D, Ramaesh K, Dhillon B, Mo ley SD, e al.
Inc eased co neal epi helial u no e con ibu es o abno mal homeos asis in he
Pax6(+/-) mouse model o ani idia. PloS One 2013;8:e71117.
[222] Ba ba o V, Nas i AA, Del Vecchio C, Fe a i S, Miglio a i A, Ra a P, e al.
Co ec ion o mu an p63 in EEC synd ome using siRNA media ed allele-speci ic
silencing es o es de ec i e s em cell unc ion. S em Cell 2016;34:1588–600.
[223] Walczysko P, Rajnicek AM, Collinson JM. Con ac -media ed con ol o adial
mig a ion o co neal epi helial cells. Mol Vis 2016;22:990–1004.
[224] Aza DT. Co neal angiogenic p i ilege: angiogenic and an iangiogenic ac o s in
co neal a ascula i y, asculogenesis, and wound healing (an Ame ican
Oph halmological Socie y hesis). T ans Am Oph halmol Soc 2006;104:264–302.
[225] Amba i BK, Nozaki M, Singh N, Takeda A, Jani PD, Su ha T, e al. Co neal
a ascula i y is due o soluble VEGF ecep o -1. Na u e 2006;443:993–7.
[226] Shalom-Feue s ein R, Se o L, De La Fo es Di onne S, Pe i I, Abe dam E,
Cama go L, e al. Plu ipo en s em cell model e eals essen ial oles o miR-450b-
5p and miR-184 in emb yonic co neal lineage speci ica ion. S em Cell 2012;30:
898–909.
[227] Ryan DG, Oli ei a-Fe nandes M, La ke RM. Mic oRNAs o he mammalian eye
display dis inc and o e lapping issue speci ici y. Mol Vis 2006;12:1175–84.
[228] Pa k JK, Peng H, Yang W, Ka snelson J, Volpe O, La ke RM. miR-184 exhibi s
angios a ic p ope ies ia egula ion o Ak and VEGF signaling pa hways. Faseb J
2017;31:256–65.
[229] Gao J, Wang Y, Zhao X, Chen P, Xie L. Mic oRNA-204-5p-Media ed egula ion o
SIRT1 con ibu es o he delay o epi helial cell cycle a e sal in diabe ic co neas.
In es Oph halmol Vis Sci 2015;56:1493–504.
[230] Lu Y, Tai PWL, Ai J, Gessle DJ, Su Q, Yao X, e al. T ansc ip ome p o iling o
neo ascula ized co neas e eals miR-204 as a mul i- a ge bio he apy deli e able
by AAVs. Mol The Nucleic Acids 2018;10:349–60.
[231] Shiels A. TRPM3_miR-204: a complex locus o eye de elopmen and disease.
Hum Genom 2020;14:7.
[232] Shaham O, Gue a K, Mo E, O en-Giladi P, G inbe g D, Xie Q, e al. Pax6 egula es
gene exp ession in he e eb a e lens h ough miR-204. PLoS Gene 2013;9:
e1003357.
[233] Xie Q, Yang Y, Huang J, Ninko ic J, Walche T, Wol L, e al. Pax6 in e ac ions
wi h ch oma in and iden i ica ion o i s no el di ec a ge genes in lens and
o eb ain. PloS One 2013;8:e54507.
L. La a e al.
The Ocula Su ace 22 (2021) 245–266
265
[234] Xie Q, Ung D, Kha izo K, Fise A, C ekl A. Gene egula ion by PAX6: s uc u al-
unc ional co ela ions o missense mu an s and ansc ip ional con ol o T pm3/
miR-204. Mol Vis 2014;20:270–82.
[235] Con e I, Ca ella S, A ellino R, Ka ali M, Ma co-Fe e es R, Bo olen a P, e al.
miR-204 is equi ed o lens and e inal de elopmen ia <em>Meis2</em>
a ge ing. P oc Na l Acad Sci Uni S a es Am 2010;107:15491–6.
[236] Mukwaya A, Jensen L, Peebo B, Lagali N. Mic oRNAs in he co nea: ole and
implica ions o ea men o co neal neo ascula iza ion. Ocul Su 2019;17:
400–11.
[237] Lapid-Go zak R, San ana NTY, Nieuwendaal CP, Mou i s MP, an de
Meulen IJE. Topical be acizumab ea men in ani idia. In Oph halmol 2018;38:
1741–6.
[238] Nowak JA, Polak L, Pasolli HA, Fuchs E. Hai ollicle s em cells a e speci ied and
unc ion in ea ly skin mo phogenesis. Cell S em Cell 2008;3:33–43.
[239] Rompolas P, G eco V. S em cell dynamics in he hai ollicle niche. Semin Cell De
Biol 2014;25–26:34–42.
[240] Fujiwa a H, Fe ei a M, Dona i G, Ma ciano DK, Lin on JM, Sa o Y, e al. The
basemen memb ane o hai ollicle s em cells is a muscle cell niche. Cell 2011;
144:577–89.
[241] Pinnamaneni N, Funde bu gh JL. Concise e iew: s em cells in he co neal
s oma. S em Cell 2012;30:1059–63.
[242] Pea on DJ, Yang Y, Dhouailly D. T ansdi e en ia ion o co neal epi helium in o
epide mis occu s by means o a mul is ep p ocess igge ed by de mal
de elopmen al signals. P oc Na l Acad Sci U S A 2005;102:3714–9.
[243] Xie H-T, Chen S-Y, Li G-G, Tseng SCG. Limbal epi helial s em/p ogeni o cells
a ac s omal niche cells by SDF-1/CXCR4 signaling o p e en di e en ia ion.
S em Cell 2011;29:1874–85.
[244] Gu -Cohen S, Yang H, Baksh SC, Miao Y, Le o se J, Ka a u RP, e al. S em
cell–d i en lympha ic emodeling coo dina es issue egene a ion. Science 2019;
366:1218–25.
[245] Kolega J, Manabe M, Sun T-T. Basemen memb ane he e ogenei y and a ia ion
in co neal epi helial di e en ia ion. Di e en ia ion 1989;42:54–63.
[246] Ljubimo AV, Bu geson RE, Bu kowski RJ, Michael AF, Sun TT, Kenney MC.
Human co neal basemen memb ane he e ogenei y: opog aphical di e ences in
he exp ession o ype IV collagen and laminin iso o ms. Lab In es 1995;72:
461–73.
[247] Polise i N, Zenkel M, Menzel-Se e ing J, K use FE, Schlo ze -Sch eha d U. Cell
adhesion molecules and s em cell-niche-in e ac ions in he limbal s em cell niche.
S em Cell 2016;34:203–19.
[248] Higa K, Ka o N, Yoshida S, Ogawa Y, Shimazaki J, Tsubo a K, e al. Aquapo in 1-
posi i e s omal niche-like cells di ec ly in e ac wi h N-cadhe in-posi i e clus e s
in he basal limbal epi helium. S em Cell Res 2013;10:147–55.
[249] Dziasko MA, A me HE, Le is HJ, Sho AJ, Tu S, Daniels JT. Localisa ion o
epi helial cells capable o holoclone o ma ion in i o and di ec in e ac ion wi h
s omal cells in he na i e human limbal c yp . PloS One 2014;9:e94283.
[250] Polise i N, Zenkel M, Menzel-Se e ing J, K use FE, Schl¨
o ze -Sch eha d U. Cell
adhesion molecules and s em cell-niche-in e ac ions in he limbal s em cell niche.
S em Cell 2016;34:203–19.
[251] Higa K, Shimmu a S, Miyashi a H, Ka o N, Ogawa Y, Kawaki a T, e al. N-cadhe in
in he main enance o human co neal limbal epi helial p ogeni o cells in i o.
In es Oph halmol Vis Sci 2009;50:4640–5.
[252] Dziasko MA, Tu SJ, Daniels JT. Limbal melanocy es suppo limbal epi helial
s em cells in 2D and 3D mic oen i onmen s. Exp Eye Res 2015;138:70–9.
[253] Funde bu gh JL, Funde bu gh ML, Du Y. S em cells in he limbal s oma. Ocul
Su 2016;14:113–20.
[254] Ra i S, Bha i K, Rencus-Laza S, Cohen-Taya Y, Schy R, E an al N, e al. PAX6
egula es melanogenesis in he e inal pigmen ed epi helium h ough eed-
o wa d egula o y in e ac ions wi h MITF. PLoS Gene 2014;10. e1004360-e.
[255] Naka su MN, Gonz´
alez S, Mei H, Deng SX. Human limbal mesenchymal cells
suppo he g ow h o human co neal epi helial s em/p ogeni o cells. In es
Oph halmol Vis Sci 2014;55:6953–9.
[256] Gou eia RM, Lepe G, Gup a S, Mohan RR, Pa e son C, Connon CJ. Assessmen o
co neal subs a e biomechanics and i s e ec on epi helial s em cell main enance
and di e en ia ion. Na Commun 2019;10:1496.
[257] Kang YG, Shin JW, Pa k SH, Oh M-J, Pa k HS, Shin J-W, e al. E ec s o low-
induced shea s ess on limbal epi helial s em cell g ow h and en ichmen . PloS
One 2014;9. e93023-e.
[258] Vicen e A, Bys ¨
om B, Linds ¨
om M, S ene i U, Ped osa Domell¨
o F. Ani idia-
ela ed ke a opa hy: s uc u al changes in naï e and ansplan ed co neal bu ons.
PloS One 2018;13:e0198822.
[259] Mo RL, Ben ley AJ, Ma in FL, Collinson JM, Dou a as P, Hill RE, e al. E ec s o
abe an Pax6 gene dosage on mouse co neal pa hophysiology and co neal
epi helial homeos asis. PloS One 2011;6:e28895.
[260] Ramaesh T, Ramaesh K, Leask R, Sp ingbe A, Riley SC, Dhillon B, e al.
Inc eased apop osis and abno mal wound-healing esponses in he he e ozygous
Pax6 +/−mouse co nea. In es Oph halmol Vis Sci 2006;47:1911–7.
[261] Espana EM, Kawaki a T, Romano A, Di Pascuale M, Smiddy R, Liu C-y, e al.
S omal niche con ols he plas ici y o limbal and co neal epi helial
di e en ia ion in a abbi model o ecombined issue. In es Oph halmol Vis Sci
2003;44:5130–5.
[262] Zhang Y, Yeh L-K, Zhang S, Call M, Yuan Y, Yasunaga M, e al. Wn /β-ca enin
signaling modula es co neal epi helium s a i ica ion ia inhibi ion o Bmp4
du ing mouse de elopmen . De elopmen (Camb idge, England) 2015;142:
3383–93.
[263] Vicen e A, Bys ¨
om B, Ped osa Domell¨
o F. Al e ed signaling pa hways in ani idia-
ela ed Ke a opa hySignaling pa hways in ani idia- ela ed ke a opa hy. In es
Oph halmol Vis Sci 2018;59:5531–41.
[264] Bausili MM, Al a ez de Toledo J, Ba aque RI, Michael R, T esse a F, de la
Paz MF. His opa hology indings o co neal bu ons in congeni al ani idia
pa ien s. Oph halmic Res 2016;56:202–6.
[265] Planella S, de la Paz MF, de Toledo JA. Lamella and pene a ing ke a oplas y in
congeni al ani idia. In: Pa ekh M, Poli B, Fe a i S, Teo ili C, Ponzin D, edi o s.
Ani idia: ecen de elopmen s in scien i ic and clinical esea ch. Cham: Sp inge
In e na ional Publishing; 2015. p. 63–74.
[266] P ochian z A, Di Na do AA. Homeop o ein signaling in he de eloping and adul
ne ous sys em. Neu on 2015;85:911–25.
[267] Lesa e B, Jolio A, P ochian z A, Volo i ch M. Di ec non-cell au onomous Pax6
ac i i y egula es eye de elopmen in he zeb a ish. Neu al De 2007;2:2.
[268] Wei ZG, Co sa elis G, Sun TT, La ke RM. Label- e aining cells a e p e e en ially
loca ed in o nical epi helium: implica ions on conjunc i al epi helial
homeos asis. In es Oph halmol Vis Sci 1995;36:236–46.
[269] La a L, No ds ¨
om K, S achon T, Langenbuche A, F ies FN, Szen m´
a y N, e al.
Exp ession o e inoic acid signaling componen s ADH7 and ALDH1A1 is educed
in ani idia limbal epi helial cells and a siRNA p ima y cell based ani idia model.
Exp Eye Res 2019;179:8–17.
[270] Ed´
en U, Fage holm P, Danyali R, Lagali N. Pa hologic epi helial and an e io
co neal ne e mo phology in ea ly-s age congeni al ani idic ke a opa hy.
Oph halmology 2012;119:1803–10.
[271] Tsai RJ, Tseng SC. E ec o s omal in lamma ion on he ou come o limbal
ansplan a ion o co neal su ace econs uc ion. Co nea 1995;14:439–49.
[272] He senbe g AJ, Shojaa i G, Funde bu gh ML, Mann MM, Du Y, Funde bu gh JL.
Co neal s omal s em cells educe co neal sca ing by media ing neu ophil
in il a ion a e wounding. PloS One 2017;12:e0171712.
[273] Shojaa i G, Khandake I, Funde bu gh ML, Mann MM, Basu R, S olz DB, e al.
Mesenchymal s em cells educe co neal ib osis and in lamma ion ia
ex acellula esicle-media ed deli e y o miRNA. S em cells ansla ional
medicine 2019;8:1192–201.
[274] Calonge M, P´
e ez I, Galindo S, Nie o-Miguel T, L´
opez-Paniagua M, Fe n´
andez I,
e al. A p oo -o -concep clinical ial using mesenchymal s em cells o he
ea men o co neal epi helial s em cell de iciency. T ansl Res 2019;206:18–40.
[275] Li Z, Bu ns AR, Rumbau RE, Smi h CW. gamma del a T cells a e necessa y o
pla ele and neu ophil accumula ion in limbal essels and e icien epi helial
epai a e co neal ab asion. Am J Pa hol 2007;171:838–45.
[276] Mas opasqua L, Nubile M, Lanzini M, Ca pine o P, Ciancaglini M, Pannellini T,
e al. Epi helial dend i ic cell dis ibu ion in no mal and in lamed human co nea:
in i o con ocal mic oscopy s udy. Am J Oph halmol 2006;142. 736-44.e2.
[277] Maye WJ, Macke MJ, K anebi e N, Messme EM, G ü e ich M, Kampik A,
e al. Dis ibu ion o an igen p esen ing cells in he human co nea: co ela ion o
in i o con ocal mic oscopy and immunohis ochemis y in di e en pa hologic
en i ies. Cu Eye Res 2012;37:1012–8.
[278] No a a M, Sho AJ, O’Callaghan AR, Daniels JT. The impac o age on he
physical and cellula p ope ies o he human limbal s em cell niche. Age (Do d ).
2013;35:289–300.
[279] Mas opasqua L, Massa o-Gio dano G, Nubile M, Sacche i M. Unde s anding he
pa hogenesis o neu o ophic ke a i is: he ole o co neal ne es. J Cell Physiol
2017;232:717–24.
[280] Mülle LJ, Ma u CF, K use F, Te o TMT. Co neal ne es: s uc u e, con en s
and unc ion. Exp Eye Res 2003;76:521–42.
[281] Shaheen BS, Baki M, Jain S. Co neal ne es in heal h and disease. Su
Oph halmol 2014;59:263–85.
[282] Nishida T, Chikama T-i, Mo ishige N, Yanai R, Yamada N, Sai o J. Pe sis en
epi helial de ec s due o neu o ophic ke a opa hy ea ed wi h a subs ance p-
de i ed pep ide and insulin-like g ow h ac o 1. Jpn J Oph halmol 2007;51:
442–7.
[283] Gao N, Lee P, Yu F-S. In aepi helial dend i ic cells and senso y ne es a e
s uc u ally associa ed and unc ional in e dependen in he co nea. Sci Rep 2016;
6:36414.
[284] Pe y VH, B own MC, Go don S. The mac ophage esponse o cen al and
pe iphe al ne e inju y. A possible ole o mac ophages in egene a ion. J Exp
Med 1987;165:1218–23.
[285] Seyed-Raza i Y, Chinne y HR, McMenamin PG. A no el associa ion be ween
esiden issue mac ophages and ne es in he pe iphe al s oma o he mu ine
co nea. In es Oph halmol Vis Sci 2014;55:1313–20.
[286] Ihna ko R, Eden U, Fage holm P, Lagali N. Congeni al ani idia and he ocula
su ace. Ocul Su 2016;14:196–206.
[287] Law enson JG, Ruskell GL. The s uc u e o co puscula ne e endings in he
limbal conjunc i a o he human eye. J Ana 1991;177:75–84.
[288] Ueno H, Fe a i G, Ha o i T, Saban DR, Ka iki eddy KR, Chauhan SK, e al.
Dependence o co neal s em/p ogeni o cells on ocula su ace inne a ion. In es
Oph halmol Vis Sci 2012;53:867–72.
[289] Higa K, Higuchi J, Kimo o R, Miyashi a H, Shimazaki J, Tsubo a K, e al. Human
co neal limbal o ganoids main aining limbal s em cell niche unc ion. S em Cell
Res 2020;49:102012.
[290] Fe a i G, Haj asouliha AR, Sad ai Z, Ueno H, Chauhan SK, Dana R. Ne es and
neo essels inhibi each o he in he co nea. In es Oph halmol Vis Sci 2013;54:
813–20.
[291] Liu L, Dana R, Yin J. Senso y neu ons di ec ly p omo e angiogenesis in esponse
o in lamma ion ia subs ance P signaling. Faseb J 2020;34:6229–43.
[292] Li Z, Bu ns AR, Han L, Rumbau RE, Smi h CW. IL-17 and VEGF a e necessa y o
e icien co neal ne e egene a ion. Am J Pa hol 2011;178:1106–16.
L. La a e al.

The Ocula Su ace 22 (2021) 245–266
266
[293] Yu CQ, Zhang M, Ma is KI, Kim C, Rosenbla MI. Vascula endo helial g ow h
ac o media es co neal ne e epai . In es Oph halmol Vis Sci 2008;49:3870–8.
[294] Agga wal S, Colon C, Khei khah A, Ham ah P. E icacy o au ologous se um ea s
o ea men o neu opa hic co neal pain. Ocul Su 2019;17:532–9.
[295] Yoon CH, Lee HJ, Pa k HY, Kim H, Kim MK, Jeoung JW, e al. E ec s o opical
au ologous se um on he ocula su ace in pa ien s wi h oxic co neal
epi heliopa hy induced by an i-glaucoma d ugs. In Oph halmol 2020;40:547–52.
[296] L´
opez-Ga cía JS, Ri as L, Ga cía-Lozano I, Mu ube J. Au ologous se um eyed ops
in he ea men o ani idic ke a opa hy. Oph halmology 2008;115:262–7.
[297] Sheha H, Tighe S, Hashem O, Hayashida Y. Upda e on Cenege min eye d ops in
he ea men o neu o ophic ke a i is. Clin Oph halmol 2019;13:1973–80.
[298] P lug elde SC, Massa o-Gio dano M, Pe ez VL, Ham ah P, Deng SX, Espanda L,
e al. Topical ecombinan human ne e g ow h ac o (Cenege min) o
neu o ophic ke a opa hy: a mul icen e andomized ehicle-con olled pi o al
ial. Oph halmology 2020;127:14–26.
[299] Mas opasqua L, Lanzini M, Dua HS, D’ U izi A, Di Nicola M, Calienno R, e al. In
i o e alua ion o co neal ne es and epi helial healing a e ea men wi h
ecombinan ne e g ow h ac o o neu o ophic ke a opa hy. Am J Oph halmol
2020;217:278–86.
[300] Vasilye a TA, Ma akhono AV, Vosk esenskaya AA, Kadyshe VV, K¨
asmann-
Kellne B, Sukhano a NV, e al. Analysis o geno ype-pheno ype co ela ions in
PAX6-associa ed ani idia. J Med Gene 2020.
[301] Law SK, Sami M, Pi i N, Coleman AL, Cap ioli J. Asymme ic pheno ype o
Axen eld-Riege anomaly and ani idia associa ed wi h a no el PITX2 mu a ion.
Mol Vis 2011;17:1231–8.
[302] I o YA, Foo z TK, Be y FB, Mi zayans F, Yu M, Khan AO, e al. Se e e molecula
de ec s o a no el FOXC1 W152G mu a ion esul in ani idia. In es Oph halmol
Vis Sci 2009;50:3573–9.
[303] Ansa i M, Rainge J, Hanson IM, Williamson KA, Sha key F, Ha ewood L, e al.
Gene ic analysis o ‘PAX6-nega i e’ indi iduals wi h ani idia o Gillespie
synd ome. PloS One 2016;11:e0153757.
[304] Landsend ES, U heim Ø A, Pede sen HR, Lagali N, Ba aas RC, U heim TP. The
gene ics o congeni al ani idia-a guide o he oph halmologis . Su Oph halmol
2018;63:105–13.
[305] Yongblah K, Al o d SC, Ryan BC, Chow RL, Howa d PL. P o ec ing Pax6 3’ UTR
om Mic oRNA-7 pa ially es o es PAX6 in isle s om an ani idia mouse model.
Mol The Nucleic Acids 2018;13:144–53.
[306] Macdonald R, Wilson SW. Dis ibu ion o Pax6 p o ein du ing eye de elopmen
sugges s disc e e oles in p oli e a i e and di e en ia ed isual cells. De Gene
E ol 1997;206:363–9.
[307] Ranaei Pi ma dan E, Soheili Z-S, Samiei S, Ahmadieh H, Mowla SJ, Nase i M,
e al. In i o e alua ion o PAX6 o e exp ession and NMDA cy o oxici y o
s imula e p oli e a ion in he mouse e ina. Sci Rep 2018;8:17700.
[308] Ba d JB, Hay ED. The beha io o ib oblas s om he de eloping a ian co nea.
Mo phology and mo emen in si u and in i o. J Cell Biol 1975;67:400–18.
[309] Ramaesh K, Ramaesh T, Du on GN, Dhillon B. E ol ing concep s on he
pa hogenic mechanisms o ani idia ela ed ke a opa hy. In J Biochem Cell Biol
2005;37:547–57.
[310] La a L, Vies enz A, S achon T, Colanesi S, Szen m´
a y N, Sei z B, e al. Human
ani idia limbal epi helial cells lack exp ession o ke a ins K3 and K12. Exp Eye
Res 2018;167:100–9.
[311] Si ak JM, Mohan R, Rineha WB, Xu P-X, Maas RL, Fini ME. Pax-6 exp ession
and ac i i y a e induced in he eepi helializing co nea and con ol ac i i y o he
ansc ip ional p omo e o ma ix me allop o einase gela inase B. De Biol
2000;222:41–54.
[312] Leipe LJ, Ou J, Walczysko P, Kuce o a R, La e y DN, Wes JD, e al. Con ol o
pa e ns o co neal inne a ion by Pax6. In es Oph halmol Vis Sci 2009;50:
1122–8.
L. La a e al.