Resea ch A icle
Role o ciliopa hy p o ein TMEM107 in eye de elopmen :
insigh s om a mouse model and e inal o ganoid
Ma ija Dubaic
1,2
, Lucie Pesko a
3
, Ma ek Hampl
1,2
, Kamila Weisso a
1,3
, Canan Celike
3
, Na alia A Shylo
4,5
, E a H uba
1
,
Michaela Ka ko a
6
, Tomas Zikmund
6
, Sco D Wea he bee
4,7
, Joze Kaise
6
, Tomas Ba a
1,3
, Ma cela Buch o a
1,2
P ima y cilia a e cellula su ace p ojec ions en iched in ecep-
o s and signaling molecules, ac ing as signaling hubs ha e-
spond o s imuli. Mal unc ions in p ima y cilia ha e been linked o
human diseases, including e inopa hies and ocula de ec s.
He e, we ocus on TMEM107, a p o ein localized o he ansi ion
zone o p ima y cilia. TMEM107 mu a ions we e ound in pa ien s
wi h Joube and Meckel–G ube synd omes. A mouse model
lacking Tmem107 exhibi ed eye de ec s such as anoph halmia and
mic oph halmia, a ec ing e ina di e en ia ion. Tmem107 ex-
p ession du ing p ena al mouse de elopmen co ela ed wi h
pheno ype occu ence, wi h enhanced exp ession in di e en i-
a ing e ina and op ic s alk. TMEM107 deficiency in e inal o ga-
noids esul ed in he loss o p ima y cilia, down- egula ion o
e ina-specific genes, and cys o ma ion. Knocking ou TMEM107
in human ARPE-19 cells p e en ed p ima y cilia o ma ion and
impai ed esponse o Smoo hened agonis ea men because o
ec opic ac i a ion o he SHH pa hway. Ou da a sugges TMEM107
plays a c ucial ole in ea ly e eb a e eye de elopmen and cil-
iogenesis in he di e en ia ing e ina.
DOI 10.26508/lsa.202302073 | Recei ed 4 Ap il 2023 | Re ised 27 Sep embe
2023 | Accep ed 28 Sep embe 2023 | Published online 20 Oc obe 2023
In oduc ion
The p ima y cilium is a cellula o ganelle ha p ojec s om he
su ace o mos cell ypes. I pe o ms senso y, mechanical, and
signal p ocessing unc ions, and egula es nume ous c i ical de-
elopmen al p ocesses, including neu ogenesis, skele ogenesis,
and kidney o ma ion (Chang e al, 2015;Ma a e al, 2016). P ima y
cilia a e also c ucial coo dina o s o he Sonic Hedgehog (Shh)
pa hway and o he signaling p ocesses (Huang u e al, 2003;
Caspa y e al, 2007;Wheway e al, 2018). Du ing ea ly eye de el-
opmen , p ima y cilia a e p esen on he su ace o he op ic
neu epi helium, su ace ec ode m, and pe iocula mesenchyme
(Lupu e al, 2018). They also play an impo an ole in he anspo
o p o eins in ol ed in isual ansduc ion in pho o ecep o s,
making cilia y signaling c i ical o bo h e inal s uc u e o ma ion
and e inal physiology in pos na al s ages (Wheway e al, 2014).
A b oad ange o de ec s known as ciliopa hies can esul om
al e a ions in cilia y biogenesis o unc ion (Lee & Gleeson, 2011).
These de ec s include ocula de o mi ies, such as e ini is pig-
men osa and macula degene a ion in humans, and eye de ec s
obse ed in se e al mouse s ains wi h ciliopa hies (Badano e al,
2006;Go i odsky e al, 2009;Qin e al, 2011;Cela e al, 2018). The
p o ein composi ion and unc ion o cilia is p ecisely con olled
h ough a specialized domain loca ed a he base o he cilium
known as he ansi ion zone (TZ) (Gonçal es & Pelle ie , 2017).
TMEM107 is a p o ein loca ed a he TZ ha has been shown o
ec ui ciliopa hy-associa ed p o eins such as MKS-1, TMEM-231
(JBTS20), and JBTS-14 (TMEM237) o his domain (Lambache e al,
2016). The TMEM107 locus has ecen ly been ound o be mu a ed
in pa ien s wi h Meckel–G ube synd ome, O o aciodigi al syn-
d ome, and Joube synd ome (Iglesias e al, 2014;Shaheen e al,
2015;Lambache e al, 2016;Shylo e al, 2016;Chinen e al, 2022).
These synd omes a e associa ed wi h al e ed p ima y cilia mo -
phology and unc ion, highligh ing he ole o TMEM107 in cilia y
unc ion.
Human pa ien s wi h TMEM107 mu a ions exhibi nume ous
de elopmen al de ec s, such as polydac yly o acial dysmo phic
ea u es (Iglesias e al, 2014;Lambache e al, 2016;Shylo e al,
2016;Chinen e al, 2022). Mouse s ains wi h mu a ions in Tmem107
demons a e simila de ec s o human pa ien s, including ex a
digi s and a spec um o c anio acial anomalies such as exence-
phaly, mic oph halmia o skele al de ec s in Tmem107
schlei
emb yos
(Ch is ophe e al, 2012). Tmem107
null
mouse emb yos display e en
s onge pheno ypes, including sho e snou s, expanded acial
midlines, cle pala es, and ex ensi e exencephaly (Cela e al, 2018).
Whe eas he associa ion be ween Tmem107 deficiency and c a-
nio acial de ec s has been p e iously desc ibed, he ole o his
gene in eye de elopmen is no ye ully unde s ood.
He e, we used mouse emb yos, e inal o ganoids, and e inal cell
cul u e models o closely in es iga e he ole o TMEM107 in eye
1
Labo a o y o Molecula Mo phogenesis, Ins i u e o Animal Physiology and Gene ics, Czech Academy o Sciences, B no, Czech Republic
2
Depa men o Expe imen al
Biology, Facul y o Science, Masa yk Uni e si y, B no, Czech Republic
3
Depa men o His ology and Emb yology, Facul y o Medicine, Masa yk Uni e si y, B no, Czech
Republic
4
Depa men o Gene ics, Yale Uni e si y, School o Medicine, New Ha en, CT, USA
5
S owe s Ins i u e o Medical Resea ch, Kansas Ci y, MO, USA
6
CEITEC -
Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no, Czech Republic
7
Biology Depa men , Fai field Uni e si y, Fai field, CT, USA
Co espondence: buch o [email protected]; [email p o ec ed]
©2023Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 1o 16
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de elopmen . We ound ha Tmem107 is specifically en iched in he
neu al e ina (NR) du ing mouse eye de elopmen . I s loss leads o
he dis inc i e ocula pheno ypes associa ed wi h p ima y cilia
de ec s, including mic oph halmia and anoph halmia; and al e ed
exp ession o c ucial ansc ip ion ac o s in ol ed in eye de el-
opmen . TMEM107-deficien human e inal o ganoid model enabled
us o de e mine i s ole in he human e ina, and i also allowed us
o s udy he ole o his gene in neu al e ina o ma ion wi hou any
influence o su ounding o closely associa ed eye s uc u es and
issues including he b ain o su ace ec ode m. We ound ha
TMEM107-deficien e inal o ganoids la gely co obo a ed he e-
sul s om he mouse model, ailing o o m neu al e ina s uc u es
and exhibi ing p ima y cilia de ec s. Finally, using he e inal cell
cul u e model, we ound ha TMEM107 is c i ical o SHH signaling
and i s loss abe an ly up- egula es he SHH pa hway. Taken o-
ge he , ou findings sugges ha TMEM107 plays a c ucial ole in eye
de elopmen and SHH signaling in bo h mice and humans, p o-
iding a be e unde s anding o eye abno mali ies ha may po-
en ially lead o he apeu ic in e en ions o ela ed condi ions.
Resul s
Loss o Tmem107 leads o dis inc i e ocula pheno ypes in
mouse emb yos
We used Tmem107
−/−
mouse model o de e mine he unc ion o
Tmem107 in eye de elopmen (Ch is ophe e al, 2012). Mu an
emb yos h ough all analyzed s ages (E10.5–E15.5) exhibi ed a a-
ie y o eye abno mali ies (Figs 1A and S1A–Fand Table S1) (Video 1
and Video 2). He e ozygous emb yos did no exhibi an abno mal
pheno ype. Two mos equen ly obse ed pheno ypes included
comple e loss o an eye (anoph halmia), obse ed in 33.3% o
examined mu an s, and abno mally small eye (mic oph halmia)
wi h 47% occu ence (Fig 1D). In nine examined cases, bo h phe-
no ypes we e p esen wi hin he same emb yo. In anoph halmic
animals, a small a ea o pigmen esidue was p esen , whe eas
emb yos exhibi ing mic oph halmia o en displayed o he de ec s,
such as o al absence o he lens (aphakia) and/o op ic ne e (ON)
hypoplasia (Fig 1A). The mice e ina wi h mic oph halmic pheno ype
was smalle and elonga ed, compa ed wi h e ina in WT animals.
Addi ional mo phome ic analyses pe o med using mic o-CT ap-
p oach e ealed dec eased e ina olume and sho ened ON in
mu an emb yos (Fig 1B and C).
Tmem107 is highly exp essed in he e ina du ing
eye de elopmen
To in es iga e he ole o Tmem107 in he de elopmen o specific
eye s uc u es, we analyzed in si u exp ession o Tmem107 using he
RNAscope app oach du ing c i ical s ages o eye de elopmen
(E10–E15). A ea ly s ages o op ic esicle ou g ow h (E10 and E11)
(Fig 2A and B), Tmem107 exp ession was de ec ed in he bilaye ed
op ic cup, whe e he inne laye ep esen s he p esump i e neu al
e ina (NR) and he ou e laye will gi e ise o he e inal pigmen
epi helium (RPE). Al hough Tmem107 exp ession was obse ed
h oughou all hese s uc u es, he signal was pa icula ly en iched
in he p esump i e NR (Fig 2A’’’’–F’’’’), whe eas he lens placode and
RPE laye exhibi ed lowe exp ession (Fig 2A’–F’and A’’–F’’). The
pa e ns o Tmem107 exp ession emained simila du ing la e s ages
(E12, E13), wi h he signal loca ed in he an e io and pos e io lens
epi helium (Fig 2C and D) and in he newly o med co nea (Fig 2C’and
D’). A E13, he o ma ion o a ganglion cell laye (GCL) is associa ed
wi h lowe exp ession o Tmem107 compa ed wi h he es o NR (Fig
2D’’). La e , a E14 and E15, he neu oblas cell laye (NCL), which
con ains neu onal p ogeni o s, exp esses high le els o Tmem107 (Fig
2E’’’’ and F’’’’), whe eas di e en ia ed neu ons o GCL exhibi low
exp ession o his gene. In e es ingly, Tmem107 mRNA exp ession in
he cilia y ma ginal zone (CMZ) was e y low compa ed wi h he es
o he NR (Figs 2F’’ and S2A–D). Thus, ou findings indica e ha
Tmem107 is s ongly exp essed du ing ea ly s ages o eye de el-
opmen (E10–E15), pa icula ly in he NR.
Key ac o s in eye de elopmen a e al e ed in
Tmem107
2/2
animals
To gain mo e insigh in o molecula changes caused by Tmem107
dele ion, we analyzed in si u exp ession o key p o eins in ol ed
in eye pa e ning. Because he pheno ypic analysis e ealed
dis inc anomalies a ec ing ce ain eye a eas including e ina,
lens, and op ic s alk, we u he ocused on he e alua ion o he
exp ession pa e ns o p o eins ha a e c i ical o mo pho-
genesis o he e ina (PAX6, SOX2), he op ic s alk (PAX2), and he
lens (SOX1). PAX6 and SOX2 a e ansc ip ion ac o s associa ed
wi h anoph halmia and mic oph halmia in humans (Ma sushima
e al, 2011). Fu he mo e, SOX1 is a key egula o exp essed in he
de eloping lens (Nishiguchi e al, 1998), and PAX2 has p e iously
been linked o he de elopmen o op ic s alk and closu e o op ic
fissu e (Bosze e al, 2021). Because i is well es ablished ha all o
hese ansc ip ion ac o s a e exp essed a he ea ly s ages o
eye de elopmen , we pe o med he analyses a E10.5 and E11.5
(Fig 3). We obse ed al e ed exp ession o PAX6 (Fig 3A–A’’
compa ed wi h Fig 3B–B’’), PAX2 (Fig 3C–C’’ compa ed wi h Fig
3D–D’’) and SOX1 (Fig 3E–E’’ compa ed wi h Fig 3F–F’’)inTmem107
−/−
emb yos al eady a E10.5 wi h mo e s iking di e ences ound a
E11.5 (Fig 3G–L). We analyzed he exp ession o PAX6, PAX2, and
SOX1 in mic oph halmia mu an s because s uc u es exp essing
hese p o eins a e missing in animals wi h anoph halmia, which
we also see in ou mu an s. In e es ingly, SOX2 exp ession in
mic oph halmic Tmem107
−/−
mu an s a E10.5 was s ill main-
ained in NR (Fig S3C–C’’ compa ed wi h Fig S3A–A’’)andp o-
ound di e ences we e ound la e a E11.5 (Fig S3F–F’’’ compa ed
wi h Fig S3D–D’’). In anoph halmic Tmem107
−/−
mu an s, he ex-
p ession o SOX2 was educed in he op ic s alk, whe eas in
he op ic cup, i was comple ely los a E10.5 and E11.5 (Fig S3B’’
and E’’).
Mo eo e , he educ ion o SOX1 exp ession was ound in he
lens o mic oph halmia mu an s, whe eas PAX2 and PAX6 we e
down- egula ed in he dis al pa o NR (Fig 3). In summa y, ou da a
indica e ha TMEM107 is impo an o ea ly eye pa e ning in mice
and i may es ablish he p ope exp ession o pi o al playe s du ing
c ucial s ages o he op ic cup and s alk mo phogenesis.
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 2o 16
Figu e 1. Tmem107
2/2
mu an s display se e e mo phological de ec s in eye egions.
(A) Mac oscopic pic u es and HE-s ained sec ions illus a ing mic oph halmia and anoph halmia in E10.5–E15.5 mu an emb yos compa ed wi h WT. (B) Mic o-CT
econs uc ion o s age E15.5 WT and Tmem107
−/−
eyes. Wall hickness is displayed as a colo g adien om blue ( he hinnes , 0 μm) o ed ( he hickes , up o 200 μm)
demons a ed in he colo legend. (C) G aphical ep esen a ion o changes in eye s uc u e size a s age E15.5 measu ed om mic o-CT scans (le — olume o eyes in mm
3
;
igh —op ic ne e leng h in mm) in Tmem107
−/−
emb yos. Pai ed, nonpa ame ic, wo- ailed es ; ns, nonsignifican ; *P< 0.05; **P< 0,01; ***P< 0,001; n = 3. (D) Va iabili y
o an eye pheno ype p esen in di e en s ages o examined mu an specimens. RPE, e inal pigmen epi helium; NR, neu al e ina; OS, op ic s alk; ON, op ic ne e. Scale
ba s: mac oscopic pic u es = 700 μm; hema oxylin–eosin (HE)-s ained sec ions = 200 μm; mic o-CT = 800 μm.
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 3o 16
Figu e 2. Tmem107 exp ession du ing eye de elopmen .
(A, B, C, D, E, F) Rep esen a ion o RNAScope signal o Tmem107 (shown in ed) a low magnifica ion in he whole eye a s ages om E10 o E15. (A’,A’’,A’’’,A’’’’,B’,B’’,B’’’,
B’’’’,C’,C’’,C’’’,C’’’’,D’,D’’,D’’’,D’’’’,E’,E’’,E’’’,E’’’’,F‘,F’’,F’’’,F’’’’)De ails o Tmem107 exp ession in lens, (A’’,B’’,C’’,D’’,E’’,F’’) in cilia y ma ginal zone egion, (A’’’,B’’’,C’’’,D’’’,
E’’’,F’’’) in op ic s alk/op ic ne e egion (OS), (A’’’’,B’’’’,C’’’’,D’’’’,E’’’’,F’’’’) and in he neu al e ina egion. Nuclei a e coun e s ained wi h DAPI (blue). NR, neu al e ina; RPE,
e inal pigmen epi helium; GCL ganglion cell laye ; NCL neu oblas cell laye ; CMZ, cilia y ma ginal zone; * au ofluo escen blood cells. Scale ba : lowe powe = 150 μm;
highe powe = 15 μm.
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 4o 16
TMEM107 deficiency leads o he ailu e o neu al e ina o ma ion
in human e inal o ganoids
Gi en he s iking e inal pheno ype obse ed in Tmem107
−/−
an-
imals, we aimed o u he es i he human TMEM107 gene is
essen ial o he de elopmen o he e ina. We used e inal
o ganoids di e en ia ed om hESCs as a model o closely in es-
iga e he oles o TMEM107 in human e inal de elopmen .
Mo eo e , his app oach allowed us o e alua e he di ec ole o
TMEM107 in e inal di e en ia ion wi hou he e ec s o su -
ounding o closely associa ed eye s uc u es and issues including
he b ain o su ace ec ode m.
We gene a ed TMEM107
−/−
hESCs using CRISPR/Cas9 app oach
(Figs S4 and S5) and di e en ia ed hem in o e inal o ganoids
using an al eady published p o ocol (Kuwaha a e al, 2015;Pesko a
e al, 2020;Celike e al, 2023). Re inal o ganoids we e analyzed a
day 30 (D30, ea ly s age) and day 150 (D150, la e s age) o he
di e en ia ion p ocess. Ea ly di e en ia ion s eps du ing he e inal
o ganoid o ma ion include he gene a ion o NR epi helium
con aining p ogeni o s ha gi e ise o pho o ecep o s (RAX+),
ganglion cells (VSX2+), and o he cell ypes o he human e ina,
whe eas he la e s age is cha ac e ized by pho o ecep o ma u-
a ion (CRX+, RHODOPSIN+) and appea ance o inne and ou e
pho o ecep o segmen s (Bu meis e e al, 1996;Fu ukawa e al,
1997b;I ie e al, 2015).
A D30 o he di e en ia ion p ocess, WT o ganoids con ained NR
epi helium, whe eas TMEM107
−/−
o ganoids lacked NR epi helium
and con ained cys ic s uc u es (Figs 4A and D and S4A–L). RT–
qPCR analysis confi med ha TMEM107
−/−
o ganoids ailed o
gene a e NR, as demons a ed by significan down- egula ion o
genes ha a e ypically exp essed in he de eloping NR s uc u e
including RAX,PAX6,SOX2,andVSX2 (Fig 4B, op ow)(Fu ukawa
e al, 1997a;Kozmik, 2008;Ma sushima e al, 2011;Bu meis e e al,
1996).
In e es ingly, scanning elec on mic oscopy (SEM) analysis
e ealed ha TMEM107
−/−
o ganoids a D30 lack p ima y cilia on
hei su ace (Fig 4C). In addi ion, we confi med he absence o
p ima y cilia using immunofluo escence s aining o p ima y cilia
Figu e 3. Key playe s o eye de elopmen a e al e ed in Tmem107
2/2
animals a E10.5–E11.5.
(A, A’’,A’’,B,B’,B’’,C,C’,C’’,D,D’,D’’,E,E’,E’’,F,F’,F’’,G,G’,G’’,H,H’,H’’,I,I’,I’’,J,J’,J’’,K,K’,K’’,L,L’,L’’)Immunohis ochemical de ec ion o PAX6 (A, A’,A’’,B,B’,
B’’,G,G’,G’’,H,H’,H’’), PAX2 (C, C’,C’’,D,D’,D’’,I,I’,I’’,J,J’,J’’), and SOX1 (E, E’,E’’,F,F’,F’’,K,K’,K’’,L,L’,L’’)p o einsinindi idualeyes uc u esinE10.5andE11.5
emb yos. (A, A’’,A’’,B,B’,B’’,C,C’,C’’,D,D’,D’’,E,E’,E’’,F,F’,F’’,G,G’,G’’,H,H’,H’’,I,I’,I’’,J,J’,J’’,K,K’,K’’,L,L’,L’’)PAX6 ( ed) exp ession in he neu al e ina, lens,
pigmen epi helium, and op ic s alk in WT (A, A’,A‘‘,G,G’,G‘‘)incompa isonwi hTmem107
−/−
animals (B, B’,B’’,H,H’,H’’). PAX2 ( ed) exp ession in op ic s alk and
dis al pa o neu al e ina in WT (C, C’,C’’,I,I’,I’’)incompa isonwi hTmem107
−/−
animals (D, D’,D’’,J,J’,J’’). SOX1 ( ed) exp ession in lens esicle o WT (E, E’,E’’)was
highe in con as wi h Tmem107
−/−
(F, F’,F’’) simila as in E11.5 WT (K, K’,K’’)incompa isonwi hTmem107
−/−
mu an s (L, L’,L’’). Nuclei a e coun e s ained wi h DAPI.
NR, neu al e ina; RPE, e inal pigmen epi helium; OS, op ic s alk. Scale ba = 200 μm.
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 5o 16

ma ke ARL13B (Fig 4D). To e eal whe he he absence o
TMEM107 leads o he impai ed ma u a ion o he e inal o ga-
noids and ailu e o gene a e pho o ecep o s and o he e inal
cell ypes, we cul u ed e inal o ganoids un il D150. TMEM107
−/−
e inal o ganoids a he la e s age comple ely ailed o gene a e
NR demons a ed by: (I) al e ed o ganoid mo phology (Fig 4A),
(II) lack o pho o ecep o ou e segmen s demons a ed by SEM
and immunofluo escence s aining o ARL13B (Fig 4C and E), (III)
significan down- egula ion o pho o ecep o ma ke s (CRX,
RHODOPSIN,RCVRN) and e inal ganglion cell ma ke (MATH5), as
demons a ed by RT–qPCR analysis (Fig 4B, bo om ow). In e -
es ingly, Hema oxylin/Eosin s aining o e inal o ganoid c oss
sec ions e ealed he p esence o cys s and Oil Red O lipid
s aining iden ified inc eased he p esence o lipids in TMEM107-
deficien o ganoids (Fig S4).
To co obo a e he pheno ype o TMEM107
−/−
o ganoids, we
used a di e en loss-o - unc ion app oach— he shRNA-media ed
knockdown o TMEM107 in human induced plu ipo en s em cells
(hiPSCs). We gene a ed len i i al pa icles con aining mChe y e-
po e and doxycycline (DOX)-inducible exp ession o shRNA o
TMEM107 down- egula ion. Upon ansduc ion, pu omycin selec-
ion, and FACS so ing, hiPSCs and he gene a ed e inal o ganoids
exp essed mChe y epo e (Fig S6A and B). DOX was applied om
D2 o he di e en ia ion p ocess, and he e inal o ganoids we e
ha es ed and analyzed a D25. We ound ~50% down- egula ion o
TMEM107 gene exp ession as de e mined by RT–qPCR in he
p esence o DOX (Fig S6C) ha led o al e a ions in p ima y cilia
o ma ion including ex emely elonga ed o e y sho cilia wi h
expanded bulges in hei ip (Fig S6D), cys o ma ion inside o he
o ganoids (Fig S6B), and ailu e o o m NR s uc u es in e inal
o ganoids (Fig S6E), hus co obo a ing he esul s gene a ed using
he knock-ou app oach.
Taken oge he , ou esul s indica e ha he absence o TMEM107
leads o he ollowing: (I) absence o p ima y cilia on ea ly-s age
o ganoids and ou e segmen s on la e s age e inal o ganoids, (II)
he down- egula ion o e ina-specific genes, (III) he ailu e o
gene a e he NR s uc u es and cell ypes in he human e inal
o ganoid model, (IV) he gene a ion o o ganoid wi h cys s con-
aining lipids.
Tmem107
2/2
animals ha e p ima y cilia de ec s in pigmen
epi helium and neu al e ina
P ima y cilia de ec in di e en o gans in Tmem107
−/−
emb yos
ha e been p e iously epo ed (Cela e al, 2018;Shylo e al, 2020).
Howe e , he po en ial cilia y anomalies in he e ina o hese
animals emain elusi e. To es whe he Tmem107
−/−
eye phe-
no ype in he mouse model is associa ed wi h p ima y cilia
Figu e 4. TMEM107 is essen ial o human
e inal de elopmen .
(A) Mo phology o e inal o ganoids a D30
and D150, as demons a ed using b igh field
mic oscopy. (B) Exp ession o e inal genes
a ea ly s age (D30—uppe ow) and la e s age
(D150—bo om ow) o he di e en ia ion
p ocess in WT and TMEM107
−/−
e inal
o ganoids, as demons a ed using RT–qPCR;
pa ame ic pai ed, wo- ailed es ; n = 3.
(C) Mic opho og aph o e inal o ganoid
su ace (D30—uppe ow, D150—bo om ow),
as demons a ed using SEM. Scale ba s
ep esen 10 μm. (D) Exp ession o PAX6
(g een), ARL13B ( ed), RAX (g een), and VSX2
( ed) in WT and TMEM107
−/−
e inal o ganoids
a D30, as demons a ed using
immunofluo escence s aining. Nuclei a e
coun e s ained wi h DAPI. (E) Exp ession o
ARL13B and CRX in WT and TMEM107
−/−
e inal o ganoids, as demons a ed using
immunofluo escence s aining. Nuclei a e
coun e s ained wi h DAPI.
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 6o 16
de ec s, we labeled cilia using an i-ARL13B an ibody, coun ed he
numbe o cilia ed cells, and measu ed he leng h o p ima y
cilia. We analyzed p ima y cilia in dis inc eye s uc u es wi h a
special ocus on RPE and NR a de elopmen al s ages E10.5, E11.5
o E12.5. Analysis o he NR egion e ealed a educed numbe o
p ima y cilia (RCC– a io o cilia ed cells) a s ages E10.5 (Fig
5A–C), E11.5 (Fig 5D–F), and E12.5 (Fig 5G–I) associa ed wi h
significan ly educed cilia leng h a E10.5 (Fig 5C’)andE12.5(Fig
5I’). In addi ion, he leng h o cilia was also educed a E11.5, bu
no wi h he s a is ical significance (Fig 5F’). Howe e , mo e
p o ound changes we e obse ed in he RPE egion, whe e we
ound a dec eased leng h and cilia numbe in all analyzed
samples o di e en de elopmen al s ages: E10.5 (Fig 5J–L’), E11.5
(Fig 5M–O’), and E12.5 (Fig 5P–R’). These da a sugges ha
Figu e 5. P ima y cilia in neu al e ina (NR) and e inal pigmen epi helium (RPE) Labeling o p ima y cilia using ARL13B ( ed) cilia y p o ein.
(A, A´, B, B’, D, D´, E, E’, G, G´, H, H’)In compa ison wi h WT animals (A, A´, D, D´, G, G´), educed numbe and leng h o p ima y cilia in NR o E10.5 (B, B’), E11.5 (E, E’), and E12.5
(H, H’)Tmem107
−/−
compa ed wi h WT emb yos. (C, C’,F,F’,I,I’)G aphs ep esen ing di e ence in cilia numbe in WT (RCC, a io o cilia ed cells) and Tmem107
−/−
NR (C, F, I)
and di e ence in cilia leng h (μm) in WT and Tmem107
−/−
NR (C’,F’,I’). (J, J’,K,K’,M,M’,N,N’,P,P’,Q,Q’)Compa ison o p ima y cilia in RPE a ea be ween WT (J, J´; M, M´; P, P´)
and Tmem107
−/−
display educed numbe and leng h o p ima y cilia in RPE o E10.5 (K, K’), E11.5 (N, N’), and E12.5 (Q, Q’). (L, L’,O,O’,R,R’)G aphs ep esen ing di e ence
in cilia numbe in WT and Tmem107
−/−
RPE (L, O, R) and di e ence in cilia leng h in WT and Tmem107
−/−
RPE (L’,O’,R’). Nuclei a e coun e s ained wi h DAPI. Scale ba s: lowe
magnifica ion = 35 μm; highe magnifica ion = 5 μm. n = numbe o measu ed cilia; nonpa ame ic unpai ed wo- ailed es ; ns, nonsignifican ; *P< 0.05; **P< 0.01; ****P<
0.0001.
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 7o 16
TMEM107 plays a ole in cilia biogenesis in i o and egula ion o
cilia y leng h in he de eloping eye.
Loss o TMEM107 leads o abe an SHH signaling in e inal cells
The e iden associa ion o p ima y cilia dis up ion wi h eye
pheno ypes in all ou TMEM107
−/−
models p omp ed us o p o-
ceed wi h he analysis o he e ec s o TMEM107 loss a he
molecula le el. Because he unc ion o he Shh pa hway ully
elies on he o ma ion o p ima y cilium (Huang u & Ande son,
2005), we aimed o closely in es iga e he e ec s o TMEM107 loss
on his signaling pa hway. Re inal o ganoids do no ep esen
a sui able model o specifically add ess a ec ed molecula
pa hways in indi idual cells, because o he he e ogenei y o
di e en cell ypes hey con ain, he e o e i is challenging o
s udy signaling pa hways using an o ganoid model. We, he e o e,
used he ARPE-19 cell line, de i ed om e inal pigmen ed epi-
helium, o add ess he e ec s o TMEM107 loss on he Shh
pa hway by gene a ing ARPE-19 TMEM107
−/−
and TMEM107
+/−
cell
lines, using CRISPR/Cas9 echnology.
Fi s , we assessed he exp ession o he c ucial componen s o
he Shh pa hway GLI1 and PTCH1 using RT–qPCR. Whe eas he e
was no significan di e ence in PTCH1 exp ession be ween WT
and mu an cells, we de ec ed ~15- old up- egula ion o GLI1
exp ession in TMEM107
−/−
cells (Fig 6A), indica ing an abe an
ac i a ion o he Shh pa hway in he absence o TMEM107.To
confi m he ac i a ion o he Shh pa hway and o e eal whe he
ARPE-19 cells possess he unc ional Shh pa hway, we ea ed
he cells using Shh ac i a o smoo hened agonis (SAG). Upon
SAG ea men , WT and TMEM107
+/−
cells up- egula ed he ex-
p ession o GLI1 and PTCH1 by ~ wo old, bu TMEM107
−/−
cells
ailed o up- egula e GLI1 and PTCH1 (Fig 6B and C). The e o e,
high GLI1 exp ession and no esponse o SAG ea men o
TMEM107
−/−
cells indica e ha he Shh pa hway is abe an ly
ac i a ed in TMEM107
−/−
cellsand ha TMEM107isc i ical o Shh
signaling.
The abe an Shh signaling in TMEM107
−/−
cells could be
explained by al e ed p ima y cilia o ma ion ha we, indeed, also
obse ed in TMEM107
−/−
e inal o ganoids. To es his, we s ained
ARPE-19 cells o he cilia y ma ke ARL13B. WT and TMEM107
+/−
o med p ima y cilia, whe eas TMEM107
−/−
comple ely ailed o o m
hese s uc u es (Fig 6D and E). To es SHH ac i a ion, we used he
SAG ea men app oach as desc ibed abo e, and assessed in si u
localiza ion o o he c i ical Shh playe s SMO and GLI2. Immuno-
fluo escence s aining e ealed ha upon SAG ea men SMO lo-
calizes in o he p ima y cilia and GLI2 becomes up- egula ed and
localized in o nuclei o WT and TMEM107
+/−
cells. TMEM107
−/−
ailed
o o m p ima y cilia and GLI2 was up- egula ed e en in he absence
o SAG (Fig 6D and E).
P e ious s udies showed down- egula ed SHH in mice wi h
Tmem107 mu a ion (Ch is ophe e al, 2012;Cela e al, 2018;Shylo
e al, 2020). Howe e , because o dis inc cilia y ansmission
o SHH in di e en o gans, bo h up- egula ion and down-
egula ion ha e been obse ed in di e en issues o he de-
eloping emb yo (Bu ne e al, 2017). In models ha lack p ima y
cilia, an up- egula ion o SHH has been obse ed in he em-
b yonic e ina (Bu ne e al,2017). To es how he al e ed
mo phology o p ima y cilia a ec ed Shh signaling in he mouse
model, we analyzed he exp ession o SHH p o ein and P ch1 RNA
a s age E12.5 (Fig 7). Whe eas in WT eyes, SHH localized o
disc e e egions in he dis al pa o he eye (Fig 7A’), Tmem107
−/−
animals exhibi ed highe exp ession o his ligand in p oximal
and dis al pa s o he NR and in op ic s alk (Fig 7B). The di -
e ences we e mos p ominen in he lens and he op ic s alk
egions (Fig 7B’and B”). On he con a y, RNAScope analysis o
P ch1 e ealed no dis inc changes in i s exp ession in he eyes
o E12.5 mu an animals (Fig 7D–D”), as compa ed wi h WT (Fig
7C–C”). These da a demons a e ha al hough he exp ession o
SHH was ele a ed, dec ease in p ima y cilia keeps he pa hway
dys unc ional in mice mu an s.
Taken oge he , ou esul s indica e ha he absence o
TMEM107 leads o he ollowing: (I) ailu e o o m p ima y cilia in
e inal pigmen ed epi helial cells, (II) abe an up- egula ion o
he Shh pa hway demons a ed by he up- egula ion o GLI1,GLI2,
and GLI3 FL, (III) incapabili y o TMEM107
−/−
cells o espond o SAG
ea men , because lack o cilia, and (IV) inc eased le el o SHH
ligands in i o.
Discussion
Ciliopa hies a e a g oup o gene ic diso de s cha ac e ized by
de ec s in he s uc u e and unc ion o cilia, which a e hai -like
o ganelles p esen on he su ace o many cells. P e ious esea ch
has shown ha ciliopa hy p o eins a e impo an o a a ie y o
de elopmen al p ocesses, including eye de elopmen (Wa e s &
Beales, 2011). The p o ein TMEM107 has been p e iously implica ed
in ciliopa hy-associa ed eye abno mali ies (Ch is ophe e al, 2012).
Howe e , he specific mechanisms by which TMEM107 unc ions in
eye de elopmen and he wide ange o ocula abno mali ies as-
socia ed wi h i s deficiency ha e no been ully elucida ed.
He e, we used mouse emb yos, e inal o ganoid, and e inal cell
cul u e models o closely in es iga e he ole o TMEM107 in eye
de elopmen . We ound ha (I) TMEM107 is specifically and s ongly
exp essed in NR o he de eloping eye; (II) loss o TMEM107 leads o
dis inc i e ocula pheno ypes including anoph halmia and mic o-
ph halmia associa ed wi h a unca ed ON; (III) he exp ession o
c ucial genes in eye de elopmen is al e ed in he absence o
TMEM107; (IV) TMEM107 is c i ical o ciliogenesis and Shh signaling,
and i s absence leads o he dis up ion o p ima y cilia and abe an
Shh signaling; and (V) TMEM107 deficiency is associa ed wi h he
gene a ion o cys s.
All examined Tmem107
−/−
mouse mu an s mani es ed eye mal-
o ma ions including anoph halmia and mic oph halmia. In e es ingly,
simila pheno ypes ha e been obse ed in humans. Pa ien s who
appea as homozygo es o compound he e ozygo es o TMEM107
mu an allele ha e been diagnosed wi h Joube (JS), Meckel–
G ube (MKS) o o o aciodigi al synd ome (OFD) (Iglesias e al, 2014;
Shaheen e al, 2015;Lambache e al, 2016;Shylo e al, 2016;Chinen
e al, 2022). All abo emen ioned synd omes ha e been ecognized
as ciliopa hies associa ed wi h eye de ec s like anoph halmia,
mic oph halmia, e inal de ec s, coloboma o lid anomalies (Ha ill
e al, 2017;Ha ill e al, 2017). Pa ien s wi h TMEM107 pa hological
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 8o 16
a ian s we e diagnosed wi h a s onge pheno ype in case o MKS
wi h bila e al anoph halmia (Shaheen e al, 2015), and milde
pheno ypes wi h OFD and JBT wi h oculomo o ap axia and e i-
nopa hy (Lambache e al, 2016), and OFD wi h s abismus (Chinen
e al, 2022). Se e i y o exhibi ed symp oms seems o be co ela ed
wi h he ype o pa ien s´ mu a ions. In he case o wo OFD pa ien s,
he sequencing analysis de ec ed a homozygous missense a ian ,
whe eas in he Joube synd ome pa ien , a compound he e ozy-
gous mu a ion con aining a ameshi dele ion and an in- ame
dele ion was disco e ed (Lambache e al, 2016). On he o he hand,
he pa ien ca ying one in onic base pai inse ion causing
ameshi and p ema u e p o ein unca ion de eloped mo e
se e e de ec s (Shaheen e al, 2015). Simila ly, in ciliopa hic mice,
di e en eye de ec s ha e been desc ibed (Bu ne e al, 2017;Fio e
e al, 2020). Homozygo ic hypomo phic Tmem107
schlei
mouse em-
b yos de elop milde pheno ype–mic oph halmia (Ch is ophe
e al, 2012), whe eas mu an mice used in his s udy exhibi mo e
se e e eye de ec s when compa ed wi h hose con aining hypo-
mo phic alleles. We ha e shown ha all o he examined mu an s
mani es eye mal o ma ions wi h he mos se e e o m—anoph halmia
(33% mu an s). One o he easons why all o he examined Tmem107-
deficien emb yos de elop se e e pheno ypes whe eas milde de ec s
a e obse ed in humans could be because o di e ences be ween
species and he ac ha ou models exhibi o al lack o TMEM107
p o ein. Al hough mu a ions occu ing in humans could lead o ex-
p ession o a unca ed e sion o he p o ein, which s ill pa ially
e ains he unc ion, he comple e loss o he p o ein may lead o mo e
se e e consequences. Ano he ac o be conside ed is ha human
emb yos ca ying TMEM107 mu a ions die a ea ly p egnancy and a e
no being diagnosed. In e es ingly, Tmem107
−/−
mice display ON hy-
poplasia,whichcanalsooccu inpa ien swi hMeckel–G ube syn-
d ome (MacRae e al, 1972). Fu he mo e, hese mu an s we e shown o
ha e o he de ec s such as exencephaly, polydac yly, and cle pala e,
which a e obse ed in human TMEM107 homozygo ic pa ien s and a e
also o en p esen in o he ciliopa hic cases (Wa e s & Beales, 2011).
Animals ca ying only one mu a ed Tmem107 allele do no exhibi any
ob ious pheno ype which co esponds o findings on human pa ien s’
pa en s who do no de elop any o he symp oms p esen in child en
(Cela e al, 2018).
Gi en he s iking eye pheno ype in Tmem107
−/−
mouse emb yos
and he lack o cu en knowledge abou in si u Tmem107 ex-
p ession in de eloping eye s uc u es, we aimed o assess Tmem107
exp ession du ing eye de elopmen . We ound an ele a ed Tmem107
exp ession in he p esump i e NR. High Tmem107 exp ession in NR
and mic o-/anoph halmia pheno ypes in he absence o his gene
indica es a c i ical ole o Tmem107 in he de elopmen o he NR.
Because eyes de elop as b ain e agina ions, one could no e ha he
obse ed eye pheno ypes could be a consequence o no p ope ly
Figu e 6. Loss o TMEM107 leads o abe an
Shh signaling in e inal cells.
(A) Exp ession o GLI1 and PTCH1 in WT,
TMEM107
+/−
, and TMEM107
−/−
ARPE-19 cells, as
demons a ed using RT–qPCR; Pai ed, wo-
ailed es ; n.s. = non-significan ; n = 3.
(B, C) Exp ession o GLI1 and PTCH1 upon SAG
ea men in WT, TMEM107
+/−
, and
TMEM107
−/−
ARPE-19 cells, as demons a ed
using RT–qPCR; Pai ed, wo- ailed es ; n.s.,
nonsignifican ; n = 3. (D) Exp ession o
ARL13B (g een) and GLI2 ( ed) upon SAG
ea men in WT, TMEM107
−/−
, and TMEM107
+/−
ARPE-19 cells, as demons a ed using
immunofluo escence s aining. (E) Exp ession
o ARL13B ( ed) and SMO (g een) upon SAG
ea men in WT, TMEM107
−/−
, and
TMEM107
+/−
ARPE-19 cells. Nuclei a e
coun e s ained wi h DAPI (blue).
TMEM107 in he eye de elopmen Dubaic e al. h ps://doi.o g/10.26508/lsa.202302073 ol 6 | no 12 | e202302073 9o 16
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