RESEARCH ARTICLE
Local e inoic acid signaling di ec s
eme gence o he ex aocula muscle
unc ional uni
Glenda E angelina ComaiID
1,2
*, Ma ke
´ a Tesařo a
´ID
3
, Vale
´ ie Dupe
´ID
4
, Mu iel Rhinn
5
,
Ped o Vallecillo-Ga cı
´aID
6
, Fabio da Sil a
7,8
, Be y Fe e
5
, Ka he ine Exelby
9
,
Pascal Dolle
´
5
, Lei Ca lsson
10
, B ian P yce
11
, F anc¸ois Spi z
12
, Sigma S icke ID
6
,
Toma
´s
ˇZikmundID
3
, Joze Kaise ID
3
, James B iscoeID
9
, And eas SchedlID
7
, No be
B. GhyselinckID
5
, Ronen Schwei ze ID
11
, Shah agim TajbakhshID
1,2
*
1S em Cells & De elopmen Uni , Ins i u Pas eu , Pa is, F ance, 2CNRS UMR 3738, Ins i u Pas eu ,
Pa is, F ance, 3Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no, Czech
Republic, 4Uni e si e
´de Rennes, CNRS, IGDR, Rennes, F ance, 5IGBMC-Ins i u de Ge
´ne
´ ique e de
Biologie Moleculai e e Cellulai e, Illki ch, F ance, 6Ins i u e o Chemis y and Biochemis y, F eie
Uni e si a
¨ Be lin, Be lin, Ge many, 7Uni e si e
´Co
ˆ e d’Azu , INSERM, CNRS, iBV, Nice, F ance, 8Di ision
o Molecula Emb yology, Ge man Cance Resea ch Cen e (DKFZ), Heidelbe g, Ge many, 9The F ancis
C ick Ins i u e, London, Uni ed Kingdom, 10 UmeåCen e o Molecula Medicine, UmeåUni e si y, Umeå,
Sweden, 11 Resea ch Di ision, Sh ine s Hospi al o Child en, Po land, Uni ed S a es o Ame ica,
12 Genomics o Animal De elopmen Uni , Ins i u Pas eu , Pa is, F ance
*[email p o ec ed] (GEC); shah @pas eu . (ST)
Abs ac
Coo dina ed de elopmen o muscles, endons, and hei a achmen si es ensu es eme -
gence o unc ional musculoskele al uni s ha a e adap ed o di e se ana omical demands
among di e en species. How hese di e en issues a e pa e ned and unc ionally assem-
bled du ing emb yogenesis is poo ly unde s ood. He e, we in es iga ed he mo phogenesis
o ex aocula muscles (EOMs), an e olu iona y conse ed c anial muscle g oup ha is c u-
cial o he coo dina ed mo emen o he eyeballs and o isual acui y. By means o lineage
analysis, we ede ined he cellula o igins o pe iocula connec i e issues in e ac ing wi h
he EOMs, which do no a ise exclusi ely om neu al c es mesenchyme as p e iously
hough . Using 3D imaging app oaches, we es ablished an in eg a i e bluep in o he EOM
unc ional uni . By doing so, we iden i ied a de elopmen al ime window in which indi idual
EOMs eme ge om a unique muscle anlage and es ablish inse ions in he scle a, which
se s hese muscles apa om classical muscle- o-bone ype o inse ions. Fu he , we dem-
ons a e ha he eyeballs a e a sou ce o di usible all- ans e inoic acid (ATRA) ha allow
hei a ge ing by he EOMs in a empo al and dose-dependen manne . Using gene ically
modi ied mice and inhibi o ea men s, we ind ha endogenous local a ia ions in he con-
cen a ion o e inoids con ibu e o he es ablishmen o endon condensa ions and a ach-
men si es ha p ecede he ini ia ion o muscle pa e ning. Collec i ely, ou esul s highligh
how global and si e-speci ic p og ams a e deployed o he assembly o muscle unc ional
uni s wi h p ecise de ini ion o muscle shapes and opog aphical wi ing o hei endon
a achmen s.
PLOS BIOLOGY
PLOS Biology | h ps://doi.o g/10.1371/jou nal.pbio.3000902 No embe 17, 2020 1 / 33
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OPEN ACCESS
Ci a ion: Comai GE, Tesařo a
´M, Dupe
´V, Rhinn M,
Vallecillo-Ga cı
´a P, da Sil a F, e al. (2020) Local
e inoic acid signaling di ec s eme gence o he
ex aocula muscle unc ional uni . PLoS Biol
18(11): e3000902. h ps://doi.o g/10.1371/jou nal.
pbio.3000902
Academic Edi o : Simon M. Hughes, King’s
College London, UNITED KINGDOM
Recei ed: Janua y 7, 2020
Accep ed: Oc obe 1, 2020
Published: No embe 17, 2020
Pee Re iew His o y: PLOS ecognizes he
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p ocess; he e o e, we enable he publica ion o
all o he con en o pee e iew and au ho
esponses alongside inal, published a icles. The
edi o ial his o y o his a icle is a ailable he e:
h ps://doi.o g/10.1371/jou nal.pbio.3000902
Copy igh : ©2020 Comai e al. This is an open
access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he pape and i s Suppo ing In o ma ion
iles.
In oduc ion
Acquisi ion o shape and pa e n du ing de elopmen depends on he o ches a ed c oss alk
be ween a a ie y o issues and cell ypes. Al hough signi ican knowledge on he mechanisms
o di e en ia ion and pa e ning wi hin indi idual issues has been a ained, much less is
known on how pa e ning o di e en adjacen issues is in eg a ed. The e eb a e musculo-
skele al sys em se es as an ideal model o s udy hese p ocesses as di e en issues including
muscle, endon and hei a achmen s need o be a icula ed in 3D o p ope unc ion [1,2].
Among he c anio acial muscles, he mo phological con igu a ion o he ex aocula mus-
cles (EOMs) has been a longs anding challenge in compa a i e ana omy and e olu iona y biol-
ogy. Besides specialized adap a ions, he basic EOM pa e n is sha ed among all e eb a e
classes [3–5] and includes 4 ec i muscles ( he supe io ec us, he medial ec us, he in e io
ec us, and he la e al ec us) and 2 oblique muscles (supe io oblique and in e io oblique) o
mo emen o he eyeball. Mos e eb a es ha e also accesso y ocula muscles ha se e o
e ac he eye ( he e ac o bulbi) o con ol eyelid ele a ion ( he le a o palpab ae supe -
io is) [3,6]. As such, he EOMs cons i u e an a che ypal and au onomous unc ional uni o
he s udy o how muscles, endons, and endon a achmen s a e in eg a ed wi h he de elop-
men o he eyeball, hei a ge o gan.
C anio acial muscles a e de i ed om c anial pa axial and p echo dal head mesode m
[3,7]. The co esponding connec i e issues, i.e., endons, bones, ca ilages, and muscle con-
nec i e issue, we e epo ed o be de i ed om c anial neu al c es cells (NCCs) [7,8].
Al hough ea ly myogenesis is NCC independen , NCCs la e egula e he di e en ia ion and
seg ega ion o muscle p ecu so s, dic a e he pa e n o muscle ibe alignmen , and ha o
associa ed skele al and endon s uc u es [9–14]. Mo eo e , dele ion o se e al genes in NCCs
demons a ed hei non-cell-au onomous oles in muscle mo phogenesis a he le el o he jaw
[12,15,16], ex aocula [17,18], and somi ic-de i ed ongue muscles [19]. Howe e , he ull
se ies o e en s d i ing mo phogenesis o c anio acial musculoskele al unc ional uni s is
unexplo ed o da e, in pa because o he ana omical complexi y o hei con igu a ion in he
head. Mo eo e , unde s anding he de elopmen al mechanisms ha allow musculoskele al
connec i i y is essen ial o unde s and he ana omical di e si ica ion ha ook place du ing
he e olu ion o he e eb a e head. Ye , p oxima e ac o s ha allow c oss- issue communica-
ion o coo dina ed eme gence o he indi idual muscle masses wi h ha o hei endons and
a achmen si es a e poo ly de ined.
Much o ou unde s anding o musculoske al de elopmen and in eg a ion in o unc ional
uni s comes om s udies in he limb. La e al pla e mesode m-de i ed muscle connec i e issue
cells and endon p imo dia es ablish a p e-pa e n ha de e mines he si es o myogenic di e -
en ia ion and pa icipa e in spli ing o he muscle masses in he limb [20–22]. Tendons con-
nec muscles o he skele on and a e o med by scle axis (Scx)-exp essing mesenchymal
p ogeni o s [23,24]. Al hough some ea u es o endon de elopmen a e au onomous, key
s ages ely on signals emana ing om muscle o ca ilage, acco ding o hei posi ioning in he
limb [2]. Bone supe s uc u es, which p o ide ancho ing poin s o endons o he skele on,
a e ini ia ed independen ly o muscle, bu hei main enance and g ow h depend on cues om
bo h endon and muscle [2]. Gi en he dis inc gene egula o y ne wo ks go e ning c anial
muscle de elopmen [25,26], and emb yonic o igins o connec i e issues in he head [7,8], i
is unclea i his logic o musculoskele al in eg a ion is conse ed in he head and how s uc-
u es ha do no in eg a e bones, such as EOMs, a e es ablished.
All- ans e inoic acid (ATRA), he biologically ac i e me aboli e o e inol ( i amin A), is a
c i ical mo phogen wi h widesp ead oles in c anio acial de elopmen [27,28]. ATRA ac s as a
ligand o nuclea e inoic acid ecep o s (RARs), which a e ligand-dependen ansc ip ional
PLOS BIOLOGY
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Funding: We acknowledge unding suppo om
he Ins i u Pas eu , Associa ion F anc¸aise con e le
Myopa hies, Agence Na ionale de la Reche che
(Labo a oi e d’Excellence Re i e, In es issemen
d’A eni ; ANR-10-LABX-73) and he Cen e
Na ional de la Reche che Scien i ique. We g a e ully
acknowledge he U echS Pho onic BioImaging
(Imagopole), C2RT, Ins i u Pas eu , suppo ed by
he F ench Na ional Resea ch Agency (F ance
BioImaging; ANR-10–INSB–04; In es men s o
he Fu u e). MT, TZ and JK acknowledge he
p ojec CEITEC 2020 (LQ1601) wi h inancial
suppo om he Minis y o Educa ion, You h and
Spo s o he Czech Republic unde he Na ional
Sus ainabili y P og amme II and Cei ec Nano+
p ojec CZ.02.01/0.0./.0.0./16_013/0001728 unde
he p og am OP RDE. MT was inancially
suppo ed by by he B no Ci y Municipali y as a
B no Ph.D. Talen Schola ship Holde . The unde s
had no ole in s udy design, da a collec ion and
analysis, decision o publish, o p epa a ion o he
manusc ip .
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
Abb e ia ions: ADH, alcohol dehyd ogenase;
ATRA, all- ans e inoic acid; ALDH1A1, Aldehyde
Dehyd ogenase 1 Family Membe A1; ALDH1A3,
Aldehyde Dehyd ogenase 1 Family Membe A3; E,
emb yonic day; EOM, ex aocula muscle; GFP,
g een luo escen p o ein; KO, knock-ou ; LHX2,
LIM Homeobox 2; Mesp1, mesode m pos e io
BHLH ansc ip ion ac o 1; mGFP, memb ane
agged GFP; mic o-CT, mic o-compu ed
omog aphy; MYF5, myogenic ac o 5; MYOD,
myogenic di e en ia ion 1; MYOG, Myogenin;
MyHC, myosin hea y chain; NCC, neu al c es cell;
PAX6, Pai ed Box 6; PAX7, Pai ed Box 7; PITX2,
Pai ed-like homeodomain ansc ip ion ac o 2;
POM, pe iocula mesenchyme; RAR, e inoic acid
ecep o ; RARE, e inoic acid esponse elemen ,
RDH, e inol dehy ogenase; RPE, e inal pigmen ed
epi helium; RXR, e inoid X ecep o ; Scx,
scle axis; Shh, Sonic Hedgehog; SMA, alpha
smoo h muscle ac in; SOX9, SRY-box con aining
gene 9; TCF4, T ansc ip ion ac o 4; Tom,
dToma o; WMIF, whole-moun
immuno luo escence; Wn 1, Wn Family
Membe 1.
egula o s ha wo k as he e odime s wi h e inoid X ecep o s (RXRs) [28,29]. ATRA is
syn he ized om e inol h ough 2 oxida ion s eps by speci ic e inol/alcohol dehyd ogenases
(RDH/ADH) and e inaldehyde dehyd ogenases (ALDH1A1, ALDH1A2, and ALDH1A3)
[28,29]. ATRA is c i ical o ea ly eye de elopmen in se e al species [30–32], in which ATRA
me abolic enzymes a e exp essed in he ea ly e ina wi h igh spa io empo al pa e ns [33]. As
such, he de eloping eye ac s as a signaling cen e nuclea ing an e io segmen mo phogenesis,
wi h pai ed-like homeodomain ansc ip ion ac o 2 (PITX2) being he po en ial majo
downs eam ATRA e ec o in pe iocula NCCs [30,34–36]. Whe he ATRA is equi ed o
mo phogenesis o he EOMs and associa ed connec i e issues emains unexplo ed.
He e, we eassessed he emb yological o igins o he connec i e issues o he pe iocula
egion and p esen he i s in eg a i e bluep in o mo phogenesis o he EOM unc ional
uni . We p o ide gene ic e idence o he exis ence o a e inoic acid signaling module ha
coo dina es he eme gence o indi idual EOMs, hei endons, and inse ion si es. We show
ha he ac ion o e inoic acid signaling in muscle pa e ning is mainly non-cell-au onomous,
h ough i s ac ion on he NCC-de i ed pe iocula mesenchyme. We p opose ha he in e ac-
ions be ween muscles, endons, and hei a achmen s a e simila o hose obse ed in he
limb, ye hey exhibi speci c hallma ks ha a e cha ac e is ic o his ana omical loca ion.
Resul s
Gene ic a e mapping o mouse pe iocula issues
Gi en he complex ana omical disposi ion o he EOMs, we i s se ou o map mo phological
landma ks and cell ela ionships du ing pa e ning o hese muscles. The pe iocula mesen-
chyme (POM) is a he e ogeneous cell popula ion su ounding he op ic cup ha gi es ise o
specialized s uc u es o he an e io segmen o he eye and connec i e issues associa ed wi h
he EOMs [37]. Wi h excep ion o he EOMs and endo helial lining o ocula blood essels
(cho oid), all connec i e issues o he POM (ca ilage, muscle connec i e issue, endons)
we e epo ed o be de i ed om NCCs in zeb a ish, chicken, and mouse emb yos [38–43].
The 4 ec i EOMs o igina e deep in he o bi , a he le el o a ib ous ing called he annulus o
Zinn, and inse in o he scle al laye o he eye [44]. Because in o ma ion on EOM endons
and a achmen si es is sca ce, we used gene ic a e mapping o eassess he emb yological o i-
gins o he issues in e ac ing wi h EOMs du ing hei mo phogenesis. We simul aneously
aced he con ibu ion o NCC (Fig 1A–1B”,S1A, and S1B Fig) and mesode mal (Fig 1C–
1D”,S1C and S1D Fig) de i a i es using Tg:Wn 1
C e
and Mesp1
C e
mice, espec i ely [45,46],
in combina ion wi h he R26
Tom
epo e [74].
As expec ed, connec i e issues a he EOM inse ion le el we e de i ed om NCCs as
assessed by dToma o exp ession (Tg:Wn 1
C e
;R26
Tom
,Fig 1A–1A”,S1A Fig). Su p isingly, we
ound ha lineage con ibu ions di e ed in do sal sec ions, whe e connec i e issues a he
EOM o igin le el we e de i ed om he c anial mesode m (Mesp1
C e
;R26
Tom
,Fig 1D–1D”,
S1D Fig). To cha ac e ize in mo e de ail he cell popula ions a ising om hese de i a i es, we
used ansc ip ion ac o 4 (TCF4) as muscle connec i e issue ma ke [47], and a Scx epo e
line (Tg:Scx-GFP+) o ma k endons and hei ea ly p ogeni o s wi h g een luo escen p o ein
(GFP) [24,48]. TCF4 was exp essed obus ly in muscle connec i e issue ib oblas s in bo h
NCC- and mesode m-de i ed egions (S1E-S1F’ Fig). Simila ly, Tg:Scx-GFP (Fig 1A–1D”,
S1A–S1D Fig) s ongly labeled he u u e EOM endons a he o igin and inse ion si es esid-
ing espec i ely in mesode m- and NCC-de i ed domains. Addi ionally, Tg:Scx-GFP (Fig 1A1,
1D1,S1G and S1H Fig) and Scx mRNA (S1I and S1J Fig) ma ked muscle connec i e issue
ib oblas s ha we e widely dis ibu ed among he muscle masses and o e lapped wi h TCF4
(S1G and S1H Fig), as desc ibed in o he egions o he ea ly emb yo [49,50]. Al oge he , hese
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indings indica e ha he EOMs de elop in close associa ion wi h connec i e issues o 2 dis-
inc emb yonic o igins: neu al c es la e ally (a he EOM inse ion) and mesode m medially
(a he EOM o igin).
De elopmen o he EOMs and hei inse ions o e lap spa io empo ally
Pa e ning o he EOMs and hei inse ions is unde s udied because o he di icul y in in e -
p e ing a complex 3D issue a angemen om issue sec ions alone. The e o e, we es ablished
an imaging pipeline ha includes whole-moun immuno luo escence (WMIF) o he pe iocu-
la egion, issue clea ing, con ocal mic oscopy, and econs uc ions o he ob ained images
in o 3D objec s. To isualize he de eloping EOMs, we used an ibodies agains myogenic di -
e en ia ion 1 (MYOD), myogenin (MYOG), and Desmin as myogenic commi men and di -
e en ia ion ma ke s and myosin hea y chain (MyHC) o label myo ibe s (Fig 2A–2C’ and S1
Video). A emb yonic day (E)11.75, he EOMs we e p esen as a single anlage (Fig 2A and 2A’)
medial o he eyeball. By E12.5, he EOM anlage spli owa ds he eyeball in o submasses co e-
sponding o he u u e 4 ec i, 2 oblique muscles, and he acceso y e ac o bulbi muscle (Fig
2B and 2B’). Fully indi idua ed muscles we e e iden by E13.5 (Fig 2C and 2C’). Thus, we
Fig 1. Lineage con ibu ions o he EOM unc ional uni . (A-B”) Neu al c es (Tg:Wn 1
C e
;R26
Tom
) and (C-D”) mesode mal (Mesp1
C e
;
R26
Tom
) lineage con ibu ions o he pe iocula egion o E13.5 emb yos, combined wi h immunos aining o dToma o (Tom), GFP (Tg:
Scx-GFP epo e ), and muscle (PAX7/MYOD/MYOG, myogenic ma ke s). Co onal sec ions a en al (A-A”, C-C”) and do sal (B-B”,
D-D”) le els. As e isks in B’,B” deno e Tom nega i i y a he endon o igin. As e isks in C’,C” deno e Tom nega i i y a he endon inse ion
si e. (A1, D1) High-magni ica ion iews o muscle a eas in panels Aand D. (n = 3 pe condi ion). a, an e io ; c, cho oid; cs, co neal s oma;
d, do sal; E, emb yonic day; eom, ex aocula muscle; l, la e al; m, medial; MCT, muscle connec i e issue; p, pos e io ; , e ina; i, endon
inse ion; o, endon o igin; , en al.
h ps://doi.o g/10.1371/jou nal.pbio.3000902.g001
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conclude ha EOM pa e ning in he mouse occu s by spli ing om a single mass o myo-
genic p ogeni o s ha a ge he eyeball, wi h he mos d ama ic mo phogene ic changes ak-
ing place be ween E11.75 and E12.5.
The EOMs inse in o he scle a, a dense ib ous laye de i ed om he POM [40,44].
PITX2 is a well-es ablished ma ke o bo h he POM and EOM p ogeni o s ([40], S2A–S2C
Fig), and oci o cell dea h in he POM we e sugges ed o label he endon a achmen posi ions
o he 4 ec i muscles [51]. To be e unde s and he de elopmen o he EOM inse ions, we
i s pe o med whole-moun immunos aining o he POM ma ke PITX2 and LysoT acke
Red s aining o de ec p og ammed cell dea h on li e emb yos [52]. WMIF o PITX2 a
E11.75 e ealed a ing o POM cells exp essing high le els o PITX2 ha o med a con inuum
wi h low-exp essing cells ex ending owa ds he base o he EOM anlage (Fig 2D and S2
Video). The pa e n o LysoT acke Red s aining in he POM- ing was highly dynamic, whe e
4 oci o apop osis we e p esen a E11.75 a he ho izons o he eye bu eg essed p og essi ely
om E12.5 onwa ds (Fig 2E). To con i m ha hese oci de ine endon a achmen posi ions
in he POM, we pe o med whole-moun imunos aining o myogenic and endon p ogeni o s
on LysoT acke -s ained emb yos. Su p isingly, we obse ed ha e en be o e muscle spli ing
ini ia ed, Scx-GFP+ condensa ions b idged he edges o he EOM anlage and he 4 LysoT acke
Red+ oci in he POM (E11.75, S3 Video), p esaging he a achmen si es o he u u e 4 ec i
muscles (E12.5, S4 Video).
We nex wan ed o unde s and he ela ionship be ween oci o apop osis in he POM and
he es ablishmen o he endon inse ion si es pe se. In he de eloping limb and jaw, bone
supe s uc u es o idges, gene a ed by a unique se o p ogeni o s ha co-exp ess Scx and
Fig 2. De elopmen al ime cou se o EOM de elopmen . (A-C) WMIF o MYOD/MYOG/Desmin (myogenic di e en ia ion ma ke s) (A, B) and
MyHC (myo ibe s) (C) a he indica ed emb yonic s ages. EOMs we e segmen ed om adjacen head s uc u es and 3D- econs uc ed in Ima is
(Bi plane). (A’-C’) EOMs a e shown as isosu aces o cla i y o isualiza ion. Medial iews as schemes (le eye). (D) WMIF o MYOD/Desmin
(labeling he EOM anlage, isosu ace) and PITX2 (labeling he POM and EOM anlage) on E11.75 emb yos (le eye). La e al and medial iews as
schemes. (E) Whole-moun LysoT acke Red (LysoR) s aining o he pe iocula egion a he indica ed s ages (le eye). The POM is delimi ed wi h
dashed lines. As e isks indica e apop o ic oci wi h educed in ensi y om E12.5 onwa ds. anl, anlage; a, an e io ; d, do sal; 3D- ec, 3D econs uc ion;
E, emb yonic day; i, in e io EOM anlage p ojec ion; io, in e io oblique; i , in e io ec us; l, la e al; lg, lac imal gland sulcus; l , la e al ec us; m,
medial; m-pom, medial pe iocula mesenchyme; m , medial ec us; p, pos e io ; pom, pe iocula mesenchyme; pom- , pe iocula mesenchyme ing;
b, e ac o bulbi; s, supe io EOM anlage p ojec ion; so, supe io oblique; s , supe io ec us; , en al; WMIF, whole-moun immuno lu o escence.
h ps://doi.o g/10.1371/jou nal.pbio.3000902.g002
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SRY-box con aining gene 9 (SOX9), p o ide a s able ancho ing poin o muscles ia endons
[53–56]. Al hough EOMs inse in o a non-bone NCC-de i ed s uc u e, ea ly ma ke s o p e-
commi ed ca ilage, such as SOX9, a e exp essed in he POM [57]. To examine he ime
cou se o de elopmen o EOM inse ions in g ea e de ail, we immunos ained Tg:Scx-GFP
and Tg:Wn 1
C e
;R26
Tom
;Scx-GFP co onal (Fig 3A–3J” and S3A–S3F’ Fig) and ans e se (S3G–
S3H” Fig) sec ions o PITX2 and SOX9. Be ween E11.5 and E13.5, PITX2 ma ked all cells o
he la e al-mos NCC-de i ed POM be ween he su ace ec ode m and e ina (Fig 3B and 3F,
and S2A–S2B” Fig), which co esponds o he POM- ing obse ed in 3D iews (Fig 2D). In
his egion, Scx-GFP exp ession was ini ially de ec ed in a sal -and-peppe pa e n (Fig 3C and
S3E Fig) bu became p og essi ely limi ed o he o ming endon ips (Fig 3G and S3F Fig).
SOX9 exp ession o e lapped wi h ha o PITX2 (Fig 3D, 3H,S3E’ and S3F’ Fig) bu became
mo e es ic ed o he inse ion si e by E13.5 and wi h a pa e n complemen a y o Scx-GFP+
(Fig 3G and S3F Fig). No ably, Scx-GFP+ SOX9+ cells could be de ec ed be ween E11.5 and
E13.5 a he in e ace be ween mu ually exclusi e Scx-GFP+ and SOX9+ cells (S3A–S3C” Fig),
esembling wha was obse ed du ing endon- o-bone a achmen o ma ion in o he egions
in he emb yo [53–56].
A he pu a i e inse ion si e, he dToma o s aining in he NCC-de i ed la e al POM ini-
ially appea ed as punc a e (Fig 3A), and eminiscen o he apop o ic domains obse ed in 3D
iews (Fig 2E). LysoT acke Red and TUNEL s aining con i med cell dea h o SOX9+ PITX2
+ POM cells (Fig 3I–3J”,S3E, S3E’ and S3G–S3H” Fig) ha we e a a highe densi y han he
mo e medial POM cells (Fig 3K,S1 Da a). Gi en ha LysoT acke Red+ cells could no longe
be seen a E13.5 (S3F and S3F’ Fig), hese da a sugges ha a he inse ion si es o he ec i
muscles in he POM, oci o apop osis ma k he places whe e cell compac ion and e inenmen
o he SOX9 exp ession pa e n will ake place.
Majo POM emodeling e en s could be de ec ed by E14.5. EOM endons co-exp essed
Scx-GFP, Tenascin, and PITX2, bu su p isingly, SOX9 exp ession became es ic ed o he
hin scle al laye and e inal pigmen ed epi helium (RPE) (Fig 3L–N’ and S3D–S3D” Fig).
Al oge he , hese esul s show ha de elopmen o he EOMs, hei endons and inse ion si es
o e lap spa io empo ally and hus, migh be egula ed in a coo dina ed manne . Mo eo e ,
simila ly o o he loca ions in he body, Scx and SOX9 show dynamic exp ession pa e ns a
he inse ion si e, bu addi ional speci c hallma ks, no ably he p esence o cell compac ion
and apop o ic oci, seem o be cha ac e is ic o his ana omical loca ion.
Abno mal EOM mo phogenesis in mu an s wi h ocula mal o ma ions
Ha ing assessed how mo phogenesis o EOMs and hei inse ion si es is coo dina ed, we se
ou o in es iga e he ole o he a ge o gan, he eyeball, on he es ablishmen o he EOM
unc ional uni . To his end, we pe o med mic o-compu ed omog aphy (mic o-CT) scans in
mouse mu an s wi h a spec um o ocula pe u ba ions. Fi s , we examined small eye (Sey)
Pax6 (Pai ed box 6) mu an emb yos (Pax6
Sey/Sey
), in which eye de elopmen is a es ed a he
op ic esicle s age [58–60]. EOM pa e ning in Pax6
Sey/+
emb yos p oceeded no mally (S4A
and S4B Fig), whe eas in he Pax6
Sey/Sey
mu an , he EOMs appea ed as a single mass on op o
a udimen a y op ic esicle (S4C Fig). As Pax6 is exp essed in he op ic esicle and o e lying
ec ode m ha o ms he lens and co nea [58], bu no in EOMs, hese obse a ions sugges a
non-cell-au onomous ole in EOM pa e ning. Simila ly, in LIM homeobox 2 (Lhx2) mu an
emb yos (Tg:Lhx2
C e
;Lhx2
l/ l
) emb yos, in which inac i a ion o he Lhx2 gene in eye commi -
ed p ogeni o cells leads o a degene a ion o he op ic esicle a E11.5 [61], EOM pa e ning
was se e ely a ec ed, and ew EOM submasses we e obse ed (S4D–S4E’ Fig). Finally, in
cyclopic emb yos esul ing om in e sion o Sonic hedgehog (Shh) egula o y egions, EOMs
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Fig 3. De elopmen al ime cou se o EOM inse ions in he POM. (A-D) Immunos aining o la e al POM in co onal sec ions o E11.75 Tg:
Wn 1
C e
;R26
Tom
;Scx-GFP emb yos o he indica ed ma ke s. B, Immunos aining o sec ion adjacen o he one shown in (A, C, D; channels spli o
cla i y). (E-H) Immunos aining o la e al POM in co onal sec ions o E13.5 Tg:Wn 1
C e
;R26
Tom
;Scx-GFP emb yos o he indica ed ma ke s. G,
Immunos aining o sec ion adjacen o he one shown in (E, F, H; channels spli o cla i y). B acke show o e lap be ween Scx-GFP and SOX9
exp ession domains (high-magni ica ion iews in S3C” Fig). The as e isk in Gpoin s o Scx-GFP-nega i e SOX9+ la e al-mos POM a he inse ion
si e. (I,J) Immunos aining o la e al POM in co onal sec ions o E12.5 Tg:Scx-GFP emb yos p e ea ed wi h LysoR. A owheads in J’ poin o LysoR
+ SOX9+ cells. (J”) TUNEL and LysoR s aining o a sec ion adjacen o he one shown in (I,J). (K) Quan i ica ion o he o al numbe o SOX9+ cells
pe squa e-mic ome e in LysoR+ egions ( ed squa e, J) and mo e medial LysoR-nega i e egions (yellow squa e, J). Mann–Whi ney es . Cell
densi y was 33% highe in he LysoR+ a ea compa ed wi h he mo e medial POM. See S1 Da a o indi idual alues. (L-N’) Immunos aining on
co onal sec ions o E14.5 Tg:Scx-GFP emb yos. (L,L’) Tnc and Scx-GFP co-localize in endons a le el o inse ion (a owhead). (M,M’) SOX9
exp ession in he POM is g ea ly educed a he inse ion si e and no longe o e laps wi h Scx-GFP (as e isk, cyan). Low le els o SOX9 exp ession in
he RPE and scle a. (N,N’) PITX2 emains exp essed in he POM a he inse ion si e o e laping wi h Scx-GFP (as e isk) and in he scle a. MyHC
(L’) and SMA (M-N’) we e used o label EOM muscle. Dashes in L-N we e d awn acco ding o GFP labeling in L’-N’. Images in A-N’ co espond o
inse ion si e o supe io ec us muscle in he POM as shown in he scheme. (n = 3 pe condi ion). a, an e io ; apop, apop osis spo s; d, do sal; E,
emb yonic day; eom, ex aocula muscle; l, la e al; LysoR, LysoT acke Red; m, medial; p, pos e io ; POM, pe iocula mesenchyme; pe, e inal
pigmen ed epi helium; s, scle a; , endon; i, endon inse ion, , en al.
h ps://doi.o g/10.1371/jou nal.pbio.3000902.g003
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unde wen spli ing and p ojec ed owa ds he cen ally loca ed ec opic eye, al hough wi h an
abno mal 3D a angemen as epo ed o human cyclopia condi ions (S4F–S4F” Fig) [7].
Toge he wi h p e ious s udies in which su gical emo al o he eye a speci ic imepoin s o
de elopmen esul s in smalle EOMs [9,62], ou obse a ions poin o he eye as a c i ical
o ganize o EOM pa e ning.
Muscle pa e ning depends on e inoic acid signaling o neu al o igin
To s udy he ole o a ge o gan de i ed cues in EOM pa e ning, we in es iga ed he ole o
e inoic acid signaling, which plays mul iple pa ac ine oles du ing emb yonic eye de elop-
men [33]. As ALDH1A1-3 a e a e-limi ing enzymes in he p oduc ion o ATRA [63], we
cha ac e ized hei exp ession in he pe iocula egion a he ime o EOM pa e ning. Be ween
E10.5 and E12.5, Aldh1a1 was exp essed s ongly in he do sal e ina and lens, Aldh1a2 was
exp essed in he empo al mesenchyme, and Aldh1a3 was mos s ongly exp essed in he en-
al e ina and RPE (S5A–S5A” Fig) [34,36]. A he p o ein le el, ALDH1A3 was de ec ed on
issue sec ions in he su ace ec ode m, p esump i e co neal epi helium, e ina, and RPE (Fig
4A). In e es ingly, ALDH1A3 was also exp essed in he op ic s alk be ween E10.5 and E12.5
and hus cen ally posi ioned wi h espec o EOM de elopmen (Fig 4A). To a ge he e i-
noic signaling pa hway (Fig 4B), we used Aldh1a3
-/-
[64] and Rdh10
-/-
[65] mu an s. We also
adminis e ed he BMS493 inhibi o (pan-RAR in e se agonis ) o p egnan emales e e y 10–
12 hou s be ween E10.5 and E11.75, i.e., p eceeding he ini ia ion o muscle spli ing (Fig 2A–
2C), and once NCC mig a ion o he POM was inalized [66,67]. Mic o-CT analysis showed
ha Aldh1a3
-/-
and BMS493- ea ed emb yos displayed eye en aliza ion and a sho ened
op ic ne e when compa ed wi h con ols (S5B Fig), bu hey e ained he o e all o ganiza ion
o he nasal capsule and o bi (S5C Fig). Ven al and la e al iews o he 3D- econs uc ed
EOMs (Fig 4C–4E), showed ha Aldh1a3
-/-
and BMS493- ea ed emb yos lacked he s anda d
3D a angemen o 4 ec i and 2 oblique muscles obse ed a E13.5 in con ol emb yos. Ne e -
heless, in all cases, EOMs o igina ed medially om he hypochiasma ic ca ilages o he p e-
sphenoid bone, indica ing ha he o e all o ien a ion o he EOMs was p ese ed (Fig 4D”‘).
Gi en ha he EOMs a e mo e a ec ed a hei inse ion han hei o igin le el upon ATRA
de iciency, his inding sugges s ha EOM pa e ning is, in pa , modula .
To analyze EOM and endon pa e ning wi h highe esolu ion, we pe o med whole-
moun immunos ainings o di e en ia ed myo ibe s and endon wi h MyHC and Tnc
(Tenascin) an ibodies (Fig 4F–4H’). On medial and la e al iews o 3D- econs uc ed EOMs,
only he e ac o bulbi and supe io ec us could be clea ly iden i ied among he non-seg e-
ga ed muscle ibe s in Aldh1a3
-/-
emb yos (Fig 4G and 4G’). As expec ed om global in alida-
ion o e inoic acid signaling, EOM pe u ba ion was mo e se e e in BMS493- ea ed
emb yos (Fig 4H and 4H’). The supe io oblique was absen o con inuous wi h he an e io
pa o he anlage, and he medial po ion o he e ac o bulbi was hicke and less clea ly iso-
la ed om he es o he anlage (Fig 4H). In bo h condi ions o ATRA de iciency, Tenascin
and Scx-GFP+ cells we e p esen a he ips o he indi idual, hough mispa e ned, muscles a
his s age (Fig 4G’, 4H’, and S5 Video). Analysis o Aldh1a3
-/-
and BMS493- ea ed emb yos
e ealed, on a e age, a 26% educ ion in he EOM olume compa ed wi h con ols (Fig 4I,S2
Da a). Howe e , MyHC+ myo ibe s we e p esen in Aldh1a3
-/-
and BMS493- ea ed emb yos
(Fig 4G and 4H), sugges ing ha EOM di e en ia ion was no o e ly a ec ed in hese condi-
ions. Ins ead, hese obse a ions sugges ha EOM ibe alignmen and seg ega ion o he
muscle masses a e dependen on e inoic acid signaling.
As dose and empo al con ol a e c i ical in he con ex o e inoic acid signaling [27,32,68],
we pe o med o he BMS493 injec ion egimes be ween E10.5 and E12.5 (S1 Table). EOM
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Fig 4. Ex aocula muscle mo phogenesis is dependen on ATRA. (A) Immunos aining o ALDH1A3 on co onal sec ions o E10.5, E11.5, and E12.5 con ol
emb yos (n = 3). (B) Scheme o e inoic acid signaling pa hway wi h key enzymes o oxida ion o e inol (Vi amin A) and e inaldehyde (pink) and mu an s/
inhibi o s used in his s udy (blue). (C-D”’) Mic o-CT-based 3D- econs uc ion o chond ogenic mesenchymal condensa ions o nasal capsule, abecula
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condensa ions and inse ion si es in he POM- ing. These pheno ypes a e al eady e iden
om E11.75, a s age in which he EOM s ill igu es as a muscle anlage. In e es ingly, analysis
o chick and mouse limb de elopmen also iden i ied a sho ime window in which limb mes-
enchyme pe u ba ions esul la e in muscle and endon pa e ning de ec s [71,94]. Mo eo e ,
concomi an muscle and endon de ec s ha e been obse ed in he chick limb and zeb a ish
jaw upon e inoic acid signaling mis egula ion [15,95]. Al oge he , hese esul s sugges ha a
ce ain ana omical loca ions, including he EOMs, endogenous local a ia ions in he concen-
a ion o e inoids con ibu e o he es ablishmen o Scx-GFP+ condensa ions, a c i ical s ep
in muscle unc ional uni assembly.
Role o RA in he in eg a ion o EOM pa e ning and inse ion si e
o ma ion
The EOMs inse in o he scle a, a non-bone s uc u e in mammals. The gene a ion o 4
a achmen poin s ha p ecisely mi o he posi ion o he 4 ec i muscles is a mo phogenesis
conund um, as de ails on hei speci ica ion a e sca se. Fou mesenchymal condensa ions
ha e been desc ibed a he pe iphe y o he de eloping op ic cup in ca and human emb yos,
in posi ions acing he loca ions o he ec i muscles [96,97]. In he mouse, a equi alen de el-
opmen al imepoin s (E11.5-E12.5) and homologous posi ions, apop o ic oci we e obse ed
and sugges ed o p o ide a achmen o he ec i muscles [51]. Using 3D imaging, we showed
ha a E11.75, he apop o ic oci a e aligned wi h Scx-GFP+ condensa ions ha p ojec medi-
ally, be o e any sign o spli ing o he EOM anlage. As he apop o ic oci dec ease in size om
E12.5 onwa ds concomi an wi h ongoing muscle spli ing and endon o ma ion, i is possi-
ble ha hey ini ially p esage he POM o u u e endon inse ions bu a e no equi ed o
a achmen pe se. These oci appea in condensed SOX9+ PITX2+ cell domains o he u u e
scle a; howe e , o ou knowledge, hei p esence has no been epo ed in supe s uc u es a
o he ana omical loca ions.
In ag eemen wi h he ole o RA in he induc ion o cell dea h in o he loca ions in he
emb yo [64], ou s udy shows ha upon pe u ba ion o RA signaling, he apop o ic oci in he
POM a e absen o se e ely educed. Howe e , i is also possible ha he loss o apop o ic oci
upon RA inhibi ion is seconda y o loss o mispa e ning o he SOX9+ PITX2+ POM- ing.
Thus, i will be o in e es o de e mine whe he null o NCC-speci ic mu a ions in PITX2
a ec he pa e ns o amoun o na u ally occu ing cell dea h in he pe iocula condensa ions.
Al hough ou wo k sheds ligh on he gene ic mechanisms ha egula e EOM inse ion, he
cellula mechanisms unde lying his p ocess emain unde ined. I is emp ing o specula e ha
he apop o ic oci a e ela ed o he disappea ance o signaling cen e s [98,99] o compac ion-
media ed cell dea h and ex usion [100]. Fu u e expe imen s di ec ed owa ds modi ying he
amoun o iming o cell dea h will be in o ma i e.
Few epo s ha e demons a ed ha mispa e ning o speci ic muscles is coupled wi h abe -
an supe s uc u es [13,70,72] o emo al o he p ospec i e endon a achmen si es [95].
Ou esul s a e in ag eemen wi h ha model, gi en ha EOM mispa e ning is concomi an
wi h abe an EOM inse ions. As mammals do no de elop a ca ilage laye wi hin he scle a
[57,101], ansien exp ession o SOX9 in he POM- ing o mouse emb yos a he ime o
EOM pa e ning is in iguing. In he de eloping limb and jaw, endon and bone a e a ached
by a ansi ional connec i e issue ha de elops om bipo en p ogeni o s ha co-exp ess Scx
and SOX9 [53–56], be o e p ogeni o s a e alloca ed o ei he ca ilage o endon lineages. Sim-
ila ly, we obse ed a ansien popula ion o Scx-GFP/SOX9 double-posi i e cells a he inse -
ion si es in he POM. In his con ex , ou da a sugges ha SOX9 exp ession may ep esen a
edeploymen o he de elopmen al module o endon a achmen , despi e he ac ha he e
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is no de ini i e ca ilage in he mammalian scle a. Gene ic s udies will be equi ed o ul i-
ma ely assess he unc ional ele ance o his popula ion in EOM a achmen .
Finally, ansc ip ome analysis e ealed he exis ence o global and egional egula o y
modules o supe s uc u e pa e ning in he limb bones, o e ing a mechanism o induce a i-
a ions in a achmen si es wi hou ha ing o ew i e he en i e skele ogenic p og am [102]. As
se e al ma ke s o he POM (Pi x2,Foxc1/2) and e inoic acid signaling modula o s (Cyp26a1/
b1) we e iden i ied as pa o speci ic limb supe s uc u e signa u es [102], i is emp ing o
specula e ha hose genes also play a conse ed ole in he gene a ion o he a achmen mod-
ule o he EOMs.
Conclusion
The de eloping eye has been p oposed o be key o ganize o c anio acial de elopmen , inde-
penden o i s ole in ision [103]. This no ion is based on he ole o he eye o p ope NCC
mig a ion o he pe iocula egion [104], he common associa ion o ocula and c anio acial
de elopmen al abno mali ies [105] and he ini ial de elopmen o an eye in blind e eb a e
species [103,106,107]. Mo eo e , as he appea ence o a "came a- ype” eye is a e eb a e inno-
a ion [108], and he EOM a e al eady p esen in lamp ey [4], i is possible ha muscles and
hei a ge issue migh ha e co-e ol ed. He e, by cha ac e izing coo dina ed pa e ning o
he EOMs, hei espec i e endons and inse ions, ou indings illus a e u he he ole o
he de eloping eye as a signaling cen e allowing in eg a ion o he EOM unc ional uni in he
POM. Ou esul s show ha he issue in e ac ions du ing he de elopmen o his c anio acial
muscle uni sha e ea u es wi h hose desc ibed in he limb bu wi h addi ional egional p op-
e ies (e.g., cell dea h, speci ica ion o a achmen s in he scle a) ha seem o ha e been speci i-
cally inco po a ed in o his g oup. Mo eo e , he capaci y o ins uc muscle pa e ning
h ough a ia ions in connec i e issue de i a i es p o ides a mechanism o explain he plas-
ici y o he musculoskele al sys em, a he ana omical and in e species le els, while ensu ing
unc ional in eg a ion du ing e olu ion. These indings also imply ha he gene a ion o mus-
culoskele al uni s do no equi e majo es uc u ing o he de elopmen al p og ams o all he
issues implica ed. Ins ead, co-op ion o a gene al p og am and simul aneous addi ion o local
ea u es appea o elabo a e musculoskele al di e si y.
Ma e ials and me hods
Mouse s ains and animal in o ma ion
Animals we e handled as pe Eu opean Communi y guidelines, and he e hics commi ee o
he Ins i u Pas eu (CETEA) app o ed p o ocols (Licence 2015–0008). The ollowing s ains
we e p e iously desc ibed: Aldh1a3
KO
[64], Mesp1
C e
[46], Tg(RARE-Hspa1b-c e/ERT2), desig-
na ed he e as Tg:RARE-C eERT2 [73], R26
Tom
(Ai9; [74]), R26
mTmG
[75], Tg:Scx-GFP [48],
My 5
nlacZ
[109], Myod
iC e
([79,110]), Ra b
lox
[111], Ra g
lox
, [112], Rdh10
KO
[65], Pax6
Sey/Sey
[59], and Tg:Wn 1
C e
[45]. R26
RAR403
([78]) mice con ain a loxP- lanked STOP sequence
ups eam o a mu a ed human RAR alpha gene, which beha es as a dominan nega i e ecep o
o all nuclea ecep o s upon C e-media ed ecombina ion. Rdh10
KO
emb yos we e ecei ed
om he labo a o y o Pascal Dolle
´.Tg:Wn 1C e;Ra b
l/ l
;Ra g
l/ l
emb yos we e ecei ed om
he labo a o y o Vale
´ ie Dupe
´.Pax6
Sey/Sey
and con ol emb yos we e ecei ed om he labo a-
o y o James B iscoe. Tg:Lhx2
C e
;Lhx2
l/ l
[61] we e ecei ed om he labo a o y o Lei Ca ls-
son. Shh(in C-6)2 emb yos we e ecei ed om he labo a o y o F anc¸ois Spi z.
To gene a e expe imen al emb yos o Mesp1
C e
o Tg:Wn 1
C e
oge he wi h Tg:Scx-GFP
and R26
Tom
lineage acings, C e/+ males we e c ossed wi h Tg:Scx-GFP;R26
Tom/Tom
emales.
Mice we e kep on a mixed gene ic backg ound C57BL/6JRj and DBA/2JRj (B6D2F1, Jan ie
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Labs). Mouse emb yos and e uses we e collec ed be ween E10 and E18.5, wi h noon on he
day o he aginal plug conside ed as E0.5. P egnan emales we e eu hanized by ce ical
disloca ion.
To induce ecombina ion wi h he Tg:RARE-C eERT2;R26
mTmG
line, 5 mg o amoxi en
(Sigma #T5648) we e adminis e ed by ga age o p egnan emales. A 25 mg/ml s ock solu ion
in 5% e hanol and 95% sun lowe seed oil was p epa ed by ho ough esuspen ion wi h ock-
ing a 4˚C.
To inhibi e inoic acid signaling, p egnan emales o ele an geno ypes we e injec ed
in ape i oneally wi h 10 mg/kg o BMS493 (Toc is, 3509), a pan-RAR in e se agonis . A
5-mg/ml BMS493 s ock solu ion in DMSO (SIGMA, D2650) was p epa ed and s o ed in single
use aliquo s a −20˚C in igh cap ubes. A he ime o injec ion, he aliquo was hawed, 200 μl
o s e ile PBS was added pe 50μl aliquo and injec ed immedia ely.
Immuno luo escence, de ec ion o cell dea h, and in si u hyb idiza ion
Emb yos we e ixed o 2.5 hou s in 4% pa a o maldhehyde (PFA; 15710, Elec on Mic oscopy
Sciences) in PBS wi h 0,2–0,5% T i on X-100 (acco ding o he emb yonic s age) a 4˚C and
washed o e nigh a 4˚C in PBS. Fo c yosec ioning, emb yos we e equilib a ed in 30%
suc ose in PBS o e nigh a 4˚C and embedded in OCT. C yosec ions (16–18 μm) we e
allowed o d y a RT o 30 minu es and washed in PBS. Immunos aining was pe o med as
desc ibed in [113]. An an i-DsRed an ibody ( abbi ) was used o enhance he R26
Tom
signal
excep when co-s aining was pe o med wi h an ibodies aised in abbi . In his case, he
endogenous epo e signal was used. An an i-GFP an ibody (chicken) was used o enhance
he R26
mTmG
and Tg:Scx-GFP signal in whole-moun and sec ion immunos ainings.
Scx in si u hyb idiza ion was pe o med as pe manu ac u e ins uc ions using he RNA-
scope Mul iplex Fluo escen V2 Assay [113] and RNAscope P obe-Mm-Scx p obe (Ca No.
439981). Sample p e- ea men s we e pe o med as desc ibed in [113]. Signal de elopmen
was ca ied ou using Opal 570 Reagen Pack (FP1488001KT, Pe kin Elme ) dilu ed 1:1,500 in
he ACD-p o ided TSA bu e and ollowed up by immunos aining.
TUNEL s aining, which ma ks double-s and b eaks, was pe o med wi h he In Si u Cell
Dea h De ec ion Ki /Fluo escein (Roche, 11 684 795 910). Slides we e i s p e ea ed wi h a
2:1 mix o E hanol:Ace ic Acid o 5 minu es a −20˚C, washed wice o 20 minu es wi h PBS
a RT, and p ocessed o TUNEL s aining as desc ibed by he manu ac u e .
Fo whole-moun immunos aining, emb yos we e ixed and washed as desc ibed he e and
dehyd a ed in 50% Me hanol in PBS and wice in 100% Me hanol, 30 minu es each a RT and
kep a −20˚C ill needed. Heads we e ehyd a ed, he pe iocula egion was mic odissec ed in
PBS, and immunos aining pe o med as desc ibed in [113]. Fo emb yos olde han E13.5, he
al e na i e p e ea men (con aining 0.1% Tween-20, 0.1% T i onX100, 0.1% Deoxychola e,
0.1% NP40, 20% DMSO in PBS) and p ima y an ibody immunolabeling s eps o he idisco
p o ocol (h ps://idisco.in o/idisco-p o ocol/) we e gene ally used. In all cases, seconda y an i-
bodies we e applied in blocking bu e as desc ibed in [113] o >4 days a 4˚C wi h ocking.
A e immunolabelling, samples washed in 0.1% Tween/PBS, dehyd a ed in 50% Me hanol in
PBS and 100% Me hanol 10 minu es each a RT, clea ed wi h a mix benzyl alcohol and benzyl
benzoa e (BABB), and moun ed o imaging as desc ibed in [114].
LysoT acke Red s aining was used o de ec ion o cell dea h in whole-moun li e issues
as i e eals lysosomal ac i i y co ela ed wi h inc eased cell dea h [52,115]. Emb yos we e
quickly dissec ed in HBSS (In i ogen, 14025–092), incuba ed in 2-ml ubes con aining 5 μM
o LysoT acke Red DND-99 (Molecula P obes, L7528) 45 minu es a 37˚C wi h ocking in
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he da k, washed wice in PBS, ixed and p ocessed o c yosec ions o whole-moun immu-
nos aining as desc ibed p e iously.
An ibodies
P ima y and seconda y an ibodies used in his s udy a e lis ed in S2 Table. To de ec di e en i-
a ing EOMs, we used a-smoo h muscle ac in (SMA), which is ansien ly exp essed in di e en-
ia ing myoblas s and myo ubes [116–118]; Desmin, an ea ly cy oskele al muscle p o ein
exp essed in myoblas s, myo ubes, and myo ibe s [116,119]; and myosin hea y chain (MyHC)
o label sa come ic myosin [120].
S a ic imaging
A Zeiss S eREO Disco e y V20 mac oscope was used o imaging he endogenous luo escence
o whole emb yos a he ime o dissec ion. Fo issue sec ions and whole-moun immunos-
aining o clea ed emb yos, a LSM700 and a LSM800 lase -scanning con ocal mic oscope wi h
ZEN so wa e (Ca l Zeiss, www.zeiss.com) we e used.
All images we e assembled in Adobe Pho oshop and InDesign (Adobe Sys ems). Volume-
3D ende ing o he Z-s ack se ies was pe o med in Ima is ( e sion 7.2.1) so wa e (Bi plane).
Fo ease o EOM isualiza ion, he Z-s ack olumes we e i s manually segmen ed o de ine
he EOM o he whole POM a ea using he Isosu ace Ima is unc ion. The signal ou side he
isosu ace was se o ze o, he co esponding channel duplica ed, and subsequen ly, a new iso-
su ace was c ea ed using au oma ic h esholding on he new channel. This new isosu ace was
used o calcula e he co esponding EOM olumes.
In si u hyb idiza ion
Whole-moun in si u hyb idiza ion wi h digoxigenin-labeled an isense mRNA p obes was pe -
o med as desc ibed p e iously in [90]. The Aldh1a1, Aldh1a2, and Aldh1a3 p obes we e p e-
iously desc ibed in [34,36].
mic o-CT analysis
The issue con as ing p o ocol has been adap ed om he o iginal p o ocol de eloped by
[121] and applied o mouse emb yos as desc ibed in [122] and [86]. Fo issue con as ing,
E13.5 emb yos we e s ained in 0.5% phospho- ungs ic acid (PTA) in 90% me hanol o 4 days,
E15.5 emb yos we e s ained in 0.7% PTA in 90% me hanol o 1 week.
The mic o-CT analysis o he emb yos was conduc ed using he GE phoenix | ome|x L 240
(GE Sensing and Inspec ion Technologies GmbH, Ge many), equipped wi h a 180 kV/15W
maximum powe nano ocus X- ay ube and la panel de ec o DXR250 wi h 2048 ×2048
pixel, 200 ×200 μm pixel size. The exposu e ime o he de ec o was 900 milliseconds in e e y
posi ion o e 360˚. Th ee p ojec ions we e acqui ed and a e aged in e e y posi ion o educ-
ion o he noise in mic o-CT da a. The accele a ion ol age was se o 60 kV and ube cu en
o 200 μA. The adia ion was il e ed by 0.2 mm o aluminium pla e. The oxel size o ob ained
olumes (depending on a size o an emb yo) appea ed in he ange o 2–6 μm. The omo-
g aphic econs uc ions we e pe o med using GE phoenix da os|x 2.0 3D CT so wa e (GE
Sensing and Inspec ion Technologies GmbH, Ge many). The EOM, eye, and ca ilages in he
emb yo head we e segmen ed by an ope a o wi h semiau oma ic ools wi hin A izo - 3D
image da a p ocessing so wa e (FEI, USA). The 3D segmen ed egion was ans o med o a
polygonal mesh as an STL ile and impo ed o VG S udio MAX 2.2 so wa e (Volume G aph-
ics GmbH, Ge many) o su ace smoo hing and 3D isualiza ion.
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Cell isola ion om he pe iocula egion and bulk cell cul u es
The pe iocula egion o Tg:RARE-C eERT2;R26
mTmG
emb yos (including he eye i sel ) was
mic odissec ed and minced wi h small scisso s inside a 2-ml Eppe ndo ube. Samples we e
incuba ed wi h 1 ml o T ypLE Exp ess (In i ogen, 12604013) o 15 minu es a 37˚C wi h
agi a ion. Samples we e esuspended by gen ly pipe ing up and down 10–15 imes using a
P1000 pipe e. Upon addi ion o 1 ml o cul u e media con aining (10 μg/ml) DNaseI (Roche,
11284932001), samples we e spun 15 minu es a 500ga RT, pelle esuspended in 400 μl o
cul u e media con aining o 20% e al bo ine se um (FBS, Gibco), 1% Penicillin-S ep omycin
(15140, Gibco), and 2% Ul ose G (15950–017, Pall Biosep a) in 50:50 DMEM:F12 (31966
and 31765, Gibco) and pla ed on indi idual wells o 8-well glass-bo om dishes (Ibidi, 80826)
coa ed wi h 1 mg/ml o Ma igel (354234, BD Biosciences). Cells we e allowed o a ach o 8
hou s a 37˚C 5% CO
2
, washed wi h PBS, and ixed o 15 minu es a RT wi h 4% PFA in PBS.
A e ixa ion, cells we e washed in PBS and pe meabilized wi h 0.5% T i on X-100 in PBS o
5 minu es a RT. A e 3 washes in PBS (5 minu es each), cells we e blocked wi h 20% goa
se um in PBS 1 hou a RT. P ima y an ibodies we e added o cells in 2% goa se um in PBS
o 2 h a RT o ON a 4˚C. Cells we e washed 3 imes wi h PBS, incuba ed wi h seconda y
an ibodies o 1 hou a RT, washed in PBS, and kep in PBS o imaging.
S a is ics
The numbe o emb yos o each geno ype used o analysis is indica ed in he igu e legends
and S1 Table. The g aphs we e plo ed, and s a is ical analyses we e pe o med using P ism8
(G aphPad So wa e, Inc). All da a poin s a e p esen ed as mean ±SEM (e o ba s). Indi id-
ual alues can be ound in S1, S2 and S3 Da a iles. S a is ical es s used o analysis a e indi-
ca ed on he espec i e igu e legends. p- alues less han 0.05 we e conside ed signi ican (�p<
0.05; ��p<0.01; ���p<0.001).
Suppo ing in o ma ion
S1 Fig. Lineage con ibu ions o he EOM unc ional uni . (A-D) NCC (Tg:Wn 1
C e
;R26
Tom
;
Scx-GFP) and mesode m (Mesp1
C e
;R26
Tom
;Scx-GFP) lineage con ibu ions o he pe iocula
egion on co onal c yosec ions o E17.5 emb yos, combined wi h immunos aining o endon
(GFP) and muscle (Tnn 3, T oponin T Type 3, di e en ia ed muscle). Sec ions a en al (A,
C) and do sal (B,D) le els. No e ha endon inse ions a he le el o he o bi a e NCC-
de i ed (A), whe eas he endon o igin (D) is mesode m-de i ed. Highe -magni ica ion iews
a he le el o he endon inse ion (A1,C1) and o igin (B1,D1) a e shown as inse s. As e isk in
D1 indica es Scx-GFP+ cells in he pe ichond ium o he hypochiasma ic ca ilage. (E-F’)
NCC (Tg:Wn 1
C e
;R26
Tom
) and mesode m (Mesp1
C e
;R26
Tom
) lineage con ibu ions o he
pe iocula egion on co onal c yosec ions o E13.5 emb yos, combined wi h immunos aining
o TCF4 and muscle (PAX7/MYOD/MYOG, myogenic ma ke s). No e ha TCF4 is
exp essed obus ly in connec i e issue ib oblas s and a lowe le els in myogenic cells. Whi e
a owheads in (E’,F’) ma k Tom+, TCF4+, myogenic ma ke -nega i e connec i e issue cells
in he NCC- and mesode m-de i ed a eas. Pink a owheads in (E’,F’) ma k myogenic cells
(TCF4
low
). (G-H’) Co onal c yosec ions o E13.5 Tg:Scx-GFP emb yos, combined wi h immu-
nos aining o TCF4 (connec i e issue, myogenic p ogeni o s) and muscle (PAX7/MYOD/
MYOG). Whi e a owheads in (G’,H’) ma k Tom+, TCF4+, myogenic ma ke -nega i e con-
nec i e issue cells in he NCC and mesode m-de i ed a eas. Pink a owheads in (G’,H’) ma k
myogenic cells (TCF4
low
). (I,J) In si u hyb idiza ion on E12.5 (I) and E13.5 (J) co onal c yo-
sec ions o Scx combined wi h immuno luo escence o muscle (PAX7/MYOD/MYOG).
High le els o Scx mRNA a e seen a he endon o igin and inse ion bu also in he bulk o he
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muscle masses (I’,J’, inse s). a, an e io ; d, do sal; l, la e al; m, medial; MCT, muscle connec i e
issue; E, emb yonic day; eom, ex aocula muscle; NCC, neu al c es cell; p, pos e io ; i, en-
don inse ion; o, endon o igin; , en al.
(TIF)
S2 Fig. Exp ession o PITX2 in he POM. (A-C) Immunos aining on E11.5 (A), E12.5 (B)
and E13.5 (C) co onal sec ions o con ol emb yos o PITX2 (EOM myogenic p ogeni o s,
POM) and SMA (di e en ia ed muscle). A owheads ma k he EOM masses and as e iks
poin o PITX2 exp ession in he medial POM. No e hinning o he la e al POM as de elop-
men p oceeds (b acke s). (n = 3 pe s age). E, emb yonic day; EOM, ex aocula muscle;
POM, pe iocula mesenchyme.
(TIF)
S3 Fig. De elopmen al iming o EOM unc ional uni componen s. (A-D’) Immunos ain-
ing o he indica ed ma ke s on co onal sec ions o E11.5 (A), E12.5 (B, E, F), E13.5 (C) and
E14.5 (D)Tg:Scx-GFP emb yos. (A’-D”) Highe -magni ica ion iews o he an e io endon
inse ions (supe io ec us) in he POM. Whi e a owheads ma k SOX9+ Scx-GFP+ cells up o
E13.5. As e iks in A’-C” poin o SOX9+ Scx-GFP-nega i e a eas. Double as e isk in D’ ma k
Scx-GFP+ SOX9-nega i e endon ips a E14.5. SOX9 exp ession emains a he scle a. (E-F’)
Immunos aining o he indica ed ma ke s on co onal sec ions o E11.75 and E13.5 Tg:Scx-
GFP emb yos p eincuba ed wi h LysoR. A owheads in E,E’ indica e SOX9+ LysoR+ cells in
he E11.75 POM. (G-H”) Immunos aining o he indica ed ma ke s on an e sal sec ions o
E12.5 Tg:Scx-GFP emb yos. (G’-H”) Highe magni ica ion iews o he la e al endon inse -
ions (la e al ec us) showing SOX9+ o PITX2+ LysoR+ a eas. MYOD/MYOG (myogenic
ma ke s) in A-D and MyHC (myo ibe s) in G-H we e used o iden i y he EOMs. (n = 3 pe
s age). a, an e io ; d, do sal; E, emb yonic day; EOM, ex aocula muscle; l, la e al; LysoR,
LysoT acke Red; m, medial; p, pos e io ; POM, pe iocula mesenchyme; s, scle a; , en al.
(TIF)
S4 Fig. EOM mo phogenesis in mu an s wi h eye de ec s. (A-C) Mic o-CT-based 3D econ-
s uc ion o EOM, eyeball, op ic ne e, and lens in E13.5 con ol (A),Pax6
Sey/+
(B), and Pax6-
Sey/Sey
emb yos (C). No e ha in he e ozygo e emb yos, EOM pa e ning p oceeds no mally
despi e ha ing a smalle e ina and lens (n = 3). (D-E) Mic o-CT-based 3D- econs uc ion o
EOM and eyeball in E18.5 con ol (A) and Tg:Lhx2
C e
;Lhx2
l/ l
(B) emb yos. A owheads high-
ligh some ex en o EOM seg ega ion in he mu an . (D’-E’) Co onal sec ions o con ol (D’)
and mu an (E’) emb yos s ained wi h MyHC (di e en ia ed muscle). A owheads indica e
indi idual EOM masses (n = 2). (F-F”) Analysis o EOM pa e ning in E15.5 emb yos con ain-
ing in e sions o Shh genomic egula o y egions (In (6-C2)). (F) Skele al p epa a ion o
mu an emb yos displaying cyclopia. (F’-F”) Mic o-CT-based 3D- econs uc ion o EOM and
eyeball o mu an emb yos (n = 2). a, an e io ; c l, con ol; d, do sal; E, emb yonic day; EOM,
ex aocula muscle; l, la e al; m, medial; mic o-CT, mic o-compu ed omog aphy; p, pos e io ;
, en al.
(TIF)
S5 Fig. EOM mo phogenesis is dependen e inoic acid signaling. (A) Whole-moun in si u
hyb idiza ion o Aldh1a1,Aldh1a2, and Aldh1a3 in E10.5, E11.5, and E12.5 wild ype emb yos
(n = 3). (A’-A”) In si u hyb idiza ion o Aldh1a2 (A’) and immunos aining o ALDH1A2
and MyHC (di e en ia ed muscle) (A”) on E12.5 co onal sec ions. ALDH1A2 is exp essed in
he empo al mesenchyme and adjacen connec i e issues (n = 3). (B) Mic o-CT-based
3D- econs uc ion o eyeball, op ic ne e and lens o E13.5 con ol, Aldh1a3
KO
and BMS493-
ea ed emb yos (n = 2 each geno ype). The lowe ow is a scheme o a sphe e i ing he
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eyeball and lens, and a cylinde o he op ic ne e. No e en aliza ion o he eyeball in
Aldh1a3
KO
and BMS493- ea ed emb yos. (C) Mic o-CT-based 3D- econs uc ion o he mes-
enchymal condensa ions o he nasal capsule and abecula ca ilage o E13.5 con ol (whi e),
Aldh1a3
KO
(yellow) and BMS493- ea ed (g een) emb yos. (D-D’) MyHC WMIF o E13.5
BMS493- ea ed emb yos as desc ibed in S1 Table. The mos se e e pheno ype ob ained in
each condi ion is shown as an isosu ace. Two di e en examples o he pheno ype obse ed
upon ea men VI (a,b) a e shown (mos and leas se e e). An Aldh1a3
KO
emb yo is shown as
compa ison. As e isk deno es ec opic duplica ed SO muscle. (D’) Highe -magni ica ion iews
om D(dashed squa es) wi h examples o adjacen , nonspli muscle masses (1,2) depic ed by
di e en ial ibe o ien a ion. (E-F) MyHC WMIF o E13.5 con ol (E) and Rdh10
KO
(F)
emb yos. No e o al absence o muscle spli ing in mu an (n = 3 pe condi ion). EOMs in D-F
we e segmen ed om adjacen head s uc u es and 3D- econs uc ed in Ima is (Bi plane). c l,
con ol; d , do sal e ina; 3D- ec, 3D- econs uc ion; e, eyelid g oo e; E, emb yonic day;
EOM, ex aocula muscle; io, in e io oblique; i , in e io ec us; le, lens; lgb, lac imal gland
bud; l , la e al ec us; mic o-CT, mic o-compu ed omog aphy; m , medial ec us; nlg, nasola-
c imal g oo e; np, nasal pi ; os, op ic s alk; b, e ac o bulbi; pe, e inal pigmen ed epi he-
lium; so, supe io oblique; s , supe io ec us; m, empo al mesenchyme; , en al e ina,
WMIF, whole-moun immuno luo escence.
(TIF)
S6 Fig. Re inoic acid signaling esponsi eness in he pe iocula egion. (A-C) Mac oscopic
iews o endogenous GFP luo escence o Tg:RARE-C eERT2;R
26mTmG
emb yos. Tam was
injec ed in o p egnan emales and emb yos analyzed a indica ed ime poin s. A oheads indi-
ca e labeling in POM (n>3 pe condi ion). (D) Immunos aining on cells isola ed om he
pe iocula egion o Tg:RARE-C eERT2;R
26mTmG
emb yos o GFP and myogenic ma ke s
(MYOD/MYOG). (E) S a egy used o de e mine esponsi eness o Tg:RARE-C eERT2
epo e in p esence o BMS493. BMS493 was injec ed o p egnan emales e e y 10–12 hou s
be ween E10 and E11.75. Recombina ion was induced by amoxi en a E10.5 (2 hou s a e
he i s BMS injec ion). (F-I) WMIF o MyHC (di e en ia ed muscle) and GFP (ATRA-
esponsi e cells) o con ol (F,G) and BMS493- ea ed emb yos (H,I). BMS493 ea men
be o e and a e amoxi en induc ion e eals a d as ic dec ease in GFP+ cells in he pe iocula
egion (I) compa ed wi h con ols (G). As e isks ma k he loca ion o he op ic ne e (n = 3).
(J) Co onal sec ions (do sal, EOM o igin) o E12.5 Tg:RARE-C eERT2;R26
Tom
;Scx-GFP
emb yos immunos ained o GFP, Tom (ATRA- esponsi e cells) and MYOD/MYOG (mus-
cle). Highe magni ica ion iews as inse s. A owheads in J1 ma k Tom-nega i e myogenic
cells, and as e isks indica e spo adic labeling in myogenic cells. A owheads in J2 ma k Tom+
Scx-GFP+ cells (n = 3). (K-L) WMIF o SMA (di e en ia ed muscle) o Myod
iC e
(con ol, K,
K’) and Myod
iC e
;R26
RAR403
(mu an emb yos, L,L’) (n = 3). EOMs in F-I and K-L’ we e seg-
men ed om adjacen head s uc u es and 3D- econs uc ed in Ima is (Bi plane). a, an e io ;
ATRA, all- ans e inoic acid; c l, con ol; d, do sal; E, emb yonic day; EOM, ex aocula
muscle; l, la e al; m, medial; p, pos e io ; POM, pe iocula mesenchyme; Tam, amoxi en; ,
en al; WMIF, whole-moun immuno luo escence.
(TIF)
S7 Fig. EOM inse ions in he POM a e al e ed upon ATRA de iciency. (A-D’) Immunos-
aining o he indica ed ma ke s on co onal E12 sec ions o con ol (A,C,C’) and
BMS493- ea ed (B,D,D’)Tg:Scx-GFP emb yos p e-incuba ed wi h LysoT acke Red (LysoR).
(C’,D’) Highe -magni ica ion iews o he POM egion. In BMS493- ea ed emb yos, LysoR
and SOX9 s aining a e absen in he POM (D’, as e isk) and SOX9 s aining also missing in he
RPE (D’, double as e isk). (E-N) WMIF o he indica ed ma ke s o E11.75 con ol (E,F,K),
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Aldh1a3
KO
(G,H,M) and BMS493- ea ed (I,J,N) emb yos p e-incuba ed wi h LysoR ( igh
eyes). In la e al iews, nei he Aldh1a3
KO
o BMS493- ea ed emb yos (H,J) show a ull
PITX2+ POM- ing as he con ols (F). A owheads in (G,H) ma k emaining exp ession o
SOX9/PITX2 in he POM o mu an o inhibi o ea ed emb yos. As e isks in (F,H,J) ma k
apop osis spo s in he POM. (K-N) Segmen ed medial iews o pe iocula egion o con ol,
Aldh1a3
KO
o BMS493- ea ed emb yos. Volumes we e unca ed in Z o cla i y. Full iew as
schemes below. The PITX2+ POM- ing is con inuous wi h he medial-POM in con ol
emb yos (K). In mu an and BMS493- ea ed emb yos, esidual PITX2 exp ession in POM is
discon inous wi h he medial-POM (blue a owheads) (M,N). (O-Q’) WMIF o SMA (di e -
en ia ed muscle), GFP (ATRA- esponsi e cells) and PITX2 (muscle p ogeni o s, POM) o
Tg:RARE-C eERT2;R26
mTmG
con ol (O,O’), Aldh1a3
KO
(P,P’) and BMS493- ea ed (Q,Q’)
emb yos. Blue a owheads in Pand Qshow educ ion o loss o ATRA- esponsi e cells in he
pe iocula egion. (O’-Q’) Single Z-planes o he segmen ed olume. Whi e a owhead in O
shows co ec PITX2+ connec i e issue p e-pa e n in he p ospec i e muscle a eas and
a ow ma ks PITX2 exp ession along he muscle masses. Blue a owheads in P’ and Q’ show
educ ion o loss o ATRA- esponsi e cells and PITX2 p e-pa e n in he medial pe iocula
mesenchyme. (R-T) Single cell plane o WMIF o E12 con ol (R), BMS493- ea ed (S) and
My 5
nlacZ/nlacZ
emb yos (T) ( igh eyes) o TCF4 (muscle connec i e issue) and SMA (di e -
en ia ed muscle). As e isks ma k posi ion o he op ic ne e. Whi e a owheads in R,T show
co ec TCF4+ connec i e issue p e-pa e n in he p ospec i e muscle a eas and whi e a ow
ma ks TCF4 exp ession along he muscle masses (R). (U-V) WMIF o GFP and β-gal (myo-
genic p ogeni o s) o E14.5 Tg:Scx-GFP;My 5
nlacZ/+
(U, con ol) and Tg:Scx-GFP;My5
nlacZ/nlacZ
(V, mu an ) emb yos (le eyes). As e isk indica es ew emaining β-gal+ cells in mu an . (U’,
V’) spli GFP channel. A owheads in (V) indica e he co ec posi ion o endon condensa-
ions o he 4 ec i muscles al hough hese a e absen in he mu an . Lowe panel, highe -mag-
ni ica ion iews. a, an e io ; ATRA, all- ans e inoic acid; anl, anlage; apop, apop osis; c l,
con ol; d, do sal; E, emb yonic day; eom, ex aocula muscle; l, la e al; m, medial; m-pom,
medial pe iocula mesenchyme; l-pom, la e al pe iocula mesenchyme; POM, pe iocula mes-
enchyme; pom- , pe iocula mesenchyme ing; p, pos e io ; pe, e inal pigmen ed epi helium;
, en al; WMIF, whole-moun immuno luo escence.
(TIF)
S8 Fig. Illus a ion depic ing he imeline o EOM and POM de elopmen in ela ion o
ATRA signaling. Summa y o he mos ele an gene ic pe u ba ions and d ug ea men s o
his s udy. ATRA, all- ans e inoic acid; EOM, ex aocula muscle; POM, pe iocula mesen-
chyme.
(TIF)
S1 Table. BMS493 injec ion egimes. The same BMS493 concen a ion (Conc BMS) was used
ac oss injec ion egimes, om I o VI. Pink cases ma k injec ion ime poin s in he mo ning
(mo), midday (mid) o e ening (e e) be ween E10.5 and E12.5. The numbe o emb yos wi h di -
e en muscle pheno ype se e i y a E13.5 a e shown: (no), no al e ed pheno ype; mild (+), muscle
mispa e ning bu o e all o ganiza ion e ained; s ong (++), s ong mispa e ning bu wi h a
minimum o 2 muscles spli ; se e e (+++), no o almos absen spli ing. n is he o al numbe o
emb yos analyzed o a ce ain ea men ype (n = 38 emb yos analyzed in o al); N exp, numbe
o imes he expe imen was epea ed. E, emb yonic day; EOM, ex aocula muscle.
(XLSX)
S2 Table. An ibodies and esou ces used in his s udy.
(XLSX)
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Es ablishmen o he ex aocula muscle unc ional uni
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S1 Da a. Excel able con aining indi idual alues o gene a e he his og am in Fig 3K.
Fo each issue sec ion, a ec angula ROI ( egion o in e es ) was de ined in Fiji (Image J) in
he LysoT acke Red-posi i e and adjacen LysoT acke Red-nega i e a eas. The numbe o
SOX9+ cells was sco ed in each ROI. Indi idual alues we e ep esen ed by he a io o
N˚cells/ROI a ea (μm2).
(XLSX)
S2 Da a. Excel able con aining indi idual alues o gene a e he his og am in Fig 4I.Fo
each emb yo, he EOM olume was calcula ed as desc ibed in he me hods sec ion. The no -
malized EOM olume is he a io o he indi idual olume alue/a e age o he con ol alues.
EOM, ex aocula muscle.
(XLSX)
S3 Da a. Excel able con aining indi idual alues o gene a e he his og am in Fig 5C.Each
da a poin ep esen s he pe cen age o myogenic and non-myogenic GFP+ cells isola ed by
bulk diges ion o he pe iocula a ea o a single Tg:RARE-C eERT2;R26
mTmG
epo e emb yo.
(XLSX)
S1 Video. Tempo al sequence o EOM pa e ning. Whole-moun immunos aining o
MYOD/MYOG/Desmin (E11.5 o E12.5) o MyHC (E13.5 o E14.5) on he de eloping EOM
o con ol emb yos. EOMs we e segmen ed om adjacen head s uc u es and 3D- econ-
s uc ed in Ima is (Bi plane). E, emb yonic day; EOM, ex aocula muscle.
(MOV)
S2 Video. O ganiza ion o he EOM unc ional uni a E11.75. Whole-moun immunos ain-
ing o MyHC (myo ibe s, Red) and PITX2 (POM and myogenic p ogeni o s, Cyan) on con-
ol emb yo. EOMs we e segmen ed om adjacen head s uc u es and 3D- econs uc ed in
Ima is (Bi plane). E, emb yonic day; EOM, ex aocula muscle; POM, pe iocula mesen-
chyme.
(MOV)
S3 Video. O ganiza ion o he EOM unc ional uni a E11.75. Whole-moun immunos ain-
ing o MyHC (myo ibe s, Cyan) and GFP ( endon p ogeni o s, G een) on Tg:Scx-GFP
emb yo. EOMs we e segmen ed om adjacen head s uc u es and 3D- econs uc ed in Ima is
(Bi plane). Apop o ic oci a e isualized by LysoT acke Red s aining. A clipping plane was
added o cla i y o isualiza ion. No e ha endon condensa ions s a o o ganize adially
and owa ds he apop o ic oci in he POM. E, emb yonic day; EOM, ex aocula muscle;
POM, pe iocula mesenchyme.
(MOV)
S4 Video. O ganiza ion o he EOM unc ional uni a E12.5. Whole-moun immunos ain-
ing o MyHC (myo ibe s, Cyan) and GFP ( endon p ogeni o s, G een) on Tg:Scx-GFP
emb yo. EOMs we e segmen ed om adjacen head s uc u es and 3D- econs uc ed in Ima is
(Bi plane). Apop o ic oci a e isualized by LysoT acke Red s aining. A clipping plane was
added o cla i y o isualiza ion. No e how endon condensa ions o he ec i muscles a e
mo e e ined a his s age and p ojec owa ds he apop o ic oci in he POM. E, emb yonic
day; EOM, ex aocula muscle; POM, pe iocula mesenchyme.
(MOV)
S5 Video. O ganiza ion o he EOM unc ional uni o E13.5 BMS493- ea ed (E10.5 !
E11.75) Tg:Scx-GFP emb yos. Whole-moun immunos aining o MyHC (myo ibe s, Red)
and GFP ( endon p ogeni o s, G een). EOMs we e segmen ed om adjacen head s uc u es
PLOS BIOLOGY
Es ablishmen o he ex aocula muscle unc ional uni
PLOS Biology | h ps://doi.o g/10.1371/jou nal.pbio.3000902 No embe 17, 2020 24 / 33
and 3D- econs uc ed in Ima is (Bi plane). No e he p esence o endon condensa ions a he
ips o mispa e ned muscle masses. E, emb yonic day; EOM, ex aocula muscle.
(MOV)
S6 Video. ATRA- esponsi eness in he pe iocula egion o E13.5 Tg:RARE-C eERT2;
R26
mTmG
con ol emb yos (Tamoxi en induc ion a E10.5). Whole-moun immunos aining
o MyHC (myo ibe s, Red), and GFP (ATRA- esponsi e cells, G een). EOMs we e segmen ed
om adjacen head s uc u es and 3D- econs uc ed in Ima is (Bi plane). EOMs a e shown as
isosu aces o cla i y o isualiza ion. ATRA, all- ans e inoic acid; E, emb yonic day; EOM,
ex aocula muscle.
(MOV)
S7 Video. ATRA- esponsi eness in he pe iocula egion o E13.5 BMS493- ea ed Tg:
RARE-C eERT2;R26
mTmG
emb yos (BMS493 ea men : E10 !E11.75; Tamoxi en induc-
ion a E10.5). Whole-moun immunos aining o MyHC (myo ibe s, Red) and GFP (ATRA-
esponsi e cells, G een). EOMs we e segmen ed om adjacen head s uc u es and 3D- econ-
s uc ed in Ima is (Bi plane). EOMs a e shown as isosu aces o cla i y o isualiza ion.
ATRA, all- ans e inoic acid; E, emb yonic day; EOM, ex aocula muscle.
(MOV)
S8 Video. ATRA- esponsi eness in he pe iocula egion o E12.5 Tg:RARE-C eERT2;
R26
mTmG
con ol emb yos (Tamoxi en induc ion a E10.5). Whole-moun immunos aining
o GFP (ATRA- esponsi e cells, G een), PITX2 (POM and myogenic p ogeni o s, Cyan) and
SMA (di e en ia ed muscle, Red). Z-planes om medial o la e al le els. ATRA, all- ans e i-
noic acid; E, emb yonic day; POM, pe iocula mesenchyme.
(MOV)
S9 Video. ATRA- esponsi eness in he pe iocula egion o E12.5 Tg:RARE-C eERT2;
R26
mTmG
;Aldh1a3
KO
emb yos (Tamoxi en induc ion a E10.5). Whole-moun immunos-
aining o GFP (ATRA- esponsi e cells, G een), PITX2 (POM and myogenic p ogeni o s,
Cyan), and SMA (di e en ia ed muscle, Red). Z-planes om medial o la e al le els. ATRA,
all- ans e inoic acid; E, emb yonic day; POM, pe iocula mesenchyme.
(MOV)
S10 Video. ATRA- esponsi eness in he pe iocula egion o E12.5 Tg:RARE-C eERT2;
R26
mTmG
BMS493- ea ed emb yos (BMS493 ea men : E10 !E11.75; Tamoxi en induc-
ion a E10.5). Whole-moun immunos aining o GFP (ATRA- esponsi e cells, G een),
PITX2 (POM and myogenic p ogeni o s, Cyan) and SMA (di e en ia ed muscle, Red). Z-
planes om medial o la e al le els. ATRA, all- ans e inoic acid; E, emb yonic day; POM,
pe iocula mesenchyme.
(MOV)
S11 Video. O ganiza ion o he EOM inse ions o E12.5 Tg:Scx-GFP con ol emb yos.
Whole-moun immunos aining o MyHC (myo ibe s, Red) and GFP ( endon p ogeni o s,
G een). EOMs we e segmen ed om adjacen head s uc u es and 3D- econs uc ed in Ima is
(Bi plane). A clipping plane has been added o cla i y o isualiza ion. E, emb yonic day;
EOM, ex aocula muscle.
(MOV)
S12 Video. O ganiza ion o he EOM inse ions o E12.5 BMS493- ea ed (E10.5 !
E11.75) Tg:Scx-GFP emb yos. Whole-moun immunos aining o MyHC (myo ibe s, Red)
and GFP ( endon p ogeni o s, G een). EOMs we e segmen ed om adjacen head s uc u es
PLOS BIOLOGY
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