GigaScience , 2024, 13 , 1–16
DOI: 10.1093/gigascience/giae037
Resea ch
Un eiling e eb a e de elopmen dynamics in og
Xenopus lae is using mic o-CT imaging
J akub Lazno sk y
1
, Michaela Ka ko a
1
, Alice Helena Reis
2
, P a la Robo ska-Ha elko a
3
, Lo ena Agos ini Maia
4
,
J an K i anek
5
, Tomas Zikm und
1
, J oze Kaise
1 ,6
, Ma cela Buc h o a
4 ,7
, and J akub Ha nos
4 ,
*
1
Cen al Eu opean Ins i u e o Tec hnology, B no Uni e si y o Tec hnology, 612 00 B no, Czech Republic
2
Depa men o Chemical Enginee ing, Columbia Uni e si y, Ne w Yo k, NY 10025, USA, and Depa men o Gene ics and De elopmen , Columbia S em Cell
Ini ia i e, Columbia Uni e si y I ing Medical Cen e , New Yo k, NY 10032, USA
3
Depa men o Zoology, Facul y o Science, Uni e si y o Sou h Bohemia, 370 05 Ceske Budejo ice, Czech Republic
4
Depa men o Expe imen al Biology, Facul y o Science, Masa yk Uni e si y, 625 00 B no, Czech Republic
5
Depa men o His ology and Emb yology, Facul y o Medicine, Masa yk Uni e si y, 625 00 B no, Czech Republic
6
Ins i u e o Physical Enginee ing, Facul y o Mechanical Enginee ing, B no Uni e si y o Technology, 616 69 B no, Czech Republic
7
Labo a o y o Molecula Mo phogenesis, Ins i u e o Animal Physiology and Gene ics, . .i., Czech Academy o Sciences, 602 00 B no, Czech Republic
∗Co espondence add ess . Jakub Ha nos , Masa yk Uni e si y Facul y o Science Bohunice Campus Building D36, Kamenice 5, B no 62500, Czech Republic.
E-mail: [email p o ec ed]
Abs ac
Bac kg ound: Xenopus lae is , he A ican clawed og, is a e sa ile e eb a e model o ganism in a ious biological disciplines, p omi-
nen ly in de elopmen al biology o s udy body plan eo ganiza ion du ing me amo phosis. Howe e , a no able gap exis s in he a ail-
abili y o comp ehensi e da ase s encompassing Xenopus ’ la e de elopmen al s ages.
Findings: This s udy u ilized mic o–compu ed omog aphy (mic o-CT), a nonin asi e 3-dimensional (3D) ima ging echnique wi h
mic ome e -scale esolu ion, o explo e he de elopmen al dynamics and mo pholog ical chang es in Xenopus lae is . Ou app oach
in ol ed gene a ing high- esolu ion images and compu ed 3D models o de eloping Xenopus specimens, spanning om p eme amo -
phosis adpoles o ully ma u e adul s. This da ase enhances ou unde s anding o e eb a e de elopmen and suppo s a ious
analyses. We conduc ed a ca e ul examina ion, analyzing body size , shape , and mo phological ea u es, ocusing on skele ogenesis,
ee h, and o gans like he b ain and gu a di e en s ages. Ou analysis yielded aluable insigh s in o 3D mo phological changes
du ing Xenopus ’ de elopmen , documen ing de ails p e iousl y un eco ded. These da ase s hold he solid po en ial o u he mo -
phological and mo phome ic analyses, including segmen a ion o ha d and so issues.
Conclusions: Ou e posi o y o mic o-CT scans e p esen s a signi ican esou ce ha can enhance ou unde s anding o Xenopus ’
de elopmen and he associa ed mo phological changes in he u u e. The widesp ead u ili y o his amphibian species, coupled
wi h he excep ional quali y o ou scans, which encompass a comp ehensi e se ies o de elopmen al s a ges, opens up ex ensi e
oppo uni ies o hei b oade esea c h applica ion. Mo eo e , hese scans can be used in i ual eali y, 3D p in ing, and educa ional
con e x s, u he e xpanding hei alue and impac .
G aphical abs ac
3D compu ed images o selec ed de elopmen al s ages o X. lae is.
Summa y : X- ay omog aphy was used o examine he A ican clawed og ( Xenopus lae is ). This ex ensi e da ase o specimens om
adpoles o adul ogs opens a en ues o no el insigh s in o he changes and de elopmen al dynamics o selec ed s uc u es, leading
e en uall y o an imp o ed unde s anding o his c ucial animal model.
Ke yw o ds: Xenopus lae is , de elopmen , e eb a es, mic o–compu ed omog aphy, mo pholog ical chang es
Recei ed: No embe 30, 2023. Re ised: Ap il 10, 2024. Accep ed: June 3, 2024
©The Au ho (s) 2024. Published by Ox o d Uni e si y P ess on behal o GigaScience. 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 ( h ps://c ea i ecommons.o g/licenses/by/4.0/ ), which pe mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any
medium, p o ided he o iginal wo k is p ope ly ci ed.
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2 | GigaScience , 2024, Vol. 13
Bac kg ound
Xenopus lae is (NCBI: xid8355), commonly known as he A ican
clawed og, se es as a undamen al model o ganism in he ield
o li e sciences. I s widesp ead adop ion, pa icula l y in biologi-
cal esea c h, can be a ibu ed o he ease o b eeding and hous-
ing, as well as he subs an ial size and manipulabili y o i s eggs
and emb yos [ 1 , 2 ]. O e he yea s, his species has been ex en-
si el y in es iga ed ac oss a ious disciplines , including gene ics ,
emb yology, and de elopmen al, cell, and egene a i e biology [ 3–
5 ]. While ini ial a emp s a ana omical desc ip ions o X. lae-
is da e back a cen u y [ 6 ], subsequen s udies ha e , o a y-
ing deg ees, concen a ed onl y on speci ic ana omical egions [ 7–
25 ]. Despi e hese aluable con ibu ions, many pas app oaches
o desc ibing og ana omy ailed o p ese e he in ica e
mo phological de ails, making hem unsui able o de ailed and
compa a i e s udies. In esponse o hese limi a ions, a de ailed
desc ip ion o an adul male og’s ana omy as a comple e o gan-
ism using a nondes uc i e mic o–compu ed omog aphy (mic o-
CT) me hod was ecen l y p o ided [ 7 ]. Mic o-CT, o be candid,
has al eady been emplo y ed o some ex en as a aluable ool in
selec ed s ages o X. lae is ’ emb yos , adpoles , and ogs o ex-
plo e localized o speci ic occu ences, such as gas ula ion [ 26 ],
c anio acial [ 27 ] and u os yle [ 28 ] de elopmen , skele al mo phol-
ogy [ 29 , 30 ], limb (skele al) egene a ion [ 31–33 ], b ain egene a-
ion [ 34 ], and he ana omy o c anial and an e io spinal ne es
[ 35 ], he head [ 36 , 37 ], and se e al in e nal o gans [ 38 ]. Besides
X. lae is i sel , mic o-CT has also been u ilized in closel y ela ed
ogs o in es iga e , o ins ance , c hond oc anium o ganiza ion
in Aly es obs e icans [ 39 ], skele on [ 40 ] and digi [ 41 ] mo phol-
ogy in Xenopus opicalis , co ical bone mo phology in a ious anu-
an amphibians [ 42 ], and ee olu ion o los mandibula ee h in
Gas o heca guen he i [ 43 , 44 ], oge he wi h c anial m usculoskele-
al s uc u es in Peloba es uscus [ 45 ], lung ea angemen s du -
ing me amo phosis in Mic ohyla issipes [ 46 ], gill o ma ion du ing
me amo phosis in Bu o bu o [ 47 ], and me abolic eo ganiza ion in
me amo phic Rana omeimon is adpoles [ 48 ]. Ne e heless, he
spa io empo al dynamics o X. lae is ’ la e de elopmen a he
le el o a whole o ganism, along wi h he compa ison be ween
adul male and emale ogs, emain la gel y unexplo ed in su i-
cien de ail.
To ill his knowledge gap and gain a mo e comp ehensi e
unde s anding o X. lae is ’ la e de elopmen a he le el o a
whole o ganism, we employed mic o-CT, a powe ul imaging
echnology o examining small objec s a he mic ome e scale
[ 49 , 50 ]. Mic o-CT u ilizes X- ays o gene a e high- esolu ion 3-
dimensional (3D) images, making i an ideal ool o in es iga ing
de elopmen al and mo phological c hanges, pa icula l y in em-
b yos , adpoles , and small animals [ 49 , 50 ]. In p inciple, mic o-CT
imaging is based on cap u ing a se ies o 2-dimensional (2D) X- ay
adiog a phs om a ious angles and hen ma hema icall y p o-
cessing hem h ough omog aphic econs uc ion, esul ing in a
3D ma ix ep esen ing olume densi y. An ad an a ge o mic o-CT
lies in i s abili y o image bones and, when combined wi h a ious
con as me hods, also so issues and blood essels wi hin he
same sample [ 49 , 50 ].
Th ough mic o-CT, we p esen high- esolu ion ima ges and
compu ed 3D models o de eloping X. lae is adpoles , ogle s ,
and adul ogs, whic h o e g ea po en ial o esea c h ields
like de elopmen al and compa a i e biology. Addi ionall y, we con-
duc ed p elimina y analyses o his da ase o illus a e i s e -
sa ili y . Speci ically , ou p elimina y examina ion co e ed mo -
phological changes in body size , shape , and skele ogenesis om
selec ed p eme amo phosis, p ome amo phosis, and climax
me amo phosis s a ges h ough he ogle s a ge up o adul hood.
Ou indings un eil p omising and opening insigh s in o he in-
ica e mo phological dynamics o la e X. lae is ’ de elopmen . To
sum up, his esea c h con ibu es a unique da ase conce ning he
de elopmen al and mo phological c hanges in X. lae is , including
he adul s ages, shedding ligh on he dynamics o e eb a e de-
elopmen and bea ing b oade implica ions o u u e analyses
o Xenopus .
Da a Desc ip ion/Sampling S a egy
In ou mic o-CT s udy o Xenopus , we employed a ca e ul sam-
pling s a egy o encompass he en i e de elopmen al spec um
o his amphibian species . T he selec ion o de elopmen al s a ges
was based on speci ic mo phological ea u es and ma ke s ha
se e as c i ical indica o s o X. lae is ’ de elopmen (see below). By
s a egicall y c hoosing 9 dis inc de elopmen al s a ges, we aimed
o ca p u e he comp ehensi e p ocess o mo phogenesis, span-
ning om p eme amo phosis adpoles o ull y ma u e adul ogs.
These s ages we e ca e ully chosen wi h e e ence o speci ic c i-
e ia, such as he p esence and dimensions o legs and he ail,
o he ea angemen s o he head, as ou lined in he ables o
Nieuwkoop and Fabe [ 51 ] and Zahn and colleagues [ 52 ].
Ou sampling included he ollowing c ucial s ages o X. lae is ’
de elopmen :
P eme amo phosis (s ages 44–45, 52, and 53, acco ding o
Nieuwkoop and Fabe , NF): These ea ly s ages p o ide insigh in o
he ini ial s ages o de elopmen on me amo phosis, as Xenopus
ans o ms om adpole o ogle s.
P ome amo phosis (NF s a ges 54 and 57): This phase ep e-
sen s an in e media e s age whe e signi ican changes in he head,
limb buds, and ail a e occu ing, leading slowly o w a d he climax
me amo phosis.
Climax me amo phosis (NF s ages 59 and 62): These s ages a e
c ha ac e ized by he mos p o ound changes, ma king he peak o
me amo phosis.
F ogle s (NF s a ge 66): F ogle s ep esen a ansi ional s a ge,
b idging he gap be ween adpoles and ully ma u e adul ogs.
Adul male and emale ogs : These adul s ages ep esen he
endpoin o Xenopus de elopmen , allowing us o obse e ully
ma u e indi iduals and o compa e di e ences be ween sexes.
We c hose o ganisms ha we e a pp oxima el y 1 yea old o his
mic o-CT s udy.
Ou selec ion o hese k e y de elopmen al s a ges was pe o med
wi h ega d o hei signi icance in he o e all de elopmen al p o-
cess o X. lae is . By examining hese speci ic s ages, we aimed
o gain a solid da ase , enabling a comp ehensi e unde s anding
o he mo phological ans o ma ions and de elopmen al mile-
s ones ha occu du ing X. lae is ’ de elopmen , om i s p ima y
aqua ic adpole phase o i s seconda y aqua ic adul og s age
[ 53 ]. This sampling s a egy p o ided a de ailed and holis ic iew
o X. lae is ’ la e de elopmen , enabling us o pe o m aluable
analyses leading o p elimina y insigh s and conclusions abou
his species’ mo phological changes h oughou i s li e cycle.
Me hods/Sou ce o Samples
Xenopus emb y os w e e gene a ed and cul i a ed ollowing s an-
da d p o ocols. B ie ly, es es we e su gicall y emo ed om anes-
he ized males (20% MS-222, Sigma-Ald ich, A5040) and ans-
e ed o cold 1 ×Ma c’s Modi ied Ringe s (MMR; 100 mM NaCl,
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Un eiling e eb a e de elopmen dynamics in og | 3
2 mM KCl, 1 mM MgSO
4
, 2 mM CaCl
2
, 5 mM HEPES, bu e ed o
pH 7.4), supplemen ed wi h 50 μg/mL gen amycin (Sigma-Ald ich,
G3632). To induce egg laying, ully ma u e Xenopus emales we e
injec ed wi h 260 U o human c ho ionic gonado opin (Me c k,
O i elle 250 G) in o he do sal lymph sac a pp oxima el y 12 o 16
hou s be o e use and we e k e p o e nigh a 18
◦C. Fo e iliza ion,
eggs we e ex ac ed om induced emales di ec ly in o a Pe i dish
and mixed wi h a piece o es es in 0.1 ×MMR a 18–21
◦C. The sub-
sequen de elopmen o Xenopus adpoles, ogle s, and adul ogs
adhe ed o he app o ed cul i a ion p o ocols (see abo e). T he
Xenopus ogle s and adul ogs we e ea ed in XenopLus (ca alog
numbe RE18001301, Tecniplas ), whic h is an ad anced, ull y au-
oma ed sys em ailo ed o housing amphibians, gua an eeing
excellen animal ca e and housing condi ions. A a designa ed
ime poin , Xenopus specimens we e anes he ized and ixed in a
bu e ed 4% pa a o maldehyde solu ion (1004965000, Me c k) o 3
hou s ( adpoles) o o e nigh ( ogle s and adul ogs) and s aged
acco ding o he ables o Nieuwkoop and Fabe [ 51 ] and Zahn and
collea gues [ 52 ]. Pho og a phs o selec ed Xenopus specimens used
o mic o-CT analysis a e p esen ed in Supplemen a y Fig. S1 .
Me hods/Mic o-CT Scanning
P io o scanning, samples we e placed in ei he a 2-mL Eppen-
do ube, a 15-mL/50-mL Falcon ube (depending on sample size),
o , in he case o he adul ogs, a 500-mL plas ic con aine . To
p e en he mo ion and d ying o he sample du ing he mic o-
CT scan, all samples we e moun ed in a 1% aga ose gel (Top-
BIO, P045). Mic o-CT measu emen s we e conduc ed using he GE
Phoenix | ome | x L 240 labo a o y sys em, which is equipped wi h
a 180 kV/15 W nano ocus X- ay ube and a 4,000-pixel ×4,000-
pixel la panel de ec o wi h a 100- μm pixel size. Scan condi-
ions o eac h s a ge a e summa ized in Supplemen a y Table S1 .
I is impo an o no e ha he X- ay sou ce a ge u ilized in his
s udy was ungs en. The subsequen omog a phic econs uc ion
was pe o med using he GE Phoenix da os | x 2.0 so wa e . T he
econs uc ed da a we e impo ed in o VG S udio MAX 2023.4
so wa e (Volume G a phics GmbH), in whic h he measu ed da a
we e segmen ed, anal yzed, and isualized using VG S udio MAX
( RRID:SCR _ 017997 ). The segmen a ion o Xenopus gu s was done
manually by an ope a o using he so wa e A izo 2020.2 (The mo
Fishe Scien i icA) (A izo 3D So wa e, RRID:SCR _ 014431 ).
All Xenopus samples we e ini iall y scanned in hei na i e
s a e o isualize bone s uc u es. Subsequen l y, specimens we e
s ained wi h 1% iodine (Pen a, 21210–11000) in a 90% me hanol
(Pen a, 17570–30250) solu ion o enhance he con as and isu-
alize so issues. As o ee h (Fig. 3 ), he sepa a ed jaws we e
scanned and s ained. The dehyd a ion and s aining imes o
eac h Xenopus de elopmen al s a ge a e de ailed in Supplemen a y
Table S2 .
Da a Quali y Con ol and Limi a ions
All analyzed samples, comp ising X. lae is adpoles , ogle s , and
adul ogs, we e uni o ml y p ese ed and handled be o e scan-
ning. The p ima y a iable a ec ing da a quali y was he a ied
oxel size o each da ase , a ising om di e en sample sizes.
The smalles sample, a adpole s age (NF 44–45), ea u ed a oxel
size o 5 μm, while he la ges sample, an adul emale og, was
scanned wi h a oxel size o 40 μm ( o a oxel size o all scanned
NF s ages, see he app op ia e column in Supplemen a y Table S1
o Fig. 1 ).
The oxel size di e ence was di ec l y co ela ed wi h sample
dimensions . T he GE L240 X- ay sou ce’s cone beam geome y and
he de ec o ’s ield o iew allo w ed smalle samples o be placed
close o he X- ay sou ce, esul ing in a smalle oxel size (highe
esolu ion) due o cone beam magni ica ion. Al hough samples
exhibi ed a iable oxel sizes, his a iabili y does no necessa -
ily limi da a analysis. La ge samples, despi e ha ing la ge oxel
sizes, s ill enabled he ecogni ion o s uc u es analyzed, as hese
s uc u es we e p opo iona el y la ge in la ge animals scanned
wi h la ge oxel sizes.
All Xenopus specimens unde wen 2 sequen ial scans. Ini ially,
na i e scans we e pe o med, ollowing he p inciple o mic o-
CT imaging o samples in hei na i e o m wi hou s aining,
enabling he isualiza ion o dense mine alized s uc u es like
bones and ee h. Subsequen ly, all scans we e s ained as de-
sc ibed in Supplemen a y Table S2 . To p e en mo emen and
d ying o samples du ing da a acquisi ion, all samples we e ixed
in pol yp opylene conical ubes wi h 1% a ga ose gel. Th oughou
da a acquisi ion, no obse able sample sh inkage occu ed. I any
negligible sh inkage o d ying did occu , we an icipa e he ol-
ume educ ion o be p opo ionall y consis en ac oss all Xenopus
samples.
Image da a inhe en ly exhibi limi a ions in e ms o use ul es-
olu ion and con as among componen s . T hese cons ain s a e
in eg al o he imaging p ocess and a e no indica i e o weak-
nesses in ou s udy bu a he in insic c ha ac e is ics o he
imaging modali y emplo y ed. I is essen ial o acknowledge ha ,
in any imaging echnique, he e a e p ac ical bounda ies o he
le el o de ail and con as achie able.
A las o X. lae is ’ La e De elopmen
In o de o p o ide a mo e comp ehensi e unde s anding o X.
lae is ’ de elopmen du ing me amo phosis and o ganogenesis,
we i s collec ed specimens ep esen ing he key og s a ges, in-
cluding p eme amo phosis, p ome amo phosis, climax me amo -
phosis , ogle s , and adul male and emale ogs (see Sampling
S a egy). Ini iall y, we pe o med na i e sample scanning o ha d
issue isualiza ion and subsequen ly s ained and scanned he
samples o so issue isualiza ion (see Mic o-CT Scanning). As
a esul , we we e able o collec and analyze all majo s ages
o X. lae is ’ la e de elopmen . The en i e collec ion o he Xeno-
pus a las is p esen ed in Fig. 1 , and all aw da a can be ound
in he GigaDB eposi o y (see he Da a A ailabili y sec ion) [ 54 ].
Subsequen ly, wi h his a las , we p o ide he e se e al examples
o speci ic analyses o X. lae is ’ la e de elopmen , including an
analysis o head de elopmen , ee h, long bone g ow h dynamics,
ossi ica ion, b ain de elopmen wi h i s associa ed ne es, and gu
mo phology.
Head De elopmen Analysis
Fi s , we del ed in o he in iguing p ocess o head de elopmen
and skull me amo phosis in climax me amo phosis adpoles,
ogle s, and adul ogs ( e e o Fig. 2 A; Supplemen a y Video S1 ).
Ou ini ial in es iga ion emplo y ed mo phome ic analysis o shed
ligh on he de elopmen al c hanges in he skull. All skulls we e
o ien ed in he same di ec ion ( he an e io skull pa heading o
he op o a page; A: an e io ; P: pos e io ; R: igh ; and L: le ) in a
op-bo om ie w. Suc h ima ges can be u he used, o example, o
e alua e how he indi idual cal a ian bones a e being ea anged
du ing me amo phosis o , o example, o shed ligh on acial e-
gion de elopmen .
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4 | GigaScience , 2024, Vol. 13
Figu e 1:
T he a las o Xenopus lae is ’ la e de elopmen . (A) 3D ende s o so issues o selec ed Xenopus de elopmen al NF s a ges. (A

) C oss sec ions
o so issues . T he sec ion le el was selec ed based on cap u ing he b ain a ea o Xenopus specimens. (A
 
) 3D ende s o he skele on. In he i s 3
condi ions (NF 44–53), he bones a e zoomed in and shown in he ed ec angle on he le . The ele an scale ba and oxel size a e depic ed on
bo om o op o each pic u e. All iews a e om do sal iew wi h he c anial side poin ing o he igh . A: an e io ; L: le ; P: pos e io ; R: igh .
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Un eiling e eb a e de elopmen dynamics in og | 5
Figu e 2:
T he anal yses o he head de elopmen . (A) The se ies o Xenopus skulls o selec ed s a ges . T he scale ba wi h alues in mm is shown on he
le bo om. (B) The skull hickness o selec ed s ages is displa yed. T he scale ba wi h alues in mm is shown on he le side. (B

) The ela i e
dis ibu ion o bone hickness o each skull is shown. The expe imen in ol ed conduc ing 3 epe i ions o each de elopmen al s age, wi h he
esul s depic ed as means accompanied by s anda d de ia ions ( ±SD). (C) The o e ie w o de eloping heads wi h highligh ed eyes is shown.
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6 | GigaScience , 2024, Vol. 13
Figu e 3:
Demons a i e in e ac i e Ske c hFab isualiza ions o 3D econs uc ions highligh ing ha Xenopus emale heads exhibi an inc ease in
olume compa ed o males. (A) Skull o Xenopus lae is adul emale (link: h ps://ske ch ab.com/3d-models/skull-o -xenopus-lae is- emale-
321240d 5a5741d39937 65d457c1594 ). (B) Skull o Xenopus lae is adul male (link: h ps://ske c h ab.com/3d-models/skull-o -xenopus-lae is-male-
192dbbceb71 4b73a1097a7cd 67e5ae ).
Nex , we pe o med he examina ion o skull hickness (Fig. 2 B–
B

; Supplemen a y Video S2 ). This analysis e ealed a nonlin-
ea ela ionship, wi h adul ogs possessing signi ican ly hicke
skulls compa ed o hei younge coun e pa s . T his in es iga ion
also sugges s a posi i e co ela ion be ween skull mass and skull
hickness.
Mo ing on, we made an in oduc o y analysis ega ding he
eye dis ance du ing de elopmen , as illus a ed in Fig. 2 C. Ou
mo phome ic anal ysis ( o an example o he measu emen ,
see Supplemen a y Fig. S2A ) e ealed a p og essi e dec ease in
eye dis ance du ing de elopmen al s a ges ( oughl y es ima ed
in Supplemen a y Fig. S2B –B

). These c hanges ac uall y occu ed
in Xenopus in 2 phases: a g adual inc ease in he p eme a-
mo phic s a ges (da a no sho wn), ollo w ed b y a peak a he
onse o climax me amo phosis (NF s ages 59 and 62), and
inall y, a dec ease o a compac head con igu a ion in ogle s
(NF s age 66) and adul ogs (Fig. 2 C and e y oughly es i-
ma ed in Supplemen a y Fig. S2B –B

). This adap a ion aligns well
wi h he og’s li e s a egy, ansi ioning om a w a e -dw elling
adpole wi h la e al eyes o an adul wi h eyes posi ioned on
op o he head o a subme ged li es yle [ 55 ], eminiscen o
c ocodilians [ 56 ].
Based on he p e ious pa a g a phs, one can also explo e he
conse a ion anal ysis o he eye/head a io wi h espec o og
sex. Findings o ou s (and o he s [ 57 ]) indica e ha Xenopus e-
male heads exhibi an inc ease in olume compa ed o males
(Fig. 3 A, B). Ho w e e , al hough ou e o was in no way di ec-
i e, he e ye dis ance in emales does no emain a 100% o
he head olume in males, bu i is ela i el y less ( o a p elim-
ina y es ima e, see Supplemen a y Table S3 ). This obse a ion,
which can be e en assessed also om he mac oscopic scale , ma y
be linked o isual pe cep ion equi emen s, as he p oximi y o
he eyes is s ill c ucial o ce ain aspec s o ision [ 58 ]. How-
e e , ou in en ion he e was jus o demons a e ha simila and
p ecise anal yses a e easible wi h mic o-CT da a. In summa y,
ou mic o-CT da ase can be u he emplo y ed o he s udy o
he head and i s associa ed o gans such as he eyes in X. lae-
is . Fo mo e in o ma ion abou he X. lae is ’ head mo phology
in gene al, we also e e o he handmade d awings by Zahn and
colleagues [ 52 ].
Too h Analysis
To enhance he dep h o ou analysis and inc ease i s p ac i-
cal applicabili y, we conduc ed a comp ehensi e examina ion o
he og ee h, wi h an indi idual da ase o an adul emale jaw.
Upon ini ial obse a ion, he p esence o ee h in X. lae is may
no be eadil y a ppa en . This is due o he ac ha ee h we e
exclusi el y ound in he maxilla, e e ed o as maxilla y ee h
(Fig. 4 A, B) and behind he maxilla y a ch on he ome al bone—
so-called ome al ee h (no shown). Addi ionall y, onl y a small
po ion o he maxilla y oo h p o uded in o he o al ca i y [ 59 ]
(Fig. 4 C). In con as , he mandible con ains no ee h (Fig. 4 D).
The shape o maxilla y ee h was gene ally uni o m (i.e., ho-
modon den i ion), ypically esembling simple conical s uc u es
(see Fig. 4 B). Xenopus ’ ee h exhibi an ac odon ype o a ach-
men , whe e hey o m an ankylo ic a achmen wi h he adja-
cen bone and a e cha ac e is ic by egula enewal, a condi ion
kno wn as polyphy odon den i ion. No ably, he phenomenon o
oo h ene wal in Xenopus is akin o wha has been p e iousl y
documen ed in o he species, such as geckos [ 60 , 61 ]. In ag ee-
men wi h his, we also no iced he e idence o he eplacemen
in he maxilla y ee h (Fig. 4 E). These anal yses e ealed he p o-
g ession o ankylosis, om he ea ly de elopmen o ee h lo-
ca ed close o he g ing i a o hei ull usion wi h he adjacen
bone (see Fig. 4 E). Ou Xenopus mic o-CT da ase hus un eils
he p e iousl y concealed den al s uc u es o X. lae is , p o ides a
high- esolu ion e ela ion o hei “hidden” ee h pa e ning, and
o e s new pe spec i es o s udying ee h and hei g ow h in
Xenopus .
Mic o-CT Da a Analysis o Long Bone
G o w h Dynamics
Nex , we assessed he g ow h dynamics o se e al long bones
(shown in adul ogs in Fig. 5 A, B and Supplemen a y Video S3 ;
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Un eiling e eb a e de elopmen dynamics in og | 7
Figu e 4: T he anal ysis o ee h in an adul og. (A) The on al ie w o an adul emale og skull is depic ed wi h an uppe maxilla y a c h wi h
maxilla y ee h isualized in y ello w. (B) The la e al and op iew on he igh hal o he maxilla y a ch wi h isualized ee h om an adul emale og.
Th ee di e en de elopmen al s a ges o ee h a e highligh ed by di e en colo s (y ello w, c y an, o ange). (C) The s ained mic o-CT scan shows ha only
a small po ion o he oo h ex ends in o he o al ca i y. T he a ows poin o ee h ows ha do no pene a e he o al ca i y. (D) T he la e al ie w o he
mandible o an adul emale og con i ms he absence o ee h in his a ea. (E) The la e al iew o he os al pa o he maxilla displays di e en
s ages o ee h du ing he eplacemen o oo h ows in de ail.
e e o Supplemen a y Fig. S3 o see how he analysis was pe -
o med). Based on ou mic o-CT anal ysis, in e ms o sex, we ound
ha adul emales can be iewed as essen iall y enla ged males,
indica ing a p opo ional g ow h pa e n (Fig. 5 C). We also in es-
iga ed whe he he de elopmen o he le and igh sides o X.
lae is is uni o m o di e gen ( Supplemen a y Fig. S4 ), and consis-
en l y, we obse ed L-R leng h symme y in he leng h o all long
bones . T he e o e, we nex ocused ou de ailed examina ion only
on one side o he og, speci icall y he le side (L).
Ou indings e ealed ha ce ain limb bones, pa icula ly
hose in he hindlegs, such as he emu and ibio ibula , exhib-
i ed ela i el y a pid g ow h, while o he bones suc h as o elimb
ones g e w a a slo w e a e (Fig. 5 C). Impo an l y, his di e ence
was no a ibu ed o absolu e bone measu emen s bu a he in
ela ion o he animal’s o e all leng h (da a no shown). This phe-
nomenon may be linked o he ogs’ u iliza ion o hei hindlimbs
o swimming, whe eas e en hei o elimbs a e p ima il y em-
plo y ed o ood handling [ 62 , 63 ]. Mo eo e , he leng h o he a sal
bone, such as as agalus and calcaneum, appea s o be less c i -
ical o escape o s a le esponses, suc h as swim kicking, com-
pa ed o he leng hs o he emu and ibio ibula , whic h a e co -
ela ed wi h he muscle mass o he high and he cal [ 7 ]. I is also
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8 | GigaScience , 2024, Vol. 13
Figu e 5:
T he anal ysis o og skele ogenesis . (A) T he adul male og wi h anal yzed bones is depic ed. (B) The adul emale og wi h anal yzed bones is
displa yed. (C) T he g a ph demons a ing he anal yzed bones h oughou he Xenopus lae is ’ la e de elopmen . T he expe imen in ol ed conduc ing 3
epe i ions o each long bone measu emen , wi h he esul s depic ed as means accompanied by s anda d de ia ions ( ±SD). Howe e , e o ba s a e
depic ed only o cases demons a ing s a is ically signi ican di e ences, while hey a e no displayed o insigni ican a ia ions. S a is ical analysis
was pe o med using a 2-way analysis o a iance (ANOVA) ollo w ed b y Tuk e y pos hoc es o mul iple compa isons;
∗∗∗∗P < 0.0001. A: an e io ; AS:
as a galus; CA: calcaneum; F: emu ; H: hume us; L: le ; MC: me aca pals; MT: me a a sals; P: pos e io ; R: igh ; RU: adioulna; TF: ibio ibula .
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Un eiling e eb a e de elopmen dynamics in og | 9
in iguing o no e ha mos bones ini ia ed hei g ow h om
oughl y he same size bu e mina ed i a di e en dimen-
sions . Fo example , me a a sals de elop a a as e a e han
me aca pals (Fig. 5 C), which aligns wi h unc ional conside a-
ions, as ou lined else whe e [ 37 ]. Toge he , based on ou mic o-
CT da a, one can make new p edic ions o u he expe imen al
es ing o (long) bone g ow h.
Ossi ica ion Analysis
Besides bone leng h, i is also easible o analyze he bone mass
using ou Xenopus da ase . Wi hin he og skele on, 2 dis inc
ypes o bones can be gene all y dis inguished in e ms o hei
de elopmen al o igin. De mal ossi ica ion, o igina ing in he de -
mis, is e iden in ce ain skull elemen s and in 2 bones o he pec-
o al gi dle, namel y, he clei h um and cla icle . Con e sel y, he
emaining componen s o he pos c anial skele on consis o ei-
he ca ilage o bones eplacing he ca ila ge, ac hie ed h ough
endoc hond al ossi ica ion [ 19 ].
As he bones o he limbs unde go de elopmen in bo h ad-
poles and adul s, hey ypically comp ise an ossi ied sec ion
alongside ca ila ge. No abl y, he ossi ica ion p ocess al ways com-
mences a he cen al egions o long bones, as shown on he ex-
ample o he emu (Fig. 6 A). By assessmen o he mass a io be-
ween ca ilage and bone (Fig. 6 B–B

; Supplemen a y Fig. S5 ), we
obse ed ha , wi h a ew excep ions, all long bones sha e ela-
i el y simila p opo ions (da a no shown). Fu he mo e, in bo h
adul males and emales, he e is a lack o dynamic g ow h akin
o he de elopmen al s a ges, wi h onl y a no iceable inc ease in
mass (Fig. 6 C). A e climax me amo phosis, no p oli e a i e os-
si ica ion occu s, and he p ocess is limi ed o he calci ica ion
o ca ila ge, i espec i e o he og’s sex (Fig. 6 C). Mo eo e , he
ela i e a io o ca ila ge o an ossi ied bone diminishes as de el-
opmen p og esses, exempli ied by he em u , hume us, and a-
dioulna (Fig. 6 C, D). T hus , esea c he s can u he ak e ad an a ge
o hese indica ions o compa e he g ow h o di e en bones in
a ious a ged animals.
Segmen a ion o Selec ed In e nal So
O gans
Ou da ase o e s signi ican po en ial no only o he e alua-
ion o ha d bones bu also o so issue such as in e nal o -
gans. P ecise segmen a ion o s uc u es is essen ial because once
he s uc u e in ocus is clea ly dis inguished, u he assessmen
o i s mo phology and in aspecies di e ences becomes conside -
ably easie and mo e accu a e . T his ool also p o ides he lexibil-
i y o desc ibe each in e nal s uc u e in de ail ei he sepa a ely
o in he con ex o i s indi idual su ounding elemen s. In ad-
di ion o s udying di e ences du ing X. lae is ’ de elopmen , seg-
men ed s uc u es can be u ilized (e.g., o in es iga ing in e sex
di e ences). Fo an illus a ion o he possible applica ion o ou
mic o-CT da a, he e we u he selec ed 2 k e y e eb a e o gans,
such as he b ain and gu .
T he b ain
I is no ewo hy ha ou mic o-CT da a allow o de ailed ob-
se a ion o he de elopmen al segmen a ion o a ious egions
o he b ain, whic h is e y ha d o dissec , especiall y om Xeno-
pus adul s (pe sonal obse a ion). Speci icall y, Fig. 7 A–E, A

–E

, and
Supplemen a y Video S4 p o ide a clea mo phological isualiza-
ion o he indi idual b ain a eas such as ce eb al hemisphe es
(cbh), ce ebellum (cbl), diencephalon (dch), medulla oblonga a
(mob), op ic lobes (opl), and spinal co d (sp), in he adpole and
adul og b ains, as well as o ollow how hese s uc u es a e de-
eloping in ime cou se. Fo mo e in o ma ion abou he X. lae is ’
b ain and i s de ailed desc ip ion, including i s egene a ion in de-
eloping adpoles, we e e eade s o he ecen publica ion [ 34 ].
Subsequen ly, we also asked whe he we could analyze no only
he b ain bu also he b ain-associa ed ne es like ne us op icus
using he mic o-CT da ase (Fig. 7 F). Indeed, we could see ha he
ne es a ac hed o ey es slo wly elonga ed and hen modi ied, as
a specimen size changed du ing de elopmen (Fig. 7 F). Toge he ,
he quali y o ou mic o-CT allows us o in es iga e no only he
indi idual b ain pa s bu also i s associa ed s uc u es.
The gu
Du ing he me amo phosis o X. lae is , he gu unde goes signi i-
can emodeling, wi h he in es ine sho ening by a pp oxima el y
75% o e an 8-day pe iod. The coiling pa e n changes, wi h
he ou e loops ini ially coiling coun e clockwise and he inne
loops coiling cloc kwise, e e sing a he ileum’s swi c hbac k poin
[ 64 , 65 ]. This emodeling is cha ac e ized by a s age-dependen
sequen ial o ganiza ion o nascen smoo h muscle cells, which
plays a c ucial ole in gu coiling mo phogenesis [ 66 ].
To ou bes knowledge, his p ocess has no been s udied us-
ing mic o-CT so a . T hus , we selec ed he gu as a second in e -
nal o gan o dissec by he mic o-CT echnique, wi h a ocus on
he whole X. lae is a las (Fig. 8 ). I is e iden om he mic o-CT
images ha i success ully depic ed he gu s uc u e wi h high
quali y and de ail (Fig. 8 and Supplemen a y Video S5 ). Fo mo e
in o ma ion abou he X. lae is gu and i s ana omy in gene al, we
e e a eade o he Xenopus illus a ions by Zahn and colleagues
[ 52 ] and suppo ing li e a u e [ 64 , 65 ].
In sum, esea c he s will be able o use ou mic o-CT da ase o
segmen and explo e se e al in e nal o gans, including he b ain
o gu , as well as associa ed s uc u es suc h as ne es, o u he
analy ical and p ac ical pu poses, as men ioned in Discussion be-
low.
Discussion
This s udy used mic o-CT o c ea e high- esolu ion 3D mod-
els o X. lae is om adpoles o adul s, e ealing de ailed de-
elopmen al and mo phological c hanges . T he mic o-CT scan
eposi o y enhances unde s anding o X. lae is ’ de elopmen and
has applica ions in esea ch, i ual eali y, 3D p in ing, and
educa ion.
The X. lae is ’ de elopmen da ase no only o e s aluable in-
sigh s in o his amphibian species bu also p esen s a weal h o
biological po en ial as i se es as a s a ing poin o compa a-
i e, egene a i e, and e olu iona y biology as well as mo pholog-
ical s udy. The da ase ’s di e se specimen ep esen a ion and he
high quali y o he scans open up a ealm o possibili ies o ad-
anced subsequen analyses. One in iguing a enue o s udy lies
in he examina ion o ha d issue g ow h. The da ase p o ides an
ideal pla o m o in es iga e especiall y he long bones o Xenopus
bu also e eb ae, o a skull. Fu he mo e, he da ase acili a es
he analysis o ee h o ca ilage, which we ha e add essed using
dedica ed ools in VG S udio Max.
Nex , his da ase lends i sel o in-dep h explo a ion by e-
sea c he s in e es ed in he ana omy o Xenopus , including in e -
nal o gans . T he da ase ’s segmen a ion capabili ies enable he
p ecise segmen a ion o indi idual s uc u es, which can hen be
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Recei ed: No embe 30, 2023. Re ised: Ap il 10, 2024. Accep ed: June 3, 2024
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