cells
Re iew
Zeb a ish Models o Au osomal Recessi e A axias
Ana Quelle-Regaldie 1, Daniel Sob ido-Cameán2,† , An ón Ba ei o-Iglesias 2,‡ , Ma ía Jesús Sob ido 3,‡
and Lau a Sánchez 1,4,*,‡
Ci a ion: Quelle-Regaldie, A.;
Sob ido-Cameán, D.;
Ba ei o-Iglesias, A.; Sob ido, M.J.;
Sánchez, L. Zeb a ish Models o
Au osomal Recessi e A axias. Cells
2021,10, 836. h ps://doi.o g/
10.3390/cells10040836
Academic Edi o :
Syl ie Schneide -Maunou y
Recei ed: 9 Ma ch 2021
Accep ed: 6 Ap il 2021
Published: 8 Ap il 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Depa men o Zoology, Gene ics and Physical An h opology, Facul y o Ve e ina y Science,
Uni e sidade de San iago de Compos ela, 27002 Lugo, Spain; [email p o ec ed]
2Depa men o Func ional Biology, CIBUS, Facul y o Biology, Uni e sidade de San iago de Compos ela,
15782 San iago de Compos ela, Spain; [email p o ec ed] (D.S.-C.); an on.ba ei [email p o ec ed] (A.B.-I.)
3Ins i u o de In es igación Biomédica de A Co uña (INIBIC), Se icio Galego de Saúde,
15006 A Co uña, Spain; [email p o ec ed]
4P eclinical Animal Models G oup, Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS),
15706 San iago de Compos ela, Spain
*Co espondence: [email p o ec ed]
† Cu en add ess: Depa men o Zoology, Uni e si y o Camb idge, Camb idge CB2 3EJ, UK.
‡ These au ho s con ibu ed equally o he manusc ip .
Abs ac : Au osomal ecessi e a axias a e much less well s udied han au osomal dominan a axias
and he e a e no clea ly de ined sys ems o classi y hem. Au osomal ecessi e a axias, which a e
cha ac e ized by neu onal and mul isys emic ea u es, ha e signi ican o e lapping symp oms wi h
o he complex mul isys emic ecessi e diso de s. The gene a ion o animal models o neu odegene -
a i e diso de s inc eases ou knowledge o hei cellula and molecula mechanisms and helps in he
sea ch o new he apies. Among animal models, he zeb a ish, which sha es 70% o i s genome wi h
humans, o e he ad an ages o being small in size and demons a ing apid de elopmen , making
hem op imal o high h oughpu d ug and gene ic sc eening. Fu he mo e, emb yo and la al
anspa ency allows o isualize cellula p ocesses and cen al ne ous sys em de elopmen
in i o
.
In his e iew, we discuss he con ibu ions o zeb a ish models o he s udy o au osomal ecessi e
a axias cha ac e is ic pheno ypes, beha io , and gene unc ion, in addi ion o commen ing on possible
ea men s ound in hese models. Mos o he zeb a ish models gene a ed o da e ecapi ula e he
main ea u es o ecessi e a axias.
Keywo ds: zeb a ish; he edi a y ecessi e a axias; neu odegene a i e diso de s; gene ic edi ion
1. In oduc ion
He edi a y a axias a e a he e ogeneous g oup o a e neu odegene a i e diso de s
caused by p og essi e degene a ion o he ce ebellum and spinoce ebella ac s esul ing in
loss o balance and coo dina ion. Classi ica ion o he edi a y a axias has been p oblema ic
due o pheno ype o e lap and he b oad numbe o synd omes ha mani es a axia as a
common symp om, such as he edi a y spas ic pa aplegias and polyneu opa hies. Howe e ,
he de elopmen o no el molecula echniques has made i possible o classi y a axias
based on gene ics [
1
]. A axias can be di ided in o au osomal dominan , au osomal ecessi e
and X-linked a axias [2,3].
Unlike au osomal dominan a axias [
4
], au osomal ecessi e a axias ha e been less
well s udied. Au osomal ecessi e a axias a e mo e di icul o classi y because, con a y o
au osomal dominan a axias, hey a e no o ganized wi h a nume ical naming sys em. The
e m spinoce ebella a axia au osomal ecessi e (SCAR) was ecen ly used o designa e
no el a axias, bu i is based only on locus disco e y and i does no include he p e iously
iden i ied and mo e equen ecessi e a axias. In addi ion, many ecessi e mul isys emic
o complex me abolic diso de s p esen a axia as a symp om [
2
,
3
]. The e a e mo e han
100 loci ela ed wi h ecessi e a axias. The de elopmen o nex gene a ion sequencing
Cells 2021,10, 836. h ps://doi.o g/10.3390/cells10040836 h ps://www.mdpi.com/jou nal/cells
Cells 2021,10, 836 2 o 28
echnology helped o classi y and disco e new ecessi e a axias [
5
]. Au osomal ecessi e
a axias a e a g oup o complex neu onal diso de s usually showing ea ly onse , in ol ing
cen al and pe iphe al ne ous sys ems. They o en a ec o he sys ems and o gans and
cause—among o he s—symp oms such as: pe iphe al senso imo o neu opa hy; py amidal
and ex apy amidal in ol emen ; oculomo o de ec s; e inopa hy; cogni i e impai men ;
me abolism abno mali ies; seizu es and hypogonadism [
2
,
6
]. The pa hogenesis o au o-
somal ecessi e a axias is associa ed wi h loss o unc ion o he ela ed p o eins, which
a e in ol ed in a b oad numbe o pa hways such as: mi ochond ial me abolism and
unc ion; me abolic homeos asis; DNA epai ; he cell cycle; RNA p ocessing; p o ein
olding; synap ic mo phology and he synap ogenesis o Pu kinje cells [3,7,8].
The use o animal models o neu odegene a i e diso de s can, a leas pa ially, ecapi -
ula e he human disease o help un a el he molecula and cellula mechanisms esponsible
and ep esen a i s s ep in de eloping he apies o diseases ha do no ye ha e a ailable
ea men s [
9
]. Fo example, in a axias, Pu kinje cell degene a ion and mo o de ici s we e
demons a ed in se e al mice models [10].
The zeb a ish (Danio e io) sha es 70% o i s genome wi h humans [
11
] and is an
eme ging animal model ha is gaining popula i y in he s udy o a wide ange o medical
condi ions, such as cance [
12
], neu ogene ic diseases [
13
], psychia y diso de s [
14
], ca -
dio ascula illnesses [
15
] and auma ic inju ies [
16
,
17
]. I s many ad an ages include: easy
gene ic manipula ion; he capaci y o pe o m high h oughpu d ug and gene ic sc eening;
op ical anspa ency ha allows o isualizing he cen al ne ous sys em (CNS) and
cellula p ocesses. In addi ion, beha io al pa e ns a e measu able and easy o e alua e,
including high o de beha io s such as memo y, condi ioned esponses and social beha -
io s [
18
]. The o ganiza ion o he CNS o he zeb a ish is simila o mammals, including
humans. Mo eo e , mos genes ela ed o neu al ac i i y o de elopmen show simila
unc ions in mammals and zeb a ish. On he o he hand, he zeb a ish model has he
ad an age o ha ing a educed CNS complexi y, which simpli ies unc ional s udies [19].
The de elopmen o zeb a ish loss o unc ion models is easily achie ed by ansien
knock-down wi h an isense mo pholinos ha block ansla ion o splicing [
20
]. Pe manen
knock-ou models a e c ea ed wi h mu agens such as N-e hyl-N-ni osou ea (ENU) [
21
],
ansgenesis o gene ic edi ion ia he use o TALENs (T ansc ip ion ac i a o -like e ec o
nucleases), zinc inge nucleases o he ecen ly disco e ed CRISPR/Cas9 echniques [22]
(Figu e 1).
In his e iew, we used he Bi d classi ica ion [
23
] o a axias (which includes ecen ly
disco e ed a axias) o sea ch z in (h ps://z in.o g/ (accessed on 27 Janua y 2021)) and
PubMed (h ps://pubmed.ncbi.nlm.nih.go / (accessed on 27 Janua y 2021)) da abases
o zeb a ish models a ge ing genes ha cause ecessi e diso de s wi h ce ebella a axia
among hei clinical mani es a ions. These da a a e compiled in Supplemen al Table S1.
We discuss he zeb a ish models c ea ed o he mo e common ecessi e a axias, ecessi e
a axias ha a e ela i ely mo e uncommon (only p esen in a ew amilies), and ecessi e
diso de s ha cause abno mali ies o ce ebella de elopmen and p ominen a axia. The
molecula basis and ele an clinical da a o he diseases discussed in he p esen e iew a e
summa ized in Table 1(mo e equen au osomal ecessi e a axias in g een, less equen
au osomal ecessi e a axias in blue and ecessi e inhe i ed diso de s ela ed o a axia
in o ange).
Cells 2021,10, 836 3 o 28
Cells 2021, 10, x FOR PEER REVIEW 3 o 29
Figu e 1. Main echniques o gene a ing zeb a ish loss o unc ion models.
Table 1. Summa y o molecula mechanisms and main clinical ea u es o he a axias discussed in
his a icle.
Disease Gene P o ein Func ion
Clinical Fea u es O he Than A axia
* Re e ences
A axia- elangiec
asia (AT) ATM
Cellula esponses o
DNA damage and
cell cycle con ol
Oculocu aneous elangiec asia,
adiosensi i i y, p edisposi ion o
lymphoid malignancies, cellula and
humo al immunode iciency
[24]
Bouche –Neuhä
use synd ome;
Spas ic
pa aplegia ype
39 (SPG39)
PNPLA6 Phospholipase Hypogonado opic hypogonadism,
cho io e inal dys ophy [25]
Ma inesco-Sjög
en synd ome SIL1
Nuclea exchange
ac o o he
endoplasmic
e iculum esiden
chape one BiP
Ca a ac s, skele al muscle myopa hy,
de elopmen al delay [26]
Spinoce ebella
a axia au osomal
ecessi e ype 20
(SCAR20)
SNX14 Au ophagosome
unc ion
In ellec ual disabili y, coa se acial
ea u es, hea ing loss, epilepsy,
mac ocephaly
[27]
A axia wi h
isola ed i amin
E de iciency
(AVED)
TTPA Dis ibu ion o
i amin E
Hypo e lexia, dec eased ib a ion
sense, ca diomyopa h, e ini is
pigmen osa
[28]
Wol am WFS1 Memb ane Men al e a da ion, seizu es, [29,30]
Figu e 1. Main echniques o gene a ing zeb a ish loss o unc ion models.
Table 1. Summa y o molecula mechanisms and main clinical ea u es o he a axias discussed in his a icle.
Disease Gene P o ein Func ion Clinical Fea u es O he Than A axia * Re e ences
A axia- elangiec asia
(AT) ATM
Cellula esponses o DNA
damage and cell
cycle con ol
Oculocu aneous elangiec asia,
adiosensi i i y, p edisposi ion o
lymphoid malignancies, cellula and
humo al immunode iciency
[24]
Bouche –Neuhäuse
synd ome; Spas ic
pa aplegia ype 39
(SPG39)
PNPLA6 Phospholipase Hypogonado opic hypogonadism,
cho io e inal dys ophy [25]
Ma inesco-Sjög en
synd ome SIL1
Nuclea exchange ac o o
he endoplasmic e iculum
esiden chape one BiP
Ca a ac s, skele al muscle myopa hy,
de elopmen al delay [26]
Spinoce ebella a axia
au osomal ecessi e
ype 20 (SCAR20)
SNX14 Au ophagosome unc ion
In ellec ual disabili y, coa se acial
ea u es, hea ing loss, epilepsy,
mac ocephaly
[27]
A axia wi h isola ed
i amin E de iciency
(AVED)
TTPA Dis ibu ion o i amin E
Hypo e lexia, dec eased ib a ion sense,
ca diomyopa h, e ini is pigmen osa [28]
Wol am synd ome
(WFS) WFS1
Memb ane a icking,
endoplasmic e iculum
s ess and calcium
homeos asis
Men al e a da ion, seizu es, psychia ic
symp oms, pigmen a y e inopa hy,
Diabe es melli us, op ic a ophy,
senso ineu al hea ing loss, u ina y ac
abno mali ies
[29,30]
Polyneu opa hy, hea ing
loss, a axia, e ini is
pigmen osa and ca a ac
(PHARC)
ABHD12 Endocannabinoid and
phospholipid me abolism
Ca a ac s, hea ing loss, e ini is
pigmen osa, demyelina ing
senso imo o polyneu opa hy
[31]
Cayman a axia ATCAY Neu al de elopmen Hypo onia, psychomo o e a da ion [32]
Spinoce ebella a axia
au osomal ecessi e
ype 25 (SCAR25)
ATG5 Au ophagy Men al e a da ion,
de elopmen al delay [33]
Cells 2021,10, 836 4 o 28
Table 1. Con .
Disease Gene P o ein Func ion Clinical Fea u es O he Than A axia * Re e ences
Spas ic pa aplegia
ype 76 (SPG76) CAPN1
Neu onal plas ici y,
mig a ion,
mic o ubula egula ion
Skele al abno mali ies, pe iphe al
neu opa hy, amyo ophy,
spas ic pa aplegia
[34]
Spinoce ebella a axia
au osomal ecessi e
(SCAR17)
CWF19L1 Cell Cycle Con ol De elopmen al delay, men al
e a da ion [35]
EAST synd ome KCNJ10 Po assium channel
Sho s a u e, epilepsy, senso ineu al
dea ness, men al e a da ion, enal
ubulopa hy
[36,37]
Po e i-Bol shause
synd ome LAMA1 Cell a achmen , mig a ion Re inal dys ophy [38]
Mi ochond ial ecessi e
a axia synd ome
(MIRAS)
POLG
Ca aly ic subuni o
mi ochond ial DNA
polyme ase
Oph halmoplegia, polyneu opa hy,
myopa hy, encephalopa hy, li e ailu e,
muscle pain, senso ineu al hea ing loss,
epilepsy
[39,40]
Panc ea ic and ce ebella
agenesis (PACA) PTF1A Panc ea ic de elopmen Neona al diabe es melli us, panc ea ic
and ce ebella agenesis [41]
Go don Holmes
synd ome RNF216 Ubiqui ina ion Cho ea, demen ia, hypogonado opic
hypogonadism [42]
Childhood-onse
neu odegene a ion wi h
a axia, dys onia, and
gaze palsy (NADGP)
SQSTM1
In acellula signalling,
oxida i e s ess esponse,
apop osis and au ophagy
Dys onia, gaze palsy, dyskinesia,
cogni i e decline [43]
Spinoce ebella a axia
au osomal ecessi e ype
7 (SCAR7), Ce oid
lipo uscinosis neu onal 2
(CLN2)
TPP1 Se ine pep idase
SCAR7: Slowly p og essi e spas ici y
CLN2: Rapidly p og essi e myoclonus,
de elopmen al eg ession, ea ly dea h,
epilepsy, loss o ision
[44]
Spinoce ebella a axia
au osomal ecessi e
ype 24 (SCAR24)
UBA5 Ubiqui in-like Ca a ac s, demyelina ing senso imo o
neu opa hy [45]
Galloway-Mowa
synd ome WDR73 Unknown
In ellec ual disabili y, sho s a u e,
mic ocephaly, acial dysmo phism,
hypo onia, neph o ic syn ome, ca a ac s,
skin hypopigmen a ion
[46]
Spinoce ebella a axia
au osomal ecessi e ype
12 (SCAR12)
WWOX DNA epai Epilepsy, men al e a da ion,
de elopmen al delay. mic ocephaly [47–49]
Niemann–Pick disease
ype C (NPC) NPC1 Choles e ol a icking
Lea ning di icul ies, neu opsychia ic
symp oms, demen ia, seizu es, dys onia,
sup anuclea gaze palsy, neona al
choles asis, hepa osplenomegaly
[50]
Ce ebella a axia and
men al e a da ion wi h
o wi hou quad upedal
locomo ion 3 (CAMRQ3)
CA8 Unknown Men al e a da ion quad upedal gai [51,52]
Joube synd ome Se e al genes Di e se unc ions
Hypo onia, De elopmen al delay,
oculomo o ap axia, b ea hing
dys egula ion, e inal abno mali ies,
enal cys s, polydac ily, hepa ic ib osis
[53]
Pon oce ebella
hypoplasia Se e al genes Di e se unc ions
Hypoplasia o he ce ebellum o he
en al pons, p og essi e mic ocephaly,
mo o neu on disease, esi a o y
insu iciency, men al e a da ion
[54]
* Ce ebella a axia is a ea u e o all he diso de s in his able. No all lis ed clinical mani es a ions a e necessa ily p esen in e e y pa ien
wi h he diso de . EAST synd ome: epilepsy, a axia, senso ineu al dea ness and ubulopa hy.
Cells 2021,10, 836 5 o 28
2. Ma e ials and Me hods
A icles we e ound in he z in da abase (h ps://z in.o g/ (accessed on
27 Janua y 2021
))
by sea ching o zeb a ish models o genes ha cause ecessi e a axias as desc ibed in he
classi ica ion o Bi d [23] and some ecessi e diso de s ha ha e p ominen a axia.
The PubMed da abase (h ps://pubmed.ncbi.nlm.nih.go / (accessed on
27 Janua y 2021
))
was used o ind addi ional models ela ed o hese diseases by sea ching o “zeb a ish”
and “name o he disease”. The a icles included in his e iew we e published be o e
27 Janua y 2021.
3. Resul s
The genes esponsible o he diseases discussed he e a e implica ed in a b oad
numbe o signaling pa hways and cellula p ocesses ha a e summa ized in Figu e 2. In
he ollowing sec ions, we will e iew he main indings ob ained wi h zeb a ish models
o each o hese genes.
Cells 2021, 10, x FOR PEER REVIEW 6 o 29
The i s a axia- elangiec asia zeb a ish model was gene a ed wi h mo pholinos and
ep oduced he ole o ATM in he DNA damage esponse [55]. A m mo phan s exhibi
no mal de elopmen bu when a adia ion dose o 8 Gy was applied o 6 hp a m mo -
phan s, abno mal de elopmen wi h e a da ion was obse ed. A m i adia ed mo -
phan s exhibi ed no pigmen a ion, a lack o yolk ex ension, and ex eme en al body
cu a u e and died by 72 hp . Highe amoun s o a m mo pholino, combined wi h adia-
ion, inc eased mo ali y o emb yos by 48 hp and induced mo e se e e cu a u e and
absence o yolk ex ension pheno ypes. This pheno ype caused by i adia ion was no
seen in emb yos injec ed wi h a con ol mo pholino, which means ha de iciency in a m
leads o inc eased adiosensi i i y [55].
Figu e 2. Main unc ions o genes ela ed o ecessi e a axias.
Recen ly, an a m zeb a ish mu an was de eloped which did no show neu o-
degene a i e de ec s bu de eloped exclusi ely as in e ile males [56]. In e ili y is a
cha ac e is ic o some AT pa ien s. Disabling he apop osis pa hway wi h a p53 mu a ion
escued he all-male pheno ype, bu no in e ili y. In addi ion, a m-p53 double mu an s
showed quick umo o ma ion, which sugges s ha a m ac s as a umo supp esso and
is in ol ed in main aining genomic s abili y. Su p isingly, a m mu an emb yos did no
display he adiosensi i i y obse ed in a m mo phan emb yos. This is p obably due o
compensa ion mechanisms [56]. These esul s indica e ha zeb a ish models o a m can
be used o analyze he unc ion o ATM in he DNA damage esponse bu no o neu-
odegene a ion.
• Bouche –Neuhäuse synd ome and spas ic pa aplegia ype 39 (SPG39).
Mu a ions in he PNPLA6 (pa a in like phospholipase domain con aining 6) gene
cause SPG39 and Bouche –Neuhäuse synd ome which exp ess simila symp oms [25].
O he synd omes ha ha e been ela ed wi h mu a ions in PNPLA6 include: (i) Oli-
e –McFa lane synd ome, he ea u es o which consis o ichomegaly, congeni al hy-
popi ui a ism and e inal degene a ion wi h cho oidal a ophy; (ii) Lau ence–Moon
synd ome, which p esen s in a simila manne , hough wi h p og essi e spinoce ebella
a axia and spas ic pa aplegia, and wi hou ichomegaly [57].
Pnpla6 zeb a ish mo phan s ha e been shown o exhibi a cu ly ail ha is
dose-dependen . The head– ail dis ance was sho e in mo phan s and hey also had
abno mal eye de elopmen and educed o ic esicle size. Mo phan eyes we e smalle
han in con ols and some o hem had unclosed eye issu es [57,58]. In addi ion, some
pnpla6 mo phan s showed midb ain–hindb ain bounda y de ec s, swelling o he pe i-
ca dium, educed hea a e and educed blood low [58]. No mal PNPLA6 human
Figu e 2. Main unc ions o genes ela ed o ecessi e a axias.
3.1. Au osomal Recessi e Ce ebella A axias
•A axia- elangiec asia (AT).
AT is an in an ile onse mul isys emic disease p oduced by mu a ions in he ATM gene
(a axia- elangiec asia mu a ed), which is in ol ed in cellula esponses o DNA damage
and cell cycle con ol [24].
The i s a axia- elangiec asia zeb a ish model was gene a ed wi h mo pholinos and
ep oduced he ole o ATM in he DNA damage esponse [
55
]. A m mo phan s exhibi no -
mal de elopmen bu when a adia ion dose o 8 Gy was applied o 6 hp a m mo phan s,
abno mal de elopmen wi h e a da ion was obse ed. A m i adia ed mo phan s exhib-
i ed no pigmen a ion, a lack o yolk ex ension, and ex eme en al body cu a u e and
died by 72 hp . Highe amoun s o a m mo pholino, combined wi h adia ion, inc eased
mo ali y o emb yos by 48 hp and induced mo e se e e cu a u e and absence o yolk
ex ension pheno ypes. This pheno ype caused by i adia ion was no seen in emb yos
injec ed wi h a con ol mo pholino, which means ha de iciency in a m leads o inc eased
adiosensi i i y [55].
Recen ly, an a m zeb a ish mu an was de eloped which did no show neu odegene a-
i e de ec s bu de eloped exclusi ely as in e ile males [
56
]. In e ili y is a cha ac e is ic
o some AT pa ien s. Disabling he apop osis pa hway wi h a p53 mu a ion escued he
all-male pheno ype, bu no in e ili y. In addi ion, a m-p53 double mu an s showed quick
umo o ma ion, which sugges s ha a m ac s as a umo supp esso and is in ol ed
Cells 2021,10, 836 6 o 28
in main aining genomic s abili y. Su p isingly, a m mu an emb yos did no display he
adiosensi i i y obse ed in a m mo phan emb yos. This is p obably due o compensa ion
mechanisms [
56
]. These esul s indica e ha zeb a ish models o a m can be used o analyze
he unc ion o ATM in he DNA damage esponse bu no o neu odegene a ion.
•Bouche –Neuhäuse synd ome and spas ic pa aplegia ype 39 (SPG39).
Mu a ions in he PNPLA6 (pa a in like phospholipase domain con aining 6) gene cause
SPG39 and Bouche –Neuhäuse synd ome which exp ess simila symp oms [
25
]. O he
synd omes ha ha e been ela ed wi h mu a ions in PNPLA6 include: (i) Oli e –McFa lane
synd ome, he ea u es o which consis o ichomegaly, congeni al hypopi ui a ism and
e inal degene a ion wi h cho oidal a ophy; (ii) Lau ence–Moon synd ome, which p esen s
in a simila manne , hough wi h p og essi e spinoce ebella a axia and spas ic pa aplegia,
and wi hou ichomegaly [57].
Pnpla6 zeb a ish mo phan s ha e been shown o exhibi a cu ly ail ha is dose-
dependen . The head– ail dis ance was sho e in mo phan s and hey also had abno mal
eye de elopmen and educed o ic esicle size. Mo phan eyes we e smalle han in con ols
and some o hem had unclosed eye issu es [
57
,
58
]. In addi ion, some pnpla6 mo phan s
showed midb ain–hindb ain bounda y de ec s, swelling o he pe ica dium, educed hea
a e and educed blood low [
58
]. No mal PNPLA6 human mRNA was able o escue he
pheno ype bu no PNPLA6 human mRNA wi hou he ca aly ic domain. This means ha
he pheno ype in pnpla6 mo phan s was due o educed es e ase ac i i y [
58
]. Likewise,
co-injec ion wi h human mRNA wi h known mu a ions o SPG39, Oli e –McFa lane syn-
d ome o Lau ence–Moon synd ome, did no escue he pheno ype [
57
]. Mo o neu ons
and spinal mo o neu on numbe s we e educed in pnpla6 mo phan zeb a ish. Abe an
mo o axon b anching was also obse ed, which seemed o be unca ed and ailed o join
he en al muscles. An inc ease in apop osis was obse ed in his model, especially in
he CNS, ha was no escued wi h co-injec ion o p53 mo pholino. Cell cul u es o pnpla6
mo phan spinal neu ons we e pe o med o con i m ha he cause o impai ed mo o
neu on de elopmen was no he p esence o a de o med body. Chime ic emb yos ans-
plan ed wi h one cell s age pnpla6 mo phan cells also de eloped axon de ec s. Mau hne
neu ons, in e neu ons and senso y neu ons showed no mo phological di e ences in pnpla6
mo phan s. BMP (bone mo phogene ic p o ein) signaling was s udied by blocking he
kinase ac i i y o BMP ecep o s wi h do somo phin, which esul ed in pheno ype escue
in pnpla6 mo phan s. This indica es ha BMP was o e exp essed in pnpla6 knock-down
and ha his caused a leas pa o he mo phan pheno ype [
58
]. Al hough hese esul s
e ealed he neu ological consequences o pnpla6 dys unc ion and a ela ion wi h BMP
o e exp ession, i is necessa y o de elop a mu an zeb a ish model o see i his will
ep oduce he pheno ype obse ed wi h mo pholinos.
•Ma inesco–Sjög en synd ome.
Ma inesco–Sjög en synd ome is a mul isys emic ea ly onse diso de caused by mu a-
ions in he SIL1 (SIL1 Nucleo ide Exchange Fac o ) gene, which encodes a nuclea exchange
ac o o he endoplasmic e iculum esiden chape one BiP (binding immunoglobulin
p o ein) [26].
Zeb a ish mo phan s o sil1 showed abno mal shape and small eyes. Bi e ingence
assays e ealed abno mal skele al muscle s uc u e a 4 dp in mo phan emb yos. The phe-
no ype was escued by he co-injec ion o sil1 wild ype mRNA. His ological examina ion
e ealed de o med be a-dys oglycan exp ession a myosep a wi h a less clea V-shaped
s uc u e and dis u bed o ma ion in sil1 mo phan s. Reduced numbe s o Pu kinje cells
we e obse ed in sil1 mo phan emb yos in compa ison wi h con ols. In e es ingly, sil1
mo phan s displayed an inc ease in le els o BiP, he lipida ed o m o Lc3 (ligh chain 3)
and caspase 3, which a e ma ke s associa ed wi h endoplasmic e iculum s ess, au ophagy
and apop osis, espec i ely [59].
Sil1 de iciency in pe iphe al ne es and neu omuscula junc ions was also s udied
using sil1 mo pholino knock-down in zeb a ish. Sil1 mo phan s demons a ed dis up ed
Cells 2021,10, 836 7 o 28
synapse o ma ion along he e ical myosep a, wi h ema kable diso ganiza ion o p esy-
nap ic b anching ac oss he muscle ibe s o he myo omes. Analysis o myelina ing
Schwann cells did no show signi ican di e ences be ween sil1 mo phan s and con ol
zeb a ish [60].
These mo phan models e ealed neu al damage, abno mali ies in he skele al muscle
and neu omuscula junc ions, and ac i a ion o endoplasmic e iculum s ess, au ophagy
and apop osis, which a e ea u es o Ma inesco–Sjög en pa ien s. The de elopmen o
a mu an zeb a ish model will assis in he sea ch o ea men s and con i m he mo -
phan pheno ypes.
•Spinoce ebella a axia au osomal ecessi e ype 20 (SCAR20).
SCAR20 is caused by mu a ions in he so ing nexin 14 gene (SNX14), which is equi ed
o au ophagosome clea ance [27].
Snx14 zeb a ish mo phan s displayed loss o neu al issue olume and educed num-
be o Pu kinje cells ha was escued wi h co-injec ion wi h human SNX14 mRNA. In
addi ion, mo pholino injec ion in a epo e line ha exp essed GFP in he hindb ain
e ealed a signi ican educ ion in hindb ain size in mo phan emb yos. An inc ease
in apop osis ac oss he neu al issue was also seen. T ansmission elec on mic oscopy
analysis o snx14 mo phan neu al cells also e ealed an accumula ion o au ophagic s uc-
u es. These da a sugges ha snx14 mu a ions caused neu onal cell dea h associa ed wi h
impai ed au ophagic deg ada ion and apop osis [27].
Su p isingly, a mo e ecen s udy using snx14 mo pholinos in zeb a ish did no de ec
mo phological de ec s o mo o neu on abno mali ies. In he same s udy, an snx14 zeb a ish
knock-ou model was examined, and no mo phological o neu onal abno mali ies we e
de ec ed, no e en when ma e nal zygo ic mu an s we e analyzed. Beha io al analysis
also showed ha mu an snx14 zeb a ish did no de elop a locomo o pheno ype. As
SNX14 mu a ions had been epo ed o dis up neu al lipid me abolism, lipids om 4 dp
zeb a ish we e ex ac ed and analyzed h ough o al a y acids lipidomic p o iling. I was
obse ed ha o al a y acids om neu al lipids (bo h sa u a ed and unsa u a ed) we e
ele a ed in snx14 knock-ou emb yos in compa ison wi h wild ype and he e ozygous
emb yos. These esul s con i m ha SNX14 has a conse ed ole in lipid biogenesis [61].
The disc epancies in hese s udies could be because B yan e al. [
61
] used sub oxic
doses o mo pholinos. Mo pholinos a e only conside ed eliable when a mu an model
suppo s mo pholino indings due o he p e iously obse ed o a ge e ec s o mo -
pholinos [
62
]. In addi ion, he p ema u e s op codon o mu an s esul ed in a nonsense
media ed decay ha up egula ed homologous gene sequences ha escue he pheno ype.
The consequence o he mu a ion a he p o ein le el could no be demons a ed in zeb a ish
because he e was no SNX14 an ibody a ailable ha could eac wi h he co esponding
zeb a ish p o ein [61].
•A axia wi h isola ed i amin E de iciency (AVED).
AVED is caused by mu a ions in he
α
ocophe ol ans e p o ein (TTPA) gene which
is esponsible o he dis ibu ion o i amin E [28].
T pa is exp essed in he zeb a ish b ain, eye and ailbud. Expe imen s wi h mo -
pholinos in zeb a ish showed signi ican de elopmen al de ec s a 1 dp along he an-
e io /pos e io axis, wi h unca ed ail and b ain and eye mal o ma ions. Howe e ,
ime lapse analysis epo ed no mal de elopmen du ing blas ula o ma ion, epiboly and
gas ula ion un il 12 hp , when he ppa mo phan eye began o display issue da kening,
indica ing he ini ia ion o imp ope head g ow h. Co-injec ion wi h a p53 mo pholino did
no a ec he pheno ype [63].
P e ious s udies wi h a i amin E-de icien die in adul zeb a ish caused a dec eased
s a le esponse ha sugges ed he occu ence o neu ological de ec s. Emb yos p oduced
by zeb a ish E deple ed adul s displayed inc eased mo ali y and mo phological abno -
mali ies such as c anial mal o ma ions, a ben an e io –pos e io axis, pe ica dial edema,
swim bladde mal o ma ions and yolk-sac edema [
64
,
65
]. Su i ing i amin E de icien
Cells 2021,10, 836 8 o 28
emb yos o 24 hp had somi e mal o ma ions and s un ed in o ma ion. A 48 hp emb yos
de eloped acu e pe ica dial and yolk sac edema. Collagen shea h no ocho d ma ke s
col2a1a (collagen, ype II, alpha 1a) and col9a2 (collagen, ype IX, alpha 2) showed ben
axes. Su p isingly, i was demons a ed ha a i amin E de icien die did no a ec
he localiza ion o exp ession o pa bu a ec ed b ain s uc u e. In addi ion, ppa was
ound o be exp essed a 24 hp in he midb ain–hindb ain bounda y and pax2a (pai ed
box 2a) (which de ines he midb ain–hindb ain bounda y) exp essed a di used signal a
12 and 24 hp [65].
Fu u e s udies wi h mu an s may suppo he disco e ies made wi h
mo pholinos and wi h a i amin E de icien die .
•Wol am synd ome (WFS).
WFS is a ju enile p og essi e neu odegene a i e diso de caused by mu a ions in
WFS1, which encodes he wol amin p o ein. This is an endoplasmic e iculum memb ane-
embedded p o ein ela ed o memb ane a icking, endoplasmic e iculum s ess and
calcium homeos asis [29,30].
Zeb a ish ha e wo pa alogous genes o WFS1,w s1a and w s1b. The i s knock-
down o w s1b was c ea ed du ing an assessmen o genes ha a e ele an o diabe es
ype 2. W s1b knock-down caused a educ ion in panc ea ic
β
-isle cells and educed insulin
exp ession, sugges ing ha WFS1 is in ol ed in glucose homeos asis [66].
A b oade s udy o zeb a ish models was pe o med by Cai ns [
67
]. Mo pholinos o
w s1a and w s1b caused abno mal eye, b ain and o oli hs, al hough he w s1b pheno ype
was mo e pa hological. Co-injec ion o he wo w s1 mo pholinos caused le hali y in mos
emb yos and p53 mo pholino co-injec ion did no escue he pheno ype. CRISPR/Cas9
models o w s1a and w s1b genes displayed delayed de elopmen and smalle o oli hs.
W s1 knock-ou s, pa icula ly he w s1b knock-ou , showed signi ican delay in neu onal
g ow h. A 24 hp , w s1b displayed sho ened mo o axons ha seemed o be co ec ed a
48 hp . Exp ession o ace ylcholine ecep o s de eloped no mally in bo h w s1 knock-ou s,
indica ing ha muscula g ow h was no mal. Ace ylcholines e ase ac i i y was educed
in w s1a and w s1b knock-ou emb yos, which sugges ed educed b ain ac i i y, bu only
was educed in he w s1b knock-ou a 12 mon hs, sugges ing de ec i e neu ogenesis in
w s1b knock-ou ish. Mo eo e , mu an s had a smalle ce ebellum han he wild ypes.
Spon aneous mo emen s o w s1b knock-ou zeb a ish we e signi ican ly highe a 24 hp
and signi ican ly lowe a 48 hp , as compa ed o bo h con ols and w s1a knock-ou s.
As no di e ences we e de ec ed in he muscle ibe s uc u e, his was associa ed wi h
p oblems ela ed o neu onal signaling in esponse o ac ile s imula ion. Mi ochond ial
a icking was a ec ed in bo h w s1 knock-ou s and mi ochond ial espi a ion was educed
in he w s1b knock-ou . Mi ochond ial complex I p o ein exp ession was educed in
w s1a and w s1b knock-ou whole emb yos and muscle and also in he b ain in w s1b
knock-ou emb yos. Howe e , Ca
2+
up ake and mi ochond ial DNA le els emained
no mal in bo h w s1 knock-ou s. Hea shock— o induce endoplasmic e iculum s ess—
inc eased he dea h a e in he wo w s1 knock-ou s, sugges ing inc eased apop osis due
o inc eased endoplasmic e iculum s ess esponse. BiP p o ein exp ession was shown
o be up egula ed in bo h hea shocked and un ea ed w s1 knock-ou s. W s1b knock-ou
adul zeb a ish displayed less locomo ion when hey we e isola ed in o de o eco d hei
beha io , which sugges ed inc eased anxie y. W s1b knock-ou adul s had a signi ican ly
lowe blood glucose le el and lowe e ili y han w s1a and wild ype ish. In addi ion,
bo h knock-ou s had a signi ican ly lowe numbe o e inal ganglion cells han wild
ype ish a 4 mon hs and his numbe dec eased e en mo e a 12 mon hs. W s1b e inal
ganglion cell numbe s we e also signi ican ly lowe han in w s1a. Less eye mo emen
and a hinne op ic ne e we e also obse ed in he w s1b knock-ou s a 12 mon hs. The
ac ha w s1b displayed he mos se e e pheno ype, eplica ing some o he symp oms o
wol am synd ome pa ien s, could be due o i s exp ession in eyes and neu al issue, unlike
w s1a which was only ound o be exp essed in muscle issue [
67
]. The use o hese models
ep oducing he pa hological mechanisms o pa ien s wi h WFS may help o ind a he apy
o Wol am synd ome.
Cells 2021,10, 836 9 o 28
3.2. Less F equen Au osomal Recessi e A axias
•Polyneu opa hy, hea ing loss, a axia, e ini is pigmen osa and ca a ac (PHARC).
PHARC is p oduced by mu a ions in ABHD12, which encodes he abhyd olase domain
con aining p o ein 12, an enzyme ela ed o endocannabinoid and phospholipid me abolism
and ha has essen ial unc ions in he cen al and pe iphe al ne ous sys ems [31].
A mo pholino o he ABHD12 gene was gene a ed as pa o a s udy on neu opa hy
candida e genes ela ed o Cha co –Ma ie–Too h disease. Abhd12 mo phan emb yos had
abe an mo o neu on axon ex ension, pa h inding and b anching, wi h an abno mal
mo phology o pe iphe al neu ons [68].
In a mo e ecen s udy, Abhd12 zeb a ish mo phan s injec ed wi h a splice block mo -
pholino displayed wo pheno ypes: mild and mode a e. The mode a e pheno ype included
se e e mic oph halmia, mic ocephaly and educed body leng h due o a signi ican e-
duc ion in head size. F om 3 dp , bo h mo phan pheno ypes did no ha e spon aneous
ee-swimming ac i i y. The mode a e pheno ype did no display any locomo o ac i -
i y in a ouch-e oked escape esponse and mild pheno ype animals exhibi ed impai ed
locomo o ac i i y simila o a axia. None o hese pheno ypes we e escued a e p53
mo pholino co-injec ion. Axonogenesis and mo o neu on axon ou g ow h was no mal
in abhd12 mo phan s a 3 dp . Howe e , myelina ion o he axonal acks was se e ely
impai ed in abhd12 mo phan s a 5 dp . Neu omas s su e a la ge educ ion in he an e-
io and pos e io la e al line and audi o y sys em in compa ison wi h con ols. No mal
Pu kinje cell di e en ia ion was obse ed, while he mo phology o he Pu kinje cell laye
was a ec ed. A s ong impai men o e inal a chi ec u e and s a i ica ion, absence o
lens cla i ica ion and a high dec ease in e ino ec al p ojec ions was also ound in abhd12
mo phan s. Injec ion o a ansla ion block mo pholino caused a se e e pheno ype wi h
exace ba ion o mo phological symp oms, a cu ed ail and he absence o spon aneous
swimming. Co-injec ion wi h abhd12 mRNA o wild ype zeb a ish emb yos o human
ABHD12 mRNA escued he pheno ype o mo phan emb yos. Howe e , co-injec ion wi h
human ABHD12 mRNA encoding one missense o nonsense mu a ion associa ed wi h
PHARC could no es o e he no mal pheno ype [
69
]. The molecula consequences o
blocking ansla ion seemed o be wo se han when blocking mRNA splicing. This may be
due o he di e en biological ole o some pa s o he unca ed p o ein.
These models ep oduced se e al o he symp oms o PHARC pa ien s, such as a axia,
dis up ion o e inal a chi ec u e and a educ ion in mechanosenso y hai cells in he inne
ea and la e al line. Howe e , i is necessa y o gene a e mu an models o con i m ha
hese indings we e no caused by o - a ge e ec s and o sea ch o he apies.
•Cayman a axia.
Cayman a axia is a ecessi e congeni al a axia es ic ed o he G and Cayman Island.
Cayman a axia is caused by mu a ions in he ATCAY gene, which encodes a neu onal
p o ein called Cay axin [32].
A cay mo phan s displayed educed mo o axon leng h in a dose-dependen manne
a 72 hp . This pheno ype was escued wi h mouse a cay mRNA and also by mouse VAchT
( esicula ace ylcholine anspo e ) mRNA, which ac s downs eam o ace ylcholine syn-
hesis and elease. This indica es ha a cay egula es neu i e ou g ow h h ough choline gic
signaling. In e es ingly, knock-down o cha (choline ace yl ans e ase, which p oduces
ace ylcholine) o acl (ATP ci a e lyase, an enzyme ha p oduces ace yl-CoA o lipoge-
nesis, choles e ol genesis and ace ylcholine syn hesis in he ci osol) genes also caused
sho e mo o neu on axons. P ima y mo o neu ons om he 18 somi e s age o zeb a ish
emb yos we e isola ed. Neu i e ou g ow h could be escued by supplying ca bachol, a
choline gic agonis . Howe e , a opine (a musca inic ecep o ) applica ion in a cay,acl and
cha knock-downs inhibi ed neu i e ou g ow h
in i o
, his pheno ype was no escued
by ca bachol. These da a con i m ha a cay unc ions h ough musca inic ace ylcholine
ecep o s and he MAPK (mi ogen-ac i a ed p o ein kinase) pa hway o egula e neu i e
ou g ow h. The subcellula loca ion o Cha changed in a cay and acl mo phan s, wi h Cha
Cells 2021,10, 836 16 o 28
op ic ec a and ce ebellum. Co-injec ion o n 216 and o ud4 mo pholinos p oduced a mo e
se e e ce ebella pheno ype, accompanied wi h an inc eased educ ion in he size o he
op ic ec a and ma ked mic oph halmia. Co-injec ion o human RNF216 o OTUD4 mRNA
in double mo phan s escued he pheno ype, bu his was no he case o co-injec ion o
mu an RNF216 o OTUD4 mRNA alone. These da a sugges ha epis a ic in e ac ions
be ween hese mu a ions con ibu e o he disease pheno ype [42].
Zeb a ish mo phan s o n 216 and o ud4 showed ce ebella damage cha ac e is ic o
Go don Holmes synd ome ha should be ep oduced in a zeb a ish mu an model. The
p esence o ce ebella abno mali ies indica es ha hese migh be in e es ing models o
sea ch o a possible ea men o his synd ome, such as he possibili y o pe o ming an
unbiased d ug sc een.
•
Childhood-onse neu odegene a ion wi h a axia, dys onia, and gaze palsy (NADGP).
Biallelic mu a ions in he SQSTM1 (seques osome 1) gene, which is equi ed o
in acellula signaling, he oxida i e s ess esponse, apop osis and au ophagy, cause
NADGP. Dominan mu a ions in SQSTM1 ha e been also ela ed wi h Page ’s disease o
he bone, amyo ophic la e al scle osis and on o empo al demen ia (ALS/FTD) [43].
Knock-down o he sqs m1 gene in zeb a ish led o abno mal mo o beha io consis en
wi h a educed ouch-e oked escape esponse. Mo o neu on axons we e seen o su e
dis up ed a bo iza ion and sho ening. Howe e , he e was no neu onal loss o Mau hne
cell de ec s in sqs m1 mo phan s. Co-injec ion o human SQSTM1 mRNA escued he
pheno ype bu his was no escued by he co-injec ion o human SQSTM1 mRNA wi h
ALS/FTD mu a ions. Mo eo e , o e exp ession o wild ype and mu an human mRNAs
did no ep oduce he mo phan pheno ype. As SQSTM1 is known o egula e he ac i i y
o mTOR ( egula es esponses o DNA damage), mTOR le els we e measu ed in sqs m1
mo phan s and inc eased le els o mTOR we e ound. T ea men wi h apamycin (an
mTOR inhibi o ) a 48 hp esul ed in amelio a ion o he mo o pheno ype o sqs m1
mo phan s [97].
In addi ion, sqs m1 knock-down in a splice si e ela ed wi h ce ebella a axia, induced
s uc u al ce ebella de ec s in 60% o he emb yos which ha we e speci ic (as no o he
pheno ypic de ec s we e ound). These ce ebella de ec s anged om deple ion o he
axonal connec ions ac oss he midline o he ce ebellum o comple e a ophy. These
ce ebella pheno ypes we e escued by he co-injec ion o human SQSTM1 mRNA, bu no
wi h he co-injec ion o human SQSTM1 mRNA wi h disease mu a ions [43].
The p esence o locomo o de ec s and ce ebella abno mali ies make hese mo pholi-
nos in e es ing o u u e s udies ha compa e hese indings wi h new mu an models and
o sea ch o ea men s ha can escue hese speci ic pheno ypes.
•
Spinoce ebella a axia au osomal ecessi e ype 7 (SCAR7) and ce oid lipo uscinosis
neu onal 2 (CLN2).
TPP1 ( ipep idyl pep idase 1) is a se ine p o ease mainly exp essed in he lysosome
and melanosome. TPP1 mu a ions ha e been linked wi h SCAR7 and CLN2. I has been
p oposed ha loss o unc ion a ian s wi h abolished TPP1 enzyme ac i i y leads o CLN2
disease, whe eas a ian s ha diminish TPP1 enzyme ac i i y lead o SCAR7 [44].
Mo pholino knock-down o pp1 in zeb a ish causes se e e de elopmen al de ec s
s a ing a 28 hp wi h an abno mal head and inc eased apop osis h oughou he body. A
52 hp , pp1 mo phan s de eloped cu ly ails and ca dio ascula de ec s such as ca diac
loop mal o ma ion, pe ica dial edema, educed ca diac con ac ili y and low blood low
h ough he hea [98].
Mo e ecen ly, a knock-ou o he pp1 gene was obse ed o ecapi ula e he pa ho-
logical and beha io al ea u es o CLN2 disease. Tpp1 mu an zeb a ish de ec s s a ed
a 48 hp wi h a cu ed body, educed head and a smalle e ina. A 72 hp , he e was
no de ec able jaw and a la e s ages he swim bladde was no p esen in pp1 mu an s.
In addi ion, pp1 mu an s died p ema u ely be o e 7 dp . S o ed le els o subuni c o
mi ochond ial ATP syn hase in lysosomes, which is a cha ac e is ic o CLN2 disease, we e
Cells 2021,10, 836 17 o 28
obse ed o be inc eased in he whole body including he eye, head, spinal co d, and
mos p ominen ly, he muscle ibe s in 48 hp pp1 mu an s. CNS examina ion e ealed
a deep absence o HuC/D-posi i e di e en ia ed neu ons in he e ina, op ic ec um and
ce ebellum o pp1 mu an s and pp1 mo phan s. Mo eo e , as ocy osis was obse ed
in he halamus and ce ebellum o pp1 mu an s a 48 hp . Selec i e apop osis was seen
in he e ina, op ic ec um, ce ebellum and spinal co d in pp1 mu an s a 48 hp . This
selec i e apop osis is simila o ha ound in human pa ien s o CLN2 disease. Dec eased
p oli e a ion was shown a 48 hp in he e ina, o eb ain, midb ain–hindb ain bounda y
and spinal co d o pp1 mu an s and pp1 mo phan s. Axonal diso ganiza ion, which is
ano he ea u e o CLN2 disease, was shown o s a a 48 hp in pp1 mu an s and pp1 mo -
phan s. Axonal diso ganiza ion in pp1 de icien zeb a ish was cha ac e ized by pos e io
commissu e de ascicula ion, absence o he ochlea ne e and almos comple e absence
o he op ic ne e, which ailed o send ibe s o he op ic ec um. No axonal de ec s we e
seen be o e 48 hp and p ima y axon ac o ma ion was no a ec ed in pp1 de icien
models, meaning ha axonal loss was seconda y o degene a i e changes. Locomo ion
was impai ed in pp1 mu an s a 72 hp . Mu an s had an abe an swimming pa e n
demons a ing many u ns, a signi ican ly inc eased hype ac i e beha io co e ing long
dis ances, and seemed o show con ulsions. A 96 hp , pp1 mu an s displayed sus ained
muscle con ac ions and los he abili y o mo e. The ouch-e oked escape esponse was
also educed in pp1 mu an s [
99
]. Seizu e ac i i y in pp1 mu an s was demons a ed
h ough single elec ode elec oencephalog aphy. T ea men wi h alp oa e, bu no wi h
pen oba bi one, signi ican ly educed seizu e- ela ed mo emen and educed he mo ali y
be ween 3 and 6 dp [
100
]. This exhaus i e esea ch and he posi i e esul s ob ained wi h
alp oa e open up he possibili y o pe o ming high h oughpu d ug sc een agains CLN2
in pp1 zeb a ish mu an s.
•Spinoce ebella a axia au osomal ecessi e ype 24 (SCAR24).
Biallelic mu a ions in he UBA5 (ubiqui in-ac i a ing enzyme o UFM1) gene we e
ecen ly seen o cause SCAR24 [
45
] and ea ly onse encephalopa hy wi h in ellec ual de i-
ciency, mic ocephaly, mo emen diso de s, and epilepsy [
101
]. S udies in ib oblas s om
a ec ed indi iduals showed ha UBA5 mu a ions impai ed he p ocess o UFMyla ion,
esul ing in an abno mal endoplasmic e iculum s uc u e [101].
Injec ion o uba5 mo pholinos in zeb a ish emb yos did no esul in a mo phological
pheno ype. Howe e , locomo ion de ec s we e obse ed in hese mo phan s. Uba5 mo -
phan s had educed mo emen s and di icul ies o exi he cho ion in compa ison wi h wild
ype la ae. The ouch-escape esponse o uba5 mo phan la ae a 72 hp was dec eased.
La ae exhibi ed looping o pinwheel swimming which esembled a seizu e-like beha io .
The spon aneous mo ili y o uba5 la ae a 5 dp was obse ed o be se e ely a ec ed [
101
].
The nex necessa y s ep will be o de elop a uba5 zeb a ish mu an line o e eal whe he
i ecapi ula es he uba5 mo pholino locomo ion de ec s. Mo e esea ch is also needed o
unde s and he cellula /molecula /physiological de ec s caused by uba5 knock-down.
•Galloway-Mowa synd ome.
Galloway-Mowa synd ome is caused by mu a ions in he WDR73 (WD epea domain 73)
gene, which has high le els o exp ession in he b ain, mainly in he ce ebellum [46].
A Galloway-Mowa synd ome zeb a ish model was gene a ed using wd 73 mo pholi-
nos. Wd 73 mo phan s demons a ed a educed head size, b ain mo phology de ec s,
hypopigmen a ion—which displayed incomple e pene ance—and a cu ed body o un-
ca ed ail egion a 1–2 dp . Wd 73 mo phan s lacked exp ession o dmbx1a, which is ela ed
o midb ain p ogeni o cells, in he ce ebellum a 48 hp bu no a 24 hp . A 48 hp , wd 73
mo phan s had a poo ly expanded and di e en ia ed midb ain. Howe e , g 8 ( ib oblas
g ow h ac o 8) exp ession, which is a ma ke o p ogeni o cells o he midb ain-hindb ain
bounda y, was equal o ha o wild ype emb yos. Mo phogenesis o he midb ain and
hindb ain was dis up ed in wd 73 mo phan s, leading o dila ed en icles. P oli e a ion
o p ogeni o cells was also educed in wd 73 mo phan s a 1 dp . Pu kinje cells in wd 73
Cells 2021,10, 836 18 o 28
mo phan s we e absen a 4 dp . This ce ebella pheno ype was escued in all he la ae
co-injec ed wi h zeb a ish wd 73 mRNA and in he majo i y o la ae co-injec ed wi h
human WDR73 mRNA. In con as , co-injec ion wi h human mRNA con aining a nonsense
mu a ion ela ed o Galloway-Mowa synd ome did no escue he pheno ype [
46
]. As
was p e iously men ioned o o he mo pholino models, he c ea ion o mu an lines ha
ecapi ula e he ce ebella pheno ype is undamen al o p og ess esea ch in his a ea,
especially when mo pholinos cause se e e mo phological de ec s such as in his case.
•Spinoce ebella a axia au osomal ecessi e ype 12 (SCAR12).
SCAR12 is p oduced by mu a ions in he WWOX (WW domain con aining oxido e-
duc ase) gene, which has unc ions in DNA epai and ac s as umo supp esso gene [
48
].
Tsu uwaka and colleagues used mo pholinos and siRNA o knock-down zeb a ish
wwox o s udy in acellula Ca
2+
dynamics. Wwox knock-down caused se e e edemas,
cu led backbones, educed body leng h, head and eye size and inc eased le hali y in
emb yos. In addi ion, hese models had al e ed Ca
2+
dynamics [
102
]. This model p esen ed
de elopmen al delay, which is a cha ac e is ic o SCAR12 and also had dis u bed Ca
2+
dynamics, which ema ks he wwox ole in DNA epai . Howe e , he CNS was no s udied
and locomo ion assays o e alua e epilepsy we e no pe o med. Fu u e s udies looking a
hese aspec s will in o m us as o whe he zeb a ish can be a good model o SCAR12.
3.3. Recessi e Inhe i ed Diso de s Rela ed o A axia
In his sec ion we discuss se e al zeb a ish models designed o model pa hologies ha
p esen a axia as one o hei symp oms: Niemann–Pick disease ype C (NPC), CAMRQ3,
Joube synd ome and pon oce ebella hypoplasia.
•Niemann–Pick disease ype C (NPC).
NPC is a a e neu odegene a i e lysosomal s o age diso de caused by mu a ions
in he NPC1 (95% o he cases) o NPC2 genes, which a e in ol ed in choles e ol a ick-
ing. The clinical diagnosis equi es ib oblas s aining wi h ilipin o de e mine whe he
accumula ion o unes e i ied choles e ol in lysosomes is p esen [50].
Mo pholino knock-down o npc1 in zeb a ish led o de elopmen al delay cha ac e ized
by a educed p og ession o epiboly. Co-injec ion wi h Npc1 mouse mRNA escued he
epiboly delay. When he npc1 mo pholino was injec ed in he yolk syncy ial laye a
1000 cell s age, epiboly was delayed mo e o en. This means ha npc1, exp essed in he
yolk syncy ial laye , is c i ical o no mal epiboly mo emen s. Analysis o mesode m
induc ion and cell a es e ealed ha bo h de elop no mally in npc1 mo phan s. Ac in
cy oskele on diso ganiza ion was also obse ed in npc1 mo phan s, wi h ac in clumping
in some cells and localized loss o ac in mic o ilamen s in o he cells. T ea men wi h
he s e oid ho mones p egnenolone and Dex in npc1 mo phan s pa ially escued epiboly
delay and ac in cy oskele on de ec s, sugges ing ha a educ ion in s e oidogenesis was,
in pa , esponsible o he epiboly delay pheno ype in npc1 mo phan s. Npc1 mo phan s
ha comple ed epiboly p esen ed a pheno ype cha ac e ized by sho e body axis, wide
no ocho d and somi es and died be o e 2 dp . Apop osis inc eased in npc1 mo phan s
a 1 dp . In addi ion, npc1 mo phan s displayed punc a e s e ol dis ibu ion in cells a e
ilipin s aining a 12 hp [103].
Since h ombocy openia is ound in some pa ien s wi h NPC1, his symp om was
also s udied by Louwe e e al. [
104
] in npc1 mo phan s. Zeb a ish injec ed wi h npc1
mo pholinos had mal o med heads and dysmo phic b ains and eyes. Filipin s aining
e ealed de ec i e in acellula p ocessing o choles e ol. S udy o h ombocy es e ealed
ha , e en wi h a low dose o mo pholino, 82% o he emb yos had an almos comple e
absence o h ombocy es in he caudal hema opoie ic issue a 3 dp . When he dose was
doubled, 90% o he emb yos had h ombocy openia and died a 5 dp . E y h ocy es
numbe s we e signi ican ly educed in npc1 mo phan s [104].
The gene a ion o a npc1 s able mu an model using CRISPR/Cas9 was able o e-
p oduce some o he symp oms o human pa ien s. Npc1 mu an zeb a ish had a educed
Cells 2021,10, 836 19 o 28
li espan; he majo i y o hem died be o e 2 mon hs pos - e iliza ion and none o hem
su i ed a e 8 mon hs. Mo eo e , npc1 mu an s had a educed body leng h and exhibi ed
a axia symp oms. His ological analyses allowed o he obse a ion o mac oscopic hep-
a omegaly and splenomegaly wi h oamy and acuola ed li e cells. Lipid accumula ion
in he li e s o npc1 mu an s and a massi e accumula ion o choles e ol in hepa ocy es was
also obse ed. A lipid p o ile analysis o li e issues e ealed signi ican di e ences in npc1
mu an s in p o iles o ce amide, diacylglyce ol, lysophospha idic acid, phospha idic acid,
phospha idylcholine, phospha idyl e hanolamine, phospha idyl se ine and iglyce ide, as
compa ed o wild ypes. In e es ingly, se en ce amides—which a e cha ac e is ic o NPC1
disease—we e accumula ed in he li e o npc1 mu an s. Finally, cal e inin exp ession was
signi ican ly educed in he ce ebellum o npc1 mu an s a wo mon hs pos - e iliza ion,
which indica es a loss o Pu kinje cells [105].
Tseng e al. [
106
] also gene a ed a CRISPR/Cas9 npc1 model ha had educed li espan
(died be o e 6 mon hs o age), in e ili y and educed body leng h compa ed o wild ype
ish. Mo eo e , adul npc1 mu an s ailed o main ain balance du ing swimming and died a
ew days a e he balance de ec was obse ed. His opa hological analyses e ealed axonal
sphe oids in he hindb ain o adul npc1 mu an s and diso ganized Pu kinje neu ons in
he ce ebellum. The i s sign o li e disease appea ed a 7 dp in npc1 mu an s. They had
bigge , da k and opaque li e s as compa ed o wild ype la ae. His opa hological analysis
o li e sec ions showed la ge hepa ocy es ull o acuole like s uc u es in npc1 mu an s.
In addi ion, ilipin s aining demons a ed choles e ol accumula ion in npc1 mu an li e
issue. Su p isingly, lipid accumula ion was no obse ed in npc1 mu an s. Filipin posi i e
s aining s a ed a 2 dp in npc1 mu an s in he yolk a ea and a 3 dp in la e al line issues.
By 5 and 7 dp , npc1 mu an s accumula ed mo e unes e i ied choles e ol h oughou he
en i e unk a ea. Choles e ol accumula ion was obse ed along he no ocho d and he
en al edge o he unk in npc1 mu an s. Applica ion o a pCS2+ plasmid wi h npc1 cDNA
and EGFP escued he unes e i ied choles e ol accumula ion in he yolk a ea. Lysosomes
in li e la al zeb a ish we e obse ed using LysoT acke Red s aining. A 5 dp , Npc1
mu an s had a s onge LysoT acke Red s aining signal compa ed o wild ype la ae,
mainly in he neu omas s and in he ol ac o y placode o npc1 mu an s a 3 dp . Based on
hese esul s, LysoT acke Red s aining was used o es d ug e icacy
in i o
. The 2HP
β
CD
compound, which was seen o be e ec i e in o he animal models and had been es ed
in humans, was es ed on 3 dp la ae. A e 3 days o ea men , a signi ican educ ion
in LysoT acke Red s aining was obse ed in npc1 mu an s. Filipin s aining also e ealed
educed accumula ion o choles e ol in npc1 mu an s. Howe e , he 2HP
β
CD ea men
did no escue he li e de ec s o imp o e su i al [
106
]. In any case, he de elopmen o
a me hod o es compounds in npc1 mu an zeb a ish is c ucial o a emp ing o disco e
new he apies o NPC.
•
Ce ebella a axia and men al e a da ion wi h o wi hou quad upedal locomo ion 3
(CAMRQ3).
Mu a ions in ca bonic anhyd ase 8 (CA8) a e ela ed o CAMRQ3 [
51
]. CA8 is a
ca aly ically inac i e iso o m belonging o he zinc-con aining me alloenzymes amily, ha
ca alyzes he e e sible hyd a ion o ca bon dioxide [52].
The ca8 gene was ound o be s ongly exp essed in he zeb a ish ne ous sys em,
and speci ically, in he Pu kinje cells, which co ela es wi h he obse ed exp ession in
Pu kinje cells in humans and mice. Ca8 mo pholinos caused de ec s in he zeb a ish head
as ea ly as 9 hp and inc eased mo ali y. A 1 dp , ca8 mo phan s had de ec s in he
head, a agile body, cu ed ail, small eye size and pe ica dial edema ha we e dose-
dependen [
52
,
107
]. As de elopmen ad anced, de ec s such as he sho ened ail, cu ed
body axis, absence o swim bladde became mo e ob ious. The swimming pa e n was
al e ed e en wi h a low mo pholino dose. Ca8 mo phan s swam a a slowe speed, showed
an inc eased u ning angle, and mos o hem swam along he pe iphe y o he Pe i dish.
Fu he mo e, ca8 mo phan s had a p onounced di icul y in balancing he body while
swimming compa ed wi h wild ype. When he concen a ion o ca8 mo pholino was
Cells 2021,10, 836 20 o 28
inc eased, he la ae p og essi ely los hei abili y o swim, un il highe doses caused a
comple e loss. The co-injec ion o a p53 mo pholino did no escue he pheno ype de ec s
o mo ali y a es. G oss mo phological changes in he ce ebella egion and a educ ion
in he size o he ce ebellum was obse ed in ca8 mo phan s. Mo eo e , ca8 mo phan s
showed abno mal muscle de elopmen and apop osis in he head egion and pe iphe y
o he ail egion [
52
]. Touch-e oked esponsi eness was also educed in ca8 mo phan s
due o de ec i e ce ebella unc ion, as no de ec s we e obse ed in he mo o neu ons
o Rohon Bea d senso y neu ons [
107
]. Ca8 zeb a ish mo phan s seem o ep oduce he
ypical ea u es obse ed in CAMRQ3. As wi h o he diso de s, i would be o in e es o
de elop a s able zeb a ish mu an model ha ep oduces hese (o simila ) pheno ypes.
•Joube synd ome.
Joube synd ome consis s o a g oup o ea ly onse ecessi e o X-linked ce ebella
a axias wi h a cha ac e is ic mid-hindb ain mal o ma ion called he mola oo h sign [
53
].
Mo e han 30 genes ha e been associa ed wi h Joube synd ome. Se e al zeb a ish models
ha e been c ea ed o s udy some o hese genes, bu mos o hem we e ocused solely on
hei unc ion in ciliogenesis o e inal de elopmen [108–114].
Mu a ions in he AHI1 (Abelson helpe in eg a ion si e 1) gene a e he mos common
cause o Joube synd ome. Knock-down o ahi1 in zeb a ish caused a cu ed body and ab-
no mali ies in he de elopmen o he eye, hindb ain (hyd ocephalus) and o oli hs, oge he
wi h de ec s in cys o ma ion in he p oneph ic kidney ubules. These mal o ma ions we e
compa able o hose o pa ien s wi h Joube synd ome. Co-injec ion o mu ine Ahi1 mRNA
e icien ly escued he pheno ype. His ological analyses e ealed de ec s in lamina ion o
he cell laye s o he e ina in ahi1 mo phan s. Re e sed ca diac looping wi h he hea in
a L-loop pa e n was ound in ahi1 mo phan s. Mo eo e , cloacal dila a ion and loss o
p oneph ic cilia was obse ed in he p oneph ic duc s [115].
Mo e ecen ly, Lessieu e al. [
116
] gene a ed an ahi1 TALEN mu an model and
showed ha mu an s had cone degene a ion and hodopsin mislocaliza ion in ods a
5 mon hs o age as well as absence o dis al p oneph ic duc cilia [
116
]. Zhu e al. [
117
] used
zeb a ish mo pholinos and CRISPR/Cas9 mu an s o analyze i ahi1 mu a ions lacking
he in ac WD40 epea s a ec axonal decussa ion. They ound ha ahi1 mo phan s and
mu an s had e inal ganglion cell axon misp ojec ion and ocula dysplasia caused by a
oxic gain o unc ion [117].
O he zeb a ish models o di e en genes ela ed o Joube synd ome, such as a
knock-down model o cep290 (cen osomal p o ein 290), also ecapi ula e ea u es o Joube
synd ome, such as ce ebella abno mali ies, hyd ocephalus, e inal de ec s, abno mali ies in
o ic ca i y de elopmen and p oneph ic cys s [
118
,
119
]. Mo eo e , cep290 mo phan s we e
used o sea ch o he apies o ciliopa hic enal disease, which esul ed in he disco e y
ha apamycin and osco i ine amelio a e he symp oms [
120
]. Mo e ecen ly, a mu an
line was de eloped o s udy e inal degene a ion. Cep290 mu an s p esen ed scoliosis and
p og essi e cone de e io a ion [114].
Simila ly, knock-down o cspp1 (cen osome and spindle pole associa ed p o ein 1),
cep104 (cen osomal p o ein 104), kiaa0556 (ka anin in e ac ing p o ein) and poc1b (p o eome
o he cen iole 1B) in zeb a ish esul ed in a ciliopa hy pheno ype, which is cha ac e is ic
o Joube synd ome [121–126].
Mos o hese Joube synd ome zeb a ish models we e no used o s udy he CNS.
In addi ion, mos o hem we e no c ea ed wi h mu an s ha we suspec would p obably
ep oduce be e he ai s o his synd ome.
•Pon oce ebella hypoplasia.
Pon oce ebella hypoplasia cons i u es a g oup o ea ly onse a e neu odegene a i e
diso de s wi h a iable symp oms o ce ebella a axia. Se e al mu a ions in di e en genes
ha e been associa ed wi h di e en o ms o pon oce ebella hypoplasia. Mu a ions in
TSEN54, a RNA splicing endonuclease subuni gene, we e associa ed wi h pon oce ebella
hypoplasia ype 6 and la e onse dominan he edi a y a axia [54].
Cells 2021,10, 836 21 o 28
Mo pholino knock-down o sen54 in zeb a ish esul ed in b ain hypoplasia and loss o
s uc u al in eg i y inside he b ain wi h an abe an mid-hindb ain bounda y. Co-injec ion
o human TSEN54 mRNA pa ially escued he b ain pheno ype. Co-injec ed emb yos
displayed milde b ain hypoplasia and a mo e de ined b ain s uc u e. In e es ingly, sen54
mo pholino abno mali ies we e no associa ed wi h neu ode elopmen al pa e ning de ec s
since exp ession o g 8 (which plays a ole in he main enance and de elopmen o he
mid-hindb ain bounda y) and o x2 (o hoden icle homeobox 2) (which unc ions in he
ea ly egionaliza ion o he mesencephalon) a 24 hp was no mal. Tsen54 mo phan s had
inc eased le els o apop osis in he b ain. A s able sen54 knock-ou model c ea ed wi h
ENU mu agenesis had a educed su i al a e wi h all he sen54 homozygous la ae
dying be o e 9 dp . Howe e , he de ec s ha caused hei le hali y we e no s udied
and i is no known i mu a ions in sen54 also caused b ain hypoplasia. In addi ion,
knock-down o he a s2 (a ginyl- RNA syn he ase 2) gene, which has been also ela ed o
pon oce ebella hypoplasia ype 6, esul ed in a simila b ain pheno ype o ha obse ed
in sen54 mo phan s, which was also pa ially escued by he co-injec ion o RARS2 human
mRNA [
127
]. The be e cha ac e iza ion o he mu an model would help o s udy he
molecula consequences o pon oce ebella hypoplasia.
Mu a ions in he SLC25A46 (solu e ca ie amily 25 membe 46) gene, which plays a
ole in mi ochond ial dynamics, ha e been also ela ed o pon oce ebella hypoplasia and
p og essi e myoclonic a axia wi h op ic a ophy and neu opa hy [128,129].
Slc25a46 was obse ed o be p ominen ly exp essed in he b ain and he spinal co d
du ing zeb a ish emb yonic de elopmen . Consis en wi h his, slc25a46 zeb a ish mo -
phan s showed a neu odegene a i e pheno ype wi h a ailu e o he de elopmen o he
midb ain and hindb ain, se e e cu ly ail mo phology and abno mal locomo ion. Mo o
neu ons had signi ican ly sho e axon ac s and many o hem ailed o inne a e he
os al myo ome a 48 hp , he spinal co d neu opil had ewe neu onal p ocesses, ewe
e inal ganglion cell axons eached he ec um a 72 hp , and e inal ganglion cell dend i es
we e also a ec ed. Degene a ion o mo o neu on e minals was also obse ed. Slc25a46
zeb a ish mo phan s p esen ed a misalloca ion o he mi ochond ia, which we e in he
p ocess o ission wi hin he cell bodies. This sugges ed ha incomple e ission o mi o-
chond ia in slc25a46 mo phan s migh inhibi anspo and dis ibu ion in o neu onal
p ocesses. Mi ochond ial dynamics we e s udied in dissocia ed neu ons om slc25a46
zeb a ish mo phan s. Mi ochond ia om mo phan s we e longe han hose o wild ype
emb yos and we e immobile. Co-injec ion wi h SLC25A46 human mRNA es o ed he size
o he mi ochond ia bu no he co-injec ion wi h mu an SLC25A46 mRNA. On he o he
hand, o e exp ession o he wild ype mRNA esul ed in mi ochond ial agmen a ion and
dis up ion o he mi ochond ial ne wo k [129,130].
In con as o he mo phan s udies, a CRISPR/Cas9 slc25a46 zeb a ish model showed
no pheno ype because o gene ic compensa ion, as was demons a ed by RNA sequencing.
Injec ion o a slc25a46 mo pholino in o an F2 homozygous slc25a46 zeb a ish CRISPR/Cas9
line did no cause a pheno ype. Howe e , F0 emb yos injec ed wi h an slc25a46 CRISPR/Cas9
cons uc had a pheno ype ha esembled slc25a46 mo phan s since hey had smalle
eyes, hea edema, a sho e unk and an inc ease in mo o neu on axon dis up ions in
compa ison wi h wild ypes. The F0 slc25a46 CRISPR/Cas9 pheno ype was escued a e
he co-injec ion o SLC25A46 human mRNA. The exis ence o a pheno ype in slc25a46
CRISPR/Cas9 mosaics o he F0 gene a ion, bu no in he homozygous F2 gene a ion,
indica es ha he gene ic compensa ion s a s a e he F0 gene a ion. RNA sequencing
showed a high numbe o se ine p o eases and hei ansc ip ion ac o s, se ine p o ease
inhibi o s, DNA-binding p o eins and a smalle numbe o anspo e s, p o eins ela ed
o immuni y ha we e exp essed di e en ially in F2 slc25a46 CRISPR/Cas9 emb yos a
48 hp [
131
]. In his case, i seems ha he slc25a46 mo phan model be e ep esen s he
main ea u es o pon oce ebella hypoplasia caused by mu a ions in SLC25A46 (and no
he mu an homozygous model ha su e s gene ic compensa ion). This mo phan model
will help o u he s udy pon oce ebella hypoplasia and sea ch o possible ea men s.
Cells 2021,10, 836 22 o 28
4. Conclusions
The majo i y o he zeb a ish models discussed he e o genes ela ed wi h ecessi e
a axias ep oduced some o he neu onal and non-neu onal pheno ypes obse ed in human
diso de s. The models ha did no de elop any neu onal symp oms o locomo o de ici s
we e: a m mo phan s and mu an s [
55
,
56
]; polg mu an s [
89
,
90
]; p 1a mo phan s, mu an s
and ansgenic lines [
91
–
96
]; wwox mo phan s and siRNA models [
102
]. In any case, a m
models de eloped o he non-neu onal symp oms o AT such as adiosensi i i y, in e ili y
and immunode iciency [
55
,
56
]. Polg mu an s, which we e analyzed in o de o s udy
mi ochond ial disease, showed educed mi ochond ial numbe s, educed espi a o y le els
in he CNS, and al e ed locomo ion [
89
,
90
]. p 1a models ep oduced he panc ea ic agenesis
o PACA [
91
–
96
]. wwox knock-down models, which demons a ed de elopmen al delay,
we e only used o s udy Ca
2+
dynamics, highligh ing he ole o wwox in DNA epai [
102
].
In addi ion, snx14 mu an s [
61
] did no ecapi ula e he snx14 mo phan mo o neu on
de ici pheno ype [27], bu e ealed a ole o his gene in lipid biogenesis [61].
Mos o he publica ions o au osomal ecessi e a axias employed mo pholinos o
cause a knock-down and a loss o unc ion e ec . The mo pholino e ec is ansien , i s
deg ada ion occu s in ew days and can some imes cause o - a ge e ec s. Fo his eason, i
is undamen al o alida e i wi h mo pholino con ols (s anda d o misma ch mo pholinos),
PCR/Wes e n blo /immunohis ochemis y and mRNA escue. As s a ed abo e o se e al
models, s able mu an lines a e necessa y o complemen mo pholino s udies because
hey a e mo e in o ma i e and migh acili a e d ug sc eening [
62
]. Howe e , mu an
models do no always ecapi ula e he pheno ypes obse ed wi h mo phan s due o gene ic
compensa ion and ma e nal mRNA escue.
Al hough zeb a ish a e no expec ed o ully ep oduce he human pheno ypes due
o hei less in ica e CNS and hei limi ed numbe o complex beha io s, hey a e e y
in o ma i e o un a elling gene unc ions and analyzing gene and molecula pa hways.
In addi ion, hei small size and ex e nal e iliza ion simpli y high h oughpu d ug o
gene ic sc eening. In his e iew, we epo ed a ious d ug ea men s ha alle ia e he
symp oms o zeb a ish models o au osomal ecessi e a axias [
70
,
79
,
90
,
97
,
100
,
103
,
106
,
120
].
Fu u e wo k should apply he use o hese models o high h oughpu d ug sc eening.
Recen echnological ad ances in gene ic edi ing, such as he de elopmen o CRISPR/Cas
echnologies [
132
], will imp o e he numbe o speci ic s able mu an lines ha be e
mimic human condi ions, while he use o no el echniques o mic oscopy and imaging,
such as supe - esolu ion o ligh -shee mic oscopy, in zeb a ish will ease pheno ypic
cha ac e iza ion.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/
10.3390/cells10040836/s1, Supplemen a y Table S1: Desc ip ion o zeb a ish models o au osomal
ecessi e a axias.
Au ho Con ibu ions:
W i ing—o iginal d a and igu e p epa a ion, A.Q.-R.; w i ing— e iew and
edi ing, D.S.-C., A.B.-I., M.J.S. and L.S. All au ho s ha e ead and ag eed o he published e sion o
he manusc ip .
Funding:
This esea ch was unded by Fondo de In es igaciones Sani a ias-Ins i u o de Salud Ca los
III (Spain), g an numbe : PI17/01582 and by he Asociación Galega de A axia (AGA).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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