Yog xav paub ntxiv:ali.ma@wecistanche.com

Sante Princiero Berlingerio^1,†, Junling Nws^2,†, Lies De Groef ³, Harold Taeter⁴, Tomas Norton⁴, Pieter Baatsen ⁵, Sara Cairoli⁶, Bianca Goffredo⁶, Peter de Witte ⁷, Lambertus van den Heuvel^1,8, Hans J. Baelde²thiab Elena Levtchenko^1,*

^{Ntu I: Lub raum thiab Lub Raum Tshaj Tawm hauv Cov Neeg Laus Zebra Fish Model of Cystinosis}

¹Laboratory of Pediatric Nephrology, KU Leuven, 3000 Leuven, Belgium; santeprinciero.berlingerio@kuleuven.be (SPB); Bert.vandenHeuvel@radboudumc.nl (LvdH)

²Department of Pathology, Leiden University Medical Center, 2300 RC Leiden, Lub Netherlands; J.He@lumc.nl (JH); j.j.baelde@lumc.nl (HJB)

³ Neural Circuit Development thiab Regeneration Research Group, KU Leuven, 3000 Leuven, Belgium; lies.degroef@kuleuven.be

⁴Pawg ntawm M3-BIORES, Division of Animal and Human Health Engineering, KU Leuven, 3000 Leuven, Belgium; harold.taeter@kuleuven.be (HT); tomas.norton@kuleuven.be (TN)

⁵Molecular Neurobiology, VIB-KU Leuven, 3000 Leuven, Belgium; pieter.baatsen@kuleuven.be

⁶Department of Pediatric Medicine, Laboratory of Metabolic Biochemistry Unit, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy; sara.cairoli@opbg.net (SC); biancamaria.goffredo@opbg.net (BG)

⁷Laboratory rau Molecular Biodiscovery, KU Leuven, 3000 Leuven, Belgium; peter.dewitte@kuleuven.be

⁸Department of Pediatric Nephrology, Radboud University Medical Center, 6525 GA Nijmegen, Lub Netherlands

^*Cov ntawv xov xwm: elena.levtchenko@uzleuven.be; Tel.: plus 32-16-34-38-22

^† Cov kws sau ntawv no tau pab sib npaug rau txoj haujlwm no.

Abstract:Cystosisyog ib qho tsis tshua muaj, kho tsis tau, autosomal recessive kab mob tshwm sim los ntawm kev hloov pauv hauv CTNS gene. Cov noob no encodes lysosomal cystine transporter cystinosis, ua rau lysosomal cystine tsub zuj zuj hauv tag nrho cov hlwb ntawm lub cev, nrog rau lub raum yog thawj cov kab mob. Kev kho tam sim no nrog cysteamine txo qis cystine tsub zuj zuj, tab sis tsis thim rov qab cov tubular tsis ua haujlwm, kev raug mob glomerular, lossis poob ntawmlub raummuaj nuj nqi. Hauv peb txoj kev tshawb fawb yav dhau los, peb tau tsim cov qauv zebrafish ntawm cystinosis los ntawm kev hloov pauv tsis tseem ceeb hauv CTNS gene thiab tau pom tias zebrafish larvae recapitulate lubraumphenotype piav nyob rau hauv tib neeg. Hauv kev tshawb fawb tam sim no, peb qhia tus neeg lauscystinosiszebrafish qauv thiab soj ntsuam lub sij hawm ntev los ntawm tus kab mob rauraumthiab extrarenal kab mob los ntawm biochemical, histological, fertility, thiab locomotor kev ua ub no kev tshawb fawb. Peb pom tias tus neeg lauscystinosiszebra ntses nthuav tawm cystine tsub zuj zuj hauv ntau lub cev, hloov lub raum morphology, tsis pom kev ntawm daim tawv nqaij pigmentation, txo fertility, hloov locomotor kev ua, thiab ocular anomalies. Zuag qhia tag nrho, peb cov ntaub ntawv qhia tias tus neeg lauscystinosiszebrafish qauv reproduces ob peb tib neeg phenotypes ntawm cystinosis thiab tej zaum yuav pab tau rau kev kawm pathophysiology thiab mus sij hawm ntev los ntawm kev kho tshiab.

Ntsiab lus:raumkab mob;cystinosis; qauv zebrafish;lub raumthiab extrarenal kev tshwm sim; cov neeg laus phenotypic nta

1. Taw qhia

CystosisNws yog ib qho autosomal recessive storage disorder uas tshwm sim los ntawm kev hloov hauv CTNS noob uas encodes lysosomal cystine proton co-transporter cystinosis, nqa cystine los ntawm lysosomal lumen mus rau cytosol. Ob peb lub cev muaj feem cuam tshuam rau tus kab mob, xws li cov leeg nqaij, testis, qhov muag, thiab lub hlwb, thiab ntawm tag nrho cov,lub raumyog thawj thiab feem ntau cuam tshuam lub cev [1]. Qhov kev kuaj mob ntau zaus thiab hnyav tshaj plaws yog cov menyuam mos nephropathiccystinosis, nyob rau hauv uas thawj qhov chaw kho mob manifestation yoglub raumFanconi Syndrome, tus cwj pwm los ntawm qhov tsis zoo ntawm cov tubular epithelial cell

(PTE) reabsorption uas ua rau cov zis poob ntawm cov amino acids, qabzib, qis molecular mus rau nruab nrab molecular hnyav proteins, thiab lwm yam metabolites. PTEC kev puas tsuaj yog ua raws li kev ua haujlwm glomerular tsis ua haujlwm thiab, thaum tsis kho, ua rau qhov kawgraumkab mob [1]. Txoj kev kho tam sim no muaj cysteamine, uas yog cystine-depleting amino thiol uas rhuav tshem tus choj disulfide ntawm cysteine, uas ua rau tsim cov amino acid cysteine ​​thiab ib qho cysteamine-cysteine ​​mixed disulfide. Qhov no tom kawg moleculecanexitlysosomesviatheaminoacidtransporterPQLC2[2]. Txawm li cas los xij, kev kho mob nrog cysteamine, txawm tias txo qis lysosomal cystine tsub zuj zuj, tsuas yog ncua kev loj hlob ntawm tus kab mob [3-5]. Yog li ntawd, ntxiv kev nkag siab ntawm pathogenesis ntawmcystinosisthiab tsim tshiab rapeuticoption tseem nyob rau hauv cov txheej txheem tshawb fawb. Ntau tus qauv tsiaj tau tsim los txhawm rau rov hais dua cov cystinosis phenotype [6,7]. Ib qho ntawm cov qauv siv feem ntau yog Ctns-/- C57BL/6 nas, uas tsim cov cystine tsub zuj zuj thiab me me proximal tubulopathy. Txawm li cas los xij, nws tsis qhia txog kev puas tsuaj glomerular thiab cov phenotype tau pom nyob ntawm qhov keeb kwm caj ces [8].

Txog rau tam sim no, cov qauv zebrafish tau dhau los ua ib qho cuab yeej zoo rau kev tshawb nrhiav tib neeg cov kab mob vim muaj ntau yam zoo xws li lawv txoj kev loj hlob sai, siab fecundity, txo tus nqi tu, thiab muaj cov cuab yeej siv tau yooj yim siv gene-editing technologies [9]. Hauv ib txoj kev tshawb fawb dhau los, peb tau nthuav tawm cov qauv ntawm cov kab mob zebrafish larvae nrog homozygous tsis muaj qhov hloov pauv hauv exon 8 ntawm ctns-/- noob, ua rau muaj kev ua haujlwm tsis zoo ntawm cystinosis [10], ua rau cystine tsub zuj zuj hauv tag nrho lub cev, nce ntxiv. larvae deformity, ncua kev loj hlob, thiabraumkev puas tsuaj. Txawm li cas los xij, cov kab mob mus sij hawm ntev hauv cov neeg laus zebrafish tsis tau kawm txog tam sim no. Nyob rau hauv txoj kev tshawb no tam sim no, peb tsom mus rau tus yam ntxwvlub raumthiab extrarenal manifestations nyob rau hauv tus neeg laus zebrafish qauv ntawmcystinosis.

Nyem rau OrganicCistanche rau lub raum kab mob

2. Cov txiaj ntsig

2.1. Cystine Accumulates hauv Ctns-/- Zebrafish

CystosisNws suav hais tias yog ib qho kev puas siab puas ntsws uas ua rau cystine tsub zuj zuj hauv txhua lub hlwb ntawm lub cev. Hauv peb txoj haujlwm dhau los, peb pom tias cystinosis (ctns−/−) zebrafish qhia cystine tsub zuj zuj pib txij li theem larval ntawm kev loj hlob thiab hauv ntau lub cev mus txog rau hnub nyoog 8 hli [9]. Txhawm rau ntsuas yog tias peb tus qauv tswj hwm cystine tsub zuj zuj ntawm cov theem tom qab ntawm tus kab mob, peb ntsuas cov qib cystine hauv tag nrho lub cev,raum, testis, ob lub qhov muag, nqaij, thiab hlwb nyob rau hauv 18-hli-laus ctns-/- zebrafsh piv nrog cov hnub nyoog-matched qus-hom txiv neej zebrafish. Peb pom ib 54-fold nce nyob rau hauv cystine cov ntsiab lus nyob rau hauv tag nrho lub cev (Daim duab 1A) thiab ib tug 146-fold nce nyob rau hauv lub raum (Daim duab 1B) ntawm ctns-/- zebrafsh piv nrog cov qus-hom zebrafsh. Tsis tas li ntawd, peb tau pom ntau cov cystine tsub zuj zuj hauv cov qe ntshav, qhov muag, cov leeg, thiab lub hlwb (Daim duab 1C–F) hauv ctns-/- zebrafish.

2.2. Lub raum Manifestation

2.2.1. Ctns-/- Zebrafish Tam sim no PTEC Kev puas tsuaj

Txij li thaumraumyog lub cev cuam tshuam loj tshaj plaws, peb tau tshuaj xyuas cov yam ntxwv ntawm lub raum siv Hematoxylin thiab Eosin (H&E) thiab Periodic Acid-Shiff (PAS) staining. Peb tau sau tseg tias muaj huab cua o, hyaline zoo li eosinophilic droplets, thiab cytoplasmic vacuoles nyob rau hauv PTEC ntawm ctns-/- zebrafish (Daim duab 2B, D) tab sis tsis nyob rau hauv qus hom zebrafish (Daim duab 2A, C). Nco ntsoov, tsis pom qhov kev puas tsuaj ntawm tubular distal. Cystine crystals tau pom yav dhau los hauv PTEC li cytoplasmic vacuoles nrog cov duab plaub lossis polymorphous duab [11]. Yog li ntawd, peb tau tshawb xyuas yog tias cov qauv no tseem muaj nyob hauv tubules ntawm zebrafish. Peb pom ntau lub cytoplasmic vacuoles hauv PTEC ntawm ctns-/- zebrafish los ntawm toluidine xiav staining (Daim duab 3B, D, qhia nrog hnub qub). Tsis tas li ntawd, peb pom qhov chaw vacuolar nrog cov duab plaub lossis polymorphous (Daim duab 3B, D, qhia nrog cov xub xub). Peb tau lees paub qhov kev tshawb pom no los ntawm kev xa hluav taws xob hluav taws xob microscopy (TEM) (Daim duab 3F). Cov qauv no tsis muaj nyob hauv PTEC ntawm cov tsiaj qus tsiaj qus (Daim duab 3A, C, E). Interestingly, ntxiv rau lub xub ntiag ntawm polymorphous-zoo li tus vacuoles, peb tau pom ib feem poob ntawm txhuam ciam teb hauv PTEC ultrastructure (Daim duab 3F).

Daim duab 1. Cystine cov ntsiab lus hauv homogenates ntawm hom tsiaj qus thiab ctns−/− zebrafish. (A–F) Cystine cov ntsiab lus hauv tag nrho lub cev (A),raum(B), testis (C), ob lub qhov muag (D), nqaij (E), thiab hlwb (F) nyob rau hauv qus-hom thiab ctns−/− 18- hli-laus txiv neej zebrafish. Cystine concentration yog qhia raws li nmol / mg protein. Txhua lub dot sawv cev rau ib tug zebrafish, rau tag nrho ntawm n=3 hom tsiaj qus thiab n=3 cents−/− 18- hli-laus txiv neej zebrafish; unpaired t-test nrog Welch qhov kho, ob-tailed: * p < 0.05;=""><0.01; ns:="" not="" significant.="">

Daim duab 2.Lub raumhistology nyob rau hauv qus-hom thiab ctns-/- zebrafish. (A, B) Cov duab sawv cev ntawmlub raumtubules ntawm qus-hom (A) thiab ctns−/− (B) 18- hli-hnub zebrafish. Cov ntsiab lus ntawm cov tubules ze ze nrog hyaline zoo li eosinophilic droplets (dub xub taub hau) thiab cytoplasmic vacuoles (*). H&E staining. Cov nplai bar sawv cev rau 50 µm. (C,D) Cov duab sawv cev ntawm PAS staining ntawm lub raum tubules ntawm cov tsiaj qus (C) thiab ctns−/− (D) 18- hli-hnub zebrafish. Cov ntsiab lus ntawm cov tubules ze ze nrog hyaline zoo li cov tee dej (dub xub taub hau) thiab cytoplasmic vacuoles (*). PAS staining. Cov nplai bar sawv cev rau 50 µm. PT: proximal tubule; DT: distal tubule.

Daim duab 3.Lub raumcystine crystals nyob rau hauv qus-hom thiab ctns-/- zebrafish PTECs. (A,B) Tus sawv cev toluidine xiav-stained duab ntawmlub raumproximal tubules nyob rau hauv qus-hom (A) thiab ctns−/− (B) 18- hli qub zebrafish. Cov nplai bar sawv cev rau 50 µm. (C, D) Daim duab 3C, D qhia qhov loj dua ntawm daim duab 3A, B. Cov ntsiab lus ntawm lub raum proximal tubules nrog cytoplasmic vacuoles ((D); *), duab plaub thiab polymorphous vacuolar qhov chaw ((D); dub xub taub hau). (E, F) Tus neeg sawv cev TEM cov duab ntawm lub raum proximal tubule ntawm cov tsiaj qus (E) thiab ctns−/− 18- hli-laus zebrafish (F). (F) Ib feem poob ntawm proximaltubulebrushborders(PB)andccumulationoftherectangularandpolymorphousvacuoles (*) tau pom nyob rau hauv lub raum proximal tubule ntawm ctns-/- zebrafish. Qhov kev pom zoo ntawm cov duab plaub nyob rau hauv qab txoj cai kaum qhia tau hais tias cov polymorphous vacuoles thiab ib tug loj vacuole nrog ncaj membrane ciam teb ntu (ib txoj kab ncaj nraim). Cov nplai bar sawv cev rau 5 µm.

2.2.2. Ctns-/- Zebrafish Qhia Glomerular Hypertrophy

Txij li thaum glomerular puas feem ntau ua raws li cov kab mob PTEC, peb tau soj ntsuam cov glomerular histologyofthectns-/- zebrafish, andweobservedthatctns-/- malezebrafishshowed significant enlargement of Bowman's capsule (Daim duab 4B, B4B) thiab 'Glomer's , G) thaum piv nrog cov txiv neej tsiaj qus (Daim duab 4A, E–G). Thesamewasconfirmedfortheglomerulartuftinfemalezebrafish nqa lub ctns-/- mutation (Daim duab 4F), thiab ib tug zoo sib xws tau pom raws li kev ntsuas ntawmtheBowman's spaceandtheBowman'scapsule (Figure4E, G). Noproliferationofmesangial cells lossis glomerulosclerosis tau pom. Ntawm cov duab TEM, peb tsis tau soj ntsuam cov kev hloov pauv ntawm glomerular hauv qab daus daim nyias nyias, podocyte ko taw txheej txheem effacement, los yog txawv txav fenestrated endothelial hlwb nyob rau hauv lub glomeruli ntawm ctns-/- zebrafish (Daim duab 4D) piv nrog cov tsiaj qus hom zebrafish (Daim duab 4C).

Daim duab 4. Glomerular morphology hauv cov tsiaj qus thiab hauv ctns−/− zebrafish. (A, B) Cov duab sawv cev ntawm cov glomerulus ntawm cov tsiaj qus (A) thiab ctns−/− (B) 18-mob zebrafish. PAS staining. Cov nplai bar sawv cev rau 50 µm. (C,D) Cov duab sawv cev TEM ntawm cov glomerulus ntawm cov tsiaj qus (ctns−/− (D) zebrafish. Cov nplais sawv cev rau 1 µm. glomerular tuft (F), thiab Bowman qhov chaw (G) ntawm qus-hom thiab zebrafish nyob rau hauv ob qho tib si genders.

2.2.3. Ctns-/- Zebrafish Zaub Nce Apoptosis ntawm PTEC Qib

Tom ntej no, peb tshawb xyuas sebcystinosistriggered cell tuag nyob rau hauv peb tus qauv zebrafish [10,12]. Peb tau ua cleaved caspase -3 immunostaining hauvlub raumcov ntaub so ntswg ntawm cov tsiaj qus thiab ctns-/- zebrafish thiab peb tau pom qhov tseem ceeb nce ntawm cleaved caspase3 protein ntau ntau hauv PTEC ntawm ctns-/- zebrafish (Daim duab 5B, D, G) piv nrog cov tsiaj qus tsiaj qus (Daim duab 5A, C, G) ob leeg. Cov lus qhia ntawm cleaved caspase-3 yog tam sim no nyob rau hauv cov hlwb uas qhia cov polymorphous cytoplasmic vacuoles (Daim duab 5B, D). Tsis muaj kev qhia ntawm cleaved caspase-3 nyob rau hauv glomeruli, ob qho tib si hauv cov tsiaj qus thiab ctns−/− zebrafish (Daim duab 5E, F).

Daim duab 5. Kev nce apoptosis hauv PTECs nrog vacuoles hauv ctns−/− zebrafish. (A, B) Cov duab sawv cev ntawm cov cleaved caspase-3 kev tiv thaiv ntawm tubules ntawm cov tsiaj qus (A) thiab ctns- qub zebrafish. Qhov ntsuas ntsuas sawv cev 50 µm. (C, D) Cov vaj huam sib luag hauv qab qhia qhov kev nthuav dav ntau dua ntawm cov thawv ntim hauv cov vaj huam sib luag sab saud. Cov ntsiab lus ntawm PTECs nrog cytoplasmic vacuoles (*). Qhov ntsuas ntsuas sawv cev 50 µm. (E, F) Cov duab sawv cev ntawm cov cleaved caspase-3 immunostaining ntawm glomerulus ntawm cov tsiaj qus (E) thiab ctns−/− (F) 18- hli-hnub zebrafish. Cov nplai bar sawv cev rau 50 µm. (G) Qhov kom muaj nuj nqis ntawm tubular caspase-3 staining zoo cheeb tsam nyob rau hauv qus-hom thiab zebrafish nyob rau hauv ob tug poj niam los txiv neej. Txhua lub dot sawv cev rau ib tug zebrafish, rau tag nrho ntawm n=3 hom tsiaj qus thiab ctns−/− 18-mob zebrafish. Ob txoj kev ANOVA nrog Fisher qhov tsawg kawg qhov sib txawv (PLSD) xeem: ** p < 0.01;="" ***="" p=""><>

2.3. Extra Renal Manifestations

2.3.1. Ctns-/- Zebrafish Qhia tus yam ntxwv Hypopigmented Spotted Spotted Skin Pattern

CystosisCov neeg mob feem ntau pom cov tawv nqaij laus ntxov ntxov, hypopigmentation, thiab cov plaub hau daj [1,13]. Interestingly, peb tau pom ib tug hypopigmented pom qauv ntawm daim tawv nqaij ntawm ctns-/- zebrafish, thaum tsiaj qus-hom ntses nthuav tawm cov qauv dub kab txaij. Cov phenotype muaj nyob rau hauv ob qho tib si poj niam txiv neej thaum lawv tag nrho lub neej (Daim duab 6A, B). Txhawm rau tshawb xyuas qhov sib txawv ntawm pigmentation, peb tau ua PAS staining ntawm daim tawv nqaij zebrafish thiab peb pom tias ctns-/- zebrafish qhia hloov pauv ntawm melanin txheej hauv cov epidermis, uas ua rau lub teeb pom kev zoo (Daim duab 6C, D).

Daim duab 6. Hypopigmented spotted skin patterned in ctns−/− zebrafish. (A, B) Cov duab sawv cev ntawm cov tsiaj qus (A) thiab ctns−/− (B) 18-mob zebrafish. Cov nplai tuav sawv cev 0.5cm. Cov xub xub dub qhia txog cov kab txaij sib txawv ntawm daim tawv nqaij. (C,D) PAS-stained duab ntawm epidermis ntawm qus-hom (C) thiab ctns−/− (D) 18- hli-hnub zebrafish. Cov xub xub dub taw qhia cov txheej melanin. Kev cuam tshuam hauv cov txheej txheem melanin muaj nyob hauv ctns-/- (D) thaum nws tsis nyob hauv cov tsiaj qus-hom zebrafish (C). Cov nplai bar sawv cev 50 µ

2.3.2. Ctns-/- Zebrafish Qhia Spermatogenic Cysts Enriched hauv Spermatozoa

Cystosispaub tias muaj kev cuam tshuam rau cov qog nqaij hlav, ua rau azoospermia thiab infertility hauv cov txiv neej cov neeg mob [14–16]. Lub hauv paus chiv keeb ntawm azoospermia tseem tsis tau paub meej, txawm hais tias spermatogenesis tau pom tias tsis zoo nyob rau hauv cov qe menyuam. Hauv kev sib piv nrog cov txiv neej cov neeg mob, cov poj niam uas muaj cystinosis yog fertile [17]. Hauv peb cov qauv zebrafish, ob leeg poj niam thiab txiv neej ctns-/- zebrafish yog fertile. Txhawm rau tshawb xyuas seb ctns-/- kev hloov pauv puas cuam tshuam rau lub cev ntawm lub qog, peb tau ua PAS staining thiab peb pom muaj cov spermatozoa nce ntxiv hauv cov spermatogenic cysts hauv ctns-/- zebrafish.

Daim duab 7. Spermatogenic cysts enriched hauv spermatozoa hauv ctns−/− zebrafish. (A, B) Tus Neeg Sawv Cev H&E stained duab ntawm testis ntawm qus-hom (A) thiab ctns−/− (B) 18- hli-hnub zebrafish. Tsaus xiav thiab puag ncig staining sawv cev rau cov phev spermatozoa sau hauv cov kab mob spermatogenic (qhia nrog cov hnub qub). Cov nplai bar sawv cev rau 200 µm.

2.3.3. Ctns−/− Poj Niam Zebrafish Show Txo Qe Production

Txhawm rau tshawb xyuas qhov ua haujlwm ntawm kev ua me nyuam, peb ntsuas cov qe qe thiab qhov feem pua ​​​​ntawm cov qe fertilized nyob rau hauv ob hom tsiaj qus thiab ctns-/- zebrafish. Peb pom tias ctns−/− poj niam qhia tias qe qe qis (Daim duab 8A) thiab qis feem pua ​​​​ntawm cov qe fertilized (Daim duab 8B) piv nrog cov poj niam qus.

Daim duab 8. Kev tsim qe thiab feem pua ​​​​ntawm cov qe fertilized hauv cov tsiaj qus thiab ctns−/− zebrafish. (A) Qe ntau lawm yog ntsuas raws li tag nrho cov qe nyob rau hauv cov qus hom thiab ctns−/− zebrafish 18- hli-hnub zebrafish. Cov qe ntau lawm yog 1.941 siab nyob rau hauv qus-hom zebrafish piv nrog ctns−/− 18 hli-laus zebrafish (tus piv=1.941); 95 feem pua ​​CI (qis khi=1.165, siab dua khi=3.234); Poisson regression tsom xam; p=0 {30}}11. (B) Qhov feem pua ​​​​ntawm cov qe fertilized yog ntsuas ntawm tag nrho cov qe. Qhov txawv ntawm cov qe fertilized yog 3.504 siab nyob rau hauv qus-hom zebrafish piv nrog ctns−/− 18-hli-hnub zebrafish (qhov txawv piv txwv=3.504); 95 feem pua ​​CI (qis khi=2.005, siab dua khi=6.124); Logistic regression tsom xam; p <0.001; n/a:="" tsis="" siv="">

2.3.4. Ctns−/− Zebrafish Zaub Txo Cov Locomotor Kev Ua Haujlwm

Txij li thaum cov nqaij ntshiv thiab tsis muaj zog tau kuaj pom nyob rau hauvcystinosiscov neeg mob, feem ntau los ntawm lub xyoo caum thib ob ntawm lub neej [1,18], peb tau soj ntsuam cov haujlwm locomotor hauv peb tus qauv zebrafish. Peb tau tshawb pom tias ctns-/- zebrafish los ntawm ob tug poj niam los txiv neej tau pom cov kev ua haujlwm hauv lub cev qis dua piv nrog cov tsiaj qus hom zebrafish, qhia txog kev ua haujlwm tsis zoo ntawm cov leeg nqaij (Daim duab 9). Txawm li cas los xij, kev soj ntsuam histological ntawm cov nqaij pob txha pob txha tsis qhia pom qhov txawv txav hauv ctns-/- zebrafish.