The neural crest is a unique cell population associated with vertebrate evolution. Neural crest cells (NCCs) are characterized by their multipotent and migratory potentials. While zebrafish is a powerful genetic model organism, the isolation and culture of zebrafish NCCs would provide a useful adjunct to fully interrogate the genetic networks that regulate NCC development. Here we report for the first time the isolation, in vitro culture, and characterization of NCCs from zebrafish embryos. NCCs were isolated from transgenic sox10:egfp embryos using fluorescence activated cell sorting and cultured in complex culture medium without feeder layers. NCC multilineage differentiation was determined by immunocytochemistry and real-time qPCR, cell migration was assessed by wound healing assay, and the proliferation index was calculated by immunostaining against the mitosis marker phospho-histone H3. Cultured NCCs expressed major neural crest lineage markers such as sox10, sox9a, hnk1, p75, dlx2a, and pax3, and the pluripotency markers c-myc and klf4. We showed that the cultured NCCs can be differentiated into multiple neural crest lineages, contributing to neurons, glial cells, smooth muscle cells, melanocytes, and chondrocytes. We applied the NCC in vitro model to study the effect of retinoic acid on NCC development. We showed that retinoic acid had a profound effect on NCC morphology and differentiation, significantly inhibited proliferation and enhanced cell migration. The availability of high numbers of NCCs and reproducible functional assays offers new opportunities for mechanistic studies of neural crest development, in genetic and chemical biology applications.
CorderoDRBrugmannSChuYBajpaiRJameMHelmsJA. Cranial neural crest cells on the move: their roles in craniofacial development. Am J Med Genet A2011; 155A: 270–9.
2.
HallBK. The neural crest as a fourth germ layer and vertebrates as quadroblastic not triploblastic. Evol Dev2000; 2: 3–5.
3.
EtcheversH. Primary culture of chick, mouse or human neural crest cells. Nat Protoc2011; 6: 1568–77.
4.
LeeGKimHElkabetzYAl ShamyGPanagiotakosGBarberiTTabarVStuderL. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol2007; 25: 1468–75.
5.
LeeGPapapetrouEPKimHChambersSMTomishimaMJFasanoCAGanatYMMenonJShimizuFVialeATabarVSadelainMStuderL. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature2009; 461: 402–6.
6.
ZhuHZonLI. Use of zebrafish models for the analysis of human disease. Curr Protoc Hum Genet2002. Chapter 15: Unit 15 3.
7.
JuCZhangKWuX. Derivation of corneal endothelial cell-like cells from rat neural crest cells in vitro. PLoS One2012; 7: e42378–e42378.
8.
RovasioRAFaasLBattiatoNL. Insights into stem cell factor chemotactic guidance of neural crest cells revealed by a real-time directionality-based assay. Eur J Cell Biol2012; 91: 375–90.
9.
JaurenaMBCarriNGBattiatoNLRovasioRA. Trophic and proliferative perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are prevented by neurotrophin 3. Neurotoxicol Teratol2011; 33: 422–30.
10.
GarcezRCTeixeiraBLSchmitt SdosSAlvarez-SilvaMTrentinAG. Epidermal growth factor (EGF) promotes the in vitro differentiation of neural crest cells to neurons and melanocytes. Cell Mol Neurobiol2009; 29: 1087–91.
11.
JiangHBTianWDLiuLKXuY. In vitro odontoblast-like cell differentiation of cranial neural crest cells induced by fibroblast growth factor 8 and dentin non-collagen proteins. Cell Biol Int2008; 32: 671–8.
12.
LiFLuoZHuangWLuQWilcoxCSJosePAChenS. Response gene to complement 32, a novel regulator for transforming growth factor-beta-induced smooth muscle differentiation of neural crest cells. J Biol Chem2007; 282: 10133–7.
13.
EvrardYAMohammad-ZadehLHoltonB. Alterations in Ca2+-dependent and cAMP-dependent signaling pathways affect neurogenesis and melanogenesis of quail neural crest cells in vitro. Dev Genes Evol2004; 214: 193–9.
14.
PrinceSWigginsTHulleyPAKidsonSH. Stimulation of melanogenesis by tetradecanoylphorbol 13-acetate (TPA) in mouse melanocytes and neural crest cells. Pigment Cell Res2003; 16: 26–34.
15.
KawakamiTSomaYKawaYItoMYamasakiEWatabeHHosakaEYajimaKOhsumiKMizoguchiM. Transforming growth factor beta1 regulates melanocyte proliferation and differentiation in mouse neural crest cells via stem cell factor/KIT signaling. J Invest Dermatol2002; 118: 471–8.
WentzelPErikssonUJ. Altered gene expression in rat cranial neural crest cells exposed to a teratogenic glucose concentration in vitro: paradoxical downregulation of antioxidative defense genes. Birth Defects Res B Dev Reprod Toxicol2011; 92: 487–97.
18.
LiJHuangXXuXMayoJBringasPJrJiangRWangSChaiY. SMAD4-mediated WNT signaling controls the fate of cranial neural crest cells during tooth morphogenesis. Development2011; 138: 1977–89.
19.
NakanishiKChanYSItoK. Notch signaling is required for the chondrogenic specification of mouse mesencephalic neural crest cells. Mech Dev2007; 124: 190–203.
20.
JiaLChengLRaperJ. Slit/Robo signaling is necessary to confine early neural crest cells to the ventral migratory pathway in the trunk. Dev Biol2005; 282: 411–21.
21.
TestazSJarovAWilliamsKPLingLEKotelianskyVEFournier-ThibaultCDubandJL. Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched- Smoothened-Gli signaling pathway. Proc Natl Acad Sci U S A2001; 98: 12521–6.
22.
CurtinEHickeyGKamelGDavidsonAJLiaoEC. Zebrafish wnt9a is expressed in pharyngeal ectoderm and is required for palate and lower jaw development. Mech Dev2011; 128: 104–15.
23.
DoughertyMKamelGShubinetsVHickeyGGrimaldiMLiaoEC. Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model. J Craniofac Surg2012; 23: 1333–7.
24.
SchlegelmilchKMohseniMKirakOPruszakJRodriguezJRZhouDKregerBTVasioukhinVAvruchJBrummelkampTRCamargoFD. Yap1 acts downstream of alpha-catenin to control epidermal proliferation. Cell2011; 144: 782–95.
ZhaoHBringasPJrChaiY. An in vitro model for characterizing the post-migratory cranial neural crest cells of the first branchial arch. Dev Dyn2006; 235: 1433–40.
27.
BritschSGoerichDERiethmacherDPeiranoRIRossnerMNaveKABirchmeierCWegnerM. The transcription factor Sox10 is a key regulator of peripheral glial development. Genes Dev2001; 15: 66–78.
SatoTOkumuraFArigaTHatakeyamaS. TRIM6 interacts with Myc and maintains the pluripotency of mouse embryonic stem cells. J Cell Sci2012; 125: 1544–55.
30.
LeeGChambersSMTomishimaMJStuderL. Derivation of neural crest cells from human pluripotent stem cells. Nat Protoc2010; 5: 688–701.
31.
HerbarthBPingaultVBondurandNKuhlbrodtKHermans-BorgmeyerIPulitiALemortNGoossensMWegnerM. Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease. Proc Natl Acad Sci U S A1998; 95: 5161–5.
32.
KuhlbrodtKHerbarthBSockEHermans-BorgmeyerIWegnerM. Sox10, a novel transcriptional modulator in glial cells. J Neurosci1998; 18: 237–50.
33.
PuschCHustertEPfeiferDSudbeckPKistRRoeBWangZBallingRBlinNSchererG. The SOX10/Sox10 gene from human and mouse: sequence, expression, and transactivation by the encoded HMG domain transcription factor. Hum Genet1998; 103: 115–23.
34.
MenegolaEBrocciaMLDi RenzoFMassaVGiaviniE. Relationship between hindbrain segmentation, neural crest cell migration and branchial arch abnormalities in rat embryos exposed to fluconazole and retinoic acid in vitro. Reprod Toxicol2004; 18: 121–30.
35.
Morriss-KayG. Retinoic acid and development. Pathobiology1992; 60: 264–70.
36.
VillanuevaSGlavicARuizPMayorR. Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction. Dev Biol2002; 241: 289–301.
37.
ElliesDLLangilleRMMartinCCAkimenkoMAEkkerM. Specific craniofacial cartilage dysmorphogenesis coincides with a loss of dlx gene expression in retinoic acid-treated zebrafish embryos. Mech Dev1997; 61: 23–36.
38.
ZhangZBalmerJELovlieAFrommSHBlomhoffR. Specific teratogenic effects of different retinoic acid isomers and analogs in the developing anterior central nervous system of zebrafish. Dev Dyn1996; 206: 73–86.
FerrettiPGeraudieJ. Retinoic acid-induced cell death in the wound epidermis of regenerating zebrafish fins. Dev Dyn1995; 202: 271–83.
41.
TolletJEverettAWSparrowMP. Development of neural tissue and airway smooth muscle in fetal mouse lung explants: a role for glial-derived neurotrophic factor in lung innervation. Am J Respir Cell Mol Biol2002; 26: 420–9.
42.
HuangHZhaoXChenLXuCYaoXLuYDaiLZhangM. Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture. Biochem Biophys Res Commun2006; 351: 321–7.
43.
MianoJMTopouzisSMajeskyMWOlsonEN. Retinoid receptor expression and all-trans retinoic acid-mediated growth inhibition in vascular smooth muscle cells. Circulation1996; 93: 1886–95.
44.
WilliamsSSMearJPLiangHCPotterSSAronowBJColbertMC. Large-scale reprogramming of cranial neural crest gene expression by retinoic acid exposure. Physiol Genomics2004; 19: 184–97.
45.
Kawai-KowaseKKumarMSHoofnagleMHYoshidaTOwensGK. PIAS1 activates the expression of smooth muscle cell differentiation marker genes by interacting with serum response factor and class I basic helix-loop-helix proteins. Mol Cell Biol2005; 25: 8009–23.
46.
JinLGanQZiebaBJGoicoecheaSMOwensGKOteyCASomlyoAV. The actin associated protein palladin is important for the early smooth muscle cell differentiation. PLoS One2010; 5: e12823–e12823.
47.
GollaschMHaaseHRiedCLindschauCMoranoILuftFCHallerH. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J1998; 12: 593–601.
48.
KimKH. A role of retinoic acid in the regulation of the morphology and the levels of intermediate filament proteins and mRNAs in PC12 cells. Exp Cell Res1992; 203: 374–82.
49.
Martinez-LiarteJHSolanoFGarcia-BorronJCJaraJRLozanoJA. Alpha-MSH and other melanogenic activators mediate opposite effects on tyrosinase and dopachrome tautomerase in B16/F10 mouse melanoma cells. J Invest Dermatol1992; 99: 435–9.
50.
RomeroCAberdamELarnierCOrtonneJP. Retinoic acid as modulator of UVB-induced melanocyte differentiation. Involvement of the melanogenic enzymes expression. J Cell Sci1994; 107: 1095–103.
51.
KawaguchiJMeePJSmithAG. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone2005; 36: 758–69.
52.
WangLMearJPKuanCYColbertMC. Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells. Dev Growth Differ2005; 47: 119–30.
53.
SalvarezzaSBRovasioRA. Exogenous retinoic acid decreases in vivo and in vitro proliferative activity during the early migratory stage of neural crest cells. Cell Prolif1997; 30: 71–80.
54.
CraneJFTrainorPA. Neural crest stem and progenitor cells. Annu Rev Cell Dev Biol2006; 22: 267–86.
