title: A zebrafish sox9 gene required for cartilage morphogenesis.
abstract: The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.
authors.affiliation: Institute of Neuroscience, University of Oregon, Eugene 97403, USA.
authors.forename: Yi-Lin
authors.initials: YL
authors.lastname: Yan
authors.forename: Craig T
authors.initials: CT
authors.lastname: Miller
authors.forename: Robert M
authors.initials: RM
authors.lastname: Nissen
authors.forename: Amy
authors.initials: A
authors.lastname: Singer
authors.forename: Dong
authors.initials: D
authors.lastname: Liu
authors.forename: Anette
authors.initials: A
authors.lastname: Kirn
authors.forename: Bruce
authors.initials: B
authors.lastname: Draper
authors.forename: John
authors.initials: J
authors.lastname: Willoughby
authors.forename: Paul A
authors.initials: PA
authors.lastname: Morcos
authors.forename: Adam
authors.initials: A
authors.lastname: Amsterdam
authors.forename: Bon-Chu
authors.initials: BC
authors.lastname: Chung
authors.forename: Monte
authors.initials: M
authors.lastname: Westerfield
authors.forename: Pascal
authors.initials: P
authors.lastname: Haffter
authors.forename: Nancy
authors.initials: N
authors.lastname: Hopkins
authors.forename: Charles
authors.initials: C
authors.lastname: Kimmel
authors.forename: John H
authors.initials: JH
authors.lastname: Postlethwait
authors.forename: Robert
authors.initials: R
authors.lastname: Nissen
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001846:Bone Development / Q000235:genetics; D002356:Cartilage / Q000002:abnormalities / Q000196:embryology* / Q000254:growth & development; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D020440:Gene Duplication; D018507:Gene Expression Regulation, Developmental; D006609:High Mobility Group Proteins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D009132:Muscles / Q000196:embryology; D009154:Mutation; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D010009:Osteochondrodysplasias / Q000196:embryology / Q000235:genetics; D010614:Pharynx / Q000196:embryology; D012326:RNA Splicing / Q000187:drug effects; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics* / Q000254:growth & development
chemical_list: D018076:DNA, Complementary; D006609:High Mobility Group Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors