The molecular genetic mechanisms of cartilageconstruction are incompletely understood. Zebrafishembryos homozygous for jellyfish (jef) mutations showcraniofacial defects and lack cartilage elements of theneurocranium, pharyngeal arches, and pectoral girdlesimilar to humans with campomelic dysplasia. We showthat two alleles of jef contain mutations in sox9a, one of twozebrafish orthologs of the human transcription factorSOX9. A mutation induced by ethyl nitrosourea changed aconserved nucleotide at a splice junction and severelyreduced splicing of sox9a transcript. A retrovirus insertioninto sox9a disrupted its DNA-binding domain. Inhibitingsplicing of the sox9a transcript in wild-type embryos withsplice site-directed morpholino antisense oligonucleotidesproduced a phenotype like jef mutant larvae, and causedsox9a transcript to accumulate in the nucleus; thisaccumulation can serve as an assay for the efficacy of amorpholino independent of phenotype. RNase-protectionassays showed that in morpholino-injected animals, thepercent of splicing inhibition decreased from 80% at 28hours post fertilization to 45% by 4 days. Homozygousmutant embryos had greatly reduced quantities of col2a1message, the major collagen of cartilage. Analysis of dlx2expression showed that neural crest specification andmigration was normal in jef (sox9a) embryos. Confocalimages of living embryos stained with BODIPY-ceramiderevealed at single-cell resolution the formation ofprecartilage condensations in mutant embryos. Besides thelack of overt cartilage differentiation, pharyngeal archcondensations in jef (sox9a) mutants lacked three specificmorphogenetic behaviors: the stacking of chondrocytesinto orderly arrays, the individuation of pharyngealcartilage organs and the proper shaping of individualcartilages. Despite the severe reduction of cartilages,analysis of titin expression showed normal musclepatterning in jef (sox9a) mutants. Likewise, calcein labelingrevealed that early bone formation was largely unaffectedin jef (sox9a) mutants. These studies show that jef (sox9a)is essential for both morphogenesis of condensations andovert cartilage differentiation.