A fish with no sex: gonadal and adrenal functions partition between zebrafish NR5A1 co-orthologs.

People with NR5A1 mutations experience testicular dysgenesis, ovotestes, or adrenal insufficiency, but we do not completely understand the origin of this phenotypic diversity. NR5A1 is expressed in gonadal soma precursor cells before expression of the sex-determining gene SRY. Many fish have two co-orthologs of NR5A1 that likely partitioned ancestral gene subfunctions between them. To explore ancestral roles of NR5A1, we knocked out nr5a1a and nr5a1b in zebrafish. Single-cell RNA-seq identified nr5a1a-expressing cells that co-expressed genes for steroid biosynthesis and the chemokine receptor Cxcl12a in 1-day postfertilization (dpf) embryos, as does the mammalian adrenal-gonadal (interrenal-gonadal) primordium. In 2dpf embryos, nr5a1a was expressed stronger in the interrenal-gonadal primordium than in the early hypothalamus but nr5a1b showed the reverse. Adult Leydig cells expressed both ohnologs and granulosa cells expressed nr5a1a stronger than nr5a1b. Mutants for nr5a1a lacked the interrenal, formed incompletely differentiated testes, had no Leydig cells, and grew far larger than normal fish. Mutants for nr5a1b formed a disorganized interrenal and their gonads completely disappeared. All homozygous mutant genotypes lacked secondary sex characteristics, including male breeding tubercles and female sex papillae, and had exceedingly low levels of estradiol, 11-ketotestosterone, and cortisol. RNA-seq showed that at 21dpf, some animals were developing as females and others were not, independent of nr5a1 genotype. By 35dpf, all mutant genotypes greatly under-expressed ovary-biased genes. Because adult nr5a1a mutants form gonads but lack an interrenal and conversely, adult nr5a1b mutants lack a gonad but have an interrenal, the adrenal, and gonadal functions of the ancestral nr5a1 gene partitioned between ohnologs after the teleost genome duplication, likely owing to reciprocal loss of ancestral tissue-specific regulatory elements. Identifying such elements could provide hints to otherwise unexplained cases of Differences in Sex Development.

BICRA, a SWI/SNF Complex Member, Is Associated with BAF-Disorder Related Phenotypes in Humans and Model Organisms.

SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.

yippee like 3 (ypel3) is a novel gene required for myelinating and perineurial glia development.

Hypomyelination, a neurological condition characterized by decreased production of myelin sheets by glial cells, often has no known etiology. Elucidating the genetic causes of hypomyelination provides a better understanding of myelination, as well as means to diagnose, council, and treat patients. Here, we present evidence that YIPPEE LIKE 3 (YPEL3), a gene whose developmental role was previously unknown, is required for central and peripheral glial cell development. We identified a child with a constellation of clinical features including cerebral hypomyelination, abnormal peripheral nerve conduction, hypotonia, areflexia, and hypertrophic peripheral nerves. Exome and genome sequencing revealed a de novo mutation that creates a frameshift in the open reading frame of YPEL3, leading to an early stop codon. We used zebrafish as a model system to validate that YPEL3 mutations are causative of neuropathy. We found that ypel3 is expressed in the zebrafish central and peripheral nervous system. Using CRISPR/Cas9 technology, we created zebrafish mutants carrying a genomic lesion similar to that of the patient. Our analysis revealed that Ypel3 is required for development of oligodendrocyte precursor cells, timely exit of the perineurial glial precursors from the central nervous system (CNS), formation of the perineurium, and Schwann cell maturation. Consistent with these observations, zebrafish ypel3 mutants have metabolomic signatures characteristic of oligodendrocyte and Schwann cell differentiation defects, show decreased levels of Myelin basic protein in the central and peripheral nervous system, and develop defasciculated peripheral nerves. Locomotion defects were observed in adult zebrafish ypel3 mutants. These studies demonstrate that Ypel3 is a novel gene required for perineurial cell development and glial myelination.

Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes.

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.

Grxcr1 Promotes Hair Bundle Development by Destabilizing the Physical Interaction between Harmonin and Sans Usher Syndrome Proteins.

Morphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.

Usherin defects lead to early-onset retinal dysfunction in zebrafish.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination.

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.

Non-manifesting AHI1 truncations indicate localized loss-of-function tolerance in a severe Mendelian disease gene.

Determination of variant pathogenicity represents a major challenge in the era of high-throughput sequencing. Erroneous categorization may result if variants affect genes that are in fact dispensable. We demonstrate that this also applies to rare, apparently unambiguous truncating mutations of an established disease gene. By whole-exome sequencing (WES) in a consanguineous family with congenital non-syndromic deafness, we unexpectedly identified a homozygous nonsense variant, p.Arg1066*, in AHI1, a gene associated with Joubert syndrome (JBTS), a severe recessive ciliopathy. None of four homozygotes expressed any signs of JBTS, and one of them had normal hearing, which also ruled out p.Arg1066* as the cause of deafness. Homozygosity mapping and WES in the only other reported JBTS family with a homozygous C-terminal truncation (p.Trp1088Leufs*16) confirmed AHI1 as disease gene, but based on a more N-terminal missense mutation impairing WD40-repeat formation. Morpholinos against N-terminal zebrafish Ahi1, orthologous to where human mutations cluster, produced a ciliopathy, but targeting near human p.Arg1066 and p.Trp1088 did not. Most AHI1 mutations in JBTS patients result in truncated protein lacking WD40-repeats and the SH3 domain; disease was hitherto attributed to loss of these protein interaction modules. Our findings indicate that normal development does not require the C-terminal SH3 domain. This has far-reaching implications, considering that variants like p.Glu984* identified by preconception screening ('Kingsmore panel') do not necessarily indicate JBTS carriership. Genomes of individuals with consanguineous background are enriched for homozygous variants that may unmask dispensable regions of disease genes and unrecognized false positives in diagnostic large-scale sequencing and preconception carrier screening.

Mutation of POC1B in a severe syndromic retinal ciliopathy.

We describe a consanguineous Iraqi family with Leber congenital amaurosis (LCA), Joubert syndrome (JBTS), and polycystic kidney disease (PKD). Targeted next-generation sequencing for excluding mutations in known LCA and JBTS genes, homozygosity mapping, and whole-exome sequencing identified a homozygous missense variant, c.317G>C (p.Arg106Pro), in POC1B, a gene essential for ciliogenesis, basal body, and centrosome integrity. In silico modeling suggested a requirement of p.Arg106 for the formation of the third WD40 repeat and a protein interaction interface. In human and mouse retina, POC1B localized to the basal body and centriole adjacent to the connecting cilium of photoreceptors and in synapses of the outer plexiform layer. Knockdown of Poc1b in zebrafish caused cystic kidneys and retinal degeneration with shortened and reduced photoreceptor connecting cilia, compatible with the human syndromic ciliopathy. A recent study describes homozygosity for p.Arg106ProPOC1B in a family with nonsyndromic cone-rod dystrophy. The phenotype associated with homozygous p.Arg106ProPOC1B may thus be highly variable, analogous to homozygous p.Leu710Ser in WDR19 causing either isolated retinitis pigmentosa or Jeune syndrome. Our study indicates that POC1B is required for retinal integrity, and we propose POC1B mutations as a probable cause for JBTS with severe PKD.

Zebrafish models in translational research: tipping the scales toward advancements in human health.

Advances in genomics and next-generation sequencing have provided clinical researchers with unprecedented opportunities to understand the molecular basis of human genetic disorders. This abundance of information places new requirements on traditional disease models, which have the potential to be used to confirm newly identified pathogenic mutations and test the efficacy of emerging therapies. The unique attributes of zebrafish are being increasingly leveraged to create functional disease models, facilitate drug discovery, and provide critical scientific bases for the development of new clinical tools for the diagnosis and treatment of human disease. In this short review and the accompanying poster, we highlight a few illustrative examples of the applications of the zebrafish model to the study of human health and disease.

Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish.

The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.

Identification of differentially expressed genes in the zebrafish hypothalamic-pituitary axis.

The vertebrate hypothalamic-pituitary axis (HP) is the main link between the central nervous system and endocrine system. Although several signal pathways and regulatory genes have been implicated in adenohypophysis ontogenesis, little is known about hypothalamic-neurohypophysial development or when the HP matures and becomes functional. To identify markers of the HP, we constructed subtractive cDNA libraries between adult zebrafish hypothalamus and pituitary. We identified previously published genes, ESTs and novel zebrafish genes, some of which were predicted by genomic database analysis. We also analyzed expression patterns of these genes and found that several are expressed in the embryonic and larval hypothalamus, neurohypophysis, and/or adenohypophysis. Expression at these stages makes these genes useful markers to study HP maturation and function.

Notch signaling regulates endocrine cell specification in the zebrafish anterior pituitary.

The vertebrate pituitary gland is a key endocrine control organ that contains six distinct hormone secreting cell types. In this study, we analyzed the role of direct cell-to-cell Delta-Notch signaling in zebrafish anterior pituitary cell type specification. We demonstrate that initial formation of the anterior pituitary placode is independent of Notch signaling. Later however, loss of Notch signaling in mind bomb (mib) mutant embryos or by DAPT treatment leads to increased numbers of lactotropes and loss of corticotropes in the anterior pars distalis (APD), increased number of thyrotropes and loss of somatotrope cell types in the posterior pars distalis (PPD), and fewer melanotropes in the posterior region of the adenohypophysis, the pars intermedia (PI). Conversely, Notch gain of function leads to the opposite result, loss of lactotrope and thyrotrope cell specification, and an increased number of corticotropes, melanotropes, and gonadotropes in the pituitary. Our results suggest that Notch acts on placodal cells, presumably as a permissive signal, to regulate progenitor cell specification to hormone secreting cell types. We propose that Notch mediated lateral inhibition regulates the relative numbers of specified hormone cell types in the three pituitary subdomains.

Sparc (Osteonectin) functions in morphogenesis of the pharyngeal skeleton and inner ear.

Sparc (Osteonectin), a matricellular glycoprotein expressed by many differentiated cells, is a major non-collagenous constituent of vertebrate bones. Recent studies indicate that Sparc expression appears early in development, although its function and regulation during embryogenesis are largely unknown. We cloned zebrafish sparc and investigated its role during development, using a mo rpholino antisense oligonucleotide-based knockdown approach. Consistent with its strong expression in the otic vesicle and developing pharyngeal cartilages, knockdown of Sparc function resulted in specific inner ear and cartilage defects that are highlighted by changes in gene expression, morphology and behavior. We rescued the knockdown phenotypes by co-injecting sparc mRNA, providing evidence that the knockdown phenotype is due specifically to impairment of Sparc function. A comparison of the phenotypes of Sparc knockdown and known zebrafish mutants with similar defects places Sparc downstream of sox9 in the genetic network that regulates development of the pharyngeal skeleton and inner ear of vertebrates.

Caveolin-1 is required for lateral line neuromast and notochord development.

Caveolae have been linked to diverse cellular functions and to many disease states. In this study we have used zebrafish to examine the role of caveolin-1 and caveolae during early embryonic development. During development, expression is apparent in a number of tissues including Kupffer's vesicle, tailbud, intersomite boundaries, heart, branchial arches, pronephric ducts and periderm. Particularly strong expression is observed in the sensory organs of the lateral line, the neuromasts and in the notochord where it overlaps with expression of caveolin-3. Morpholino-mediated downregulation of Cav1alpha caused a dramatic inhibition of neuromast formation. Detailed ultrastructural analysis, including electron tomography of the notochord, revealed that the central regions of the notochord has the highest density of caveolae of any embryonic tissue comparable to the highest density observed in any vertebrate tissue. In addition, Cav1alpha downregulation caused disruption of the notochord, an effect that was enhanced further by Cav3 knockdown. These results indicate an essential role for caveolin and caveolae in this vital structural and signalling component of the embryo.

Fgf-dependent otic induction requires competence provided by Foxi1 and Dlx3b.

BACKGROUND: The inner ear arises from a specialized set of cells, the otic placode, that forms at the lateral edge of the neural plate adjacent to the hindbrain. Previous studies indicated that fibroblast growth factors (Fgfs) are required for otic induction; in zebrafish, loss of both Fgf3 and Fgf8 results in total ablation of otic tissue. Furthermore, gain-of-function studies suggested that Fgf signaling is not only necessary but also sufficient for otic induction, although the amount of induced ectopic otic tissue reported after misexpression of fgf3 or fgf8 varies among different studies. We previously suggested that Foxi1 and Dlx3b may provide competence to form the ear because loss of both foxi1 and dlx3b results in ablation of all otic tissue even in the presence of a fully functional Fgf signaling pathway. RESULTS: Using a transgenic line that allows us to misexpress fgf8 under the control of the zebrafish temperature-inducible hsp70 promoter, we readdressed the role of Fgf signaling and otic competence during placode induction. We find that misexpression of fgf8 fails to induce formation of ectopic otic vesicles outside of the endogenous ear field and has different consequences depending upon the developmental stage. Overexpression of fgf8 from 1-cell to midgastrula stages leads to formation of no or small otic vesicles, respectively. Overexpression of fgf8 at these stages never leads to ectopic expression of foxi1 or dlx3b, contrary to previous studies that indicated that foxi1 is activated by Fgf signaling. Consistent with our results we find that pharmacological inhibition of Fgf signaling has no effect on foxi1 or dlx3b expression, but instead, Bmp signaling activates foxi1, directly and dlx3b, indirectly. In contrast to early activation of fgf8, fgf8 overexpression at the end of gastrulation, when otic induction begins, leads to much larger otic vesicles. We further show that application of a low dose of retinoic acid that does not perturb patterning of the anterior neural plate leads to expansion of foxi1 and to a massive Fgf-dependent otic induction. CONCLUSION: These results provide further support for the hypothesis that Foxi1 and Dlx3b provide competence for cells to respond to Fgf and form an otic placode.

pitx3 defines an equivalence domain for lens and anterior pituitary placode.

Hedgehog signaling is required for formation and patterning of the anterior pituitary gland. However, the role of Hedgehog in pituitary precursor cell specification and subsequent placode formation is not well understood. We analyzed pituitary precursor cell lineages and find that pitx3 and distal-less3b (dlx3b) expression domains define lens and pituitary precursor positions. We show that pitx3 is required for pituitary pre-placode formation and cell specification, whereas dlx3b and dlx4b are required to restrict pituitary placode size. In smoothened mutant embryos that cannot transduce Hedgehog signals, median pituitary precursors are mis-specified and form an ectopic lens. Moreover, overexpression of sonic hedgehog (shh) blocks lens formation, and derivatives of lens precursors express genes characteristic of pituitary cells. However, overexpression of shh does not increase median pituitary placode size nor does it upregulate patched (ptc) expression in pituitary precursors during early somitogenesis. Our study suggests that by the end of gastrulation, pitx3-expressing cells constitute an equivalence domain of cells that can form either pituitary or lens, and that a non-Hedgehog signal initially specifies this placodal field. During mid-somitogenesis, Hedgehog then acts on the established median placode as a necessary and sufficient signal to specify pituitary cell types.

Fgf3 and Fgf8 dependent and independent transcription factors are required for otic placode specification.

The vertebrate inner ear develops from the otic placode, an ectodermal thickening that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to signals from adjacent tissues to form the placode. Members of the Fgf family of growth factors and the Dlx family of transcription factors have been implicated in this signal-response pathway. We show that compromising Fgf3 and Fgf8 signaling blocks ear development; only a few scattered otic cells form. Removal of dlx3b, dlx4b and sox9a genes together also blocks ear development, although a few residual cells form an otic epithelium. These cells fail to form if sox9b function is also blocked. Combined loss of Fgf signaling and the three transcription factor genes, dlx3b, dlx4b and sox9a, also completely eliminates all indications of otic cells. Expression of sox9a but not dlx3b, dlx4b or sox9b requires Fgf3 and Fgf8. Our results provide evidence for Fgf3- and Fgf8-dependent and -independent genetic pathways for otic specification and support the notion that Fgf3 and Fgf8 function to induce both the otic placode and the epithelial organization of the otic vesicle.

A zebrafish sox9 gene required for cartilage morphogenesis.

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.