In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.

Understanding how developmental systems evolve after genome amplification is important for discerning the origins of vertebrate novelties, including neural crest, placodes, cartilage and bone. Sox9 is important for the development of these features, and zebrafish has two co-orthologs of tetrapod SOX9 stemming from an ancient genome duplication event in the lineage of ray-fin fish. We have used a genotype-driven screen to isolate a mutation deleting sox9b function, and investigated its phenotype and genetic interactions with a sox9a null mutation. Analysis of mutant phenotypes strongly supports the interpretation that ancestral gene functions partitioned spatially and temporally between Sox9 co-orthologs. Distinct subsets of the craniofacial skeleton, otic placode and pectoral appendage express each gene, and are defective in each single mutant. The double mutant phenotype is additive or synergistic. Ears are somewhat reduced in each single mutant but are mostly absent in the double mutant. Loss-of-function animals from mutations and morpholino injections, and gain-of-function animals injected with sox9a and sox9b mRNAs showed that sox9 helps regulate other early crest genes, including foxd3, sox10, snai1b and crestin, as well as the cartilage gene col2a1 and the bone gene runx2a;however, tfap2a was nearly unchanged in mutants. Chondrocytes failed to stack in sox9a mutants, failed to attain proper numbers in sox9b mutants and failed in both morphogenetic processes in double mutants. Pleiotropy can cause mutations in single copy tetrapod genes, such as Sox9, to block development early and obscure later gene functions. By contrast, subfunction partitioning between zebrafish co-orthologs of tetrapod genes, such as sox9a and sox9b, can relax pleiotropy and reveal both early and late developmental gene functions.

Disruption of signaling pathways such as those mediated by Shh or Pdgf causes craniofacial disease, including cleft palate. The role that microRNAs play in modulating palatogenesis, however, is completely unknown. We show, in zebrafish, that the microRNA Mirn140 negatively regulates Pdgf signaling during palatal development and we provide a mechanism for how disruption of Pdgf signaling causes palatal clefting. The pdgf-receptor alpha (pdgfra) 3' UTR contains a Mirn140 binding site functioning in the negative regulation of Pdgfra protein levels in vivo. Both pdgfra mutants and Mirn140-injected embryos share panoply of facial defects including clefting of the crestderived cartilages that develop in the roof of the larval mouth. Concomitantly, the oral ectoderm beneath where these cartilages develop loses pitx2 and shha expression. Mirn140 modulates Pdgfmediated attraction of cranial neural crest cells to the oral ectoderm, where crest-derived signals are necessary for oral ectodermal gene expression. Both Mirn140 loss-of-function and pdgfra overexpression alters palatal shape and causes neural crest cells to accumulate around the optic stalk, a source of the ligand Pdgfaa. Conserved molecular genetics and expression patterns of mirn140 and pdgfra suggest that their regulatory interactions are ancient methods of palatogenesis that provide a candidate mechanism for cleft palate.Cleft palate and other craniofacial diseases are common in humans and have complex cellular and genetic etiologies. In amniotes, the palate serves to separate the nasal and oral cavities and is generated through an intricate series of morphogenic events that include early neural crest cell migration and cell-cell signaling during the formation of facial prominences, as well as later generation and fusion of palatal shelves. While later events involving palatal shelves have not been described in zebrafish, palatal precursors migrate both rostral and caudal to the eye to condense upon the oral ectoderm in amniotes 1 as well as zebrafish 2,3 and evidence continues to accumulate that the early signaling environment governing palatogenesis is also largely equivalent [3][4][5][6] . For instance, Hh signaling is crucial for palatogenesis in humans and zebrafish 3,4,7 . Zebrafish and amniotes also share expression patterns of palatogenic genes such as Shh 4,8 , Fgf8 4,9,10 and Pdgf receptor alpha (Pdgfra) [11][12][13] .In mouse, the Pdgf family consists of four soluble ligands, Pdgfa, Pdgfb, Pdgfc, and Pdgfd as well as two receptor tyrosine kinases, Pdgfra and Pdgfrb 14 . Pdgf signaling regulates a myriad of biological processes as demonstrated by analyses of mouse Pdgf ligand and receptor mutants 14 . Mice null for Pdgfra have a facial clefting phenotype that includes cleft palate 12,13 . This facial phenotype is fully recapitulated in mice doubly mutant for Pdgfa and Pdgfc 15. Most Pdgfc mutants have cleft palate 15 MicroRNAs (miRNAs) provide a unique mechanism for modulating signaling pathways [17][18][19][20] . Skeletogenic, including...

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Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a model for development and human health. We used RADtag population genomics to identify sex-linked polymorphisms. After verifying this "RAD-sex" method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, "male genotypes" became males and some "female genotypes" also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture.. . .researchers using standard lines of zebrafish that have long been maintained in laboratories are often plagued by severe sex ratio distortions. . .. C. Lawrence, J. P. Ebersole, and R. V. Kesseli (2008) C ONSIDERING the fundamental importance of sex for species propagation, it is surprising that primary sexdetermining mechanisms are not strongly conserved among animal taxa (Bull 1983;Charlesworth 1996;Ming et al. 2011;Bachtrog et al. 2014). Closely related species or even populations of the same species can have different sexdetermining mechanisms (Takehana et al. 2007;Ross et al. 2009;Kobayashi et al. 2013;Heule et al. 2014;Larney et al. 2014). Zebrafish (Danio rerio) is a popular model for studies of vertebrate development, behavior, physiology, evolution, disease, and human health (Mills et al. 2007;Seth et al. 2013;Braasch et al. 2014;Ota and Kawahara 2014;Wilkinson et al. 2014), but researchers struggle with highly variable and distorted sex ratios, and investigations into the genetic nature of zebrafish sex determination are conflicting. To help understand these issues, we conducted a population genomic study of sex determination in multiple zebrafish strains. Zebrafish exhibit...

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.

To understand the hierarchy of developmental controls underlying axis specification in vertebrate embryos, it is helpful to identify relationships between regulatory molecules and the genes that give axial cells their differentiated phenotypes. This work reports the cloning and expression pattern of one of these differentiation genes, a type I1 collagen (col2al) gene from the zebrafish Danio rerio. Along the embryonic axis, col2al is expressed dynamically in three rows that are each a single cell wide: the notochord and the rows of cells immediately dorsal and ventral to it-the floor plate of the central nervous system, and the hypochord. In addition, col2al is expressed in the pharyngeal arches, the epithelium of the otic capsule, and in the mesenchyme of the neurocranium. Experiments probed the expression pattern of col2al relative to that of known or potential regulators of axis development, including axial, sonic hedgehog, twist, and cyclops. The results showed that the spatial and temporal pattern of col2al expression in axial mesoderm follows the expression of twist closer than other genes tested. In cyclops embryos, which lack an intact floor plate, col2al expression was usually low, but not missing in cells in the ventral spinal cord. Because col2al expression reveals abnormalities in the notochord of cyclopsbz6 embryos, and less col2al-expressing mesenchyme accumulates rostra1 to the notochord in cyclops embryos, the effects of the cyclopsbz6 mutation are not confined to the central nervous system. 0 1995 Wiley-Liss, Inc.