Many genes are members of large families that have arisen during evolution through gene duplication events. Our increasing understanding of gene organization at the scale of whole genomes is revealing further evidence for the extensive retention of genes that arise during duplication events of various types. Duplication is thought to be an important means of providing a substrate on which evolution can work. An understanding of gene duplication and its resolution is crucial for revealing mechanisms of genetic redundancy. Here, we consider both the theoretical framework and the experimental evidence to explain the preservation of duplicated genes.

Splitting pairs: the diverging fates of duplicated genes.

https://www.cell.com/trends/genetics/pdf/S0168-9525(04)00213-6.pdf

Subfunction partitioning, the teleost radiation and the annotation of the human genome

The zebrafish has emerged as an important model system with which to investigate cancer, particularly for validating genomics data and for undertaking screens for oncogenes and drivers of tumour progression and metastasis. This Review outlines what we have learned and could still learn from cancer research using the zebrafish.

Zebrafish cancer: the state of the art and the path forward

https://www.cell.com/current-biology/pdf/S0960-9822(03)00910-2.pdf

Shroom induces apical constriction and is required for hingepoint formation during neural tube closure.

http://uoneuro.uoregon.edu/~cristian/Canestro_files/pdf%20publications/Rodriguez-Mari%2005GEP%20zebrafish%20AMH.pdf

Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development

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.

A zebrafish sox9 gene required for cartilage morphogenesis

Zebrafish Hagoromo mutants upregulate fgf8 post-embryonically and develop neuroblastoma

Erratum: A zebrafish sox9 gene required for cartilage morphogenesis (Development vol. 129 (5065-5079))

BackgroundCraniofacial birth defects result from defects in cranial neural crest (NC) patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1) signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified.ResultsThrough screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage.ConclusionThis work represents a systematic identification of approximately 25% of the essential genes required for craniofacial development. The identification of zebrafish models for two human disease syndromes indicates that homologs to the other genes are likely to also be relevant for human craniofacial development. The initial characterization of wdr68 suggests an important role in craniofacial development for the highly conserved Wdr68-Dyrk1 protein complexes.

A zebrafish screen for craniofacial mutants identifies wdr68as a highly conserved gene required for endothelin-1 expression

The invention features methods and reagents for introducing a mutation into a gene in a cell. The method includes contacting the cell with a recombinant retrovirus including: (i) branch-point sequence; (ii) a polypyrimidine tract; (iii) a splice acceptor; (iv) a splice donor; and (v) viral long-terminal repeats, wherein the splice acceptor and the splice donor flank nucleic acid sequence encoding a stop codon that is in frame with the splice acceptor; and allowing the retrovirus to integrate into a gene of the cell. Integration of the retrovirus into the gene introduces a mutation into the gene.

Adam Amsterdam

Papers

A zebrafish sox9 gene required for cartilage morphogenesis

Zebrafish Hagoromo mutants upregulate fgf8 post-embryonically and develop neuroblastoma

Erratum: A zebrafish sox9 gene required for cartilage morphogenesis (Development vol. 129 (5065-5079))

A zebrafish screen for craniofacial mutants identifies wdr68as a highly conserved gene required for endothelin-1 expression

Retroviral vectors and methods for production and use thereof