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Uveal melanoma (UM) is the most common primary malignancy of the adult eye but lacks any FDA-approved therapy for the deadly metastatic disease. Thus, there is a great need to dissect the driving mechanisms for UM and develop strategies to evaluate potential therapeutics. Using an autochthonous zebrafish model, we previously identified MITF, the master melanocyte transcription factor, as a tumor suppressor in GNAQsupQ209L/sup-driven UM. Here, we show that zebrafish mitfa-deficient GNAQsupQ209L/sup-driven tumors significantly up-regulate neural crest markers, and that higher expression of a melanoma-associated neural crest signature correlates with poor UM patient survival. We further determined how the mitfa-null state, as well as expression of GNAQsupQ209L/sup, YAPsupS127A;S381A/sup, or BRAFsupV600E/suponcogenes, impacts melanocyte lineage cells before they acquire the transformed state. Specifically, examination 5 days post-fertilization showed that mitfa-deficiency is sufficient to up-regulate pigment progenitor and neural crest markers, while GNAQsupQ209L/supexpression promotes a proliferative phenotype that is further enhanced by YAPsupS127A;S381A/supco-expression. Finally, we show that this oncogene-induced proliferative phenotype can be used to screen chemical inhibitors for their efficacy against the UM pathway. Overall, this study establishes that a neural crest signature correlates with poor UM survival, and describes an in vivo assay for preclinical trials of potential UM therapeutics.

Supplementary Data from Zebrafish Hagoromo Mutants Up-Regulate fgf8 Postembryonically and Develop Neuroblastoma

We screened an existing collection of zebrafish insertional mutants for cancer susceptibility by histologic examination of heterozygotes at 2 years of age. As most mutants had no altered cancer predisposition, this provided the first comprehensive description of spontaneous tumor spectrum and frequency in adult zebrafish. Moreover, the screen identified four lines, each carrying a different dominant mutant allele of Hagoromo previously linked to adult pigmentation defects, which develop tumors with high penetrance and that histologically resemble neuroblastoma. These tumors are clearly neural in origin, although they do not express catecholaminergic neuronal markers characteristic of human neuroblastoma. The zebrafish tumors result from inappropriate maintenance of a cell population within the cranial ganglia that are likely neural precursors. These neoplasias typically remain small but they can become highly aggressive, initially traveling along cranial nerves, and ultimately filling the head. The developmental origin of these tumors is highly reminiscent of human neuroblastoma. The four mutant Hagoromo alleles all contain viral insertions in the fbxw4 gene, which encodes an F-box WD40 domain–containing protein. However, although one allele clearly reduced the levels of fbxw4 mRNA, the other three insertions had no detectable effect on fbw4 expression. Instead, we showed that all four mutations result in the postembryonic up-regulation of the neighboring gene, fibroblast growth factor 8 (fgf8). Moreover, fgf8 is highly expressed in the tumorigenic lesions. Although fgf8 overexpression is known to be associated with breast and prostate cancer in mammals, this study provides the first evidence that fgf8 misregulation can lead to neural tumors. (Mol Cancer Res 2009;7(6):841–50)

Cutaneous melanoma (CM) and uveal melanoma (UM) both originate from the melanocytic lineage but are primarily driven by distinct oncogenic drivers, BRAF/NRAS or GNAQ/GNA11, respectively. The melanocytic master transcriptional regulator, MITF, is essential for both CM development and maintenance, but its role in UM is largely unexplored. Here, we use zebrafish models to dissect the key UM oncogenic signaling events and establish the role of MITF in UM tumors. Using a melanocytic lineage expression system, we showed that patient-derived mutations of GNAQ (GNAQQ209L) or its upstream CYSLTR2 receptor (CYSLTR2L129Q) both drive UM when combined with a cooperating mutation, tp53M214K/M214K. The tumor-initiating potential of the major GNAQ/11 effector pathways, YAP, and phospholipase C-β (PLCβ)–ERK was also investigated in this system and thus showed that while activated YAP (YAPAA) induced UM with high potency, the patient-derived PLCβ4 mutation (PLCB4D630Y) very rarely yielded UM tumors in the tp53M214K/M214K context. Remarkably, mitfa deficiency was profoundly UM promoting, dramatically accelerating the onset and progression of tumors induced by Tg(mitfa:GNAQQ209L);tp53M214K/M214K or Tg(mitfa:CYSLTR2L129Q);tp53M214K/M214K. Moreover, mitfa loss was sufficient to cooperate with GNAQQ209L to drive tp53–wild type UM development and allowed Tg(mitfa:PLCB4D630Y);tp53M214K/M214K melanocyte lineage cells to readily form tumors. Notably, all of the mitfa−/− UM tumors, including those arising in Tg(mitfa:PLCB4D630Y);tp53M214K/M214K;mitfa−/− zebrafish, displayed nuclear YAP while lacking hyperactive ERK indicative of PLCβ signaling. Collectively, these data show that YAP signaling is the major mediator of UM and that MITF acts as a bona fide tumor suppressor in UM in direct opposition to its essential role in CM.

Despite the pivotal role of MYC in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and many other cancers, the mechanisms underlying MYC-mediated tumorigenesis remain inadequately understood. Here we utilized a well-characterized zebrafish model of Myc-induced T-ALL for genetic studies to identify novel genes contributing to disease onset. We found that heterozygous inactivation of a tricarboxylic acid (TCA) cycle enzyme, dihydrolipoamide S-succinyltransferase (Dlst), significantly delayed tumor onset in zebrafish without detectable effects on fish development. DLST is the E2 transferase of the α-ketoglutarate (α-KG) dehydrogenase complex (KGDHC), which converts α-KG to succinyl-CoA in the TCA cycle. RNAi knockdown of DLST led to decreased cell viability and induction of apoptosis in human T-ALL cell lines. Polar metabolomics profiling revealed that the TCA cycle was disrupted by DLST knockdown in human T-ALL cells, as demonstrated by an accumulation of α-KG and a decrease of succinyl-CoA. Addition of succinate, the downstream TCA cycle intermediate, to human T-ALL cells was sufficient to rescue defects in cell viability caused by DLST inactivation. Together, our studies uncovered an important role for DLST in MYC-mediated leukemogenesis and demonstrated the metabolic dependence of T-lymphoblasts on the TCA cycle, thus providing implications for targeted therapy.

Talin (tln) binds and activates integrins to couple extracellular matrix–bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1fl02k mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin β1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgβ1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.—Wu, Q., Zhang, J., Koh, W., Yu, Q., Zhu, X., Amsterdam, A., Davis, G. E., Arnaout, M. A., Xiong, J.-W. Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

Uveal melanoma (UM) is the most common primary intraocular cancer and has a high incidence of metastasis, which lacks any effective treatment. Here, we present zebrafish models of UM, which are driven by melanocyte-specific expression of activating GNAQ or GNA11 alleles, GNAQ/11 Q209L , the predominant initiating mutations for human UM. When combined with mutant tp53, GNAQ/11 Q209L transgenics develop various melanocytic tumors, including UM, with near complete penetrance. These tumors display nuclear YAP localization and thus phenocopy human UM. We show that GNAQ/11 Q209L expression induces profound melanocyte defects independent of tp53 mutation, which are apparent within 3 days of development. First, increases in melanocyte number, melanin content, and subcellular melanin distribution result in hyperpigmentation. Additionally, altered melanocyte migration, survival properties, and evasion of normal boundary cues lead to aberrant melanocyte localization and stripe patterning. Collectively, these data show that GNAQ/11 Q209L is sufficient to induce numerous protumorigenic changes within melanocytes., National Institute of General Medical Sciences (Grant T32GM007287), National Cancer Institute (Grant P30‐CA14051)

Hepatic steatosis is the initial stage of nonalcoholic fatty liver disease (NAFLD) and may predispose to more severe hepatic disease, including hepatocellular carcinoma. Endoplasmic reticulum (ER) stress has been recently implicated as a novel mechanism that may lead to NAFLD, although the genetic factors invoking ER stress are largely unknown. During a screen for liver defects from a zebrafish insertional mutant library, we isolated the mutant cdipthi559Tg/+ (hi559). CDIPT is known to play an indispensable role in phosphatidylinositol (PtdIns) synthesis. Here we show that cdipt is expressed in the developing liver, and its disruption in hi559 mutants abrogates de novo PtdIns synthesis, resulting in hepatomegaly at 5 days postfertilization. The hi559 hepatocytes display features of NAFLD, including macrovesicular steatosis, ballooning, and necroapoptosis. Gene set enrichment of microarray profiling revealed significant enrichment of endoplasmic reticulum stress response (ERSR) genes in hi559 mutants. ER stress markers, including atf6, hspa5, calr, and xbp1, are selectively up-regulated in the mutant liver. The hi559 expression profile showed significant overlap with that of mammalian hepatic ER stress and NAFLD. Ultrastructurally, the hi559 hepatocytes display marked disruption of ER architecture with hallmarks of chronic unresolved ER stress. Induction of ER stress by tunicamycin in wild-type larvae results in a fatty liver similar to hi559, suggesting that ER stress could be a fundamental mechanism contributing to hepatic steatosis. Conclusion: cdipt-deficient zebrafish exhibit hepatic ER stress and NAFLD pathologies, implicating a novel link between PtdIns, ER stress, and steatosis. The tractability of hi559 mutant provides a valuable tool to dissect ERSR components, their contribution to molecular pathogenesis, and evaluation of novel therapeutics of NAFLD. (HEPATOLOGY 2011;)

Aneuploidy is a hallmark of human cancers, but most mouse cancer models lack the extensive aneuploidy seen in many human tumors. The zebrafish is becoming an increasingly popular model for studying cancer. Here we report that malignant peripheral nerve sheath tumors (MPNSTs) that arise in zebrafish as a result of mutations in either ribosomal protein ( rp ) genes or in p53 are highly aneuploid. Karyotyping reveals that these tumors frequently harbor near-triploid numbers of chromosomes, and they vary in chromosome number from cell to cell within a single tumor. Using array comparative genomic hybridization, we found that, as in human cancers, certain fish chromosomes are preferentially overrepresented, whereas others are underrepresented in many MPNSTs. In addition, we obtained evidence for recurrent subchromosomal amplifications and deletions that may contain genes involved in cancer initiation or progression. These focal amplifications encompassed several genes whose amplification is observed in human tumors, including met , cyclinD2 , slc45a3 , and cdk6 . One focal amplification included fgf6a . Increasing fgf signaling via a mutation that overexpresses fgf8 accelerated the onset of MPNSTs in fish bearing a mutation in p53 , suggesting that fgf6a itself may be a driver of MPNSTs. Our results suggest that the zebrafish is a useful model in which to study aneuploidy in human cancer and in which to identify candidate genes that may act as drivers in fish and potentially also in human tumors.

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase II alpha-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.

Background— Cardiac repolarization, the process by which cardiomyocytes return to their resting potential after each beat, is a highly regulated process that is critical for heart rhythm stability. Perturbations of cardiac repolarization increase the risk for life-threatening arrhythmias and sudden cardiac death. Although genetic studies of familial long-QT syndromes have uncovered several key genes in cardiac repolarization, the major heritable contribution to this trait remains unexplained. Identification of additional genes may lead to a better understanding of the underlying biology, aid in identification of patients at risk for sudden death, and potentially enable new treatments for susceptible individuals. Methods and Results— We extended and refined a zebrafish model of cardiac repolarization by using fluorescent reporters of transmembrane potential. We then conducted a drug-sensitized genetic screen in zebrafish, identifying 15 genes, including GINS3 , that affect cardiac repolarization. Testing these genes for human relevance in 2 concurrently completed genome-wide association studies revealed that the human GINS3 ortholog is located in the 16q21 locus, which is strongly associated with QT interval. Conclusions— This sensitized zebrafish screen identified 15 novel myocardial repolarization genes. Among these genes is GINS3 , the human ortholog of which is a major locus in 2 concurrent human genome-wide association studies of QT interval. These results reveal a novel network of genes that regulate cardiac repolarization.

Zebrafish carrying heterozygous mutations for 17 different ribosomal protein ( rp ) genes are prone to developing malignant peripheral nerve sheath tumors (MPNSTs), a tumor type that is seldom seen in laboratory strains of zebrafish. Interestingly, the same rare tumor type arises in zebrafish that are homozygous for a loss-of-function point mutation in the tumor suppressor gene p53 . For these reasons, and because p53 is widely known to be mutated in the majority of human cancers, we investigated the status of p53 in the rp +/− MPNSTs. Using monoclonal antibodies that we raised to zebrafish p53, we found that cells derived from rp +/− MPNSTs are significantly impaired in their ability to produce p53 protein even in the presence of a proteasome inhibitor and γ-irradiation. Although the coding regions of the p53 gene remain wild type, the gene is transcribed, and overall protein production rates appear normal in rp +/− MPNST cells, p53 protein does not get synthesized. This defect is observed in all MPNSTs we examined that were derived from our 17 zebrafish lines with rp gene mutations. To date, studies of p53 in malignancies have focused predominantly on either p53 gene mutations or the aberrant posttranslational regulation of the p53 protein. Our results show that the appropriate amount of numerous ribosomal proteins is required for p53 protein production in vivo and that disruption of this regulation most likely contributes to tumorigenesis.

Epithelial integrity requires the adhesion of cells to each other as well as to an underlying basement membrane. The modulation of adherence properties is crucial to morphogenesis and wound healing, and deregulated adhesion has been implicated in skin diseases and cancer metastasis. Here, we describe zebrafish that are mutant in the serine protease inhibitor Hai1a (Spint1la),which display disrupted epidermal integrity. These defects are further enhanced upon combined loss of hai1a and its paralog hai1b. By applying in vivo imaging, we demonstrate that Hai1-deficient keratinocytes acquire mesenchymal-like characteristics, lose contact with each other, and become mobile and more susceptible to apoptosis. In addition, inflammation of the mutant skin is evident, although not causative of the epidermal defects. Only later, the epidermis exhibits enhanced cell proliferation. The defects of hai1 mutants can be phenocopied by overexpression and can be fully rescued by simultaneous inactivation of the serine protease Matriptase1a(St14a), indicating that Hai1 promotes epithelial integrity by inhibiting Matriptase1a. By contrast, Hepatocyte growth factor (Hgf), a well-known promoter of epithelial-mesenchymal transitions and a prime target of Matriptase1 activity, plays no major role. Our work provides direct genetic evidence for antagonistic in vivo roles of Hai1 and Matriptase1a to regulate skin homeostasis and remodeling.

Uveal melanoma is the most common intraocular cancer, with an estimated 2000 cases diagnosed yearly in the U.S. Tumors arise from melanocytes in the choroid, iris, and ciliary body. Treatment typic...

In order to rapidly identify a substantial fraction of the genes with a unique and essential role in vertebrate development, the laboratory of Nancy Hopkins at MIT has performed a large insertional mutagenesis screen in zebrafish using a pseudotyped retroviral vector as the mutagen. We have recovered mutations in about one-quarter of the embryonic essential genes in this organism, and have identified the mutated genes in nearly all of these (333). As the ease of gene identification allowed us to clone the mutated genes for nearly all of the mutants rather than prioritizing based upon the initially observed phenotypes, this has provided an unbiased view of the diversity of genes required for vertebrate development as well as a large collection of mutants to be screened for more specific phenotypes. In collaboration with other labs, we have screened the insertional mutant for the development of a variety of organs and cell types, as well as phenotypes that could represent disease models, such as cystic kidney and hepatomegaly. Furthermore, while all of these mutants are embryonic lethal in their homozygous state, we are investigating the heterozygous adults for additional phenotypes, such as cancer predisposition.

Hepatomegaly is a sign of many liver disorders. To identify zebrafish mutants to serve as models for hepatic pathologies, we screened for hepatomegaly at day 5 of embryogenesis in 297 zebrafish lines bearing mutations in genes that are essential for embryonic development. Seven mutants were identified, and three have phenotypes resembling different liver diseases. Mutation of the class C vacuolar protein sorting gene vps18results in hepatomegaly associated with large, vesicle-filled hepatocytes,which we attribute to the failure of endosomal-lysosomal trafficking. Additionally, these mutants develop defects in the bile canaliculi and have marked biliary paucity, suggesting that vps18 also functions to traffic vesicles to the hepatocyte apical membrane and may play a role in the development of the intrahepatic biliary tree. Similar findings have been reported for individuals with arthrogryposis-renal dysfunction-cholestasis(ARC) syndrome, which is due to mutation of another class C vps gene. A second mutant, resulting from disruption of the tumor suppressor gene nf2,develops extrahepatic choledochal cysts in the common bile duct, suggesting that this gene regulates division of biliary cells during development and that nf2 may play a role in the hyperplastic tendencies observed in biliary cells in individuals with choledochal cysts. The third mutant is in the novel gene foie gras, which develops large, lipid-filled hepatocytes, resembling those in individuals with fatty liver disease. These mutants illustrate the utility of zebrafish as a model for studying liver development and disease, and provide valuable tools for investigating the molecular pathogenesis of congenital biliary disorders and fatty liver disease.

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.

The zebrafish, originally an object of study as an inexpensive and prolific vertebrate embryological model with a plethora of genetic tricks, has over the past decade moved to large-scale chemical mutagenesis and recently came of age as a high throughput transgenic model with a sequenced genome nearing completion. Insertional mutagenesis, gene trapping and enhancer detection are all contributing to the increasing speed with which research in this biomedical model is progressing. We review here some of the recent developments in the emerging field of zebrafish developmental genomics and transgenesis. Developmental Dynamics 234:255–268, 2005. © 2005 Wiley-Liss, Inc.

We completed a large insertional mutagenesis screen in zebrafish to identify genes essential for embryonic and early larval development. We isolated 525 mutants, representing lesions in approximately 390 different genes, and we cloned the majority of these. Here we describe 315 mutants and the corresponding genes. Our data suggest that there are roughly 1,400 embryonic-essential genes in the fish. Thus, we have mutations in approximately 25% of these genes and have cloned approximately 22% of them. Re-screens of our collection to identify mutants with specific developmental defects suggest that approximately 50 genes are essential for the development of some individual organs or cell types. Seventy-two percent of the embryonic-essential fish genes have homologues in yeast, 93% have homologues in invertebrates (fly or worm), and 99% have homologues in human. Yeast and worm orthologues of genes that are essential for early zebrafish development have a strong tendency to be essential for viability in yeast and for embryonic development in the worm. Thus, the trait of being a genetically essential gene is conserved in evolution. This mutant collection should be a valuable resource for diverse studies of cell and developmental biology.

To identify genes required for development of the brain and somites, we performed a pilot screen of gynogenetic haploid zebrafish embryos produced from mothers mutagenized by viral insertion. We describe an efficient method to identify new mutations and the affected gene. In addition, we report the results of a small-scale screen that identified five genes required for brain development, including novel alleles of nagie oko, pou5f1, ribosomal protein L36, and n-cadherin, as well as a novel allele of the laminin g1 gene that is required for normal skeletal muscle fiber organization and somite patterning.

Mutations in ESCO2, one of two EStablishment of COhesion factors necessary for proper Sister Chromatid Cohesion (SCC), cause a spectrum of developmental defects in the autosomal recessive disorder Roberts Syndrome (RBS), warranting in vivo analysis of the consequence of cohesion dysfunction. Through a genetic screen in zebrafish targeting embryonic lethal mutants with increased genomic instability, we have identified an esco2 mutant zebrafish. Utilizing the natural transparency of zebrafish embryos, we have developed a novel technique to observe chromosome dynamics within a single cell during mitosis in a live vertebrate embryo. Within esco2 mutant embryos, we observed premature chromatid separation, a unique chromosome scattering, prolonged mitotic delay, and genomic instability in the form of anaphase bridges and micronuclei formation. Cytogenetic studies indicated complete chromatid separation and high levels of aneuploidy within mutant embryos. Amongst aneuploid spreads, we observed predominantly decreases in chromosome number, suggesting either cells with micronuclei or micronuclei themselves are eliminated. We also demonstrated that the genomic instability leads to p53-dependent neural tube apoptosis. Surprisingly, while many cells require Esco2 to establish cohesion, 10-20% of cells have only weakened cohesion in the absence of Esco2, suggesting that compensatory cohesion mechanisms exist in these cells that undergo a normal mitotic division. These studies provide a unique in vivo vertebrate view of the mitotic defects and consequences of cohesion establishment loss, and they provide a compensation-based model to explain the RBS phenotypes.

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 intosox9a disrupted its DNA-binding domain. Inhibiting splicing of thesox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jefmutant 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 injef (sox9a) mutants. These studies show that jef(sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.

To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.

Dysfunctional mucus barriers can result in important pulmonary and gastrointestinal conditions, but model systems to study the underlying causes are largely missing. We identified and characterized five mucin homologues in zebrafish, and demonstrated a strategy for fluorescence labeling of one selected mucin. These tools can be used for in vivo experiments and in pharmacological and genetic screens to study the dynamics and mechanisms of mucosal physiology., National Institute of Environmental Health Sciences (Grant P30-ES002109), Johnson & Johnson. Corporate Office of Science and Technology, National Institutes of Health (U.S.) (Grant CA106416), Kathy and Curt Marble Cancer Research Fund, David H. Koch Institute for Integrative Cancer Research at MIT (Zebrafish Core Facility)

The zebrafish, Danio rerio , has three types of pigment cells (melanophores, xanthophores and iridophores) and, in adult fish, these cells are organized into a stripe pattern. The mechanisms underlying formation of the stripe pattern are largely unknown. We report here the identification and characterization of a novel dominant zebrafish mutation, hagoromo ( hag ), which was generated by insertional mutagenesis using a pseudotyped retrovirus. The hag mutation caused disorganized stripe patterns. Two hag mutant alleles were isolated independently and proviruses were located within the fifth intron of a novel gene, which we named hag , encoding an F-box/WD40-repeat protein. The hag gene was mapped to linkage group (LG)13, close to fgf8 and pax2.1 . Amino acid sequence similarity, conserved exon–intron boundaries and conserved synteny indicated that zebrafish hag is an ortholog of mouse Dactylin , the gene mutated in the Dactylaplasia ( Dac ) mouse [1]. The Dac mutation is dominant and causes defects in digit formation in fore- and hindlimbs. This study revealed that the hag locus is important for pattern formation in fish but is involved in distinct morphogenetic events in different vertebrates.

Not really finished (nrf), a larval-lethal mutation in zebrafish generated by retroviral insertion, causes specific retinal defects. Analysis of mutant retinae reveals an extensive loss of photoreceptors and their precursors around the onset of visual function. These neurons undergo apoptosis during differentiation, affecting all classes of photoreceptors, suggesting an essential function of nrf for the development of all types of photoreceptors. In the mutant, some photoreceptors escape cell death, are functional and, as judged by opsin expression, belong to at least three classes of cones and one class of rods. The protein encoded by nrf is a close homologue of human Nuclear Respiratory Factor 1 and avian Initiation Binding Repressor, transcriptional regulators binding the upstream consensus sequence RCGCRYGCGY. At 24 hours of development, prior to neuronal differentiation, nrf is expressed ubiquitously throughout the developing retina and central nervous system. At 48 hours of development, expression of nrf is detected in the ganglion cell layer, in the neurons of the inner nuclear layer, and in the optic nerve and optic tracts, and, at 72 hours of development, is no longer detectable by in situ hybridization. Mutants contain no detectable nrf mRNA and die within 2 weeks postfertilization as larvae with reduced brain size. On the basis of its similarity with NRF-1 and IBR, nrf is likely involved in transcriptional regulation of multiple target genes, including those that encode mitochondrial proteins, growth factor receptors and other transcription factors. This demonstrates the power of insertional mutagenesis as a means for characterizing novel genes necessary for vertebrate retinal development.

Background: Linear DNA-based and Tol2-mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long-term transgene stability unpredictable with variable expression. Targeted, site-directed transgene integration into pre-determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site-directed transgenesis. Results: We report the implementation of the phiC31 integrase-mediated attP/attB recombination for site-directed zebrafish transgenics of attB-containing transgene vectors into single genomic attP landing sites. We generated Tol2-based single-insertion attP transgenic lines and established their performance in phiC31 integrase-catalyzed integration of an attB-containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue-specific transgene applications as well as long-term stability of phiC31-mediated transgenes. Conclusions: Our results establish phiC31 integrase-controlled site-directed transgenesis into single, genomic attP sites as space-, time-, and labor-efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community. Developmental Dynamics 242:949–963, 2013. © 2013 Wiley Periodicals, Inc.

The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers., Kathy and Curt Marble Cancer Research Fund, Arthur C. Merrill, National Institutes of Health (U.S.) (Grant CA106416), National Institutes of Health (U.S.) (Grant ROI RR020833), National Institutes of Health (U.S.) (Grant 1F32GM095213-01)

An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximately 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.

We have generated transgenic (Tg) lines of zebrafish in which the green fluorescent protein (GFP)-encoding gfp cDNA is driven by the Xenopus laevis ef1 α enhancer/promoter; Tg embryos from most of these lines show detectable fluorescence throughout their body. We have investigated the copy number of the Tg genes in fluorescent and nonfluorescent lines, in order to determine how this affects the production of detectable levels of GFP in the zebrafish embryo. Additionally, we have injected purified recombinant GFP into embryos to determine the intracellular GFP concentration required for detection, both when all of the cells in the embryo contain GFP and when only a few do.

The proto-oncogene MYC has been implicated in the pathogenesis of many human cancers, including hematological and solid malignancies1. In the majority of T-ALL cases, MYC is aberrantly expressed downstream of activated NOTCH1 mutations. Despite the pivotal role of MYC in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and many other cancers, the mechanisms underlying MYC-mediated tumorigenesis remain inadequately understood2. Here we utilized a well-characterized zebrafish model of Myc-induced T-ALL for genetic studies to identify novel genes contributing to disease onset. We found that heterozygous inactivation of a tricarboxylic acid (TCA) cycle enzyme, dihydrolipoamide S-succinyltransferase (Dlst), significantly delayed tumor onset. Concordant with our zebrafish results, RNAi knockdown of DLST led to decreased cell viability and induction of apoptosis in human T-ALL cell lines. DLST is the E2 transferase of the -ketoglutarate dehydrogenase complex (KGDHC), which converts -ketoglutarate (-KG) to succinyl-CoA in the TCA cycle3. Polar metabolomics profiling revealed that the TCA cycle was disrupted by DLST knockdown in human T-ALL cells, as demonstrated by an accumulation of -KG and its precursor glutamine, as well as a loss of succinyl-CoA. Addition of succinate, the downstream TCA cycle intermediate, to human T-ALL cells was sufficient to rescue defects in cell viability caused by DLST inactivation. Taken together, our studies identify DLST as an important mediator of MYC-driven leukemogenesis and provide compelling evidence for the metabolic dependence of T-ALL cells on the TCA cycle. 1. Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene 1999 May 13; 18(19): 3004-3016. 2. Sharma VM, Calvo JA, Draheim KM, Cunningham LA, Hermance N, Beverly L, et al. Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc. Molecular and cellular biology 2006 Nov; 26(21): 8022-8031. 3. Sheu KF, Blass JP. The alpha-ketoglutarate dehydrogenase complex. Ann N Y Acad Sci 1999; 893: 61-78. Citation Format: Nicole Anderson, Dun Li, H.L. Peng, Marc Mansour, Fabrice Laroche, Evisa Gjini, Daniel Helman, Itrat Harrold, Le Meng, Takaomi Sanda, Adam Amsterdam, Donna Neuberg, Travis Denton, Anurag Singh, A Thomas Look, Hui Feng. The TCA cycle transferase DLST is critical for MYC-mediated leukemogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1180.

The formation of the central nervous system depends on the coordinated development of neural and glial cell types that arise from a common precursor. Using an existing group of zebrafish mutants generated by viral insertion, we performed a “shelf-screen” to identify genes necessary for astroglial development and axon scaffold formation. We screened 274 of 315 viral insertion lines using antibodies that label axons (anti-Acetylated Tubulin) and astroglia (anti-Gfap) and identified 25 mutants with defects in gliogenesis, glial patterning, neurogenesis, and axon guidance. We also identified a novel class of mutants affecting radial glial cell numbers. Defects in astroglial patterning were always associated with axon defects, supporting an important role for axon-glial interactions during axon scaffold development. The genes disrupted in these viral lines have all been identified, providing a powerful new resource for the study of axon guidance, glio- and neurogenesis, and neuron-glial interactions during development of the vertebrate CNS., National Institutes of Health (U.S.) (Grant T32MH020051), National Institutes of Health (U.S.) (Grant F32NS043872)

Background: Photoreceptors of the retina are highly compartmentalized cells that function as the primary sensory neurons for receiving and initiating transmission of visual information. Proper morphogenesis of photoreceptor neurons is essential for their normal function and survival. We have characterized a zebrafish mutation, cannonball, that completely disrupts photoreceptor morphogenesis. Results: Analysis revealed a non-sense mutation in cytoplasmic dynein heavy chain 1 (dync1h1), a critical subunit in Dynein1, to underlie the cannonball phenotypes. Dynein1 is a large minus-end directed, microtubule motor protein complex that has been implicated in multiple, essential cellular processes. In photoreceptors, Dynein1 is thought to mediate post-Golgi vesicle trafficking, while Dynein2 is thought to be responsible for outer segment maintenance. Surprisingly, cannonball embryos survive until larval stages, owing to wild-type maternal protein stores. Retinal photoreceptor neurons, however, are significantly affected by loss of Dync1h1, as transmission electron microscopy and marker analyses demonstrated defects in organelle positioning and outer segment morphogenesis and suggested defects in post-Golgi vesicle trafficking. Furthermore, dosage-dependent antisense oligonucleotide knock-down of dync1h1 revealed outer segment abnormalities in the absence of overt inner segment polarity and trafficking defects. Consistent with a specific function of Dync1h1 within the outer segment, immunolocalization showed that this protein and other subunits of Dynein1 and Dynactin localized to the ciliary axoneme of the outer segment, in addition to their predicted inner segment localization. However, knock-down of Dynactin subunits suggested that this protein complex, which is known to augment many Dynein1 activities, is only essential for inner segment processes as outer segment morphogenesis was normal. Conclusions: Our results indicate that Dynein1 is required for multiple cellular processes in photoreceptor neurons, including organelle positioning, proper outer segment morphogenesis, and potentially post-Golgi vesicle trafficking. Titrated knock-down of dync1h1 indicated that outer segment morphogenesis was affected in photoreceptors that showed normal inner segments. These observations, combined with protein localization studies, suggest that Dynein1 may have direct and essential functions in photoreceptor outer segments, in addition to inner segment functions., National Institutes of Health (U.S) ( training grant in Vision Sciences T32EY014536 ), National Institutes of Health (U.S) ( R01EY03222 ), National Institutes of Health (U.S) (R01EY014167 ), National Eye Institute (Core Facilities grant P30EY001931)

Eukaryotes have numerous checkpoint pathways to protect genome fidelity during normal cell division and in response to DNA damage. Through a screen for G2/M checkpoint regulators in zebrafish, we identified ticrr (for TopBP1-interacting, checkpoint, and replication regulator), a previously uncharacterized gene that is required to prevent mitotic entry after treatment with ionizing radiation. Ticrr deficiency is embryonic-lethal in the absence of exogenous DNA damage because it is essential for normal cell cycle progression. Specifically, the loss of ticrr impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry and mitotic catastrophe. We show that the human TICRR ortholog associates with TopBP1, a known checkpoint protein and a core component of the DNA replication preinitiation complex (pre-IC), and that the TICRR–TopBP1 interaction is stable without chromatin and requires BRCT motifs essential for TopBP1's replication and checkpoint functions. Most importantly, we find that ticrr deficiency disrupts chromatin binding of pre-IC, but not prereplication complex, components. Taken together, our data show that TICRR acts in association with TopBP1 and plays an essential role in pre-IC formation. It remains to be determined whether Ticrr represents the vertebrate ortholog of the yeast pre-IC component Sld3, or a hitherto unknown metazoan replication and checkpoint regulator.

We screened an existing collection of zebrafish insertional mutants for cancer susceptibility by histologic examination of heterozygotes at 2 years of age. As most mutants had no altered cancer predisposition, this provided the first comprehensive description of spontaneous tumor spectrum and frequency in adult zebrafish. Moreover, the screen identified four lines, each carrying a different dominant mutant allele of Hagoromo previously linked to adult pigmentation defects, which develop tumors with high penetrance and that histologically resemble neuroblastoma. These tumors are clearly neural in origin, although they do not express catecholaminergic neuronal markers characteristic of human neuroblastoma. The zebrafish tumors result from inappropriate maintenance of a cell population within the cranial ganglia that are likely neural precursors. These neoplasias typically remain small but they can become highly aggressive, initially traveling along cranial nerves, and ultimately filling the head. The developmental origin of these tumors is highly reminiscent of human neuroblastoma. The four mutant Hagoromo alleles all contain viral insertions in the fbxw4 gene, which encodes an F-box WD40 domain–containing protein. However, although one allele clearly reduced the levels of fbxw4 mRNA, the other three insertions had no detectable effect on fbw4 expression. Instead, we showed that all four mutations result in the postembryonic up-regulation of the neighboring gene, fibroblast growth factor 8 (fgf8). Moreover, fgf8 is highly expressed in the tumorigenic lesions. Although fgf8 overexpression is known to be associated with breast and prostate cancer in mammals, this study provides the first evidence that fgf8 misregulation can lead to neural tumors. (Mol Cancer Res 2009;7(6):841–50), National Cancer Institute (U.S.) (Grant CA106416)

Defining the genetic pathways essential for hematopoietic stem cell (HSC) development remains a fundamental goal impacting stem cell biology and regenerative medicine. To genetically dissect HSC emergence in the aorta-gonad-mesonephros (AGM) region, we screened a collection of insertional zebrafish mutant lines for expression of the HSC marker, c-myb. Nine essential genes were identified, which were subsequently binned into categories representing their proximity to HSC induction. Using overexpression and loss-of-function studies in zebrafish, we ordered these signaling pathways with respect to each other and to the Vegf, Notch, and Runx programs. Overexpression of vegf and notch is sufficient to induce HSCs in the tbx16 mutant, despite a lack of axial vascular organization. Although embryos deficient for artery specification, such as the phospholipase C gamma-1 (plcγ1) mutant, fail to specify HSCs, overexpression of notch or runx1 can rescue their hematopoietic defect. The most proximal HSC mutants, such as hdac1, were found to have no defect in vessel or artery formation. Further analysis demonstrated that hdac1 acts downstream of Notch signaling but upstream or in parallel to runx1 to promote AGM hematopoiesis. Together, our results establish a hierarchy of signaling programs required and sufficient for HSC emergence in the AGM.

We have characterized 28 zebrafish lines with heterozygous mutations in ribosomal protein (rp) genes, and found that 17 of these are prone to develop zebrafish malignant peripheral nerve sheath tumors (zMPNST). Heterozygotes from the vast majority of tumor-prone rp lines were found to be growth-impaired, though not all growth-impaired rp lines were tumor-prone. Significantly, however, the rp lines with the greatest incidence of zMPNSTs all displayed a growth impairment. Furthermore, heterozygous cells from one tumor-prone rp line were out-competed by wild-type cells in chimeric embryos. The growth impairment resulting from heterozygosity for many rp genes suggests that a global defect in protein translation exists in these lines, raising the possibility that a translation defect that precedes tumor development is predictive of tumorigenesis.

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers., Author Summary EpCAM is a well-established marker for carcinomas of epithelial origin and a potential target for immunotherapy. In vitro analyses have implicated EpCAM in a plethora of different cellular processes, such as adhesion, motility, proliferation, differentiation, and signaling. Strikingly, depending on the context, EpCAM displayed rather opposite effects, either promoting or attenuating cell–cell adhesion versus cell migration and tissue invasion, a phenomenon described as the “double-face” of EpCAM. However, the in vivo relevance of its different effects remained largely unclear. Here, we present the first genetic analysis of EpCAM function in vivo, based on loss-of-function mutants in the zebrafish. As it is in mammals, zebrafish EpCAM is expressed in simple epithelia. Mutant embryos display defects both in epithelial morphogenesis and in epithelial integrity. Reduced epithelial morphogenesis is accompanied, and possibly caused, by an extension of apical junctional complexes and compromised basal protrusive activity. Furthermore, mutant epithelia display alterations in the relative abundance of adherence junction versus tight junction components. In addition, EpCAM tightly cooperates with E-cadherin and has a previously unrecognized trans effect on the morphogenesis and integrity of underlying cell layers. Cell differentiation and proliferation in EpCAM mutants are not, or only secondarily, affected. During later development and adulthood, EpCAM is largely dispensable, reinforcing its suitability as a target for anti-carcinoma immunotherapy with minimal side effects.

We review here some recent developments in the field of insertional mutagenesis in zebrafish. We highlight the advantages and limitations of the rich body of retroviral methodologies, and we focus on the mechanisms and concepts of new transposon-based mutagenesis approaches under development, including prospects for conditional 'gene trapping' and 'gene breaking' approaches.

Checkpoint genes maintain genomic stability by arresting cells after DNA damage. Many of these genes also control cell cycle events in unperturbed cells. By conducting a screen for checkpoint genes in zebrafish, we found that dtl/cdt2 is an essential component of the early, radiation-induced G2/M checkpoint. We subsequently found that dtl/cdt2 is required for normal cell cycle control, primarily to prevent rereplication. Both the checkpoint and replication roles are conserved in human DTL. Our data indicate that the rereplication reflects a requirement for DTL in regulating CDT1, a protein required for prereplication complex formation. CDT1 is degraded in S phase to prevent rereplication, and following DNA damage to prevent origin firing. We show that DTL associates with the CUL4–DDB1 E3 ubiquitin ligase and is required for CDT1 down-regulation in unperturbed cells and following DNA damage. The cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels. In contrast, the early G2/M checkpoint defect appears to be Cdt1-independent. Thus, DTL promotes genomic stability through two distinct mechanisms. First, it is an essential component of the CUL4–DDB1 complex that controls CDT1 levels, thereby preventing rereplication. Second, it is required for the early G2/M checkpoint.

Background: Craniofacial 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. Results: Through 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. Conclusion: This 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., National Institutes of Health (U.S.)

It has been nearly a decade since the completion of two large-scale chemical mutagenesis screens in zebrafish, and two years since the completion of a large-scale insertional mutagenesis. In this article, we use the accumulated data from these screens to compare the efficiency of each mutagen to isolate mutants and to identify mutated genes, and argue that the two mutagens target the same set of genes. We then review how both forward genetic screens and reverse genetic techniques, such as morpholinos and TILLING, and transgenics are being used to develop models of human disease.