MITF deficiency accelerates GNAQ-driven uveal melanoma.

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-ß (PLCß)­ERK was also investigated in this system and thus showed that while activated YAP (YAPAA) induced UM with high potency, the patient-derived PLCß4 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 tp53­wild 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 PLCß 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.

A vertebrate gene, ticrr, is an essential checkpoint and replication regulator.

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.

Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

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 tln1(fl02k) 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 ß1b (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 Itgß1b 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.

Comparative oncogenomic analysis of copy number alterations in human and zebrafish tumors enables cancer driver discovery.

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.

Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system.

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.

Highly aneuploid zebrafish malignant peripheral nerve sheath tumors have genetic alterations similar to human cancers.

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.

Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development.

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.

Lack of de novo phosphatidylinositol synthesis leads to endoplasmic reticulum stress and hepatic steatosis in cdipt-deficient zebrafish.

UNLABELLED: 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.

The epithelial cell adhesion molecule EpCAM is required for epithelial morphogenesis and integrity during zebrafish epiboly and skin development.

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.

MITF deficiency and oncogenic GNAQ each promote proliferation programs in zebrafish melanocyte lineage cells.

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 GNAQQ209L -driven UM. Here, we show that zebrafish mitfa-deficient GNAQQ209L -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 GNAQQ209L , YAPS127A;S381A , or BRAFV600E oncogenes, 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 GNAQQ209L expression promotes a proliferative phenotype that is further enhanced by YAPS127A;S381A co-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.

A genetic screen in zebrafish defines a hierarchical network of pathways required for hematopoietic stem cell emergence.

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 (plcgamma1) 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.

Loss of p53 synthesis in zebrafish tumors with ribosomal protein gene mutations.

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 gamma-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.

The identification of zebrafish mutants showing alterations in senescence-associated biomarkers.

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated beta-galactosidase (SA-beta-gal) in our current study. We validated the use of embryonic SA-beta-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-beta-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-beta-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-beta-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms.

Essential genes for astroglial development and axon pathfinding during zebrafish embryogenesis.

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.

Drug-sensitized zebrafish screen identifies multiple genes, including GINS3, as regulators of myocardial repolarization.

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.

Mutagenesis strategies in zebrafish for identifying genes involved in development and disease.

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.

Uveal melanoma driver mutations in GNAQ/11 yield numerous changes in melanocyte biology.

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/11Q209L , the predominant initiating mutations for human UM. When combined with mutant tp53, GNAQ/11Q209L 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/11Q209L 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/11Q209L is sufficient to induce numerous protumorigenic changes within melanocytes.

Many ribosomal protein genes are cancer genes in zebrafish.

We have generated several hundred lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation. Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression.

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.

Identification of zebrafish insertional mutants with defects in visual system development and function.

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.