Nancy Hopkins

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.

A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney

Polycystic kidney disease (PKD) is a common human genetic illness. It is characterized by the formation of multiple kidney cysts that are thought to result from over-proliferation of epithelial cells. Zebrafish larvae can also develop kidney cysts. In an insertional mutagenesis screen in zebrafish, we identified 12 genes that can cause cysts in the glomerular-tubular region when mutated and we cloned 10 of these genes. Two of these genes, vhnf1 (tcf2) and pkd2, are already associated with human cystic kidney diseases. Recently, defects in primary cilia have been linked to PKD. Strikingly, three out of the 10 genes cloned in this screen are homologues of Chlamydomonas genes that encode components of intraflagellar transport (IFT) particles involved in cilia formation. Mutation in a fourth blocks ciliary assembly by an unknown mechanism. These results provide compelling support for the connection between cilia and cystogenesis. Our results also suggest that lesions in genes involved in cilia formation and function are the predominant cause of cystic kidney disease, and that the genes identified here are excellent candidates for novel human PKD genes.

A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.

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 vps18 results 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.

Zebrafish foxi one modulates cellular responses to Fgf signaling required for the integrity of ear and jaw patterning.

We identified four insertional alleles of foxi one (foo), an embryonic lethal mutation in zebrafish that displays defects in both otic placode and the jaw. In foo/foo embryos the otic placode is split into two smaller placodes and mutant embryos show a dorsoventral (DV) cartilage defect manifested as a reduced hyomandibular and reduced third and fourth branchial arches. We identified foxi one (foo), the zebrafish ortholog of Foxi1 (FREAC6, FKHL10, HFH-3, Fkh10) and a member of the forkhead domain transcriptional regulator family, as the gene mutated in foo/foo embryos. foo is expressed in otic placode precursor cells, and foo/foo embryos lack placodal pax8 expression and have disorganized otic expression of pax2.1 and dlx3. Third stream neural crest cell migration, detected by dlx2 and krox20 expression, is aberrant in that it invades the otic placode territory. foo is expressed in pharyngeal pouch endoderm and is required for pouch expression of pax8 and proper patterning of other markers in the pouch such as nkx2.3. In foo/foo embryos, we observed a failure to maintain fgf3 expression in the pouches, followed by apoptosis of neural crest cells in adjacent arches. We conclude that foo expression is essential for pax8 expression probably downstream of Fgf signaling in a conserved pathway jointly required for integrity of patterning in the otic placode and pharyngeal pouches. We propose that correct placement of survival/proliferation cues is essential for shaping the pharyngeal cartilages and that evolutionary links between jaw and ear formation can be traced to Fgf-Foxi1-Pax8 pathways.

Mutations affecting pigmentation and shape of the adult zebrafish.

Abstract Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish.

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 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.

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 TopBP1s 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.

Requirements for green fluorescent protein detection in transgenic zebrafish embryos.

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 alpha 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 non-fluorescent 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.

Many Ribosomal Protein Mutations Are Associated With Growth Impairment and Tumor Predisposition in Zebrafish

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. Developmental Dynamics 238:76–85, 2009.

Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis

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.