Harma Feitsma

Zebrafish as a cancer model.

The zebrafish has developed into an important model organism for biomedical research over the last decades. Although the main focus of zebrafish research has traditionally been on developmental biology, keeping and observing zebrafish in the lab led to the identification of diseases similar to humans, such as cancer, which subsequently became a subject for study. As a result, about 50 articles have been published since 2000 in which zebrafish were used as a cancer model. Strategies used include carcinogenic treatments, transplantation of mammalian cancer cells, forward genetic screens for proliferation or genomic instability, reverse genetic target-selected mutagenesis to inactivate known tumor suppressor genes, and the generation of transgenics to express human oncogenes. Zebrafish have been found to develop almost any tumor type known from human, with similar morphology and, according to gene expression array studies, comparable signaling pathways. However, tumor incidences are relatively low, albeit highly comparable between different mutants, and tumors develop late in life. In addition, tumor spectra are sometimes different when compared with mice and humans. Nevertheless, the zebrafish model has created its own niche in cancer research, complementing existing models with its specific experimental advantages and characteristics. Examples of these are imaging of tumor progression in living fish by fluorescence, treatment with chemical compounds, and screening possibilities not only for chemical modifiers but also for genetic enhancers and suppressors. This review aims to provide a comprehensive overview of the state of the art of zebrafish as a model in cancer research. (Mol Cancer Res 2008;6(5):685–94)

Targeted Mutation Reveals Essential Functions of the Homeodomain Transcription Factor Shox2 in Sinoatrial and Pacemaking Development

Background— Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. Methods and Results— To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2−/− embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. Conclusions— From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

Accurate SNP and mutation detection by targeted custom microarray-based genomic enrichment of short-fragment sequencing libraries

Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction for next-generation sequencing. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we explored the use of short fragment libraries (85–110 bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. High enrichment specificity (60–75%) was obtained with a relative even base coverage. Up to 98% of the target-sequence was covered more than 20× at an average coverage depth of about 200×. To verify the accuracy of SNP/mutation detection, we evaluated 384 known non-reference SNPs in the targeted regions. At ∼200× average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls, mostly due to low coverage. Using the same settings, a total of 1197 novel candidate variants were detected. Verification experiments revealed only eight false positive calls, indicating an overall false positive rate of less than 1 per ∼200 000 bp. Taken together, short fragment libraries provide highly efficient and flexible enrichment of exonic targets and yield relatively even base coverage, which facilitates accurate SNP and mutation detection. Raw sequencing data, alignment files and called SNPs have been submitted into GEO database http://www.ncbi.nlm.nih.gov/geo/ with accession number GSE18542.

Planar cell polarity defects and defective Vangl2 trafficking in mutants for the COPII gene Sec24b

Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles in many aspects of their development, in particular, controlling alignment of various types of epithelial cells. Disrupted PCP has been linked to developmental defects in animals and to human pathology. Neural tube closure defects (NTD) and disorganization of the mechanosensory cells of the organ of Corti are commonly known consequences of disturbed PCP signaling in mammals. We report here a typical PCP phenotype in a mouse mutant for the Sec24b gene, including the severe NTD craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. In addition, we observed genetic interaction between Sec24b and the known PCP gene, scribble. Sec24b is a component of the COPII coat protein complex that is part of the endoplasmic reticulum (ER)-derived transport vesicles. Sec24 isoforms are thought to be directly involved in cargo selection, and we present evidence that Sec24b deficiency specifically affects transport of the PCP core protein Vangl2, based on experiments in embryos and in cultured primary cells.

Zebrafish with Mutations in Mismatch Repair Genes Develop Neurofibromas and Other Tumors

Defective mismatch repair (MMR) in humans causes hereditary nonpolyposis colorectal cancer. This genetic predisposition to colon cancer is linked to heterozygous familial mutations, and loss-of-heterozygosity is necessary for tumor development. In contrast, the rare cases with biallelic MMR mutations are juvenile patients with brain tumors, skin neurofibromas, and café-au-lait spots, resembling the neurofibromatosis syndrome. Many of them also display lymphomas and leukemias, which phenotypically resembles the frequent lymphoma development in mouse MMR knockouts. Here, we describe the identification and characterization of novel knockout mutants of the three major MMR genes, mlh1, msh2, and msh6, in zebrafish and show that they develop tumors at low frequencies. Predominantly, neurofibromas/malignant peripheral nerve sheath tumors were observed; however, a range of other tumor types was also observed. Our findings indicate that zebrafish mimic distinct features of the human disease and are complementary to mouse models.

Mlh1 Deficiency in Zebrafish Results in Male Sterility and Aneuploid as Well as Triploid Progeny in Females

In most eukaryotes, recombination of homologous chromosomes during meiosis is necessary for proper chromosome pairing and subsequent segregation. The molecular mechanisms of meiosis are still relatively unknown, but numerous genes are known to be involved, among which are many mismatch repair genes. One of them, mlh1, colocalizes with presumptive sites of crossing over, but its exact action remains unclear. We studied meiotic processes in a knockout line for mlh1 in zebrafish. Male mlh1 mutants are sterile and display an arrest in spermatogenesis at metaphase I, resulting in increased testis weight due to accumulation of prophase I spermatocytes. In contrast, females are fully fertile, but their progeny shows high rates of dysmorphology and mortality within the first days of development. SNP-based chromosome analysis shows that this is caused by aneuploidy, resulting from meiosis I chromosomal missegregation. Surprisingly, the small percentage of progeny that develops normally has a complete triploid genome, consisting of both sets of maternal and one set of paternal chromosomes. As adults, these triploid fish are infertile males with wild-type appearance. The frequency of triploid progeny of mlh1 mutant females is much higher than could be expected for random chromosome segregation. Together, these results show that multiple solutions exist for meiotic crossover/segregation problems.

An ENU-Mutagenesis Screen in the Mouse: Identification of Novel Developmental Gene Functions

Background Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development. Methodology/Principal Findings Mice were mutagenized with N-ethyl-N-nitrosurea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib. Conclusions/Significance Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

Highly Efficient ENU Mutagenesis in Zebrafish

ENU (N-ethyl-N-nitrosourea) mutagenesis is a widely accepted and proven method to introduce random point mutations in the genome. Because there are no targeted knockout strategies available for zebrafish so far, random mutagenesis is currently the preferred method in both forward and reverse genetic approaches. To obtain high-density mutagenized zebrafish, six consecutive ENU treatments are applied at weekly intervals to adult male zebrafish by bathing them in ENU solution. With this procedure an average germ line mutation load of one mutation every 1.0 x 10(5)-1.5 x 10(5) basepairs is reached routinely in our lab.

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

A novel mutant allele of Ncx1: a single amino acid substitution leads to cardiac dysfunction

The biological role and structure-function relationship of the Na(+)Ca(2+) exchanger NCX1 have been the subject of much investigation. Subtle mutagenesis to study the function of a protein seems only feasible in in vitro systems, but genetic forward screens have the potential to provide in vivo models to study single amino acid substitutions. In a genetic screen in mouse, we have isolated a mutant line carrying a novel mutant allele of the mouse Ncx1 gene. In this allele, a point mutation causes the substitution of a highly conserved asparagine residue (N874) with lysine. Accepted models for NCX1 structure propose that the affected amino acid is located in one of the reentrant membrane loops and experiments in vitro have identified N874 as critical for the ion transport function of NCX1. We found severe circulation defects and defective placentation in homozygous Ncx1(N87K4) mutant embryos, making the phenotype essentially indistinguishable from those of previously described null mutants. By ex vivo analysis, we demonstrated intrinsic functional abnormalities of cardiomyocytes. Western blot analysis and immunohistochemistry demonstrated normal levels and subcellular localization of the altered protein, ruling out the possibility that the abnormalities are a mere consequence of a major disturbance of protein structure. This study confirms and extends studies in vitro indicating the significance of amino acid N874 for the function of the NCX1 protein. It provides an in vivo model for this mutation and demonstrates the potential of forward genetic screens in a mammalian system.