Lisette A. Maddison

Conditional Deletion of Rb Causes Early Stage Prostate Cancer

Prostate cancer remains the second leading cause of cancer-related death for men in the United States. Mutations in tumor suppressor genes including retinoblastoma (Rb), p53, and PTEN have been linked to the development of prostate cancer in man and mouse models, and loss of heterozygosity of the Rb locus has been observed in up to 60% of clinical cases. In this study we demonstrate that conditional somatic deletion of even a single Rb allele in the epithelial cells of the mouse prostate causes focal hyperplasia, thereby establishing a causal relationship between Rb loss and development of early stage prostate cancer. As a consequence of Rb ablation we observed increased expression of E2F target genes and a concomitant increase in proliferation in the epithelial compartment. However, by 52 weeks of age these lesions had not become malignant and represent an early stage of the disease. Nevertheless, the multifocal nature of the phenotype in the mice closely resembled multifocality of clinical disease. Taken together, our data demonstrated that loss of pRB-mediated cell cycle control directly caused the initiation of proliferative prostate disease but was insufficient to cause malignancy. Establishment of this early initiation model will aid efforts to thoroughly characterize early prostate disease as well as the elucidation of molecular mechanisms that cooperate with Rb loss to facilitate progression and metastasis.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.

Conditional control of gene function by an invertible gene trap in zebrafish

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.

Nutrient Excess Stimulates β-Cell Neogenesis in Zebrafish

Persistent nutrient excess results in a compensatory increase in the β-cell number in mammals. It is unknown whether this response occurs in nonmammalian vertebrates, including zebrafish, a model for genetics and chemical genetics. We investigated the response of zebrafish β-cells to nutrient excess and the underlying mechanisms by culturing transgenic zebrafish larvae in solutions of different nutrient composition. The number of β-cells rapidly increases after persistent, but not intermittent, exposure to glucose or a lipid-rich diet. The response to glucose, but not the lipid-rich diet, required mammalian target of rapamycin activity. In contrast, inhibition of insulin/IGF-1 signaling in β-cells blocked the response to the lipid-rich diet, but not to glucose. Lineage tracing and marker expression analyses indicated that the new β-cells were not from self-replication but arose through differentiation of postmitotic precursor cells. On the basis of transgenic markers, we identified two groups of newly formed β-cells: one with nkx2.2 promoter activity and the other with mnx1 promoter activity. Thus, nutrient excess in zebrafish induces a rapid increase in β-cells though differentiation of two subpopulations of postmitotic precursor cells. This occurs through different mechanisms depending on the nutrient type and likely involves paracrine signaling between the differentiated β-cells and the precursor cells.

Using retroviruses as a mutagenesis tool to explore the zebrafish genome.

We review different uses of the retroviral mutagenesis technology as the tool to manipulate the zebrafish genome. In addition to serving as a mutagen in a phenotype-driven forward mutagenesis screen as it was originally adapted for, retroviral insertional mutagenesis can also be exploited in reverse genetic approaches, delivering enhancer- and gene-trap vectors for the purpose of examining gene expression patterns and mutagenesis, making sensitized mutants amenable for chemical and genetic modifier screens, and producing gain-of-function mutations by epigenetically overexpressing the retroviral-inserted genes. From a technology point of view, we also summarize the recent advances in the high-throughput cloning of retroviral integration sites, a pivotal step toward identifying mutations. Lastly, we point to some potential directions that retroviral mutagenesis might take from the lessons of studying other model organisms.

PhiC31 integrase induces efficient site-specific excision in zebrafish

Site-specific recombinases catalyze recombination between specific targeting sites to delete, insert, invert, or exchange DNA with high fidelity. In addition to the widely used Cre and Flp recombinases, the phiC31 integrase system from Streptomyces phage may also be used for these genetic manipulations in eukaryotic cells. Unlike Cre and Flp, phiC31 recognizes two heterotypic sites, attB and attP, for recombination. While the phiC31 system has been recently applied in mouse and human cell lines and in Drosophila, it has not been demonstrated whether it can also catalyze efficient DNA recombination in zebrafish. Here we show that phiC31 integrase efficiently induces site-specific deletion of episomal targets as well as chromosomal targets in zebrafish embryos. Thus, the phiC31 system can be used in zebrafish for genetic manipulations, expanding the repertoire of available tools for genetic manipulation in this vertebrate model.

A gain-of-function screen in zebrafish identifies a guanylate cyclase with a role in neuronal degeneration

Manipulation of gene expression is one of the most informative ways to study gene function. Genetic screens have been an informative method to identify genes involved in developmental processes. In the zebrafish, loss-of-function screens have been the primary approach for these studies. We sought to complement loss-of-function screens using an unbiased approach to overexpress genes with a Gal4-UAS based system, similar to the gain-of-function screens in Drosophila. Using MMLV as a mutagenic vector, a cassette containing a UAS promoter was readily inserted in the genome, often at the 5′ end of genes, allowing Gal4-dependent overexpression. We confirmed that genes downstream of the viral insertions were overexpressed in a Gal4-VP16 dependent manner. We further demonstrate that misexpression of one such downstream gene gucy2F, a membrane-bound guanylate cyclase, throughout the nervous system results in multiple defects including a loss of forebrain neurons. This suggests proper control of cGMP production is important in neuronal survival. From this study, we propose that this gain-of-function approach can be applied to large-scale genetic screens in a vertebrate model organism and may reveal previously unknown gene function.

Overnutrition induces β-cell differentiation through prolonged activation of β-cells in zebrafish larvae

Insulin from islet β-cells maintains glucose homeostasis by stimulating peripheral tissues to remove glucose from circulation. Persistent elevation of insulin demand increases β-cell number through self-replication or differentiation (neogenesis) as part of a compensatory response. However, it is not well understood how a persistent increase in insulin demand is detected. We have previously demonstrated that a persistent increase in insulin demand by overnutrition induces compensatory β-cell differentiation in zebrafish. Here, we use a series of pharmacological and genetic analyses to show that prolonged stimulation of existing β-cells is necessary and sufficient for this compensatory response. In the absence of feeding, tonic, but not intermittent, pharmacological activation of β-cell secretion was sufficient to induce β-cell differentiation. Conversely, drugs that block β-cell secretion, including an ATP-sensitive potassium (K ATP) channel agonist and an L-type Ca(2+) channel blocker, suppressed overnutrition-induced β-cell differentiation. Genetic experiments specifically targeting β-cells confirm existing β-cells as the overnutrition sensor. First, inducible expression of a constitutively active K ATP channel in β-cells suppressed the overnutrition effect. Second, inducible expression of a dominant-negative K ATP mutant induced β-cell differentiation independent of nutrients. Third, sensitizing β-cell metabolism by transgenic expression of a hyperactive glucokinase potentiated differentiation. Finally, ablation of the existing β-cells abolished the differentiation response. Taken together, these data establish that overnutrition induces β-cell differentiation in larval zebrafish through prolonged activation of β-cells. These findings demonstrate an essential role for existing β-cells in sensing overnutrition and compensating for their own insufficiency by recruiting additional β-cells.

Generating Conditional Mutations in Zebrafish Using Gene-trap Mutagenesis

While several mutagenesis methods have been successfully applied in zebrafish, these mutations do not allow tissue- or temporal-specific functional analysis. We have developed a strategy that will allow tissue- or temporal-specific disruption of genes in zebrafish. This strategy combines gene-trap mutagenesis and FlEx modules containing target sites for site-specific recombinases. The gene-trap cassette is highly mutagenic in one orientation and nonmutagenic in the opposite orientation, with different fluorescent proteins as indicators of the orientation. The inclusion of the FlEx modules allows two rounds of stable inversion mediated by the Cre and Flp recombinases. This gene-trap cassette can be easily delivered via transposons. Through large-scale community-wide efforts, broad genome coverage can be obtained. This should allow investigation of cell/tissue-specific gene function of a wide range of genes.

Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner

Insulin resistance creates an environment that promotes β-cell failure and development of diabetes. Understanding the events that lead from insulin resistance to diabetes is necessary for development of effective preventional and interventional strategies, and model systems that reflect the pathophysiology of disease progression are an important component toward this end. We have confirmed that insulin enhances glucose uptake in zebrafish skeletal muscle and have developed a zebrafish model of skeletal muscle insulin resistance using a dominant-negative IGF-IR. These zebrafish exhibit blunted insulin signaling and glucose uptake in the skeletal muscle, confirming insulin resistance. In young animals, we observed an increase in the number of β-cells and normal glucose tolerance that was indicative of compensation for insulin resistance. In older animals, the β-cell mass was reduced to that of control with the appearance of impaired glucose clearance but no elevation in fasting blood glucose. Combined with overnutrition, the insulin-resistant animals have an increased fasting blood glucose compared with the control animals, demonstrating that the β-cells in the insulin-resistant fish are in a vulnerable state. The relatively slow progression from insulin resistance to glucose intolerance in this model system has the potential in the future to test cooperating genes or metabolic conditions that may accelerate the development of diabetes and provide new therapeutic targets.