Wenbiao Chen

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75–99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.

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.

Exocrine Gland Dysfunction in MC5-R-Deficient Mice: Evidence for Coordinated Regulation of Exocrine Gland Function by Melanocortin Peptides

The effects of pituitary-derived melanocortin peptides are primarily attributed to ACTH-mediated adrenocortical glucocorticoid production. Identification of a widely distributed receptor for ACTH/MSH peptides, the melanocortin-5 receptor (MC5-R), suggested non-steroidally mediated systemic effects of these peptides. Targeted disruption of the MC5-R produced mice with a severe defect in water repulsion and thermoregulation due to decreased production of sebaceous lipids. High levels of MC5-R was found in multiple exocrine tissues, including Harderian, preputial, lacrimal, and sebaceous glands, and was also shown to be required for production and stress-regulated synthesis of porphyrins by the Harderian gland and ACTH/MSH-regulated protein secretion by the lacrimal gland. These data show a requirement for the MC5-R in multiple exocrine glands for the production of numerous products, indicative of a coordinated system for regulation of exocrine gland function by melanocortin peptides.

Active medulloblastoma enhancers reveal subgroup-specific cellular origins

Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

Ribeye is the only known protein specific to synaptic ribbon, but its function is unclear. We show that the teleost fish, Fugu and zebrafish, have two ribeye genes, ribeye a and ribeye b. Whole-mount in situ hybridization revealed that ribeye a is expressed in tissues containing synaptic ribbons, including the pineal gland, inner ear, and retina. Ribeye b is absent in the pineal gland. In the retina, ribeye a is expressed in both photoreceptors and bipolar cells, whereas ribeye b is detected only in photoreceptors. To study the function of Ribeye a in retina, we depleted it by morpholino antisense oligos. Fish deficient in Ribeye a lack an optokinetic response and have shorter synaptic ribbons in photoreceptors and fewer synaptic ribbons in bipolar cells. Their bipolar cells still target Syntaxin-3 proteins to the inner plexiform layer and have abundant vsx1 mRNA. However, they lack large synaptic terminals and show increased apoptosis. Rod bipolar cells are fewer in number and/or deficient in PKCα. Recovery of Ribeye a levels rescues the optokinetic response, increases the number of PKCα-positive bipolar cells, and stops apoptosis. We conclude that Ribeye a is important for late steps in bipolar cell development.

FlEx‐based transgenic reporter lines for visualization of Cre and Flp activity in live zebrafish

Site‐specific recombinases such as Cre and Flp are invaluable tools for genetic manipulations, but their usage in zebrafish has been limited. Incorporating recently developed flip‐excision (FlEx) design that allows stable inversions, we have established zebrafish reporter lines that express bright and ubiquitous EGFP, but switch to express mCherry in the presence of Cre or Flp. Here, we demonstrate the stable inversion in the reporter lines, both in somatic cells and in the germ line by Cre or Flp, and the subsequent reinversion using the other recombinase. Using the reporter lines, we characterized cardiomyocyte‐specific Cre lines and neuronal progenitor‐specific and tamoxifen‐dependent Cre lines. We also used the reporter lines for screening Cre‐ and Flp‐based enhancer trap lines. Similar to the widely used Cre reporter lines in mice, these FlEx‐based reporter lines will facilitate the use of recombinases for genetic manipulations in zebrafish. genesis 47:484–491, 2009.

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.

Circadian Modulation of Dopamine Levels and Dopaminergic Neuron Development Contributes to Attention Deficiency and Hyperactive Behavior

Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children and adults. While ADHD patients often display circadian abnormalities, the underlying mechanisms are unclear. Here we found that the zebrafish mutant for the circadian gene period1b (per1b) displays hyperactive, impulsive-like, and attention deficit-like behaviors and low levels of dopamine, reminiscent of human ADHD patients. We found that the circadian clock directly regulates dopamine-related genes monoamine oxidase and dopamine β hydroxylase, and acts via genes important for the development or maintenance of dopaminergic neurons to regulate their number and organization in the ventral diencephalic posterior tuberculum. We then found that Per1 knock-out mice also display ADHD-like symptoms and reduced levels of dopamine, thereby showing highly conserved roles of the circadian clock in ADHD. Our studies demonstrate that disruption of a circadian clock gene elicits ADHD-like syndrome. The circadian model for attention deficiency and hyperactive behavior sheds light on ADHD pathogenesis and opens avenues for exploring novel targets for diagnosis and therapy for this common psychiatric disorder.

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.

Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish

Significance The hormone leptin homeostatically maintains long-term fat stores in mammals. Made by adipocytes in proportion to total adipose mass, leptin functions by regulating behavioral, autonomic, and endocrine circuits in the CNS to control energy intake and expenditure. As leptin signals nutritional sufficiency, it also acts as a gating factor for reproductive maturation and competence. Defective leptin signaling in mammals results in hyperphagia, obesity, diabetes, and infertility. Much less is known about leptin in nonmammalian vertebrates; however, the teleost leptin homologue is not primarily expressed in adipocytes. Here we show that zebrafish leptin is not required for adipostasis, food intake, or reproduction. However, we show here that, as in mammals, zebrafish leptin retains a role in the regulation of glucose homeostasis. Leptin is the primary adipostatic factor in mammals. Produced largely by adipocytes in proportion to total adipose mass, the hormone informs the brain regarding total energy stored as triglycerides in fat cells. The hormone acts on multiple circuits in the brain to regulate food intake, autonomic outflow, and endocrine function to maintain energy balance. In addition to regulating adipose mass, mammalian leptin also plays a role in the regulation of glucose homeostasis and as a gating factor in reproductive competence. Leptin-deficient mice and people exhibit early onset profound hyperphagia and obesity, diabetes, and infertility. Although leptin and the leptin receptor are found in fish, the hormone is not expressed in adipose tissue, but is found in liver and other tissues. Here, we show that adult zebrafish lacking a functional leptin receptor do not exhibit hyperphagia or increased adiposity, and exhibit normal fertility. However, leptin receptor-deficient larvae have increased numbers of β-cells and increased levels of insulin mRNA. Furthermore, larval zebrafish have been shown to exhibit β-cell hyperplasia in response to high fat feeding or peripheral insulin resistance, and we show here that leptin receptor is required for this response. Adult zebrafish also have increased levels of insulin mRNA and other alterations in glucose homeostasis. Thus, a role for leptin in the regulation of β-cell mass and glucose homeostasis appears to be conserved across vertebrates, whereas its role as an adipostatic factor is likely to be a secondary role acquired during the evolution of mammals.