The Paf1 complex and P-TEFb have reciprocal and antagonist roles in maintaining multipotent neural crest progenitors.

Multipotent progenitor populations are necessary for generating diverse tissue types during embryogenesis. We show the RNA polymerase-associated factor 1 complex (Paf1C) is required to maintain multipotent progenitors of the neural crest (NC) lineage in zebrafish. Mutations affecting each Paf1C component result in near-identical NC phenotypes; alyron mutant embryos carrying a null mutation in paf1 were analyzed in detail. In the absence of zygotic paf1 function, definitive premigratory NC progenitors arise but fail to maintain expression of the sox10 specification gene. The mutant NC progenitors migrate aberrantly and fail to differentiate appropriately. Blood and germ cell progenitor development is affected similarly. Development of mutant NC could be rescued by additional loss of positive transcription elongation factor b (P-TEFb) activity, a key factor in promoting transcription elongation. Consistent with the interpretation that inhibiting/delaying expression of some genes is essential for maintaining progenitors, mutant embryos lacking the CDK9 kinase component of P-TEFb exhibit a surfeit of NC progenitors and their derivatives. We propose Paf1C and P-TEFb act antagonistically to regulate the timing of the expression of genes needed for NC development.

Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish.

Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.

Intracellular Calcium Mobilization Is Required for Sonic Hedgehog Signaling.

Graded Shh signaling across fields of precursor cells coordinates patterns of gene expression, differentiation, and morphogenetic behavior as precursors form complex structures, such as the nervous system, the limbs, and craniofacial skeleton. Here we discover that intracellular calcium mobilization, a process tightly controlled and readily modulated, regulates the level of Shh-dependent gene expression in responding cells and affects the development of all Shh-dependent cell types in the zebrafish embryo. Reduced expression or modified activity of ryanodine receptor (RyR) intracellular calcium release channels shifted the allocation of Shh-dependent cell fates in the somitic muscle and neural tube. Mosaic analysis revealed that RyR-mediated calcium mobilization is required specifically in Shh ligand-receiving cells. This work reveals that RyR channels participate in intercellular signal transduction events. As modulation of RyR activity modifies tissue patterning, we hypothesize that alterations in intracellular calcium mobilization contribute to both birth defects and evolutionary modifications of morphology.

A hyperactivating proinflammatory RIPK2 allele associated with early-onset osteoarthritis.

Osteoarthritis (OA) is a common debilitating disease characterized by abnormal remodeling of the cartilage and bone of the articular joint. Ameliorating therapeutics are lacking due to limited understanding of the molecular pathways affecting disease initiation and progression. Notably, although a link between inflammation and overt OA is well established, the role of inflammation as a driver of disease occurrence is highly disputed. We analyzed a family with dominant inheritance of early-onset OA and found that affected individuals harbored a rare variant allele encoding a significant amino acid change (p.Asn104Asp) in the kinase domain of receptor interacting protein kinase 2 (RIPK2), which transduces signals from activated bacterial peptidoglycan sensors through the NF-κB pathway to generate a proinflammatory immune response. Functional analyses of RIPK2 activity in zebrafish embryos indicated that the variant RIPK2104Asp protein is hyperactive in its signaling capacity, with augmented ability to activate the innate immune response and the NF-κB pathway and to promote upregulation of OA-associated genes. Further we show a second allele of RIPK2 linked to an inflammatory disease associated with arthritis also has enhanced activity stimulating the NF-κB pathway. Our studies reveal for the first time the inflammatory response can function as a gatekeeper risk factor for OA.

Precise Editing of the Zebrafish Genome Made Simple and Efficient.

We present simple and efficient methods for creating heritable modifications of the zebrafish genome. Precisely modified alleles are generated by homologous recombination between the host genome and dsDNA donor molecules, stimulated by the induction of chromosomally targeted double-strand breaks. Several kilobase-long tracts of genome sequence can be replaced. Tagging donor sequences with reporter genes that can be subsequently excised improves recovery of edited alleles by an order of magnitude and facilitates recovery of recessive and phenotypically silent conditional mutations. We generate and demonstrate functionality of (1) alleles with a single codon change, (2) an allele encoding an epitope-tagged version of an endogenous protein, (3) alleles expressing reporter proteins, and (4) a conditional allele in which an exon is flanked by recombinogenic loxP sites. Our methods make recovery of a broad range of genome editing events very practicable, significantly advancing applicability of the zebrafish for studying normal biological processes and modeling diseases.

Targeting human microRNA genes using engineered Tal-effector nucleases (TALENs).

MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise control over the expression of critical protein coding genes. Despite significant progress in our understanding of how miRNAs function in mice, there remains a fundamental need to be able to target and edit miRNA genes in the human genome. Here, we report a novel approach to disrupting human miRNA genes ex vivo using engineered TAL-effector (TALE) proteins to function as nucleases (TALENs) that specifically target and disrupt human miRNA genes. We demonstrate that functional TALEN pairs can be designed to enable disruption of miRNA seed regions, or removal of entire hairpin sequences, and use this approach to successfully target several physiologically relevant human miRNAs including miR-155*, miR-155, miR-146a and miR-125b. This technology will allow for a substantially improved capacity to study the regulation and function of miRNAs in human cells, and could be developed into a strategic means by which miRNAs can be targeted therapeutically during human disease.

Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.

SHIP2, a factor associated with diet-induced obesity and insulin sensitivity, attenuates FGF signaling in vivo.

SH2-domain-containing inositol phosphatase 2 (SHIP2) belongs to a small family of phosphoinositide 5-phosphatases that help terminate intracellular signaling initiated by activated receptor tyrosine kinases. Mammalian SHIP2 is viewed primarily as an attenuator of insulin signaling and has become a prominent candidate target for therapeutic agents that are designed to augment insulin signaling. Despite this view, no signaling pathway has yet been demonstrated as being affected directly by SHIP2 function in vivo, and in vitro studies indicate that the protein may function in multiple signaling pathways. Here, we analyze the role of a SHIP2 family member in the early zebrafish embryo where developmental and gene expression defects can be used to assay specific signaling pathways. The zebrafish ship2a transcript is maternally supplied, and inhibiting the expression of its protein product results in the expansion of dorsal tissue fates at the expense of ventral ones. We show that the developmental defects are the result of perturbation of fibroblast growth factor (FGF) signaling in the early embryo. Loss of Ship2a leads to an increased and expanded expression of outputs of FGF-mediated signaling, including FGF-dependent gene expression and activated mitogen-activated protein kinase (MAPK) signaling. Our findings demonstrate that Ship2a attenuates the FGF signaling pathway in vivo and functions in the establishment of normal tissue patterning in the early embryo. We suggest that modulation of FGF signaling may be a principal function of SHIP2 in mammals.

Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle.

Mutations affecting the seemingly unrelated gene products, SepN1, a selenoprotein of unknown function, and RyR1, the major component of the ryanodine receptor intracellular calcium release channel, result in an overlapping spectrum of congenital myopathies. To identify the immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebrafish embryo. These studies demonstrate the two proteins are required for the same cellular differentiation events and are needed for normal calcium fluxes in the embryo. SepN is physically associated with RyRs and functions as a modifier of the RyR channel. In the absence of SepN, ryanodine receptors from zebrafish embryos or human diseased muscle have altered biochemical properties and have lost their normal sensitivity to redox conditions, which likely accounts for why mutations affecting either factor lead to similar diseases.

An interacting network of T-box genes directs gene expression and fate in the zebrafish mesoderm.

T-box genes encode transcription factors that play critical roles in generating the vertebrate body plan. In many developmental fields, multiple T-box genes are expressed in overlapping domains, establishing broad regions in which different combinations of T-box genes are coexpressed. Here we demonstrate that three T-box genes expressed in the zebrafish mesoderm, no tail, spadetail, and tbx6, operate as a network of interacting genes to regulate region-specific gene expression and developmental fate. Loss-of-function and gain-of-function genetic analyses reveal three kinds of interactions among the T-box genes: combinatorial interactions that generate new regulatory functions, additive contributions to common developmental pathways, and competitive antagonism governing downstream gene expression. We propose that T-box genes, like Hox genes, often function within gene networks comprised of related family members.

A protein disulfide isomerase expressed in the embryonic midline is required for left/right asymmetries.

Although the vertebrate embryonic midline plays a critical role in determining the left/right asymmetric development of multiple organs, few genes expressed in the midline are known to function specifically in establishing laterality patterning. Here we show that a gene encoding protein disulfide isomerase P5 (PDI-P5) is expressed at high levels in the organizer and axial mesoderm and is required for establishing left/right asymmetries in the zebrafish embryo. pdi-p5 was discovered in a screen to detect genes down-regulated in the zebrafish midline mutant one-eyed pinhead and expressed predominantly in midline tissues of wild-type embryos. Depletion of the pdi-p5 product with morpholino antisense oligonucleotides results in loss of the asymmetric development of the heart, liver, pancreas, and gut. In addition, PDI-P5 depletion results in bilateral expression of all genes known to be expressed asymmetrically in the lateral plate mesoderm and the brain during embryogenesis. The laterality defects caused by pdi-p5 antisense treatment arise solely due to loss of the PDI-P5 protein, as they are reversed when treated embryos are supplied with an exogenous source of the PDI-P5 protein. Thus the spectrum of laterality defects resulting from depletion of the PDI-P5 protein fully recapitulates that resulting from loss of the midline. As loss of PDI-P5 does not appear to interfere with other aspects of midline development or function, we propose that PDI-P5 is specifically involved in the production of midline-derived signals required to establish left/right asymmetry.

Headwaters of the zebrafish -- emergence of a new model vertebrate.

The understanding of vertebrate development has advanced considerably in recent years, primarily due to the study of a few model organisms. The zebrafish, the newest of these models, has risen to prominence because both genetic and experimental embryological methods can be easily applied to this animal. The combination of approaches has proven powerful, yielding insights into the formation and function of individual tissues, organ systems and neural networks, and into human disease mechanisms. Here, we provide a personal perspective on the history of zebrafish research, from the assembly of the first genetic and embryological tools through to sequencing of the genome.