CRISPR/Cas9 Gene Disruption Studies in F0 Xenopus Tadpoles: Understanding Development and Disease in the Frog.

CRISPR/Cas9 has become the favorite method for gene knockouts in a range of vertebrate model organisms due to its ease of use and versatility. Gene-specific guide RNAs can be designed to a unique genomic sequence and used to target the Cas9 endonuclease, which causes a double-stranded break at the desired locus. Repair of the breaks through non-homologous end joining often results in the deletion or insertion of several nucleotides, which frequently result in nonsense mutations. Xenopus frogs have long been an excellent model organism in which to study gene function, and they have proven to be useful in gene-editing experiments, especially the diploid species, X. tropicalis. In this chapter, we present our protocols for gene disruption in Xenopus, which we regularly use to investigate developmental processes and model human genetic disease.

I-SceI-Mediated Transgenesis in Xenopus.

Transgenic frogs can be very efficiently generated using I-SceI meganuclease, a nuclease with an 18-bp recognition site. The desired transgene must be flanked by I-SceI sites, in either a plasmid or a polymerase chain reaction (PCR) product. After a short in vitro digestion with the meganuclease, the complete reaction is injected into fertilized eggs, where the enzyme mediates genomic integration by an unknown mechanism. Posttransgenesis development is typically normal, and up to 70% of the embryos integrate the transgene.

Cryopreservation of Xenopus Sperm and In Vitro Fertilization Using Frozen Sperm Samples.

The cryopreservation of Xenopus sperm allows for a significant reduction of the number of animals that must be kept, more efficient archiving of genetically altered (GA) lines, and easy exchange of lines with other laboratories, leading to improvements in animal welfare and cost efficiency. In this protocol, sperm from Xenopus laevis or Xenopus tropicalis are frozen using straightforward techniques and standard laboratory equipment. Testes are macerated in Leibovitz's L-15 medium, mixed with a simple cryoprotectant made from egg yolk and sucrose, and frozen slowly overnight in a polystyrene box at -80°C. Unlike mouse sperm, Xenopus sperm can be stored at -80°C rather than in liquid nitrogen, further reducing costs. The frozen sperm are then used for in vitro fertilization.

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

Xenopus Resources: Transgenic, Inbred and Mutant Animals, Training Opportunities, and Web-Based Support.

Two species of the clawed frog family, Xenopus laevis and X. tropicalis, are widely used as tools to investigate both normal and disease-state biochemistry, genetics, cell biology, and developmental biology. To support both frog specialist and non-specialist scientists needing access to these models for their research, a number of centralized resources exist around the world. These include centers that hold live and frozen stocks of transgenic, inbred and mutant animals and centers that hold molecular resources. This infrastructure is supported by a model organism database. Here, we describe much of this infrastructure and encourage the community to make the best use of it and to guide the resource centers in developing new lines and libraries.

Husbandry of Xenopus tropicalis.

Xenopus tropicalis combine the advantages of X. laevis, for example using explants and targeted gain of function, with the ability to take classical genetics approaches to answering cell and developmental biology questions making it arguably the most versatile of the model organisms. Against this background, husbandry of X. tropicalis is less well developed than for its larger, more robust relative. Here we describe the methods used to keep and breed these frogs successfully.

The hitchhiker's guide to Xenopus genetics.

A decade after the human genome sequence, most vertebrate gene functions remain poorly understood, limiting benefits to human health from rapidly advancing genomic technologies. Systematic in vivo functional analysis is ideally suited to the experimentally accessible Xenopus embryo, which combines embryological accessibility with a broad range of transgenic, biochemical, and gain-of-function assays. The diploid X. tropicalis adds loss-of-function genetics and enhanced genomics to this repertoire. In the last decade, diverse phenotypes have been recovered from genetic screens, mutations have been cloned, and reverse genetics in the form of TILLING and targeted gene editing have been established. Simple haploid genetics and gynogenesis and the very large number of embryos produced streamline screening and mapping. Improved genomic resources and the revolution in high-throughput sequencing are transforming mutation cloning and reverse genetic approaches. The combination of loss-of-function mutant backgrounds with the diverse array of conventional Xenopus assays offers a uniquely flexible platform for analysis of gene function in vertebrate development.

The secreted integrin ligand nephronectin is necessary for forelimb formation in Xenopus tropicalis.

While limb regeneration has been extensively studied in amphibians, little is known about the initial events in limb formation in metamorphosing anurans. The small secreted integrin ligand nephronectin (npnt) is necessary for development of the metanephros in mouse. Although expressed in many tissues, its role in other developmental processes is not well-studied. Here we show that a transgene insertion that disrupts this gene ablates forelimb formation in Xenopus tropicalis. Our results suggest a novel role for integrin signalling in limb development, and represent the first insertional phenotype to be cloned in amphibians.

Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6.

Mechanisms coupling heart function and cardiac morphogenesis can be accessed in lower vertebrate embryos that can survive to swimming tadpole stages on diffused oxygen. Forward genetic screens in Xenopus tropicalis have identified more than 80 mutations affecting diverse developmental processes, including cardiac morphogenesis and function. In the first positional cloning of a mutation in X. tropicalis, we show that non-contractile hearts in muzak (muz) embryos are caused by a premature stop codon in the cardiac myosin heavy chain gene myh6. The mutation deletes the coiled-coil domain responsible for polymerization into thick filaments, severely disrupting the cardiomyocyte cytoskeleton. Despite the lack of contractile activity and absence of a major structural protein, early stages of cardiac morphogenesis including looping and chamber formation are grossly normal. Muz hearts subsequently develop dilated chambers with compressed endocardium and fail to form identifiable cardiac valves and trabeculae.

Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes.

Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed fluorescence in situ hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes. Developmental Dynamics 238:1398-1406, 2009. (c) 2009 Wiley-Liss, Inc.

Identification of a structural and functional domain in xNAP1 involved in protein-protein interactions.

xNAP1 (Xenopus nucleosome assembly protein) belongs to the family of nucleosome assembly proteins (NAPs) and shares 92% identity with human and mouse NAP1. NAPs have been reported to have a role in nucleosome assembly, cell cycle regulation, cell proliferation and transcriptional control, although the precise function of NAP1 is still not clear. Here we report the identification of a putative domain of xNAP1 by limited proteolysis. This domain has been mapped in the xNAP1 protein sequence to residues 38-282 and thus lacks the acidic sequences at the N- and C-termini. We have studied this domain and related fragments in vitro and by a functional assay involving over-expression of the protein in Xenopus laevis embryos. Analytical ultracentrifugation shows that removal of the acidic N- and C-terminal regions does not prevent the formation of larger multimers, which are predominantly hexadecamers. Injection of mRNA encoding the full-length xNAP1 or the putative domain and other related constructs into Xenopus embryos gave identical phenotypes. These results are discussed in relation to protein-protein interactions between NAP1 octamers and a possible 'squelching' mechanism.

Genetic screens for mutations affecting development of Xenopus tropicalis.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Zygotic nucleosome assembly protein-like 1 has a specific, non-cell autonomous role in hematopoiesis.

Nucleosome assembly proteins (NAPs) bind core histones, facilitate chromatin remodeling, and can act as transcriptional coactivators. We previously described the isolation of a Xenopus NAP1-like (xNAP1L) cDNA, which encodes a member of this protein family. Its zygotic expression is restricted to neural cells, the outer cells of the ventral blood island (VBIs), and the ectoderm overlying the blood precursors. Here, we report that depletion of zygotic xNAP1L in embryos produces no obvious morphologic phenotype, but ablates alpha-globin mRNA expression in the VBIs. Transcript levels of the hematopoietic precursor genes SCL and Xaml (Runx-1) are also reduced in the VBIs. SCL expression can be rescued by injection of xNAP1L mRNA into the ectoderm, showing that the effect of xNAP1L can be non-cell autonomous. Fli1 and Hex, genes expressed in hemangioblasts but subsequently endothelial markers, were unaffected, suggesting that xNAP1L is required for the hematopoietic lineage specifically. Our data are consistent with a requirement for xNAP1L upstream of SCL, and injection of SCL mRNA into xNAP1L-depleted embryos rescues alpha-globin expression. Thus, xNAP1L, which belongs to a family of proteins previously believed to have general roles, has a specific function in hematopoiesis.

Xenopus nucleosome assembly protein becomes tissue-restricted during development and can alter the expression of specific genes.

Nucleosome assembly proteins have been identified in all eukaryotic species investigated to date and their suggested roles include histone shuttle, histone acceptor during transcriptional chromatin remodelling and cell cycle regulator. To examine the role of these proteins during early development we have isolated the cDNA encoding Xenopus NAP1L, raised an antibody against recombinant xNAP1L and examined the expression pattern of this mRNA and protein. Expression in adults is predominantly in ovaries. This maternal protein remains a major component of xNAP1L within the embryo until swimming tadpole stages. xNAP1L mRNA is initially throughout the embryo but by gastrula stages it is predominantly in the presumptive ectoderm. Later, mRNA is detected in the neural crest, neural tube, eyes, tailbud and ventral blood islands. In order to test whether xNAP1L has a potential role in gene regulation we overexpressed this protein in animal pole explants and tested the effect on expression of a series of potential target genes. The mRNA encoding the transcription factor GATA-2 was markedly up-regulated by this overexpression. These data support a role for xNAP1L in tissue-restricted gene regulation.