The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to "cut and paste" embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying "leapfrog transplants" (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

The fertilized frog egg contains all the materials needed to initiate development of a new organism, including stored RNAs and proteins deposited during oogenesis, thus the earliest stages of development do not require transcription. The onset of transcription from the zygotic genome marks the first genetic switch activating the gene regulatory network that programs embryonic development. Zygotic genome activation occurs after an initial phase of transcriptional quiescence that continues until the midblastula stage, a period called the midblastula transition, which was first identified in Xenopus. Activation of transcription is programmed by maternally supplied factors and is regulated at multiple levels. A similar switch exists in most animals and is of great interest both to developmental biologists and to those interested in understanding nuclear reprogramming. Here we review in detail our knowledge on this major switch in transcription in Xenopus and place recent discoveries in the context of a decades old problem.

Gene inactivation is an important tool for correlation of phenotypic and genomic data, allowing researchers to infer normal gene function based on the phenotype when the gene is impaired. New and better approaches are needed to overcome the shortfalls of existing methods for any significant acceleration of scientific progress. We have adapted the CRISPR/Cas system for use in Xenopus tropicalis and report on the efficient creation of mutations in the gene encoding the enzyme tyrosinase, which is responsible for oculocutaneous albinism. Biallelic mutation of this gene was detected in the F0 generation, suggesting targeting efficiencies similar to that of TALENs. We also find that off-target mutagenesis seems to be negligible, and therefore, CRISPR/Cas may be a useful system for creating genome modifications in this important model organism.

Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as "open chromatin." Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), but DNA in nucleosomes or higher-order chromatin fibers is less accessible to the nuclease. The DNase-seq method uses high-throughput sequencing to permit the interrogation of DNase hypersensitive sites (DHSs) across the entire genome and does not require prior knowledge of histone modifications, transcription factor binding sites, or high quality antibodies to identify potentially active regions of chromatin. Here, discontinuous iodixanol gradients are used as a gentle preparation of the nuclei from Xenopus embryos. Short DNase I digestion times are followed by size selection of digested genomic DNA, yielding DHS fragments. These DNA fragments are subjected to real-time quantitative polymerase chain reaction (qPCR) and sequencing library construction. A library generation method and pipeline for analyzing DNase-seq data are also described.

Germ layer formation is among the earliest differentiation events in metazoan embryos. In triploblasts, three germ layers are formed, among which the endoderm gives rise to the epithelial lining of the gut tube and associated organs including the liver, pancreas and lungs. In frogs (Xenopus), where early germ layer formation has been studied extensively, the process of endoderm specification involves the interplay of dozens of transcription factors. Here, we review the interactions between these factors, summarized in a transcriptional gene regulatory network (GRN). We highlight regulatory connections conserved between frog, fish, mouse, and human endodermal lineages. Especially prominent is the conserved role and regulatory targets of the Nodal signaling pathway and the T-box transcription factors, Vegt and Eomes. Additionally, we highlight network topologies and motifs, and speculate on their possible roles in development.

It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.

Wnt genes encode secreted growth factors that exhibit potent effects on both embryonic and postembryonic development in vertebrates and invertebrates. Recently, the dishevelled (dsh), shaggy/zeste-white 3, and armadillo genes have been shown to participate in Wnt (wingless; wg) signaling in Drosophila. Vertebrate genes that have sequence similarities to all of these Drosophila genes have been identified. To determine whether these structurally conserved components of insect wg signaling represent a functionally conserved Wnt signaling pathway in vertebrates, we investigated the role of Drosophila dsh in Xenopus Wnt signaling. Xenopus embryos ectopically injected with Drosophila dsh mRNA developed duplicated axes similar to those seen in embryos injected with Wnt mRNAs. The involvement of dsh function in the Wnt signaling pathway in Xenopus was demonstrated using two assays which are specifically sensitive to Wnt signaling: synergistic induction of dorsal mesoderm with bFGF and the specific induction of a Wnt-responsive reporter gene. These findings support the notion that the intracellular response to the Wnt signal has been conserved during evolution to such an extent that its components may be interchanged between distantly related species.

Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid-blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation-deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.

Although Wnt/β-catenin signaling is generally conserved and well understood, the regulatory mechanisms controlling context-specific direct Wnt target gene expression in development and disease are still unclear. The onset of zygotic gene transcription in early embryogenesis represents an ideal, accessible experimental system to investigate context-specific direct Wnt target gene regulation. We combine transcriptomics using RNA-seq with genome-wide β-catenin association using ChIP-seq to identify stage-specific direct Wnt target genes. We propose coherent feedforward regulation involving two distinct classes of direct maternal Wnt target genes, which differ both in expression and persistence of β-catenin association. We discover that genomic β-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfβ signaling. Our results support a new paradigm for direct Wnt target gene co-regulation with context-specific mechanisms that will inform future studies of embryonic development and more widely stem cell-mediated homeostasis and human disease.