A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs.

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.

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes.

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce 'leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.

A catalog of Xenopus tropicalis transcription factors and their regional expression in the early gastrula stage embryo.

Gene regulatory networks (GRNs) involve highly combinatorial interactions between transcription factors and short sequence motifs in cis-regulatory modules of target genes to control cellular phenotypes. The GRNs specifying most cell types are largely unknown and are the subject of wide interest. A catalog of transcription factors is a valuable tool toward obtaining a deeper understanding of the role of these critical effectors in any biological setting. Here we present a comprehensive catalog of the transcription factors for the diploid frog Xenopus tropicalis. We identify 1235 genes encoding DNA-binding transcription factors, comparable to the numbers found in typical mammalian species. In detail, the repertoire of X. tropicalis transcription factor genes is nearly identical to human and mouse, with the exception of zinc finger family members, and a small number of species/lineage-specific gene duplications and losses relative to the mammalian repertoires. We applied this resource to the identification of transcription factors differentially expressed in the early gastrula stage embryo. We find transcription factor enrichment in Spemann's organizer, the ventral mesoderm, ectoderm and endoderm, and report 218 TFs that show regionalized expression patterns at this stage. Many of these have not been previously reported as expressed in the early embryo, suggesting thus far unappreciated roles for many transcription factors in the GRNs regulating early development. We expect our transcription factor catalog will facilitate myriad studies using Xenopus as a model system to understand basic biology and human disease.

Developmentally regulated long non-coding RNAs in Xenopus tropicalis.

Advances in RNA sequencing technologies have led to the surprising discovery that a vast number of transcripts emanate from regions of the genome that are not part of coding genes. Although some of the smaller ncRNAs such as microRNAs have well-characterized functions, the majority of long ncRNA (lncRNA) functions remain poorly understood. Understanding the significance of lncRNAs is an important challenge facing biology today. A powerful approach to uncovering the function of lncRNAs is to explore temporal and spatial expression profiling. This may be particularly useful for classes of lncRNAs that have developmentally important roles as the expression of such lncRNAs will be expected to be both spatially and temporally regulated during development. Here, we take advantage of our ultra-high frequency (temporal) sampling of Xenopus embryos to analyze gene expression trajectories of lncRNA transcripts over the first 3 days of development. We computationally identify 5689 potential single- and multi-exon lncRNAs. These lncRNAs demonstrate clear dynamic expression patterns. A subset of them displays highly correlative temporal expression profiles with respect to those of the neighboring genes. We also identified spatially localized lncRNAs in the gastrula stage embryo. These results suggest that lncRNAs have regulatory roles during early embryonic development.

Genome-wide view of TGFß/Foxh1 regulation of the early mesendoderm program.

Nodal/TGFß signaling regulates diverse biological responses. By combining RNA-seq on Foxh1 and Nodal signaling loss-of-function embryos with ChIP-seq of Foxh1 and Smad2/3, we report a comprehensive genome-wide interaction between Foxh1 and Smad2/3 in mediating Nodal signaling during vertebrate mesendoderm development. This study significantly increases the total number of Nodal target genes regulated by Foxh1 and Smad2/3, and reinforces the notion that Foxh1-Smad2/3-mediated Nodal signaling directly coordinates the expression of a cohort of genes involved in the control of gene transcription, signaling pathway modulation and tissue morphogenesis during gastrulation. We also show that Foxh1 may function independently of Nodal signaling, in addition to its role as a transcription factor mediating Nodal signaling via Smad2/3. Finally, we propose an evolutionarily conserved interaction between Foxh1 and PouV, a mechanism observed in Pou5f1-mediated regulation of pluripotency in human embryonic stem and epiblast cells.

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton.

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.

Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system.

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.

Histone deacetylase 1 maintains lineage integrity through histone acetylome refinement during early embryogenesis.

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.

CRISPR-Cas9 Mutagenesis in Xenopus tropicalis for Phenotypic Analyses in the F0 Generation and Beyond.

CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Xenopus tropicalis Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR-Cas9 can be used to create either mosaic loss-of-function Xenopus embryos that display F0 generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR-Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.

Uncovering the mesendoderm gene regulatory network through multi-omic data integration.

Mesendodermal specification is one of the earliest events in embryogenesis, where cells first acquire distinct identities. Cell differentiation is a highly regulated process that involves the function of numerous transcription factors (TFs) and signaling molecules, which can be described with gene regulatory networks (GRNs). Cell differentiation GRNs are difficult to build because existing mechanistic methods are low throughput, and high-throughput methods tend to be non-mechanistic. Additionally, integrating highly dimensional data composed of more than two data types is challenging. Here, we use linked self-organizing maps to combine chromatin immunoprecipitation sequencing (ChIP-seq)/ATAC-seq with temporal, spatial, and perturbation RNA sequencing (RNA-seq) data from Xenopus tropicalis mesendoderm development to build a high-resolution genome scale mechanistic GRN. We recover both known and previously unsuspected TF-DNA/TF-TF interactions validated through reporter assays. Our analysis provides insights into transcriptional regulation of early cell fate decisions and provides a general approach to building GRNs using highly dimensional multi-omic datasets.

Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus.

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.

Control of zygotic genome activation in Xenopus.

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.

Anterograde regulation of mitochondrial genes and FGF21 signaling by hepatic LSD1.

Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than 1000 nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here, we show that histone demethylase LSD1 KO from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in the nucleus. Lsd1 KO reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPß and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in the liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.

Foxh1/Nodal Defines Context-Specific Direct Maternal Wnt/ß-Catenin Target Gene Regulation in Early Development.

Although Wnt/ß-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 ß-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 ß-catenin association. We discover that genomic ß-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfß 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.

Sox17 and ß-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and ß-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and ß-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses ß-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/ß-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and ß-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

DNase-seq to Study Chromatin Accessibility in Early Xenopus tropicalis Embryos.

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.

Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis.

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.

Endodermal Maternal Transcription Factors Establish Super-Enhancers during Zygotic Genome Activation.

Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus.

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.

Foxh1 Occupies cis-Regulatory Modules Prior to Dynamic Transcription Factor Interactions Controlling the Mesendoderm Gene Program.

The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-ß signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied by Foxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.