Identification of in vivo Hox13-binding sites reveals an essential locus controlling zebrafish brachyury expression.

During early embryogenesis, the vertebrate embryo extends from anterior to posterior because of the progressive addition of cells from a posteriorly localized neuromesodermal progenitor (NMp) population. An autoregulatory loop between Wnt and Brachyury/Tbxt is required for NMps to retain mesodermal potential and, hence, normal axis development. We recently showed that Hox13 genes help to support body axis formation and to maintain the autoregulatory loop, although the direct Hox13 target genes were unknown. Here, using a new method for identifying in vivo transcription factor-binding sites, we identified more than 500 potential Hox13 target genes in zebrafish. Importantly, we found two highly conserved Hox13-binding elements far from the tbxta transcription start site that also contain a conserved Tcf7/Lef1 (Wnt response) site. We show that the proximal of the two elements is sufficient to confer somitogenesis-stage expression to a tbxta promoter that, on its own, only drives NMp expression during gastrulation. Importantly, elimination of this proximal element produces shortened embryos due to aberrant formation of the most posterior somites. Our study provides a potential direct connection between Hox13 and regulation of the Wnt/Brachyury loop.

Hif1α and Wnt are required for posterior gene expression during Xenopus tropicalis tail regeneration.

Regeneration of complex tissues is initiated by an injury-induced stress response, eventually leading to activation of developmental signaling pathways such as Wnt signaling. How early injury cues are interpreted and coupled to activation of these developmental signals and their targets is not well understood. Here, we show that Hif1α, a stress induced transcription factor, is required for tail regeneration in Xenopus tropicalis. We find that Hif1α is required for regeneration of differentiated axial tissues, including axons and muscle. Using RNA-sequencing, we find that Hif1α and Wnt converge on a broad set of genes required for posterior specification and differentiation, including the posterior hox genes. We further show that Hif1α is required for transcription via a Wnt-responsive element, a function that is conserved in both regeneration and early neural patterning. Our findings indicate that Hif1α has regulatory roles in Wnt target gene expression across multiple tissue contexts.

Nutrient availability contributes to a graded refractory period for regeneration in Xenopus tropicalis.

Xenopus tadpoles are a unique model for regeneration in that they exhibit two distinct phases of age-specific regenerative competence. In Xenopus laevis, young tadpoles fully regenerate following major injuries such as tail transection, then transiently lose regenerative competence during the "refractory period" from stages 45-47. Regenerative competence is then regained in older tadpoles before being permanently lost during metamorphosis. Here we show that a similar refractory period exists in X. tropicalis. Notably, tadpoles lose regenerative competence gradually in X. tropicalis, with full regenerative competence lost at stage 47. We find that the refractory period coincides closely with depletion of maternal yolk stores and the onset of independent feeding, and so we hypothesized that it might be caused in part by nutrient stress. In support of this hypothesis, we find that cell proliferation declines throughout the tail as the refractory period approaches. When we block nutrient mobilization by inhibiting mTOR signaling, we find that tadpole growth and regeneration are reduced, while yolk stores persist. Finally, we are able to restore regenerative competence and cell proliferation during the refractory period by abundantly feeding tadpoles. Our study argues that nutrient stress contributes to lack of regenerative competence and introduces the X. tropicalis refractory period as a valuable new model for interrogating how metabolic constraints inform regeneration.

Gradient expectations: Revisiting Charles Manning Child's theory of metabolic regionalisation in developmental patterning and regeneration.

Charles Manning Child introduced one of several early models to explain how an organism can both establish and re-establish positional identity during embryogenesis and regeneration. In his gradient theory model, tissues along an axis exhibit graded levels of metabolic activity demonstrated through their differential susceptibility to metabolic inhibitors. While Child's work was difficult to place in a mechanistic framework in his own time, technological advances and recent discoveries in both embryos and regenerating organisms make his early work on redox signalling as a positional cue newly pertinent.

Chromatin accessibility analysis reveals distinct functions for HDAC and EZH2 activities in early appendage regeneration.

Xenopus tropicalis tadpoles have the capacity for scarless regeneration of appendages including the limb and tail. Following injury, transcriptional programs must be activated and inactivated with high spatial and temporal resolution to result in a properly patterned appendage. Functional studies have established that histone-modifying enzymes that act to close chromatin are required for regeneration, but the genomic regions sensitive to these activities are not fully established. Here we show that early inhibition of HDAC or EZH2 activity results in incomplete tail regeneration. To identify how each of these perturbations impacts chromatin accessibility, we applied an assay for transposase-accessible chromatin (ATAC-seq) to HDAC or EZH2-inhibited regenerating tadpoles. We find that neither perturbation results in a global increase in chromatin accessibility, but that both inhibitors have targeted effects on chromatin accessibility and gene expression. Upon HDAC inhibition, regulatory regions neighbouring genes associated with neuronal regeneration are preferentially accessible, whereas regions associated with immune response and apoptosis are preferentially accessible following EZH2 inhibition. Together, these results suggest distinct roles for these two chromatin-closing activities in appendage regeneration.

Tissue disaggregation and isolation of specific cell types from transgenic Xenopus appendages for transcriptional analysis by FACS.

BACKGROUND: Xenopus embryos and tadpoles are versatile models for embryological, cell biological, and regenerative studies. Genomic and transcriptomic approaches have been increasingly employed in these frogs. Most of these genome-wide analyses have profiled tissues in bulk, but there are many scenarios where isolation of single cells may be advantageous, including isolation of a preferred cell type, or generation of a single-cell suspension for applications such as scRNA-Seq. RESULTS: Here we present a protocol for the disaggregation of complex tail and limb bud tissue, and use cell type-specific fluorescence in transgenic X. tropicalis appendages to isolate specific cell populations using fluorescence activated cell sorting (FACS). Our protocol addresses a specific challenge in Xenopus embryos and tadpoles: the storage of maternal yolk platelets in each cell, which can introduce light scatter and thereby false positives into FACS analysis. CONCLUSIONS: Here we gate against both nontransgenic and ubiquitously transgenic animals to reduce both false positives and false negatives. We use the Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP)Papal transgenic line as a test case to demonstrate that nucleic acid preparations made from sorted cells are high quality and specific. We anticipate this method will be adaptable to study various cell types that have transgenic reporter lines to better profile cell types of interest.

A temporally resolved transcriptome for developing "Keller" explants of the Xenopus laevis dorsal marginal zone.

BACKGROUND: Explanted tissues from vertebrate embryos reliably develop in culture and have provided essential paradigms for understanding embryogenesis, from early embryological investigations of induction, to the extensive study of Xenopus animal caps, to the current studies of mammalian gastruloids. Cultured explants of the Xenopus dorsal marginal zone ("Keller" explants) serve as a central paradigm for studies of convergent extension cell movements, yet we know little about the global patterns of gene expression in these explants. RESULTS: In an effort to more thoroughly develop this important model system, we provide here a time-resolved bulk transcriptome for developing Keller explants. CONCLUSIONS: The dataset reported here provides a useful resource for those using Keller explants for studies of morphogenesis and provide genome-scale insights into the temporal patterns of gene expression in an important tissue when explanted and grown in culture.

Extreme nuclear branching in healthy epidermal cells of the Xenopus tail fin.

Changes in nuclear morphology contribute to the regulation of complex cell properties, including differentiation and tissue elasticity. Perturbations of nuclear morphology are associated with pathologies that include progeria, cancer and muscular dystrophy. The mechanisms governing nuclear shape changes in healthy cells remain poorly understood, partially because there are few models of nuclear shape variation in healthy cells. Here, we introduce nuclear branching in epidermal fin cells of Xenopus tropicalis as a model for extreme variation of nuclear morphology in a diverse population of healthy cells. We found that nuclear branching arises within these cells and becomes more elaborate during embryonic development. These cells contain broadly distributed marks of transcriptionally active chromatin and heterochromatin, and have active cell cycles. We found that nuclear branches are disrupted by loss of filamentous actin and depend on epidermal expression of the nuclear lamina protein Lamin B1. Inhibition of nuclear branching disrupts fin morphology, suggesting that nuclear branching may be involved in fin development. This study introduces the nuclei of the Xenopus fin as a powerful new model for extreme nuclear morphology in healthy cells to complement studies of nuclear shape variation in pathological contexts.This article has an associated First Person interview with the first author of the paper.

Advancing genetic and genomic technologies deepen the pool for discovery in Xenopus tropicalis.

Xenopus laevis and Xenopus tropicalis have long been used to drive discovery in developmental, cell, and molecular biology. These dual frog species boast experimental strengths for embryology including large egg sizes that develop externally, well-defined fate maps, and cell-intrinsic sources of nutrients that allow explanted tissues to grow in culture. Development of the Xenopus cell extract system has been used to study cell cycle and DNA replication. Xenopus tadpole tail and limb regeneration have provided fundamental insights into the underlying mechanisms of this processes, and the loss of regenerative competency in adults adds a complexity to the system that can be more directly compared to humans. Moreover, Xenopus genetics and especially disease-causing mutations are highly conserved with humans, making them a tractable system to model human disease. In the last several years, genome editing, expanding genomic resources, and intersectional approaches leveraging the distinct characteristics of each species have generated new frontiers in cell biology. While Xenopus have enduringly represented a leading embryological model, new technologies are generating exciting diversity in the range of discoveries being made in areas from genomics and proteomics to regenerative biology, neurobiology, cell scaling, and human disease modeling.

HEB and E2A function as SMAD/FOXH1 cofactors.

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We identified a new motif, termed SMAD complex-associated (SCA), that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.

Transcriptional dynamics of tail regeneration in Xenopus tropicalis.

In contrast to humans, many amphibians are able to rapidly and completely regenerate complex tissues, including entire appendages. Following tail amputation, Xenopus tropicalis tadpoles quickly regenerate muscle, spinal cord, cartilage, vasculature and skin, all properly patterned in three dimensions. To better understand the molecular basis of this regenerative competence, we performed a transcriptional analysis of the first 72 h of tail regeneration using RNA-Seq. Our analysis refines the windows during which many key biological signaling processes act in regeneration, including embryonic patterning signals, immune responses, bioelectrical signaling and apoptosis. Our work provides a deep database for researchers interested in appendage regeneration, and points to new avenues for further study.

RNA sequencing reveals a diverse and dynamic repertoire of the Xenopus tropicalis transcriptome over development.

The Xenopus embryo has provided key insights into fate specification, the cell cycle, and other fundamental developmental and cellular processes, yet a comprehensive understanding of its transcriptome is lacking. Here, we used paired end RNA sequencing (RNA-seq) to explore the transcriptome of Xenopus tropicalis in 23 distinct developmental stages. We determined expression levels of all genes annotated in RefSeq and Ensembl and showed for the first time on a genome-wide scale that, despite a general state of transcriptional silence in the earliest stages of development, approximately 150 genes are transcribed prior to the midblastula transition. In addition, our splicing analysis uncovered more than 10,000 novel splice junctions at each stage and revealed that many known genes have additional unannotated isoforms. Furthermore, we used Cufflinks to reconstruct transcripts from our RNA-seq data and found that ∼13.5% of the final contigs are derived from novel transcribed regions, both within introns and in intergenic regions. We then developed a filtering pipeline to separate protein-coding transcripts from noncoding RNAs and identified a confident set of 6686 noncoding transcripts in 3859 genomic loci. Since the current reference genome, XenTro3, consists of hundreds of scaffolds instead of full chromosomes, we also performed de novo reconstruction of the transcriptome using Trinity and uncovered hundreds of transcripts that are missing from the genome. Collectively, our data will not only aid in completing the assembly of the Xenopus tropicalis genome but will also serve as a valuable resource for gene discovery and for unraveling the fundamental mechanisms of vertebrate embryogenesis.

Developmental enhancers are marked independently of zygotic Nodal signals in Xenopus.

To determine the hierarchy of transcriptional regulation within the in vivo vertebrate embryo, we examined whether developmental enhancers were influenced by Nodal signaling during early embryogenesis in Xenopus tropicalis. We find that developmental enhancers, defined by the active enhancer chromatin marks H3K4me1 and H3K27ac, are established as early as blastula stage and that Smad2/3 only strongly associates with these regions at gastrula stages. Significantly, when we perturb Nodal signaling using the drug SB431542, most enhancers remain marked, including at genes known to be sensitive to Nodal signaling. Overall, as enhancers are in an active conformation prior to Nodal signaling and are established independently of Nodal signaling, we suggest that many developmental enhancers are marked maternally, prior to exposure to extrinsic signals.

Chromatin immunoprecipitation and deep sequencing in Xenopus tropicalis and Xenopus laevis.

Chromatin immunoprecipitation and deep sequencing (ChIP-SEQ) represents a powerful tool for identifying the genomic targets of transcription factors, chromatin remodeling factors, and histone modifications. The frogs Xenopus laevis and Xenopus tropicalis have historically been outstanding model systems for embryology and cell biology, with emerging utility as highly accessible embryos for genome-wide studies. Here we focus on the particular strengths and limitations of Xenopus cell biology and genomics as they apply to ChIP-SEQ, and outline a methodology for ChIP-SEQ in both species, providing detailed strategies for sample preparation, antibody selection, quality control, sequencing library preparation, and basic analysis.

Protocol for tail vein injection in Xenopus tropicalis tadpoles.

Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here, we describe a high-throughput protocol for tail vein injection to introduce fluorescent tracers into tadpoles, which we have previously used to effectively inject morpholinos and molecular antagonists. We describe steps for safely positioning tadpoles onto agarose double-coated plates, draining media, injecting into the ventral tail vein, rehydrating plates, and sorting tadpoles by fluorescence with minimal injury for high-throughput experiments. For complete details on the use and execution of this protocol, please refer to Kakebeen et al.,1 Patel et al.,2 and Patel et al.3.

DNA methylation and chromatin accessibility predict age in the domestic dog.

Across mammals, the epigenome is highly predictive of chronological age. These "epigenetic clocks," most of which have been built using DNA methylation (DNAm) profiles, have gained traction as biomarkers of aging and organismal health. While the ability of DNAm to predict chronological age has been repeatedly demonstrated, the ability of other epigenetic features to predict age remains unclear. Here, we use two types of epigenetic information-DNAm, and chromatin accessibility as measured by ATAC-seq-to develop age predictors in peripheral blood mononuclear cells sampled from a population of domesticated dogs. We measured DNAm and ATAC-seq profiles for 71 dogs, building separate predictive clocks from each, as well as the combined dataset. We also use fluorescence-assisted cell sorting to quantify major lymphoid populations for each sample. We found that chromatin accessibility can accurately predict chronological age (R2 ATAC = 26%), though less accurately than the DNAm clock (R2 DNAm = 33%), and the clock built from the combined datasets was comparable to both (R2 combined = 29%). We also observed various populations of CD62L+ T cells significantly correlated with dog age. Finally, we found that all three clocks selected features that were in or near at least two protein-coding genes: BAIAP2 and SCARF2, both previously implicated in processes related to cognitive or neurological impairment. Taken together, these results highlight the potential of chromatin accessibility as a complementary epigenetic resource for modeling and investigating biologic age.

Mitochondrial leak metabolism induces the Spemann-Mangold Organizer via Hif-1α in Xenopus.

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F1Fo ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when ß-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling.

Chromatin and transcriptional signatures for Nodal signaling during endoderm formation in hESCs.

The first stages of embryonic differentiation are driven by signaling pathways hardwired to induce particular fates. Endoderm commitment is controlled by the TGF-ß superfamily member, Nodal, which utilizes the transcription factors, SMAD2/3, SMAD4 and FOXH1, to drive target gene expression. While the role of Nodal is well defined within the context of endoderm commitment, mechanistically it is unknown how this signal interacts with chromatin on a genome wide scale to trigger downstream responses. To elucidate the Nodal transcriptional network that governs endoderm formation, we used ChIP-seq to identify genomic targets for SMAD2/3, SMAD3, SMAD4, FOXH1 and the active and repressive chromatin marks, H3K4me3 and H3K27me3, in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that while SMAD2/3, SMAD4 and FOXH1 associate with DNA in a highly dynamic fashion, there is an optimal bivalent signature at 32 gene loci for driving endoderm commitment. Initially, this signature is marked by both H3K4me3 and H3K27me3 as a very broad bivalent domain in hESCs. Within the first 24h, SMAD2/3 accumulation coincides with H3K27me3 reduction so that these loci become monovalent marked by H3K4me3. JMJD3, a histone demethylase, is simultaneously recruited to these promoters, suggesting a conservation of mechanism at multiple promoters genome-wide. The correlation between SMAD2/3 binding, monovalent formation and transcriptional activation suggests a mechanism by which SMAD proteins coordinate with chromatin at critical promoters to drive endoderm specification.

Elevated pentose phosphate pathway flux supports appendage regeneration.

A fundamental step in regeneration is rapid growth to replace lost tissue. Cells must generate sufficient lipids, nucleotides, and proteins to fuel rapid cell division. To define metabolic pathways underlying regenerative growth, we undertake a multimodal investigation of metabolic reprogramming in Xenopus tropicalis appendage regeneration. Regenerating tissues have increased glucose uptake; however, inhibition of glycolysis does not decrease regeneration. Instead, glucose is funneled to the pentose phosphate pathway (PPP), which is essential for full tail regeneration. Liquid chromatography-mass spectrometry (LC-MS) metabolite profiling reveals increased nucleotide and nicotinamide intermediates required for cell division. Using single-cell RNA sequencing (scRNA-seq), we find that highly proliferative cells have increased transcription of PPP enzymes and not glycolytic enzymes. Further, PPP inhibition results in decreased cell division specifically in regenerating tissue. Our results inform a model wherein regenerating tissues direct glucose toward the PPP, yielding nucleotide precursors to drive regenerative cell proliferation.

BMP antagonists and FGF signaling contribute to different domains of the neural plate in Xenopus.

In ectodermal explants from Xenopus embryos, inhibition of BMP signaling is sufficient for neural induction, leading to the idea that neural fate is the default state in the ectoderm. Many of these experiments assayed the action of BMP antagonists on animal caps, which are relatively naïve explants of prospective ectoderm, and different results have led to debate regarding both the mechanism of neural induction and the appropriateness of animal caps as an assay system. Here we address whether BMP antagonists are only able to induce neural fates in pre-patterned explants, and the extent to which neural induction requires FGF signaling. We suggest that some discrepancies in conclusion depend on the interpretations of sox gene expression, which we show not only marks definitive neural tissue, but also tissue that is not yet committed to neural fates. Part of the early sox2 domain requires FGF signaling, but in the absence of organizer signaling, this domain reverts to epidermal fates. We also reinforce the evidence that ectodermal explants are naïve, and that explants that lack any dorsal prepattern are readily neuralized by BMP antagonists, even when FGF signaling is inhibited.