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.

Micronutrient imbalance and common phenotypes in neural tube defects.

Neural tube defects (NTDs) are among the most common birth defects, with a prevalence of close to 19 per 10,000 births worldwide. The etiology of NTDs is complex involving the interplay of genetic and environmental factors. Since nutrient deficiency is a risk factor and dietary changes are the major preventative measure to reduce the risk of NTDs, a more detailed understanding of how common micronutrient imbalances contribute to NTDs is crucial. While folic acid has been the most discussed environmental factor due to the success that population-wide fortification has had on prevention of NTDs, folic acid supplementation does not prevent all NTDs. The imbalance of several other micronutrients has been implicated as risks for NTDs by epidemiological studies and in vivo studies in animal models. In this review, we highlight recent literature deciphering the multifactorial mechanisms underlying NTDs with an emphasis on mouse and human data. Specifically, we focus on advances in our understanding of how too much or too little retinoic acid, zinc, and iron alter gene expression and cellular processes contributing to the pathobiology of NTDs. Synthesis of the discussed literature reveals common cellular phenotypes found in embryos with NTDs resulting from several micronutrient imbalances. The goal is to combine knowledge of these common cellular phenotypes with mechanisms underlying micronutrient imbalances to provide insights into possible new targets for preventative measures against NTDs.

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.

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.

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.

Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors.

Vertebrate appendage regeneration requires precisely coordinated remodeling of the transcriptional landscape to enable the growth and differentiation of new tissue, a process executed over multiple days and across dozens of cell types. The heterogeneity of tissues and temporally-sensitive fate decisions involved has made it difficult to articulate the gene regulatory programs enabling regeneration of individual cell types. To better understand how a regenerative program is fulfilled by neural progenitor cells (NPCs) of the spinal cord, we analyzed pax6-expressing NPCs isolated from regenerating Xenopus tropicalis tails. By intersecting chromatin accessibility data with single-cell transcriptomics, we find that NPCs place an early priority on neuronal differentiation. Late in regeneration, the priority returns to proliferation. Our analyses identify Pbx3 and Meis1 as critical regulators of tail regeneration and axon organization. Overall, we use transcriptional regulatory dynamics to present a new model for cell fate decisions and their regulators in NPCs during regeneration.

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.

More Than Just a Bandage: Closing the Gap Between Injury and Appendage Regeneration.

The remarkable regenerative capabilities of amphibians have captured the attention of biologists for centuries. The frogs Xenopus laevis and Xenopus tropicalis undergo temporally restricted regenerative healing of appendage amputations and spinal cord truncations, injuries that are both devastating and relatively common in human patients. Rapidly expanding technological innovations have led to a resurgence of interest in defining the factors that enable regenerative healing, and in coupling these factors to human therapeutic interventions. It is well-established that early embryonic signaling pathways are critical for growth and patterning of new tissue during regeneration. A growing body of research now indicates that early physiological injury responses are also required to initiate a regenerative program, and that these differ in regenerative and non-regenerative contexts. Here we review recent insights into the biophysical, biochemical, and epigenetic processes that underlie regenerative healing in amphibians, focusing particularly on tail and limb regeneration in Xenopus. We also discuss the more elusive potential mechanisms that link wounding to tissue growth and patterning.