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.
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.
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.
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