Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs.
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ABSTRACT:
SUBMITTER: Aztekin C
PROVIDER: S-EPMC8217717 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1? and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1? expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO4-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.
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Project description:Tissue regeneration is of fast growing importance in the development of biomedicine, particularly organ replacement therapies. Unfortunately, many human organs cannot regenerate. Anuran Xenopus laevis has been used as a model to study regeneration as many tadpole organs can regenerate. In particular, the tail, which consists of many axial and paraxial tissues, such as spinal cord, dorsal aorta and muscle, commonly present in vertebrates, can fully regenerate when amputated at late embryonic stages and most of the tadpole stages. Interestingly, between stage 45 when feeding begins to stage 47, the Xenopus laevis tail cannot regenerate after amputation. This period, termed "refractory period", has been known for about 20 years. The underlying molecular and genetic basis is unclear in part due to the difficult to carry out genetic studies in this pseudo-tetraploid species. Here we compared tail regeneration between Xenopus laevis and the highly related diploid anuran Xenopus tropicalis and found surprisingly that Xenopus tropicalis lacks the refractory period. Further molecular and genetic studies, more feasible in this diploid species, should reveal the basis for this evolutionary divergence in tail regeneration between two related species and facilitate the understanding how tissue regenerative capacity is controlled, thus with important implications for human regenerative medicine.
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Project description:Redox state sustained by reactive oxygen species (ROS) is crucial for regeneration; however, the interplay between oxygen (O2), ROS and hypoxia-inducible factors (HIF) remains elusive. Here we observe, using an optic-based probe (optrode), an elevated and steady O2 influx immediately upon amputation. The spatiotemporal O2 influx profile correlates with the regeneration of Xenopus laevis tadpole tails. Inhibition of ROS production but not ROS scavenging decreases O2 influx. Inhibition of HIF-1? impairs regeneration and stabilization of HIF-1? induces regeneration in the refractory period. In the regeneration bud, hypoxia correlates with O2 influx, ROS production, and HIF-1? stabilization that modulate regeneration. Further analyses reveal that heat shock protein 90 is a putative downstream target of HIF-1? while electric current reversal is a de facto downstream target of HIF-1?. Collectively, the results show a mechanism for regeneration via the orchestration of O2 influx, ROS production, and HIF-1? stabilization.
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Project description:Cells within salamander limbs retain memories that inform the correct replacement of amputated tissues at different positions along the length of the arm, with proximal and distal amputations completing regeneration at similar times. We investigated the possibility that positional memory is associated with variation in transcript abundances along the proximal-distal limb axis. Transcripts were deeply sampled from Ambystoma mexicanum limbs at the time they were administered fore arm vs upper arm amputations, and at 19 post-amputation time points. After amputation and prior to regenerative outgrowth, genes typically expressed by differentiated muscle cells declined more rapidly in upper arms while cell cycle transcripts were expressed more highly. These and other expression patterns suggest upper arms undergo more robust tissue remodeling and cell proliferation responses after amputation, and thus provide an explanation for why the overall time to complete regeneration is similar for proximal and distal amputations. Additionally, we identified candidate positional memory genes that were expressed differently between fore and upper arms that encode a surprising number of epithelial proteins and a variety of cell surface, cell adhesion, and extracellular matrix molecules. Also, genes were discovered that exhibited different, bivariate patterns of gene expression between fore and upper arms, implicating dynamic transcriptional regulation for the first time in limb regeneration. Finally, 43 genes expressed differently between fore and upper arm samples showed similar transcriptional patterns during retinoic acid-induced reprogramming of fore arm blastema cells into upper arm cells. Our study provides new insights about the basis of positional information in regenerating axolotl limbs.
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Project description:Extrinsic cues and intrinsic competence act in concert for cell fate determination in the developing vertebrate retina. However, what controls competence and how precise is the control are largely unknown. We studied the regulation of competence by examining the order in which individual retinal progenitor cells (RPCs) generate daughters. Experiments were performed in Xenopus laevis, whose full complement of retinal cells is formed in 2 days. We lineage-labeled RPCs at the optic vesicle stage. Subsequently we administered a cell cycle marker, 5-bromodeoxyuridine (BrdU) at early, middle or late periods of retinogenesis. Under these conditions, and in this animal, BrdU is not cleared by the time of analysis, allowing cumulative labeling. All retinal cell types were generated throughout nearly the entire retinogenesis period. When we examined the order that individual RPCs generated daughters, we discovered a regular and consistent sequence according to phenotype: RGC, Ho, CPr, RPr, Am, BP, MG. The precision of the order between the clones supports a model in which RPCs proceed through stepwise changes in competence to make each cell type, and do so unidirectionally. Because every cell type can be generated simultaneously within the same retinal environment, the change in RPC competence is likely to be autonomous.
| S-EPMC2673759 | biostudies-literature
Project description:The ability to fully restore damaged or lost organs is present in only a subset of animals. The Xenopus tadpole tail is a complex appendage, containing epidermis, muscle, nerves, spinal cord, and vasculature, which regenerates after amputation. Understanding the mechanisms of tail regeneration may lead to new insights to promote biomedical regeneration in non-regenerative tissues. Although chromatin remodeling is known to be critical for stem cell pluripotency, its role in complex organ regeneration in vivo remains largely uncharacterized. Here we show that histone deacetylase (HDAC) activity is required for the early stages of tail regeneration. HDAC1 is expressed during the 1(st) two days of regeneration. Pharmacological blockade of HDACs using Trichostatin A (TSA) increased histone acetylation levels in the amputated tail. Furthermore, treatment with TSA or another HDAC inhibitor, valproic acid, specifically inhibited regeneration. Over-expression of wild-type Mad3, a transcriptional repressor known to associate in a complex with HDACs via Sin3, inhibited regeneration. Similarly, expression of a Mad3 mutant lacking the Sin3-interacting domain that is required for HDAC binding also blocks regeneration, suggesting that HDAC and Mad3 may act together to regulate regeneration. Inhibition of HDAC function resulted in aberrant expression of Notch1 and BMP2, two genes known to be required for tail regeneration. Our results identify a novel early role for HDAC in appendage regeneration and suggest that modulation of histone acetylation is important in regenerative repair of complex appendages.
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Project description:Amphibian eggs normally require meiotic maturation to be competent for fertilization. A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation. Immature oocytes do not bind sperm, but injection of total egg poly(A)+ mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation. Using an expression cloning approach we have isolated a single cDNA from egg poly(A)+ mRNA that can induce sperm binding in immature oocytes. The cDNA was found to encode Xenopus Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control. This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.
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Project description:Appropriate thyroid hormone (TH) signaling through thyroid hormone receptors (TRs) is essential for vertebrate development. Amphibian metamorphosis is initiated and sustained through the action of TH on TRs, which are conserved across vertebrates. TRs heterodimerize with retinoid X receptors (RXRs) on thyroid hormone response elements (TREs) in the genome; however, in most cell line and adult animal studies, RXR ligands do not affect expression of TR target genes. We used a quantitative, precocious metamorphosis assay to interrogate the effects of the RXR agonist bexarotene (Bex) and the RXR antagonist UVI 3003 (UVI) on T3-induced resorption phenotypes in Xenopus laevis tadpoles 1 week postfertilization. Bex potentiated gill and tail resorption, and UVI abrogated T3 action. These results held in transgenic tadpoles bearing a TRE-driven luciferase reporter. Therefore, we used poly-A-primed RNA sequencing transcriptomic analysis to determine their effects on T3-induced gene expression. We also assayed the environmental pollutant tributyltin (TBT), which is an RXR agonist. We found that the proteases that carry out resorption were potentiated by Bex and TBT but were not significantly inhibited by UVI. However, several transcription factors from multiple families (sox4, fosl2, mxd1, mafb, nfib) were all inhibited by UVI and potentiated by Bex and TBT. All required T3 for induction. Time course analysis of gene expression showed that although the agonists could potentiate within 12 hours, the antagonist response lagged. These data indicate that the agonists and antagonist are not necessarily functioning through the same mechanism and suggest that RXR liganding may modulate TH competence in metamorphic signaling.
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