The Regulation of Growth in Developing, Homeostatic, and Regenerating Tetrapod Limbs: A Minireview.
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ABSTRACT:
SUBMITTER: Wells KM
PROVIDER: S-EPMC8763381 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned.
2022-06-15 | GSE196485 | GEO
Project description:BackgroundStudies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs.ResultsWe used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-?1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1.ConclusionsOur study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem cell markers in regeneration.
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Project description:BackgroundFollowing amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs.ResultsWe identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation.ConclusionOur data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.
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Project description:Treatment with midkine inhibitor iMDK inhibits limb regeneration. To elucidate the transcriptional mechanisms of iMDK treatment, we sequenced regenerating limbs treated with either DMSO or iMDK (bulk) in biological triplicate at 11 days post-amputation (dpa).
2020-01-15 | GSE132325 | GEO
Project description:Axolotls and other salamanders can regenerate entire limbs after amputation as adults, and much recent effort has sought to identify the molecular programs controlling this process. While targeted mutagenesis approaches like CRISPR/Cas9 now permit gene-level investigation of these mechanisms, genetic screening in the axolotl requires an extensive commitment of time and space. Previously, we quantified CRISPR/Cas9-generated mutations in the limbs of mosaic mutant axolotls before and after regeneration and found that the regenerated limb is a highfidelity replicate of the original limb (Flowers et al. 2017). Here, we circumvent aforementioned genetic screening limitations and present methods for a multiplex CRISPR/Cas9 haploid screen in chimeric axolotls (MuCHaChA), which is a novel platform for haploid genetic screening in animals to identify genes essential for limb regeneration.
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Project description:HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet "DNA loops". Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.
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Project description:Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus in which the BMP inhibitor Noggin can be over-expressed at any time during development. We have previously shown that activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles. In the current study, we have taken advantage of this transgenic line to directly compare gene expression in same stage regenerating vs. non-regenerating hind limb buds. Using Affymetrix gene chip analysis, we have identified genes whose expression levels are linked to regenerative success. These include the BMP inhibitor Gremlin and the stress protein Hsp60 (no blastema in zebrafish). Analysis of overrepresented Gene Ontology functional groupings suggests that successful regeneration in the Xenopus hind limb depends on induction of stress response pathways. Furthermore, as expected, genes involved in embryonic development and growth are also significantly over-represented in regenerating early hind limb buds. Keywords: Differential expression, regeneration
2008-06-30 | GSE9813 | GEO
Project description:Ectopic retinoic acid (RA) has been hypothesized to reprogram the positional identity of cells in developing and regenerating limbs to a single positional value corresponding to the posterior-ventral-proximal (PVPr) position on the limb. We tested this hypothesis by using RA to reprogram the information of blastema cells that were induced to form at different positions around the limb circumference. We observed that RA treatment of blastemas in anterior and dorsal locations, but not posterior and ventral locations, resulted in the induction of complete ectopic limbs. These position-specific differences in limb induction are probably due to differences in the positional disparity between the RA-reprogrammed blastema cells and the cells at the periphery of the wound. These observations are consistent with the hypothesis that RA treatment reprograms the information in blastema cells to the PVPr position on the limb, since anterior and dorsal positions have the largest disparity and posterior and ventral have the smallest disparity from the PVPr identity.
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Project description:Distinct gene expression dynamics in developing and regenerating crustacean limbs
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Project description:Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.
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