Gene expression profile of the regeneration epithelium during axolotl limb regeneration

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.
Developmental Dynamics (Impact Factor: 2.38). 07/2011; 240(7):1826-40. DOI: 10.1002/dvdy.22669
Source: PubMed


Urodele amphibians are unique among adult vertebrates in their ability to regenerate missing limbs. The process of limb regeneration requires several key tissues including a regeneration-competent wound epidermis called the regeneration epithelium (RE). We used microarray analysis to profile gene expression of the RE in the axolotl, a Mexican salamander. A list of 125 genes and expressed sequence tags (ESTs) showed a ≥1.5-fold expression in the RE than in a wound epidermis covering a lateral cuff wound. A subset of the RE ESTs and genes were further characterized for expression level changes over the time-course of regeneration. This study provides the first large scale identification of specific gene expression in the RE.

1 Follower
50 Reads
  • Source
    • "Given that urodele amphibians are the only vertebrates capable of completely regenerating limbs as adults, unraveling the mechanisms of this ideal form of vertebrate regeneration has long been a goal of regeneration biology. Using high-throughput sequencing and microarray analyses, several laboratories have recently generated lists of genes associated with the regeneration of particular tissues and structures in urodeles (Campbell et al., 2011; Holman et al., 2012; Knapp et al., 2013; Monaghan et al., 2009, 2012; Stewart et al., 2013; Wu et al., 2013); however, the identification of molecules and pathways implicated in regeneration has greatly outpaced the rate at which their function can be characterized in the animal. The axolotl is a urodele widely used in regeneration studies as it is easily maintained and bred in the laboratory. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability.
    Development 04/2014; 141(10). DOI:10.1242/dev.105072 · 6.46 Impact Factor
  • Source
    • "Several studies have used highly parallel 454 pyrosequencing to identify axolotl sequences which are used to generate a large-scale feature axolotl microarray [14,15,17,18]. However, 454 pyrosequencing has relatively lower overall transcriptome coverage when compared to Illumina/Solexa platforms [19-21]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Salamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing. The sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization. The results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration.
    BMC Genomics 07/2013; 14(1):434. DOI:10.1186/1471-2164-14-434 · 3.99 Impact Factor
  • Source
    • "This approach was the most similar to ours in the design and also the same microarray platform was used. The identified regeneration-specific gene set included 58 candidates with putative human orthologs [9]. 16 of these genes (Aldh1a1, Dnase1l3, Dsg1, Dync1i1, Gp2, Klf2, Krt5, Msx1, Msx2, Psca, S100P, Tecta, Tgm1, Tgm5, Umod, Wnt5a) were shared between this gene-set and our 112 regeneration-specific/enriched candidates with putative human orthologs. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression - early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.
    PLoS ONE 05/2013; 8(5):e61352. DOI:10.1371/journal.pone.0061352 · 3.23 Impact Factor
Show more