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Comparative Transcriptomics of Limb Regeneration: Identification of Conserved Expression Changes Among Three Species of Ambystoma [RNA-Seq]

ABSTRACT: Transcriptome studies are revealing the complex gene expression basis of limb regeneration in the primary salamander model – Ambystoma mexicanum (axolotl). To better understand this complexity, there is need to extend analyses to additional salamander species. Using microarray and RNA-Seq, we performed a comparative transcriptomic study using A. mexicanum and two other ambystomatid salamanders: A. andersoni, and A. maculatum. Salamanders were administered forelimb amputations and RNA was isolated and analyzed to identify 405 non-redundant genes that were commonly, differentially expressed 24 hours post amputation. Many of the upregulated genes are predicted to function in wound healing and developmental processes, while many of the downregulated genes are typically expressed in muscle. The conserved transcriptional changes identified in this study provide a high-confidence dataset for identifying factors that simultaneous orchestrate wound healing and regeneration processes in response to injury, and more generally for identifying genes that are essential for salamander limb regeneration.

ORGANISM(S): Ambystoma maculatum Ambystoma mexicanum Ambystoma andersoni

PROVIDER: GSE116777 | GEO | 2018/07/10

REPOSITORIES: GEO

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Project description:Study abstract: Axolotl salamanders (Ambystoma mexicanum) remain aquatic in their natural state, during which biomechanical forces on their diarthrodial limb joints are likely reduced relative to salamanders living on land. However, even as sexually mature adults, these amphibians can be induced to metamorphose into a weight-bearing terrestrial stage by environmental stress or the exogenous administration of thyroxine hormone. In some respects, this aquatic to terrestrial transition of axolotl salamanders through metamorphosis may model developmental and changing biomechanical skeletal forces in mammals during the prenatal to postnatal transition at birth and in the early postnatal period. To assess differences in the appendicular skeleton as a function of metamorphosis, anatomical and gene expression parameters were compared in skeletal tissues between aquatic and terrestrial axolotls that were the same age and genetically full siblings. The length of long bones and area of cuboidal bones in the appendicular skeleton, as well as the cellularity of cartilaginous and interzone tissues of femorotibial joints were generally higher in aquatic axolotls compared to their metamorphosed terrestrial siblings. A comparison of steady state mRNA transcripts encoding aggrecan core protein (ACAN), type II collagen (COL2A1), and growth and differentiation factor 5 (GDF5) in femorotibial cartilaginous and interzone tissues did not reveal any significant differences between aquatic and terrestrial axolotls. RNAseq samples: Total RNA was isolated from whole body tissue samples of Mexican axolotl salamanders (Ambystoma mexicanum) at the following developmental stages: Embryo at the tail bud stage, newly hatched larva, larva at the limb bud stage, juvenile at 8.5 centimeters, and adult using variations of guanidinium-based protocols. RNA quantity, purity, and integrity of both the individual samples and the resulting pool were determined with an Agilent 2100 Bioanalyzer using the Eukaryotic Total RNA nano series II analysis kit. The pooled RNA sample was poly-A selected and used for Illumina random priming directional library prep. Four lanes were sequenced only on one end providing single end reads and 4 lanes were sequenced at both ends giving paired-end reads. The library was sequenced on an Illumina HiSeq 2000 for 75bp reads producing 147,248,512 single end reads and 2 x 153,254,667 paired-end reads.

Project description:Salamander limb regeneration is dependent upon tissue interactions that are local to the amputation site. Communication among limb epidermis, peripheral nerves, and mesenchyme coordinate cell migration, cell proliferation, and tissue patterning to generate a blastema, a mass of progenitor cells that forms missing limb structures. An outstanding question is how molecular cross-talk between these tissues gives rise to the regeneration blastema. To identify genes associated with epidermis-nerve-mesenchymal interactions during limb regeneration, we examined histological and transcriptional changes during the first week following injury in the wound epidermis and subjacent cells between three injury types; 1) a flank wound on the side of the animal that will not regenerate a limb, 2) a denervated limb that will not regenerate a limb, and 3) an innervated limb that will regenerate a limb. Early, histological and transcriptional changes were highly similar between the three injury types, presumably because a common wound-healing program is employed across anatomical locations. However, we identified transcripts that were enriched in the limb compared to the flank and are associated with vertebrate limb development. Many of these genes were activated before blastema outgrowth and in situ hybridization showed that some of these genes were expressed in specific tissue types including the epidermis, peripheral nerve, and mesenchyme. We also identified a relatively small group of transcripts that were more highly expressed in innervated limbs versus denervated limbs. These transcripts encode for proteins that are associated with myelination of peripheral nerves, epidermal maintenance, and cell proliferation, suggesting that denervation affects myelinating Schwann cells, epidermal cell function, and proliferation of mesenchymal cells. Overall, our study identifies limb-specific and nerve-dependent genes that are upstream of regenerative growth, and thus promising candidates for the regulation of blastema formation. We used microarray analysis to determine the gene expression changes that take place during limb regeneration, flank wound healing, and an denervated amputated limb. Epidermal tissue and cells adhered to the epidermis were collected as samples. Two harvested samples was pooled for each animal. Four biological replicates were collected from uninjured epidermis (D0) and at 1, 3, and 7 days post injury.

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Comparative Transcriptomics of Limb Regeneration: Identification of Conserved Expression Changes Among Three Species of Ambystoma [RNA-Seq]

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