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Evolutionarily divergent mTOR remodels translatome for tissue regeneration

ABSTRACT: An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

ORGANISM(S): Ambystoma mexicanum

PROVIDER: GSE185593 | GEO | 2023/05/08

REPOSITORIES: GEO

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Project description:Salamanders, such as the Mexican axolotl, are some of the few vertebrates fortunate in their ability to regenerate diverse structures after injury. Unlike mammals they are able to regenerate a fully functional spinal cord after injury. However, the early signals required to initiate a pro-regenerative response after spinal cord injury is not well understood. To address this question we developed a spinal cord injury model in axolotls and used in vivo imaging of ion sensitive dyes and determined that spinal cord injury induces a rapid and dynamic change in the resting membrane potential of ependymoglial cells. Prolonged depolarization of ependymoglial cells after injury inhibits glial cell proliferation and subsequent axon regeneration. Using transcriptional profiling we identified c-Fos as a key voltage sensitive early response gene that is expressed specifically in the ependymoglial cells after injury. This data establishes that dynamic changes in the membrane potential after injury are essential for regulating the specific spatiotemporal expression of c-Fos that is critical for promoting faithful spinal cord regeneration in axolotl. All axolotls used in these experiments were bred in the axolotl facility at the University of Minnesota under the IACUC protocol #1201A08381. Axolotls of 2â3 cm were used for all in vivo experiments, and animals were kept in separate containers and fed daily with artemia; water was changed daily. Animals were anesthetized in 0.01% p-amino benzocaine (Sigma) before microinjection was performed. Experimental Design: Ivermectin injection Ivermectin or vehicle only (water) was pressure injected into the central canal of the spinal cord, and this was visualized by the addition of Fast Green into the solution. Directly after injection, a portion of the spinal cord was surgically removed and the animals were placed back into water in individual containers. One day post injury (1dpi) animals were anesthetized again and the area of the injury was removed. Tissue from 10 animals were pooled for each microarray replicate.

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.

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Evolutionarily divergent mTOR remodels translatome for tissue regeneration

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