Transcriptome analysis of axolotl spinal cord and limb regeneration

Regeneration is a relatively widespread phenomenon in nature, although different organisms exhibit different abilities to reconstitute missing structures. Due to the diversity in the extent of damage the organisms can repair it has been debated for a long time whether those abilities are evolutionary traits that arose independently in multiple organisms or whether they represent a by-product of more basic processes. To date, due to constant increase in the amount of available genomic information this question can be approached by means of comparative genomics by comparing several taxa that have different regenerative capabilities. Two relatively closely related salamander species, newt, Notophthalmus viridescens, and the Mexican axolotl, Ambystoma mexicanum, offer a unique opportunity to compare two organisms with well-known regenerative capabilities. Despite their importance for regeneration research, relatively little sequence information was available until recently, owing mainly to the large sizes of the respective genomes. In this work I aimed to create a comprehensive transcriptome assembly of the axolotl by sequencing and then assembling the sequence data from a number of tissues and developmental stages. I also incorporated available sequence information that mostly comes from cDNA libraries sequenced previously. I assessed the completeness of the transcriptome by comparing it to a set of available axolotl sequences and found that 96% of those have homologs in the assembly. Additionally, I found that 7,568 of 7,695 protein families common to vertebrates are also represented in the transcriptome. In order to turn the assembly from a merely collection of sequences into a valuable and useful resource for the entire research community I first annotated the sequences, predicted the open reading frames and protein domains and additionally put together multiple bits of information available for each sequence including but not limited to time-course and tissue- specific expression data and in situ hybridization results. The assembly was thereafter made available for the entire axolotl research community through a web portal I developed. Not only does the web portal provide access to the transcriptome data, it is also equipped with an engine for automated data retrieval, which could facilitate automated cross-species bioinformatics analyses. The study crossed the boundary between pure bioinformatics and biology as the transcriptome allowed for computational comparison of the axolotl and the newt in order to identify salamander-specific genes possibly implicated in regeneration and subsequent functional analysis thereof in the lab. Since regeneration closely resembles embryonic development in terms of genes involved in both processes, I first identified approximately 200 homologous contigs in axolotl and newt, which had a predicted open reading frame, but did not have homologs in non-regenerating species. The expression profile of one of those candidate genes suggested that it had a role in regeneration. I studied the molecular function of that gene using CRISPR/Cas system to confirm that it was protein-coding and to create knock-out animals to study the effect of gene knock-down and knock-out. Knock-out animals exhibited significant delays in both, limb development and tail regeneration. The exact mechanism causing this delay is currently being investigated