Transcriptomic and proteomic analysis of Hemidactylus frenatus during initial stages of tail regeneration

Abstract

Epimorphic regeneration of appendages is a complex and complete phenomenon found in selected animals. Hemidactylus frenatus, house gecko has the remarkable ability to regenerate the tail tissue upon autotomy involving epimorphic regeneration mechanism. This study has identified and evaluated the molecular changes at gene and protein level during the initial stages, i.e., during the wound healing and repair mechanism initiation stage of tail regeneration. Based on next generation transcriptomics and De novo analysis the transcriptome library of the gecko tail tissue was generated. A total of 254 genes and 128 proteins were found to be associated with the regeneration of gecko tail tissue upon amputation at 1, 2 and 5-day post amputation (dpa) against control, 0-dpa through differential transcriptomic and proteomic analysis. To authenticate the expression analysis, 50 genes were further validated involving RTPCR. 327 genes/proteins identified and mapped from the study showed association for Protein kinase A signaling, Telomerase BAG2 signaling, paxillin signaling, VEGF signaling network pathways based on network pathway analysis. This study empanelled list of transcriptome, proteome and the list of genes/proteins associated with the tail regeneration.

Understanding the complexity of Epimorphic Regeneration in zebrafish: A Transcriptomic and Proteomic approach.

“…Pathway analysis of the differentially expressed genes/proteins exhibited association of wide range of canonical pathways, disease and functions and network pathways. It was well documented from our earlier studies for association of several diverse network pathways such as cytoskeleton remodeling pathway 12,42 , Immune response based alternative complement pathway 12 ,Integrin mediated cell adhesion and migration pathway 42 , VEGF signaling pathway 42 , cellular morphology and embryonic development network pathway 43 , cellular assembly and organization network pathway and organ & organismal development pathway 43 for the regeneration of zebrafish caudal fin regeneration 12 , brittle star arm regeneration 42 and lizard tail regeneration 43 .…”

“…Next generation sequencing analysis using Illumina HiSeq 2000 was performed for the total RNA extracted from each time point of regenerating tissue with biological duplicates 43,42 . The obtained transcripts of all the tissues were assembled for de novo transcriptome analysis and subjected for functional annotation using Blastxalong with Danio rerio database.…”

“…The obtained transcripts of all the tissues were assembled for de novo transcriptome analysis and subjected for functional annotation using Blastxalong with Danio rerio database. Furthermore, the gene sequences obtained through NGS analysis were submitted in the NCBI repository and their accession numbers were obtained 42,43 . Differential expressions of mRNA transcripts were analyzed by FKPM software having 0hpa transcripts ascontrol and those with more than 1 log fold changes at each time point of regeneration.…”

“…Total protein of all time points of regenerating caudal fin tissues were extracted and quantified usingAmido black assay 12,44,43 with bovine serum albumin as standard.iTRAQ based quantitative proteomics was carried out for all the time points of regeneration against control (0hpa) in succession to a trypsin based 10% SDS gel digestion 12,43 . All the digested peptides were labelled withiTRAQ 4-plex and purified using C-18 spin columns (Thermo Scientific).…”

“…The copyright holder for this preprint (which this version posted May 21, 2021. ; https://doi.org/10.1101/2021.05.20.445069 doi: bioRxiv preprint reriodatabase 43,42 . Proteins having more than one log fold change in the selected time points were recruited for the study.…”