De novo transcriptome sequencing of axolotl blastema for identification of differentially expressed genes during limb regeneration

Abstract

BackgroundSalamanders 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.ResultsThe 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.ConclusionThe 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.

A microRNA-mRNA expression network during oral siphon regeneration in Ciona

“…2A). These two pathways have previously been implicated in the regulation of programmed cell death and proliferation during regeneration (Gilbert et al, 2013;Godwin et al, 2013;Ho and Whitman, 2008;Lévesque et al, 2007;Rao et al, 2009;Stoick-Cooper et al, 2007;Wu et al, 2013). The miRDeep2 program (Friedländer et al, 2012) was used to align and quantify miRNA reads.…”

“…The ability to detect transcript level changes in a comprehensive and unbiased manner can identify previously unknown coordination between signaling pathways, transcription factors and effector genes required for regeneration. There have been several transcriptome-wide studies of gene expression of both mRNA (Campbell et al, 2011;Hamada et al, 2015;Knapp et al, 2013;Love et al, 2011;Monaghan et al, 2009;Schebesta et al, 2006;Stewart et al, 2013;Wu et al, 2013) and microRNA (miRNA) (Gearhart et al, 2015;Holman et al, 2012;Hutchins et al, 2016;Thatcher et al, 2008) during appendage regeneration. However, none of these studies connected the expression profiles of miRNA to those of mRNA.…”

Temporal Expression Pattern of Peptides in the Regenerating Caudal Fin of Teleost Fish Poecilia latipinna With Special Emphasis on krt15 and myl-1

“…It is previously known that regeneration involves controlled expression of peptides and transcripts of signalling molecules at each stage of epimorphosis [18,19,31]. Therefore, to identify the stage-specific expression of peptides, we performed two dimensional gel electrophoresis.…”

“…There are reports on proteomic profile in regeneration models to unravel the conserved and diverse mechanisms amongst them [16][17][18][19]. It includes the regeneration in axololtl [20,21], Xenopus laevis froglet limb-bud [22,23] and zebrafish caudal fin regeneration [24].…”

First de-novo transcriptome assembly of a South American frog, Oreobates cruralis, enables population genomic studies of Neotropical amphibians

“…The "general function" category (3,166; 24.37%) represented the largest group, 454 followed by "defense mechanisms" (1,421; 10.94%). Our results showed that genes related to defense 455 functions may be relatively abundant in the transcriptome of O. cruralis, particularly compared to the 456 seven anurans studied by Huang et al (2016) and also to A. mexicanum (Wu et al 2013 Although large genome size renders complete genome sequencing practically unfeasible in many 515 species, such as most amphibians, transcriptome sequencing represents a cost-effective alternative to 516 obtain a large amount of genome-wide data. This can allow advances in the study of ecological and 517 evolutionary processes beyond the limits imposed by the use of small panels of markers.…”