Identification of differentially expressed genes in 4-day axolotl limb blastema by suppression subtractive hybridization

Abstract

The goal of our study was the identification of up-regulated genes during axolotl (Ambystoma mexicanum) hindlimb regeneration 4 days after amputation using suppression subtractive hybridization (SSH). Approximately 400 clones that harbored upregulated genes in regenerating blastema tissue were selected for sequence analysis. A BLAST homology search against NCBI non-redundant database and an ambystoma EST database revealed 102 clones that showed homology to known sequences in GenBank with annotated function, 31 were known genes without known function, 74 were novel and 72 belonged to mitochondrial sequences. Differential expression of Hmox1, Orc4L, Pls3, Fen-1, Mcm7 and Mmp3/10a was confirmed using qRT-PCR analysis. Among all genes, only Mmp3/10a has been previously described as involved in limb regeneration. Other important identified genes belong to the group of cell cycle regulators (Orc4L, Nasp, Skp1A and Mcm7, the latter being a possible proliferative marker), those involved in protein synthesis and transport (Sec63, Srp72, Sara2) and V- ATPase pump. The novel genes we identified might be important for the process of blastema formation and the onset of cell proliferation in a regenerating limb.

Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl

“…This enhancement was correlated with up regulation of IDH3B, SDHA, NDUFB3 and NDUFB8, enzymes involved in glycolysis and OxPhos, and was prevented by OxPhos inhibition, suggesting that LIN28a enhances regeneration and repair by up regulating glycolysis and OxPhos metabolic reactions. Consistent with this idea, Gorsic et al [104] detected significant up regulation of the transcripts for cytochromes b and c and intense antibody staining to these cytochromes in the epidermis and underlying tissue of 4 dpa regenerating axolotl limbs. By extension, the LIN28 protein might promote blastema formation through a similar up regulation of glycolysis and OxPhos enzymes and thus ATP production.…”

Gene expression profiles of lens regeneration and development inXenopus laevis

“…Given the large range of studies that have been carried out, we restricted our analysis to those using Xenopus (King et al, 2003; Ishino et al, 2003; Tazaki et al, 2005; Grow et al, 2006; Pearl et al, 2008), other amphibians (axolotl: Schnapp et al, 2005; Gorsic et al, 2008; and the newt: Atkinson et al, 2006; Makarev et al, 2007), as well as fish (zebrafish: Padhi et al, 2004; Schebesta et al, 2006; Monaghan et al, 2007; and medaka: Katogi et al, 2004). We also included studies examining regeneration in the planarian, ( Schmidtea mediterranea ) for which an extensive characterization has been undertaken (Sánchez-Alvarado et al, 2002; Reddien et al, 2005).…”

“…This cutoff was chosen as it would include all exact matches (e.g., E = 0) as well as those that may have had issues related to misalignments due to sequencing errors, or would include genes that are very closely related. For those comparisons with studies examining other species (e.g., Schnapp et al, 2005; Schebesta et al, 2006; Roddy et al, 2008; Padhi, et al, 2004; Monaghan et al, 2007; Lien et al, 2006; Katogi et al, 2004; Gorsic, et al, 2008; Atkinson et al, 2006) significant matches were accepted with expect values ranging as high as 1×10 -20 , that extended over spans of at least 50 amino acids, and in those cases where the sequences had already been identified (annotated) with homologous gene names.…”

Advances in Decoding Axolotl Limb Regeneration

“…For example, the single four-days-post-amputation time point (very early blastema) was chosen to compare to unamputated limbs in a “suppression subtractive hybridization” screen performed in [30]. This screen yielded 279 sequence-able clones that were likely specific to or highly enriched in early blastema.…”