Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

Abstract

Background: Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration.

Results: Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences.

Conclusion: Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

Figure 1

Histology of innervated and denervated limbs at 5 and 14 dpa. Eosin and hematoxylin staining of DL5 (A), NR5 (C), DL14 (E), and NR14 (G) limbs. Higher magnification inset pictures are provided for each image (B, D, F, H). Scale Bar A = 500 μm; B = 50 μm.

Figure 2

Schematic of matrix metalloproteinase gene expression. Matrix metalloproteinase gene expression is represented in each box. A) Microarray results are represented by fold change (FC) from day 0. B) Normalized counts are represented from the 454 cDNA sequencing experiment. Figures 2 to 4 were created using GenMAPP [101]. Figure 2 was modified from a MAPP originally created by Gladstone Institutes.

Figure 3

Schematic of collagen gene expression. Collagen gene expression is represented in each box. A) Microarray results are represented by fold change (FC) from day 0. B) Normalized counts are represented from the 454 cDNA sequencing experiment.

Figure 4

Schematic of down-regulated muscle contraction genes. Striated muscle contraction genes that were downregulated during limb regeneration. Each gene is represented by two boxes that denote proportional expression among Day 0, NR5, DL5, NR14, and DL14 samples. The left box reports hybridization intensity from the microarray experiment and the right box reports normalized count data from the 454 cDNA sequencing experiment. Figure 4 was modified from a MAPP originally created by Joanna Fong and Nathan Salomonis.

Figure 5

Clustering of genes identified as significant from the comparison of NR14 and DL14 limbs. Fold change values are relative to baseline levels at Day 0. Blue-coded genes are cell cycle associated; orange-coded genes localize to the lysosome; green-coded genes are associated with inflammatory responses; red-coded genes are matrix metalloproteinases; purple-coded genes are associated with muscle. Genes coded * are associated with inflammation and localize to the lysosome.

Figure 6

Bar graph showing contig lengths. Distribution of sequence lengths for each of the 61,127 contigs.

Figure 7

Bar graph showing percent coverage of human proteins. The distribution of the percent coverage for each of the unique 9411 human proteins with presumptive salamander orthologs.