Fig. 4

Transcriptomic analyses suggest an antagonistic expression pattern for amphiregulin. Failed regenerates that had undergone 5 amputations and sibling control limbs that had never been injured were amputated proximally and harvested at 3 days post-amputation. a Overview of RNA-sequencing strategy. b Heatmap showing the results of k-means clustering (k = 2) of significantly differentially expressed genes between repeatedly amputated limbs and control limbs. Several genes with known roles in regenerative processes are shown. c Gene Ontology analyses of genes that are downregulated in the repeated amputation condition. Panel on the left shows the top 10 most significantly enriched Biological Processes. Panel on the right is a treemap of all significantly enriched Biological Processes. d Gene Ontology analyses of genes that are upregulated in the repeated amputation condition. Panel on the left shows the top 10 most significantly enriched Biological Processes. Panel on the right is a treemap of all significantly enriched Biological Processes. “A”: forward locomotion; “B”: multicellular organismal process; “C”: directional locomotion; “D”: developmental process. A full list of significantly enriched Biological Processes can be found in Supplementary Table 2. e qRT-PCR showing normalized areg expression at 3 dpa in normally-regenerating control limbs (first amputation) and in limbs compromised by repeat amputation (sixth amputation). Error bars are SEM. N = 8 limbs per condition. Asterisks (**) denotes p < 0.01. f qRT-PCR showing normalized areg expression at 3 dpa in normally-regenerating control limbs (first amputation) and in limbs amputated twice and thrice. Error bars are SEM. N = 4-5 limbs per condition. Asterisks (**) denotes p < 0.01; Asterisk (*) denotes p < 0.05; n.s. denotes not significant. g Plot showing the 11 genes with the highest initiation ratio (ratio of early expression to late expression). Expression data for panel g were obtained from [32]