Figure 1.
Overview of the stages of lizard tail regeneration.
(A) Anolis carolinensis lizard with a regenerating tail (distal to arrow). (B-E) Histology of the 10 dpa (B), 15 dpa (C), 20 dpa (D), and 25 dpa (E) regenerating tail by Gomori's trichrome stain, with which connective tissues and collagen stain green-blue, muscle, keratin, and cytoplasm stain red, and nuclei are black. (F) Immunohistochemistry of myosin heavy chain in a 25 dpa regenerating tail using the MY-32 antibody. e, wound epithelium; v, blood vessels; m, muscle; ct, cartilaginous tissue. Composites: B-F. Scale bars in black: 200 µm.
Figure 2.
Transcriptomic analysis of gene expression in the 25 dpa regenerating lizard tail.
(A) 25 dpa regenerated tail tissue was divided into five equal sized segments (S1-S5) with S1 representing the most distal regenerating tip, and total RNA was extracted for RNA-Seq analysis. (B) A heatmap showing 326 genes that were differentially expressed, i.e., displayed significant differences between any two segments in the regenerating tail as determined by Cuffdiff (p<0.05). Genes were clustered by Jensen-Shannon divergence of the log10(FPKM+1) value into two major groups, as shown in the dendrogram on the left. 129 genes displayed increased expression distally towards the tail tip (Cluster II) while 197 displayed increased expression proximally (Cluster I). This clustering also demonstrated that the distal-most regenerating tail tip (S1) was the outlier among these samples. (C) Venn diagram of differentially expressed genes identified by DESeq2 and Cuffdiff2. (D-E) A treemap overview of differentially expressed genes in (D) Cluster I and (E) Cluster II based on representative Gene Ontology Biological Processes. The relative sizes of the treemap boxes are based on the |log10(p-value)| of the respective GO term. Related terms are visualized with the same color, with the representative category for each color group denoted in the legend.
Table 1.
Selected Genes Ontology categories represented along the regenerating tail axis.
Figure 3.
MAPK/FGF and Wnt pathway genes differentially expressed in the 25 dpa regenerating lizard tail.
(A, B) Based on RNA-Seq analysis described in Figure 2, the heatmaps show the 10 MAPK/FGF pathway genes (A), or 9 Wnt pathway genes (B) defined by KEGG, that were differentially expressed, i.e., displayed significant differences between any two segments in the regenerating tail as determined by Cuffdiff2 (p<0.05), along with previously identified Wnt inhibitors. A diagram summarizing the tail segment(s) with highest expression level for each MAPK/FGF (A) or Wnt (B) pathway gene is also shown. Differentially expressed genes are denoted with an asterisk.
Table 2.
Novel and uncharacterized transcripts in the regenerating tail.
Figure 4.
The 25 dpa regenerating tail has limited relative expression of stem cell markers.
(A-D) Heatmap showing gene expression of satellite cell (A) and embryonic (B), mesenchymal (C), and hematopoietic stem cell markers in lizard embryos (n = 2), satellite cells (n = 3), and 25 dpa regenerating tail sections (n = 5). Differentially expressed genes along the regenerating tail axis are denoted with an asterisk.
Figure 5.
Histological and RNA-Seq analysis of proliferation in the 25 dpa regenerating tail.
(A-E) MCM2 immunohistochemistry of the 25 dpa regenerating tail (brown nuclei), counterstained with hematoxylin (blue nuclei). (A) MCM2 is expressed throughout the regenerating tail, indicating a lack of a single proliferative zone. The condensing cartilage tube (B), ependymal core (C), developing muscles near the proximal base (D) and tip (E) of the regenerating tail are shown. (F) A heatmap showing gene expression of proliferative markers in the regenerating tail, the embryos, and satellite cells. DE genes along the regenerating tail axis are denoted with an asterisk. Composites: A. Scale bars in red: 200 µm (A) and 20 µm (B-E).