Comparative proteomic analysis of tail regeneration in the green anole lizard, Anolis carolinensis

Abstract

As amniote vertebrates, lizards are the most closely related organisms to humans capable of appendage regeneration. Lizards can autotomize, or release their tails as a means of predator evasion, and subsequently regenerate a functional replacement. Green anoles (Anolis carolinensis) can regenerate their tails through a process that involves differential expression of hundreds of genes, which has previously been analyzed by transcriptomic and microRNA analysis. To investigate protein expression in regenerating tissue, we performed whole proteomic analysis of regenerating tail tip and base. This is the first proteomic data set available for any anole lizard. We identified a total of 2,646 proteins - 976 proteins only in the regenerating tail base, 796 only in the tail tip, and 874 in both tip and base. For over 90% of these proteins in these tissues, we were able to assign a clear orthology to gene models in either the Ensembl or NCBI databases. For 13 proteins in the tail base, 9 proteins in the tail tip, and 10 proteins in both regions, the gene model in Ensembl and NCBI matched an uncharacterized protein, confirming that these predictions are present in the proteome. Ontology and pathways analysis of proteins expressed in the regenerating tail base identified categories including actin filament-based process, ncRNA metabolism, regulation of phosphatase activity, small GTPase mediated signal transduction, and cellular component organization or biogenesis. Analysis of proteins expressed in the tail tip identified categories including regulation of organelle organization, regulation of protein localization, ubiquitin-dependent protein catabolism, small GTPase mediated signal transduction, morphogenesis of epithelium, and regulation of biological quality. These proteomic findings confirm pathways and gene families activated in tail regeneration in the green anole as well as identify uncharacterized proteins whose role in regrowth remains to be revealed. This study demonstrates the insights that are possible from the integration of proteomic and transcriptomic data in tail regrowth in the green anole, with potentially broader application to studies in other regenerative models.

Keywords: Anole; Anolis carolinensis; Lizard; Proteome; Proteomic; Regeneration; Reptile; Squamate; Tail.

Figure 1.. Identified proteins in the green anole regenerating tail at 25 DPA.

A Venn diagram of the number of proteins unique to the tail base (red), tail tip (blue), and number of proteins shared between the two regenerating segments (purple).

Figure 2.. Gene Ontology enrichment for proteins present in the regenerating tail base.

The top 10 most highly represented biological process, cellular compartment, and molecular function GO categories in the green anole regenerating tail base. Enrichment scores, calculated as −log10(adjusted p-value), are shown for each GO term. Full data set available in Supplementary Table S3.

Figure 3.. Biological processes enriched in the regenerating tail base are summarized into major functional categories as a treemap.

Each rectangle shows a single, representative GO term where size is determined by the absolute log10 p-values of related, enriched biological processes within each group. Legend for biological processes terms summarized by letters: A, developmental process; B, metabolism; C, cellular process; D, cell-cell adhesion; E, localization; F, biological adhesion; G, multicellular organismal process; H, locomotion; I, methylation; J, establishment or maintenance of cell polarity; K, establishment of cell polarity. All enriched GO terms can be viewed in Supplementary Tables S3 and S4.

Figure 4.. Gene Ontology enrichment for proteins unique to the regenerating tail tip.

The top 10 most highly represented biological process, cellular compartment, and molecular function GO categories in the green anole regenerating tail tip. GO terms are displayed on the x-axis and enrichment scores, calculated as −log10(adjusted p-value), are shown on the y-axis. Full data set available in Supplementary Table S5.

Figure 5.. Biological processes enriched to the regenerating tail tip are summarized into major functional categories as a treemap.

Each rectangle shows a single, representative GO term where size is determined by the absolute log10 p-values of related, enriched biological processes within each group. Legend for biological processes terms summarized by letters: A, regulation of autophagy; B, autophagy; C, developmental process; D, biological regulation; E, growth; F, regulation of growth; G, regulation of neural precursor cell proliferation. All enriched GO terms can be viewed in Supplementary Tables S5 and S6.

Figure 6.. Gene Ontology enrichment for proteins shared between the regenerating tail base and tip.

The top 10 most highly represented biological process, cellular compartment, and molecular function GO categories for shared proteins in the green anole regenerating tail base and tip. GO terms are displayed on the x-axis and enrichment scores, calculated as −log10(adjusted p-value), are shown on the y-axis. Full data set available in Supplementary Table S7.

Figure 7.. Biological processes enriched in and shared in the regenerating tail base and tip are summarized into major functional categories as a treemap.

Each rectangle shows a single, representative GO term where size is determined by the absolute log10 p-values of related, enriched biological processes within each group. Legend for biological processes terms summarized by letters: A, cellular component organization or biogenesis; B, regulation of locomotion; C, biological adhesion; D, cellular process; E, localization; F, locomotion; G, metabolism; H, multicellular organismal process; I, intermediate filament-based process; J, protein folding; K, sperm-egg recognition; L, establishment or maintenance of cell polarity; M, response to stimulus. All enriched GO terms can be viewed in Supplementary Tables S7 and S9.