Multi-species comparisons of snakes identify coordinated signalling networks underlying post-feeding intestinal regeneration

Abstract

Several snake species that feed infrequently in nature have evolved the ability to massively upregulate intestinal form and function with each meal. While fasting, these snakes downregulate intestinal form and function, and upon feeding restore intestinal structure and function through major increases in cell growth and proliferation, metabolism and upregulation of digestive function. Previous studies have identified changes in gene expression that underlie this regenerative growth of the python intestine, but the unique features that differentiate this extreme regenerative growth from non-regenerative post-feeding responses exhibited by snakes that feed more frequently remain unclear. Here, we leveraged variation in regenerative capacity across three snake species-two distantly related lineages ( Crotalus and Python) that experience regenerative growth, and one ( Nerodia) that does not-to infer molecular mechanisms underlying intestinal regeneration using transcriptomic and proteomic approaches. Using a comparative approach, we identify a suite of growth, stress response and DNA damage response signalling pathways with inferred activity specifically in regenerating species, and propose a hypothesis model of interactivity between these pathways that may drive regenerative intestinal growth in snakes.

Keywords: NRF2; RNAseq; insulin signalling; proteomics; stress response; unfolded protein response.

Figure 1.

Divergent species that experience post-feeding regenerative growth exhibit similar gene expression responses. (a) The Burmese python and prairie rattlesnake both exhibit regenerative organ growth after feeding, despite being separate by roughly 90 million years of divergence. (b,c) Venn diagrams of DE (d,e) genes in the Burmese python, prairie rattlesnake and diamondback watersnake in pairwise comparisons between (b) fasted and 1 DPF, and (c) 1 DPF and 4 DPF. (d) Alluvial plots summarizing the number of upregulated (p < 0.05), downregulated (p < 0.05) and not DE (p > 0.05) genes for fasted versus 1 DPF and 1 DPF versus 4 DPF pairwise comparisons in the Burmese python, prairie rattlesnake and diamondback watersnake. Ribbon width represents the number of genes exhibiting a specified pattern of expression across the two pairwise comparisons (i.e. upregulated in fasted versus 1 DPF and downregulated in 1 DPF versus 4 DPF). Genes that were not DE in both pairwise comparisons are not shown.

Figure 2.

Canonical pathway and URM activation inferences based on comparisons of fasted versus 1 DPF RNAseq data. (a) Canonical pathways enrichment of DE genes between fasted and 1 DPF based on genes shared uniquely between the two regenerating species (‘regen’, left column) and genes shared between all three species (‘all’, right column). Black outlines denote p < 0.05. (b) Predicted URM activity based on regen and all gene sets. Cells with a black outline indicate significant enrichment and predicted activity (p < 0.05 and |z| > 1).

Figure 3.

Overlapping canonical pathway predictions characterizing regenerative and feeding responses and a hypothesis model for regenerative growth in snakes. Networks showing the overlap in genes underlying canonical pathways with predicted activity from analyses of (a) DE genes shared between all three species and (b) DE genes shared between the python and rattlesnake but not the watersnake. A connection between two pathways indicates that at least 50% of the genes underlying the significant prediction of activity in one of the pathways also underlie the prediction of the other pathway, whereas pathways that are not connected to any others (circles with grey outlines) do not share greater than 50% of the genes underlying their prediction with any other pathway. Dotted circles represent manual annotation of pathways with similar functions. (c) A hypothesis model for how the integration of growth and stress response signalling drive regenerative growth in snakes.

Figure 4.

Proteomic comparison of python and watersnake intestine following feeding. (a) Numbers of proteins that exhibited significant changes in abundance in pairwise comparisons. (b) Venn diagram of proteins showing significant changes in abundance between fasted and 4 DPF in the python and watersnake. (c) GO term characterization of proteins with significant changes in abundance between fasted and 4 DPF in the python only. Asterisks denote significant enrichment of a category (p < 0.05), and terms with likely involvement in regeneration phenotypes are bolded. (d) URM activity inferred from significant changes in protein abundance between fasted and 4 DPF (p < 0.1), and significant DE genes between fasted and 1 DPF (p < 0.05). Only URMs with significantly inferred activity in the python from both protein and RNA data are shown.