DOI: 10.5061/dryad.70rxwdc8k
title: Wnt signaling restores evolutionary loss of regenerative potential in <em>Hydra</em>

description: The regenerative potential of animals varies widely, even among
 closely-related species. In a comparative study of regeneration across the
 Hydra genus, we found that while most species exhibit robust whole-body
 regeneration, Hydra oligactis and other members of the Oligactis clade
 consistently fail to regenerate their feet. To investigate the mechanisms
 underlying this deficiency, we analyzed transcriptional responses during
 head and foot regeneration in H. oligactis. Our analysis revealed that the
 general injury response in H. oligactis lacks activation of Wnt signaling,
 a pathway essential for Hydra vulgaris foot regeneration. Notably,
 transient treatment with a Wnt agonist in H. oligactis triggered a
 foot-specific transcriptional program, successfully rescuing foot
 regeneration. Our transcriptional profiling also revealed dlx2 as a likely
 high-level regulator of foot regeneration, dependent on Wnt signaling
 activation. Our study establishes a comparative framework for
 understanding the molecular basis of regeneration and its evolutionary
 loss in closely-related species.
description: # Wnt signaling restores evolutionary loss of regenerative potential in
 *Hydra* ## Description of repository: This repository contains a backup
 for supplementary and source data files from: “Wnt signaling restores
 evolutionary loss of regenerative potential in *Hydra*” doi:
 [https://doi.org/10.1101/2025.03.18.643955](https://doi.org/10.1101/2025.03.18.643955). This repository also contains a backup of a GitHub repository containing the files and instructions for raw data analysis from this work. ## Abstract from our work: In a comparative study of regeneration across the *Hydra* genus, we found that while most species exhibit robust whole-body regeneration, *Hydra oligactis* and other members of the Oligactis clade consistently fail to regenerate their feet. To investigate the mechanisms underlying this deficiency, we analyzed transcriptional responses during head and foot regeneration in *H. oligactis*. Our analysis revealed that the general injury response in *H. oligactis* lacks activation of Wnt signaling, a pathway essential for *Hydra vulgaris* foot regeneration. Notably, transient treatment with a Wnt agonist in *H. oligactis* triggered a foot-specific transcriptional program, successfully rescuing foot regeneration. Our transcriptional profiling also revealed *dlx2* as a likely high-level regulator of foot regeneration, dependent on Wnt signaling activation. Our study establishes a comparative framework for understanding the molecular basis of regeneration and its evolutionary loss in closely-related species. ## Description of the files and directories contained in this repository: Notes: column names and other acronyms in files: * hpa: hours post amputation * ID: granScript name in *H. oligactis* transcriptomic reference. * logFC: base 2 logarithmic fold change from glmTreat test. * unshrunk.logFC: base 2 logarithmic fold change without shrinkage applied. * logCPM: logaratimic counts per million from edgeR. * Pvalue: probability of effect value from the glmTreat test. * FDR: false discovery rate from adjusted p-value from glmTreat test. * Full_name: long gene name from reciprocal blasting results against Swiss prot database. * Gene_name: short name from reciprocal blasting results against Swiss prot database. * H_sapiens: name of the human ortholog from the OrthoFinder pipeline. * P_Name: human protein name. * G_Name: human gene name. * GO_BP: Gene Ontology Biological Process annotated terms for the gene. * Description: annotated gene function. * H_vulgaris105: transcript ID of *H. vulgaris* ortholog from Jack F Cazet, Adrienne Cho, Celina E Juliano (2021). Generic injuries are sufficient to induce ectopic Wnt organizers in *Hydra* eLife 10:e60562. * FR: foot regeneration * HR: head regeneration * hpg: hours post grafting * ALP: alsterpaullone-treated aboral injured tissue. * N: number of genes in maSigPro Cluster * X: number of genes in Gene Ontology term. * Gene_ratio: Gene enrichmente quotient, N/X. * P: p-value of enrichment test. * logPvalue: logarithmic p-value of enrichment test. * P-adj: adjusted p-value for multicomparison test. * logQvalue: logarithmic adjusted p-value. * attrib ID: ID for the enriched gene ontology terms. * attrib name: description for Gene ontology terms. * sort.rotation: rotation value for factors in PC1. * cluster.mSP: number of clusters from maSigPro analysis. * LOD: base 10 logarithm of odds ratio. **Source_data_SEC_1.tar.gz folder contains the data used to plot all the figures in this work as described below:** Source_data_1.txt: Text file containing the accession numbers and sequences for CO1 used to build the phylogenetic tree. Source_data_2.xlsx: Excel workbook containing regeneration data for different Hydra species, each contained in a different sheet within the workbook: *H. oligactis* (Innsbruck), *H. vulgaris*, *H. oxycnida*, *H. hymanae*, *H. viridissima* and *H. oligactis* (Swiss). Each sheet contains head and foot regeneration data for three batches amputated at 50% of the body of at least 23 animals for up to 144 hours post-amputation. It also contains foot regeneration data for three batches of at least 23 animals amputated at 25% and 75% of body length. Source_data_3.xlsx: Excel workbook containing differentially expressed gene tables for the comparison of different conditions, each one contained in a single sheet of this workbook: FootvF0.up, containing genes upregulated in homeostatic foot tissue compared to foot regenerating tissue at 0 hours post amputation (hpa). FootvH0.up, containing genes upregulated in homeostatic foot tissue compared to head regenerating tissue at 0 hours post amputation (hpa). FootvHead.up, containing genes upregulated in homeostatic foot tissue compared to homeostatic head tissue. HeadvF0.up, containing genes upregulated in homeostatic head tissue compared to foot regenerating tissue at 0 hours post amputation (hpa).HeadvH0.up, containing genes upregulated in homeastic head tissue compared to head regenerating tissue at 0 hours post amputation (hpa). HeadvFoot.up, containing genes upregulated in homeostatic head tissue compared to homeostatic foot tissue. Cells with NA values in sheet columns represent Not Available values. Source_data_4.csv: CSV file containing normalized log2CPM for all mapped transcripts for DMSO foot and head regeneration experiments performed on *H. oligactis*. Cells with NA values in sheet columns represent Not Available values. Source_data_5.xlsx: Excel workbook containing log2 Fold Change values for different tissue comparisons, each contained in a different sheet of this workbook: HRvFR_3hpa, containing log2 Fold Change values for the comparison of head regeneration versus foot regeneration at 3 hours post amputation (hpa), controlled by the 0 hpa timepoint for each tissue. HRvFR_12hpa, containing log2 Fold Change values for the comparison of head regeneration versus foot regeneration at 12 hpa controlled by the 0 hpa timepoint for each tissue. HRvFR_24hpa, containing log2 Fold Change values for the comparison of head regeneration versus foot regeneration at 24 hpa controlled by the 0 hpa timepoint for each tissue. HRvFR_48hpa, containing log2 Fold Change values for the comparison of head regeneration versus foot regeneration at 48 hpa, controlled by the 0 hpa timepoint for each tissue. Cells with NA values in sheet columns represent Not Available values. Source_data_6.xlsx: Excel workbook containing log2 CPM for selected foot-specific genes across the time course of regeneration in the different in *H. vulgaris* and *H. oligactis*. Source_data_7.csv: CSV file containing FPKM for each individual sample (columns) and for all H. oligactis transcripts used in the Orthoclust analysis. **Source_data_SEC_2.tar.gz folder contains the data used to plot all the figures in this work as described below:** Source_data_8: CSV file containing FPKM for each individual sample (columns) and for all *H. vulgaris* transcripts used in the Orthoclust analysis. Source_data_9. Excel workbook containing secondary axis formation frequency from grafting regenerating tissue in *H. vulgaris* (AEP )and *H. oligactis* (OLI) at the indicated timepoints for each species as contained in the separate sheets: OLI 0hpa (0 hours post amputation in *H. oligactis*), OLI 3hpa (3 hours post amputation in *H. oligactis*), OLI 8hpa (8 hours post amputation in *H. oligactis*), OLI 16 hpa (16 hours post amputation in *H. oligactis*), OLI 24hpa (24 hours post amputation in *H. oligactis*), OLI 48 hpa (48 hours post amputation in *H. oligactis*), AEP 0hpa (0 hours post amputation in *H. vulgaris*), AEP 3hpa (3 hours post amputation in *H. vulgaris*), AEP 8hpa (8 hours post amputation in *H. vulgaris*), AEP 16 hpa (16 hours post amputation in *H. vulgaris*), AEP 24hpa (24 hours post amputation in *H. vulgaris*) and AEP 48 hpa (48 hours post amputation in H. vulgaris). Each sheet contains axis formation frequency (Lateral axis formation), batch size (Total grafted), percentage of lateral axis (Percentage %) across 6 different timepoints post grafting (24, 48, 72, 96, 120, and 144 hours)for 3 different batches. Source_data_10. CSV file containing foot regeneration and ectopic head formation raw data and percentages for *H. oligactis* treated with the different ALP concentrations (2.5, 5 and 10 uM). The column 'treatment' specifies ALP or DMSO concentrations. 'Polyps w/regenerated feet' specifies the number of Hydra polyps with basal disk regeneration at 144 hours post-amputation. 'Regenerated Ectopic Heads' specifies the number of Hydra polyps with an ectopic head formed at the aboral side. 'batch.size' indicates the number of Hydra in each batch. 'notes' describe specific treatment details. 'percentage{%} Foot regeneration' specifies the percentage of Hydra with regenerated basal disks per batch. 'percentage{%} Ectopic heads' specifies the percentage of ectopic heads regenerated at the aboral site. The rows below show the average Foot regeneration and ectopic head regeneration percentages for DMSO and different ALP concentrations. Source_data_11. Excel workbook containing foot regeneration and head regeneration percentages for *H. vulgaris* after 12 hours of iCRT14 treatment (in sheet: iCRT AEP) and in DMSO controls (in sheet: DMSO AEP for iCRT exp). Foot and head regeneration are shown for 24, 48, 72, and 96 hours post amputation as 'Regenerated basal disk' and 'Two tentacle buds' respectively for 3 batches each. The average foot and head regeneration percentages are also shown. Source_data_12. CSV file containing normalized log2CPM for foot regeneration under DMSO (DMSO{hpa}{#of sample}) and Alsterpaullone (ALP{hpa}{#of sample}) treatments at the different time points across the timecourse of regeneration. Cells with NA values in sheet columns represent Not Available values. Source_data_13. Excel workbook containing fold change gene tables for the timepoints comparing foot regeneration in DMSO versus Alsterpaullone at 24 hours post amputation (in sheet: DMSOvsALP_24hpa) and DMSO versus Alsterpaullone at 48 hours post amputation (in sheet: DMSOvsALP_48hpa). Source_data_14. Excel workbook containing Gene Ontology enriched terms and their stats for maSigPro modules 4 (in sheet: GO_module_4) and module 7 (in sheet: GO_module_7). **Supplementary_Data_Files.tar.gz folder contains the data used to plot all the figures in this work as described below:** Supplementary_Data_1.xlsx. An Excel workbook containing two lists for foot-specific genes (Foot-specific genes sheet) and head-specific genes (Head-specific genes sheet) in *H. oligactis*. Cells with NA values in sheet columns represent Not Available values. Supplementary_Data_2.csv. Comma-separated values file containing genes loaded onto PC1 from a Principal Component Analysis from Head regeneration RNA-seq samples in H. oligactis. Cells with NA values in sheet columns represent Not Available values. Supplementary_Data_3.csv. Comma-separated values file containing genes loaded onto PC1 from Principal Component Analysis from Foot regeneration RNA-seq samples in H. oligactis. Cells with NA values in sheet columns represent Not Available values. Supplementary_data_4.xlsx. An Excel workbook containing lists of Differentially Expressed Genes along with their functional annotations from all comparisons listed in the study. Each comparison is contained in a separate sheet: FR-specific_3hpa, contains genes upregulated specifically in foot regenerating tissue compared to head regenerating tissue at 3 hours post amputation (hpa). FR-specific_12hpa contains genes upregulated specifically in foot regenerating tissue compared to head regenerating tissue at 12 hpa. FR-specific_24hpa contains genes upregulated specifically in foot regenerating tissue compared to head regenerating tissue at 24 hpa. FR-specific_48hpa contains genes upregulated specifically in foot regenerating tissue compared to head regenerating tissue at 48 hpa. HR-specific_3hpa contains genes upregulated specifically in head regenerating tissue compared to foot regenerating tissue at 3 hpa. HR-specific_12hpa contains genes upregulated specifically in head regenerating tissue compared to foot regenerating tissue at 12 hpa. HR-specific_24hpa contains genes upregulated specifically in head regenerating tissue compared to foot regenerating tissue at 24 hpa. HR-specific_48hpa contains genes upregulated specifically in head regenerating tissue compared to foot regenerating tissue at 48 hpa. Cells with NA values in sheet columns represent Not Available values. Supplementary_data_5.xlsx. An Excel workbook containing ortholog transcripts between H. oligactis and *H. vulgaris* that were clustered by the OrthClust pipeline, along with their annotations and expression levels in foot and head regeneration time courses. Each separate sheet contains the transcripts grouped in each cluster from cluster 1 to 9. Cells with NA values in sheet columns represent Not Available values. Supplementary_data_6.csv. Comma-separated values file containing 1,773 genes with differential expression patterns grouped into 9 modules by the maSigPro pipeline. Cells with NA values in sheet columns represent Not Available values. Supplementary_data_7.xlsx. Excel workbook containing Gene Ontology (GO) term enrichment analysis for all 9 modules in Supplementary_data_6.csv. Each separate sheet contains the enrichment results for each of the 9 modules obtained with the maSigPro pipeline. Supplementary_data_8.fa. Fasta format file containing all the assembled transcripts used as transcriptomic reference for *H. oligactis* used in our study. **GITHUB_dryad.tar.gz** Directory containing the scripts and files required to analyze a time course of regeneration in Hydra oligactis from RNA-seq data as presented in our manuscript [https://doi.org/10.1101/2025.03.18.643955](https://doi.org/10.1101/2025.03.18.643955). This folder is a backup for the GitHub repository [https://github.com/cejuliano/oligactis_foot_regeneration](https://github.com/cejuliano/oligactis_foot_regeneration). The repository is divided into two sections based on the type of computer environment in which the scripts in each section can be executed. The cluster folder contains scripts meant to be executed on a computing cluster running Ubuntu that uses the Slurm workload manager. The local contains scripts meant to be executed on macOS Sonoma (14.4.1) running R (4.3.3) on RStudio (2024.04.1). Cluster scripts perform raw read trimming, mapping, and expression estimation. Local scripts calculate differential gene expression analysis, coexpression networks, and differential gene expression patterns. A Readme file with detailed instructions on how to run these pipelines is included in this folder.