Wnt signaling restores evolutionary loss of regenerative potential in Hydra

Wnt signaling restores evolutionary loss of regenerative potential in Hydra | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Wnt signaling restores evolutionary loss of regenerative potential in Hydra View ORCID Profile Sergio E. Campos , Sahar Naziri , Jackson Crane , View ORCID Profile Jennifer Tsverov , View ORCID Profile Ben D. Cox , Craig Ciampa , View ORCID Profile Celina E. Juliano doi: https://doi.org/10.1101/2025.03.18.643955 Sergio E. Campos 1 Department of Molecular and Cellular Biology, University of California , Davis, Davis, CA 95616, United States 2 Centro de Investigación sobre el Envejecimiento, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CIE-Cinvestav) , Sede Sur, Mexico City, 14330, Mexico Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sergio E. Campos Sahar Naziri 1 Department of Molecular and Cellular Biology, University of California , Davis, Davis, CA 95616, United States 3 Department of Neuroscience and Developmental Biology, Faculty of Life Sciences, University of Vienna , Djerassiplatz 1, 1030, Vienna, Austria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jackson Crane 1 Department of Molecular and Cellular Biology, University of California , Davis, Davis, CA 95616, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer Tsverov 1 Department of Molecular and Cellular Biology, University of California , Davis, Davis, CA 95616, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jennifer Tsverov Ben D. Cox 1 Department of Molecular and Cellular Biology, University of California , Davis, Davis, CA 95616, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ben D. Cox Craig Ciampa 1 Department of Molecular and Cellular Biology, University of California , Davis, Davis, CA 95616, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Celina E. Juliano 1 Department of Molecular and Cellular Biology, University of California , Davis, Davis, CA 95616, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Celina E. Juliano For correspondence: cejuliano{at}ucdavis.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract 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. Introduction Regeneration is defined as the restoration of lost or injured tissues, appendages or organs 1 . While most animals have some capacity to regenerate, this potential varies widely across animals. Thus, a fundamental question is: how are some animals capable of regenerating large portions of their body after injury, while others exhibit only a fraction of this capacity? Mapping animal regenerative abilities on the phylogenetic tree suggests a complex evolutionary history, with numerous gains and losses. Regardless, it is likely that regenerative abilities were present at the dawn of animals and have been largely lost by vertebrates, especially mammals 2 . Many studies have already advanced our understanding of the genes and pathways that control regeneration in highly regenerative animals 3 , 4 . However, we have little understanding of the mechanisms that drive loss of regenerative potential. Comparative approaches between closely related organisms with contrasting regeneration abilities offer a promising avenue to both reveal the mechanisms of regeneration, as well as uncover how regeneration has been shaped throughout evolution. Cnidarians, a group that includes sea anemones, jellyfish, and corals, exhibit high regenerative potential. Furthermore, given their phylogenetic relationship as sister group to bilaterians 5 , research in cnidarians is critical to understand the evolutionary history of regeneration. The cnidarian polyp Hydra vulgaris is capable of whole-body regeneration and is a well-established research organism for studying the molecular mechanisms of regeneration. The body plan of Hydra is arranged around a single body axis, with a head, composed of a tentacle ring around a hypostome or mouth at the oral end; and the peduncle and an adhesive basal disk (foot) at the aboral end. These structures are connected by a cylindrical body column. When bisected, both halves of H. vulgaris are capable of regeneration: the top half regenerates a foot, and the bottom half regenerates a head. However, a few studies on other Hydra species suggest that regenerative potential varies in the genus, a topic that remains largely unexplored 6 , 7 . The Wnt signaling pathway plays a key role in regeneration across a diverse range of species 8 , 9 . In H. vulgaris , the maintenance of tissue patterning in the uninjured animals relies on a Wnt signaling organizing center located at the oral end. Upon bisection perpendicular to the oral-aboral axis in H. vulgaris , the process of regeneration includes the formation of a new Wnt organizer on the oral-facing wound which will direct the regeneration of a new head 9 – 11 . However, injury-induced transcriptional activation of Wnt ligands occurs at both sides of the wound, and treatment with a Wnt inhibitor impairs or delays both head and foot regeneration 11 – 13 . In planarians, a similar regeneration pattern is observed after bisection, with Wnt activation required at both sides of the wound during the early injury response, followed by restriction to tail regeneration where it directs posterior development 8 , 14 . However, in acoels, early Wnt activation after bisection is restricted to aboral wound sites where it similarly directs posterior development, 15 exemplifying variation in the role of Wnt signaling during whole body regeneration. In vertebrates, Wnt signaling is rapidly upregulated early in regeneration and Wnt inhibition results in impaired regeneration of mouse digits, demonstrating its importance even in animals with more limited regeneration abilities 16 , 17 . In H. vulgaris , injury-induced bZIP transcription factors (TFs) likely drive the transcription of Wnt ligands, which in turn are maintained by a self-reinforcing Wnt signaling amplification loop at the oral wound, creating the head organizer 12 , 18 – 20 . In contrast, the molecular mechanisms responsible for foot specification at the aboral wound are less understood. While injury-induced Wnt signaling is necessary for this process, the Wnt signal is quickly down-regulated, giving rise to a foot-specific program through mechanisms that remain unclear 12 , 13 . In this study, we discovered that foot regeneration ability has been lost in the Oligactis clade of the Hydra genus, including the laboratory species Hydra oligactis ( Fig. 1A ). We therefore reasoned that a comparative study of H. vulgaris and H. oligactis foot regeneration could uncover the regulatory mechanisms required to initiate foot regeneration in Hydra . Towards this goal, we performed RNA-seq over a time course of H. oligactis regeneration and compared the results to existing data sets from H. vulgaris . Our analysis revealed that injury-induced Wnt signaling activation is significantly attenuated in H. oligactis , resulting in slower head regeneration and a foot regeneration block. Using pharmacological manipulation, we demonstrated that activation of the Wnt pathway rescues H. oligactis foot regeneration. This approach also allowed us to identify key transcriptional regulators involved in foot formation, providing new insights into the regulatory control of foot regeneration in Hydra . Download figure Open in new tab Figure 1. Foot regeneration ability was lost in the Oligactis clade. (A) Maximum Likelihood phylogenetic tree of the Hydra genus based on mitochondrial cytochrome oxidase 1 sequences. The genus is divided into four clades: Oligactis, Vulgaris, Braueri, and Viridissima 27 . A red triangle marks the likely point of foot regeneration loss in the Oligactis clade. Asterisks denote strains used in amputation experiments. (B-C) Following 50% bisection, 90% of H . oligactis fail to regenerate their feet, resulting in a stably footless phenotype. (D-E) Peroxidase staining correlates with foot morphology in H. oligactis . The assay was performed on 44 H. oligactis polyps 9 days after 50% bisection (5 with feet, 39 footless), confirming the presence or absence of a foot in all cases. Red triangle indicates peroxidase staining in the foot. Scale bars: 1 mm. (F) Amputation strategy to evaluate foot regeneration abilities across the Hydra genus. The top half of each bisected animal was visually monitored for foot regeneration. (G-K) Foot regeneration kinetics following amputations at various positions along the oral-aboral axis for (G) H. oligactis , (H) H. vulgaris , (I) H. oxycnida , (J) H. viridissima and (K) H. hymanae . Source data file 1. Text file containing the accession numbers and sequences for CO1 used to build the phylogenetic tree. Source data file 2. Excel workbook containing head and foot regeneration data for the experiments shown in Figure 1 G-K . Results Aboral regenerative potential varies across the Hydra genus The common laboratory Hydra species, H. vulgaris , can regenerate a complete head or foot from body column tissue. By contrast, it was previously reported that H. oligactis has a foot regeneration deficiency 6 . To investigate this further, we first characterized the foot regeneration abilities of our H. oligactis laboratory strain (Innsbruck 12) for which we recently assembled a high-quality genome 12 . We bisected H. oligactis midway between the head and foot (50% bisection), perpendicular to the oral-aboral axis, and monitored the rate of foot regeneration for the top half ( Fig. 1 B-G). We found that after 6 days, only ∼10% of the animals regenerated their feet ( Fig. 1B, C, G ), while the remaining animals became stably footless, characterized by a flat aboral end ( Fig. 1C ) and an inability to stick to surfaces. We also evaluated the absence or presence of a foot using a standard colorimetric assay that reveals the activity of a foot-specific peroxidase at seven days post bisection 21 . We confirmed that morphologically footless H. oligactis also lacked foot peroxidase staining ( Figure 1D-E ). In contrast to H. oligactis , we confirmed that in the same conditions, H. vulgaris shows complete foot regeneration after bisection, consistent with the published literature ( Fig. 1H ). We next asked if the rate of successful foot regeneration in H. oligactis depends on the location of the cut along the oral-aboral axis. To test this, we performed bisections at three different positions along the body column. In addition to mid-body bisection (50% of body length), we also bisected at 25% body length from the head, just below the tentacle ring, and at 75% body length from the head, just above the peduncle and foot ( Fig. 1F ). We performed the same bisections in H. vulgaris as a positive control for foot regeneration. Consistent with previous literature, H. vulgaris exhibited nearly 100% successful foot regeneration after all three amputations, but with different kinetics depending on the level of the amputation ( Fig 1H ) 6 , 22 – 24 . Also consistent with previous literature, the success rate of H. oligactis foot regeneration depended on the location of the cut; after 75% bisection, ∼65% of animals successfully regenerated their feet, while foot regeneration did not occur after 25% bisection ( Fig. 1G ) (Hoffmeister, 1991). We next tested foot regenerative abilities across the Hydra genus to better understand the evolutionary history of this trait. While a handful of regeneration studies have been done in different Hydra species 6 , 7 , 25 , 26 , no systematic study of foot regeneration ability has been done across the genus on validated species. We therefore performed foot amputation assays ( Fig. 1 F) on three additional Hydra species to cover each phylogenetic clade ( Fig. 1A, I -K) 27 , as well as on the Swiss laboratory strain of H. oligactis 28 ( Fig. S1 ) to ensure that our observations were not specific to the Innsbruck 12 strain of H. oligactis . Our experiments revealed that both H. oligactis and H. oxycnida , which are in the Oligactis clade, show similar foot regeneration defect profiles ( Fig. 1G, I , Fig. S1 ) . By contrast, H. viridissima had robust foot regeneration ability, with faster kinetics than H. vulgaris ( Fig. 1J ). H. hymanae, a member of another distinct group within the Hydra genus phylogeny, the Braueri clade, exhibited slightly reduced foot regenerative ability, which varied depending on the bisection location. While 75% bisections resulted in nearly 100% successful foot regeneration, 25% bisections led to only ∼60% regeneration ( Fig. 1K ). Overall, our survey of foot regeneration abilities across the Hydra genus revealed variability that enables comparative studies. Furthermore, given the relatively high rate of foot regeneration in all species, except those in the Oligactis clade, we conclude that the common ancestor of the Hydra genus likely had robust foot regeneration ability, which was lost by the ancestor of the Oligactis clade. Download figure Open in new tab Supplementary Figure 1. The Hydra oligactis Swiss strain displays a foot regeneration defect like the Innsbruck strain. Approximately 5% of H. oligactis Swiss strain polyps bisected at 50% body length successfully regenerated their feet (solid orange line), while the remaining ∼95% remained footless. In polyps bisected at 25% body length, 100% remained footless (light orange dotted line). Polyps bisected at 75% body length exhibited a higher foot regeneration potential, with over 60% successfully regenerating feet. See Figure 1F for diagram of amputation sites. The foot transcriptional program largely fails to be activated during H. oligactis regeneration To identify key aspects of the transcriptional response that result in limited foot regeneration in H. oligactis , we generated RNA-seq libraries from the regenerating tips of bisected H. oligactis polyps during a time course of both head regeneration and failed foot regeneration at 0, 3, 12, 24, and 48 hours post amputation (hpa) ( Fig. 2A ). For the foot regeneration time course, it was not possible to determine which samples would successfully regenerate their feet a priori , therefore these samples contained a mixture of both successful and unsuccessful regeneration. However, given the low rate of successful foot regeneration after 50% bisection (∼10%, Fig. 1G ), most of the signal is predicted to come from animals that will ultimately fail to regenerate their feet. To define the transcriptional end point of successful regeneration, we also collected RNA-seq data for head and foot tissue in uninjured animals. We used these data to identify head- and foot-specific genes in H. oligactis by determining the differentially expressed genes in uninjured animals between the foot, head, and body column tissue. The regenerating head and foot tissue at 0 hpa were used as uninjured body column for this analysis, given that these tissues were taken just above and below the plane of a 50% bisection right after injury ( Fig. 2B ) . This approach uncovered 404 foot-specific genes and 987 head-specific genes in H. oligactis ( Figure 2C , D; Supplementary data file 1). Download figure Open in new tab Figure 2. Foot-specific gene transcription is largely absent during H. oligactis regeneration. (A) Strategy for RNA-seq library preparation over a regeneration time course: Regenerating animals were incubated in 0.05% DMSO, allowing these samples to serve as controls for the data shown in Figure 5B . (B) RNA-seq strategy for different regions of homeostatic H. oligactis . F0 and H0 correspond to the 0 hpa foot and head regeneration samples in panel A. (C-D) Venn diagrams showing the differential gene expression strategy used to identify 404 foot-specific genes (C) and 987 head-specific genes (D) in H. oligactis . (E) PCA plot of H. oligactis oral-facing wound gene expression showing movement along the PC1 axis toward the homeostatic head profile during regeneration. (F) PCA plot of H. oligactis aboral-facing wound gene expression showing no progression along the PC1 axis toward the homeostatic foot profile during regeneration. (G-J) Comparison of log 2 fold change (log 2 FC) transcript abundance between H. oligactis head regeneration and foot regeneration at 3 hpa (G), 12 hpa (H), 24 hpa (I) and 48 hpa (J). Red dots indicate differentially expressed head-specific genes at the oral-facing wound and blue dots indicate differentially expressed foot-specific genes at the aboral-facing wound. (K) Heatmap of foot-specific gene expression in H. vulgaris (data from Cazet et al., 2021), showing upregulation by 12 hpa during foot regeneration. Values represent Z-scores calculated from log₂ counts per million (logCPM). (L) Heatmap of H. oligactis expression for the same 8 genes shown in panel K. These genes do not exhibit rapid upregulation during regeneration. Source data file 3. Excel workbook containing differentially expressed gene tables for the conditions compared in Figure 2 C-D . Source data file 4. CSV file containing normalized log2CPM used to plot Figure 2 E-F Source data file 5. Excel workbook containing log2 Fold Change values from comparisons in Figure 2 G-J . Source data file 6. Excel workbook containing log2 CPM for selected foot-specific genes in H. vulgaris and H. oligactis shown in Figure 2 K-L . To analyze the transcriptional changes that occur over the course of H. oligactis head regeneration and failed foot regeneration, we used Principal Component Analysis (PCA) ( Fig. 2E, F ). In these analyses, we included homeostatic head and foot RNA-seq libraries which represent a successful regeneration end point. Notably, we found that H. oligactis exhibits nearly 100% head regeneration after bisection, but the process takes up to 72 hpa to complete. This is approximately 24 hours longer than in H. vulgaris , as determined morphologically by the presence of tentacles ( Fig. S2 ). Our RNA-seq time course of H. oligactis head regeneration ended at 48 hours and our PCA analysis confirms that head regeneration is not transcriptionally complete at this point in H. oligactis . Although differences between homeostatic head tissue and regenerating head tissue explain 83.34% of the variation among samples (PC1), regenerating head tissue exhibited a progressive upregulation of key PC1-associated genes, such as wnt3 (Supplementary Data File 2), suggesting a shift toward head identity ( Fig. 2E ). Download figure Open in new tab Supplementary Figure 2. Head regeneration occurs at a slower rate in H. oligactis as compared to H. vulgaris. By 48 hpa, approximately 90% of bisected H. vulgaris regenerate their heads as determined by the appearance of tentacle buds (blue line plot), whereas only about 20% of H. oligactis achieve head regeneration within the same time frame (orange line plot). For failed foot regeneration, we did not observe a progressive shift of regenerating tissue toward the homeostatic foot transcriptional signature. Like head regeneration, the most variation (79.53%) for failed foot regeneration was explained by PC1, which denotes the difference in the expression profiles between homeostatic foot tissue and the regenerating samples. Importantly, 93 out of 100 of the topmost genes explaining variation within PC1 correspond to foot-specific genes (Supplementary data file 3). Whereas only 4.12% of the variation is explained by PC2, which shows changes in the wounded tissue occurring during the time course of regeneration, but the regeneration time points did not change along the PC1 axis ( Fig. 2F ). This suggests that the gene expression profile of the failed foot regeneration samples do not resemble that of the homeostatic foot at any timepoint. We next examined the expression dynamics of head-specific and foot-specific transcripts over the time course of H. oligactis head regeneration and failed foot regeneration respectively. To identify differentially expressed head and foot-specific genes we compared gene expression on each side of the wound at each time point. We found that in H. oligactis head regeneration, only a small number of head-specific genes showed tissue-specific upregulation at 12 hpa, with larger transcriptional changes becoming evident only after 24 hpa ( Fig. 2 G-J ; Supplementary data file 4 containing lists of tissue-specific genes at each timepoint). This contrasts with H. vulgaris , which shows significant structure-specific gene expression by 8 hpa for both head and foot regeneration 12 . For example, in H. vulgaris , several canonical Wnt pathway genes, including Wnt ligands become specific to the oral wound by 8 hpa 12 . In H. oligactis, the expression of head-associated Wnt genes became specific to the oral wound by 48 hpa and overall, 472 out of the 987 (47.8%) head-specific genes were expressed at this timepoint ( Fig. 2 J). In contrast, we identified only 63 out of 404 (15.6%) foot-specific genes as up-regulated in failed foot regenerating tissue at 48 hpa ( Fig. 2G-J ). Therefore, this analysis showed that by 48 hpa, head regeneration in H. oligactis although incomplete, is progressing appropriately, whereas foot regeneration is largely failing at the transcriptional level. To gain a deeper insight into the differences in foot regeneration potential between H. vulgaris and H. oligactis, we compared the injury-induced transcriptional activation of seven foot-specific genes that were identified in our previous transcriptional profiling of H. vulgaris . These genes were upregulated specifically at the aboral wound site by 12 hpa and were enriched in the foot as determined using the single cell expression atlas (Cazet et al., 2021; Siebert et al., 2019; the transcript IDs for genes discussed in this study can be found in Table S1). This analysis showed that transcriptional activation of these seven genes is weak in H. oligactis as compared to H. vulgaris ( Fig. 2K-L ) . In addition, we compared the expression of foot-specific genes from H. oligactis to the expression of their orthologs in H. vulgaris (154 genes) and found lower expression in H. oligactis at every time point, including 0 hpa ( Fig. S3 ). The same comparison performed with a random gene set did not show expression differences ( Fig. S3 ). These data suggested that the initial foot competency of the H. oligactis tissue is lower ( Fig. S3 ). Download figure Open in new tab Supplementary Figure 3. Foot regeneration genes are expressed at lower levels in H. oligactis compared to H. vulgaris. (A) Boxplots showing expression levels of 154 orthologs from the foot-specific gene list identified between H. oligactis and H. vulgaris reference transcriptomes. (B) Boxplots of 65 randomly selected orthologs between H. oligactis and H. vulgaris plotted as controls. The log2 counts per million (CPM) for these transcripts were plotted over the course of foot regeneration in H. vulgaris (pink boxplots) and H. oligactis (orange boxplots). Foot-specific genes were consistently expressed at lower levels in H. oligactis as compared to H. vulgaris , including at 0hpa. Injury-induced transcriptional activation of Wnt pathway genes is reduced in H. oligactis as compared to H. vulgaris We next compared the early injury-induced transcriptional response between H. vulgaris and H. oligactis to identify differences that could lead to downstream failure of foot regeneration in H. oligactis . To accomplish this, we compared the H. oligactis regeneration RNA-seq data collected in this study to our previously published H. vulgaris regeneration RNA-seq data set, collected for both head and foot regeneration at 0, 3, 8, and 12 hpa 12 . To establish a framework to compare the gene expression patterns of H. oligactis and H. vulgaris we used OrthoClust, a computational method that groups gene expression patterns based in gene orthology across multiple species into discrete clusters 30 . We first looked for orthologous genes by conducting a reciprocal BLAST analysis of the RNA-seq datasets for each species, obtaining 9,371 transcripts for H. oligactis with a direct ortholog in H. vulgaris. We next used OrthoClust to compare the expression data of these transcripts over the course of regeneration to identify conserved co-expression modules across the two Hydra species. With this method we identified nine gene co-expression clusters containing 566 transcripts from H. vulgaris and 489 transcripts from H. oligactis (Supplementary data file 5). While most of the OrthoClust modules contained transcripts that decreased in expression during the regeneration time course in both species, transcripts in clusters 5 and 6 showed an up regulation in head regenerating tissue in both Hydra species ( Fig. S4 , Fig. 3A, B ). Cluster 6 contains several genes involved in canonical Wnt signaling. Notably, in H. oligactis head regeneration, cluster 6 genes such as wnt3, wnt7, and brachyury were delayed in their transcriptional activation, which correlates with the extended time frame of head regeneration in this species ( Fig. 3A-H Fig. S2 ). Download figure Open in new tab Supplementary Figure 4. Gene co-expression analysis using the OrthoClust pipeline identified nine distinct clusters of orthologous genes with varying expression patterns between H. oligactis and H. vulgaris. Red ribbon plots depict the expression patterns of clustered genes in oral regenerating tissue, while blue ribbon plots represent expression patterns in aboral injured tissue. Expression profiles for H. vulgaris clusters are shown on the left, corresponding profiles for H. oligactis are shown on the right. Note that the H. oligactis RNA-seq data spans a longer time frame (0-48 hpa) as compared to the H. vulgaris data (0-12 hpa). Download figure Open in new tab Figure 3. Delayed transcriptional activation of Wnt signaling genes during regeneration in H. oligactis as compared to H. vulgaris . (A) Ribbon plots showing normalized expression patterns for genes in cluster 6, identified with OrthoClust, in both H. vulgaris (left panels) and H. oligactis (right panels) over the first 12 hours of regeneration. Expression patterns at the oral-facing wound are shown in red and expression patterns at the aboral-facing wound are shown in blue. (B) Ribbon plots showing normalized expression patterns for cluster 6 genes in the H. oligactis regeneration time course extended to 48h shows that cluster 6 genes are upregulated between 24 and 48 hours. (C-J) RNA-seq expression profiles for wnt3 (C, D), wnt7 (E, F), brachyury (G, H) and wnt9/10C (I, J), during head (red) and foot (blue) regeneration in H. vulgaris (left panels; data from Cazet et al., 2021) and H. oligactis (right panels; this study, Figure 2 ). Note the extended time course for H. oligactis . (K) Confocal images of fluorescent RNA in situ hybridizations for wnt3 in homeostatic (left) and regenerating tissue in H. vulgaris (top) and H. vulgaris (bottom) at 24 hpa (middle) and 60 hpa (right). Scale bars: 500 µm. (L) Experimental strategy used to evaluate head organizer activity acquisition during head regeneration. (M) Kinetics of secondary axis induction following grafting of injured oral tissue onto host animals at indicated times post-amputation (0, 3, 8, 16, 24 and 48 hpa). The blue line represents H. vulgaris and the orange line represents H. oligactis . Source data file 9. Excel workbook containing secondary axis formation frequency from grafting in H. vulgaris and H. oligactis Figure 3M . We found that some cluster 6 genes also behaved differently during foot regeneration when comparing the two species. In our previous study, we found that Wnt pathway genes are transiently upregulated from 0-3 hpa during H. vulgaris foot regeneration. This included the Wnt ligands wnt3, wnt7 , and wnt9/10C as well as the conserved Wnt signaling target brachyury 11 – 13 ( Fig. 3C-I ). By contrast, wnt3, wnt7 , and brachyury were not significantly upregulated at 3pha in H. oligactis foot regeneration ( Fig. 3D,F ,H). Noteworthy, wnt9/10c is significantly upregulated in both Hydra species at 3 hpa during foot regeneration, although absolute levels of transcript are lower in H. oligactis (Fig. I,J). Altogether, these results revealed that injury-induction of Wnt signaling is either absent or highly reduced in H. oligactis as compared to H. vulgaris . The formation of the head organizer is delayed in H. oligactis The hypostome in Hydra species has a conserved function as an oral organizer 31 . While it is almost certain that Wnt signaling plays a conserved role in directing axial patterning across the Hydra genus, the expression of Wnt ligands specifically in the hypostome has not been formally tested in H. oligactis . To address this, we performed fluorescent RNA in situ hybridization (FISH) to analyze the expression of wnt3 in H. oligactis . We found that like H. vulgaris , wnt3 was expressed at the hypostome of H. oligactis , similar to H. vulgaris ( Fig. 3K ). Next, we used FISH to investigate the temporal and spatial expression of wnt3 over the course of head regeneration. In contrast to H. vulgaris , the expression of wnt3 was not detected by FISH in H. oligactis at 24 hpa. However, by 60 hpa, the expression of wnt3 became apparent in H. oligactis ( Fig. 3K ). These results are consistent with slower head regeneration kinetics in H. oligactis ( Fig. S2 ) and suggest that delayed activation of Wnt signaling may contribute to differences in head regeneration timing between these species. Given the morphological delay of head regeneration we observed in H. oligactis ( Fig. S2 ), along with the reduced levels of Wnt gene transcription ( Fig. 3D, F, H, J , K), we hypothesized that formation of the oral organizer is delayed in H. oligactis as compared to H vulgaris . Tissue grafting is a classical method for determining the timing of head organizer formation: regenerating head tissue is grafted onto the body column of a host animal to test if the donor tissue can induce a secondary axis. Using this approach, previous studies demonstrated that regenerating H. vulgaris head tissue acquires organizing ability as early as 8 hpa, which also correlates with high levels of Wnt signaling pathway gene transcripts at this timepoint 12 , 32 . To test the timing of head organizer formation in H. oligactis, we conducted classic grafting experiments. We also performed these experiments in H. vulgaris as a positive control. We bisected Hydra polyps and grafted the tissue from the oral injury site onto a host polyp at 0, 3, 8, 16, 24 and 48 hpa ( Fig. 3L ). We then quantified the frequency of secondary axis formation at 5 days post grafting ( Fig. 3M ). While we observed organizer ability in regenerating H. vulgaris head tissue as early as 8 hpa as expected, regenerating H. oligactis did not begin showing organizer activity until 16 hpa. H. oligactis head organizing ability was reduced as compared to H. vulgaris until 48 hpa (two-way ANOVA, p species = 2.55 X 10 -5 ), at which time a similar capacity to direct secondary axis formation in both species was observed. Overall, these results support the conclusion that injury fails to strongly activate Wnt signaling in early stages of regeneration in H. oligactis , which may contribute to slower head regeneration kinetics. Transient pharmacological activation of Wnt signaling during the injury response rescues foot regeneration in H. oligactis We next asked how injury-induced Wnt expression impacts foot regeneration abilities in Hydra . Given a previous study showing that treatment with a Wnt inhibitor negatively impacts H. vulgaris foot regeneration 13 , we hypothesized that the lack of injury-induced Wnt expression contributes to H. oligactis foot regeneration failure. To test this, we pharmacologically activated Wnt signaling during the injury phase of H. oligactis foot regeneration using alsterpaullone (ALP), a Wnt signaling agonist that stabilizes β-catenin and induces oral patterning in H. vulgaris 10 . We transiently treated the upper halves of 50% bisected H. oligactis with different concentrations of ALP (2.5, 5, and 10 µM) for 3 hours after injury, to mimic the short window of Wnt signaling activation in H. vulgaris foot regeneration. We then monitored the animals over 6 days for the presence or absence of foot regeneration ( Fig. 4A ). Treatment of regenerating H. oligactis with ALP, regardless of the concentration, resulted in a partial but significant rescue of foot regeneration, with the concentration of 5 µM having the largest effect ( Fig. 4B-D ). Notably, at 5 µM, ∼7% of animals regenerated a second head at the aboral wound site instead of a foot, with this percentage increasing to 20% at 10 µM ALP ( Fig. S5 ). The appearance of the two-headed phenotype suggests that a precise balance in Wnt activation is required to determine whether foot or head regeneration occurs at the aboral wound site. In addition, the higher occurrence of two-headed Hydra in the 10 µM ALP treatment likely explains the reduced number of animals that regenerated a foot under this concentration. Overall, this result supports the conclusion that a transient burst of Wnt signaling activation during the generic injury response is essential for foot regeneration in Hydra . Download figure Open in new tab Supplementary Figure 5. High Wnt signaling activation induces ectopic head regeneration at the aboral wound site. (A) Bar plot showing the percentage of ectopic head regeneration in H. oligactis upper halves treated with ALP at different concentrations for 3 hours. Ectopic head formation at the aboral wound site was assessed 6 days after injury. (B) Representative image of ectopic head regenerated at the aboral wound site in H. oligactis treated with 10 µM ALP. The image was taken 14 days post-injury. Scale bar: 1 mm. Download figure Open in new tab Figure 4. Transient activation of Wnt signaling rescues foot regeneration in H. oligactis . (A) Experimental strategy for treating the top halves of 50% bisected H. oligactis with the Wnt agonist Alsterpaullone (ALP). (B) Bar graph showing the percentage of successful foot regeneration in H. oligactis following a 3-hour post-bisection treatment with DMSO (grey bar) or ALP at 2.5 µM, 5 µM, and 10 µM (green bars). Statistical comparisons of DMSO versus ALP treatments were conducted using a two-tailed t-test (p-values: 2.5 µM vs DMSO = 0.02935, 5 µM vs DMSO = 0.01026, and 10 µM vs DMSO = 0.02302). (C) Representative image of H. oligactis showing failed foot regeneration in DMSO-treated control group. (D) Representative image of H. oligactis showing successful foot regeneration after treatment with 5 µM ALP. Scale bars: 500 µm. (E) Experimental strategy to assess foot and head regeneration in H. vulgaris after treatment with the Wnt inhibitor iCRT14. (F) Line graph showing foot regeneration kinetics in H. vulgaris treated with iCRT14 (pink) and DMSO controls (black). (G) Line graph showing head regeneration kinetics in H. vulgaris treated with iCRT14 (pink) and DMSO controls (black). Source data file 10. Excel workbook containing foot regeneration percentages for H. oligactis treated with the different ALP concentrations shown in Figure 4B . Source data file 11. Excel workbook containing foot regeneration percentages for H. vulgaris after 12 hours of iCRT14 treatment in Figure 4 F-G . Transient inhibition of Wnt signaling in H. vulgaris mimics the regeneration phenotype of H. oligactis Inhibition of Wnt/beta-catenin signaling by iCRT14 in H. vulgaris inhibits both head and foot regeneration upon continuous treatment over the course of regeneration (Gufler et al., 2018). Given our findings that in H. oligactis the injury response does not elicit a strong Wnt signaling response, here we sought to mimic that effect in H. vulgaris by transiently treating animals with iCRT14 for 12 hours after bisection ( Fig. 4E ) . This treatment caused H. vulgaris regeneration to more closely resemble H. oligactis regeneration. Specifically, we found that transient inhibition of Wnt signaling in H. vulgaris resulted in reduced foot regeneration (two-way ANOVA, p treatment = 0.002824) and an apparent delay in head regeneration ( Fig. 4F, G , Fig. S6 ). Notably, this treatment produced stably footless H. vulgaris mimicking the H. oligactis phenotype ( Fig. S6 ). Our results support the conclusion that a lack of injury-induced Wnt signaling significantly contributes to foot regeneration deficiency in H. oligactis . Download figure Open in new tab Supplementary Figure 6. Wnt inhibition blocks foot regeneration and delays head regeneration in H. vulgaris . H. vulgaris polyps were pre-treated with 5 µM iCRT14 for 2 hours prior to 50% bisection and maintained in iCRT14 for 12 hours after injury. Representative images illustrate foot regeneration (top) and head regeneration (bottom) over the time course. Foot regeneration is inhibited by iCRT14 compared to DMSO treated animals (See Figure 4F ), with inhibited animals becoming stably footless (red triangle points at a stably footless aboral end in treated Hydra at 72 hpa). Head regeneration showed a delay in the first appearance of tentacles in iCRT14-treated samples as compared to DMSO treated animals (See Figure 4G ). Transient activation of Wnt signaling triggers the transcription of foot-specific genes in H. oligactis Our finding that transient activation of Wnt signaling rescues foot regeneration in H. oligactis is consistent with published literature showing that injury-induced Wnt activation is necessary for Hydra vulgaris foot regeneration 12 , 13 , 20 . However, the mechanisms by which Wnt signaling promotes foot regeneration is unknown. To shed light on this, we generated RNA-seq libraries from a time course of H. oligactis foot regenerating fragments after a 3-hour treatment with 5 µM ALP ( Fig. 5A ). These data revealed that ALP treatment elicited the transient upregulation of Wnt pathway genes such as tcf , wnt3, axin1, wntless and sFRP3 , demonstrating that ALP treatment transcriptionally activated Wnt signaling as expected ( Fig. S7 ). Furthermore, PCA of ALP-treated (rescued) and DMSO-treated (control) samples revealed that by 48 hpa, transient activation of Wnt promoted transcriptional changes in the direction of the homeostatic foot along the PC1 axis ( Fig. 5B ). Download figure Open in new tab Supplementary Figure 7. ALP treatment induces the transcriptional activation of Wnt pathway genes. RNA expression plots show log 2 counts per million (log 2 CPM) for Wnt pathway genes tcf, wnt3, axin1, wntless and sFRP . The blue line represents expression in DMSO-treated failed foot regenerating tissue; the green line represents expression in ALP-treated foot regenerating tissue. Download figure Open in new tab Figure 5. Wnt-dependent activation of the foot regeneration transcription factor dlx2 . (A) Experimental strategy for producing RNA-seq libraries from ALP-treated aboral-facing wound tissue. (B) PCA plot showing transcriptional trajectories during regeneration in DMSO-treated (blue dotted line; data from Figure 2 ) and ALP-treated samples (green dotted line). (C, D) Comparison of average log 2 fold change (log 2 FC) in transcript abundance between failed foot regeneration (DMSO-treated control animals) and rescued foot regeneration (ALP-treated animals) at 24 hpa (C) and 48 hpa (D). Blue dots represent foot-specific genes enriched in failed foot regeneration tissue; green dots represent foot-specific genes enriched in ALP-treated rescued foot regenerating tissue. (E) Gene expression modules for failed foot regeneration (DMSO-treated control animals, blue dashed line) and rescued foot regeneration (ALP-treated animals, green dashed line) identified using maSigPro. (F) Gene Ontology enrichment analysis for genes in module 7. (G) Gene Ontology enrichment analysis for genes in module 4. (H) RNA expression plot showing dlx2 expression in log 2 counts per million (log 2 CPM) over time. The blue line represents DMSO-treated failed foot regenerating tissue; the green line represents ALP-treated foot regenerating tissue. (I-L) Confocal images of RNA in situ hybridizations for dlx2 in untreated H. oligactis and H. vulgaris , showing intact polyps and aboral regenerating tissue at 24 hpa. Scales bars: 500 µm. Source data file 12. CSV file containing normalized log2CPM used to plot data shown in Figure 5 B and H. Source data file 13. Excel workbook containing fold change gene tables for the timepoints comparing foot regeneration in DMSO and ALP in Figure 5C and D . Source data file 14. Excel workbook containing Gene Ontology enriched terms for maSigPro modules shown in Figure 5L and M . To characterize the transcriptional changes elicited by ALP treatment, we performed differential gene expression analysis comparing ALP-treated and control samples. We found that Wnt pathway activation induced the transcriptional activation of foot-specific genes, with 52 and 134 foot-specific genes being upregulated in ALP treatment conditions as compared to control conditions by 24 hpa and 48 hpa respectively ( Fig. 5C-D ) . In contrast, just a few foot-specific genes were upregulated in control conditions as compared to ALP treatment conditions at these same timepoints ( Fig. 5C-D ) . To improve our understanding of the mechanisms which transient ALP treatment promotes foot regeneration, we used maSigPro, an R package that analyses time course expression data in different conditions through a regression approach to identify differential gene expression patterns 33 . This analysis uncovered 1,773 genes with differential expression patterns grouped into 9 modules ( Fig. 5E ; Supplementary data file 6). The expression pattern of module 5 showed upregulation at 3 hpa only in ALP-treated tissue, followed by downregulation at later timepoints, and included Wnt signaling pathway genes ( tcf, axin1, lgr5, sp5, and nphp3 ), confirming the validity of this method. In addition, several TFs whose orthologs are involved in cell differentiation and development were included in module 5, such as foxl1 , dmrta2, 14-3-3zeta, aatf, rfx1 and tle3 . These TFs are also expressed in the homeostatic Hydra , but are not foot specific. To better understand the functions related to each module we performed Gene Ontology (GO) term enrichment analysis for all modules (Supplementary data file 7). Module 5 was enriched for gene expression regulatory functions, including transcription factors and coactivators. Contrastingly, module 2 showed downregulation in response to ALP at 3 hpa of transcriptional regulators involved in the generic injury response, such as bZIP TFs creb, jun and fos 12 , 34 ( Fig. 5E , S8). We also identified modules with foot-specific genes upregulated in the ALP-treated samples at 12 hpa (module 7) and 24 hpa (module 4) ( Fig. 5E-G ). GO analysis of module 7 showed enrichment for morphogenesis-related functions ( Fig. 5F ), including β-catenin and genes in the BMP and Notch pathways. Moreover, the foot-specific TF dlx2 , also part of module 7, showed upregulation at 12 hpa in response to ALP, making it the earliest foot-specific TF to be transcriptionally activated upon Wnt activation ( Fig. 5H ). A previous study showed that dlx2 is essential for foot regeneration in H. vulgaris , highlighting its likely role as a key regulator of foot regeneration (Ferenc et al., 2021). Since dlx2 was the earliest foot-specific transcription factor activated by ALP, we used FISH to examine its spatial expression in H. oligactis and H. vulgaris . In uninjured H. oligactis , dlx2 expression was restricted to a small peduncle region, whereas in H. vulgaris , it extended to the basal disk ( Fig. 5I-L ), suggesting differences in dlx2 regulation under homeostatic conditions. At 24 hpa, dlx2 was clearly expressed at the aboral injury site in H. vulgaris but was undetectable in H. oligactis ( Fig. 5K-L ), reinforcing its correlation with foot regeneration potential. GO analysis of module 4 revealed enrichment for terms related to extracellular matrix organization ( Fig. 5G ). Notably, Wnt signaling has been found to induce extracellular matrix remodeling as part of the tissue patterning that occurs during H. vulgaris regeneration (Veschgini et al., 2023). In addition, this module included TFs gata3 and nk2, previously identified as foot-specific in H. vulgaris, as well as other TFs that are upregulated temporarily at 24 hpa with ALP treatment ( Fig. S9 ). Overall, these findings highlight the hierarchical gene regulatory network underlying foot regeneration in Hydra , where transient Wnt activation triggers a transcriptional cascade that sequentially activates non-tissue specific transcription factors, followed by foot-specific factors—starting with dlx2 —and ultimately genes involved in morphological changes such as extracellular matrix remodeling, leading to successful tissue regeneration. Discussion While previous work identified a role for injury-induced Wnt signaling in H. vulgaris foot regeneration, the specific contribution of early Wnt signaling and the mechanisms driving foot regeneration have remained unclear 13 . To investigate this, we used H. oligactis , a species with limited foot regeneration capacity, and performed comparative transcriptomics to identify key regulatory mechanisms. Our findings revealed that weak injury-induced Wnt signaling activation in H. oligactis contributes to its reduced foot regeneration potential. Short-term treatment with the Wnt signaling agonist ALP partially rescued foot regeneration in H. oligactis , establishing Wnt signaling as a key regulator of Hydra foot regeneration. Furthermore, differential gene expression analysis of ALP-treated H. oligactis indicated that increased Wnt signaling promoted the expression of several TFs associated with early foot regeneration in H. vulgaris , including nk-2, foxd2-like and dlx2 . In H. vulgaris, these TFs exhibit foot-specific expression between 8 and 12 hpa 12 . Notably, dlx2 was the earliest upregulated foot-specific TF in H. oligactis following ALP-treatment, suggesting this TF as a key regulator of foot regeneration. The molecular basis for weaker injury-induced Wnt signaling in H. oligactis compared to H. vulgaris remains unclear. However, one possibility is the presence of a stronger Wnt inhibitory environment in H. oligactis . In Hydra, it is hypothesized that the head organizer establishes a morphogenetic gradient through a locally self-reinforcing Wnt signal and a secreted long-range inhibitory signal, which determines cell fate along the oral-aboral axis 9 , 35 . Previous studies suggest that this head inhibitory signal is a secreted Wnt antagonist 19 , 36 , though its precise identity remains unknown. Notably, a prior study demonstrated that H. oligactis has higher levels of head inhibitor compared to H. vulgaris . If this inhibitor is indeed a Wnt antagonist, this would be consistent with our findings that injury-induced Wnt signaling is attenuated in H. oligactis . In addition, we found that foot regeneration potential in H. oligactis increases with greater distance from the head, further supporting the hypothesis that a Wnt-inhibitory gradient emanating from the head influences the strength of injury-induced Wnt induction and, consequently, foot regenerative capacity. In addition to a strong Wnt inhibitory environment in H. oligactis , reduced transcriptional activation of injury-induced Wnt signaling genes may also contribute to its impaired foot regeneration. In H. vulgaris , bZIP TFs are implicated in activating the expression of Wnt signaling genes during the general injury response 12 , 20 . Although H. oligactis demonstrates injury-induced transcription of bZIP TFs comparable to H. vulgaris ( Fig. S8 ), it is possible that in H. oligactis , bZIP TFs fail to activate the transcription of Wnt genes due to reduced protein stability or weaker binding affinity to Wnt gene regulatory regions. Supporting this, previous research revealed stronger CREB-binding activity in nuclear extracts from H. oligactis injured oral tissue compared to injured aboral tissue, whereas H. vulgaris showed equal CREB binding activity at both wound sides 37 . Moreover, our data indicate regulatory feedback between Wnt signaling and bZIP TFs. ALP treatment causes down regulation of bZIP TFs in H. oligactis , consistent with the extended bZIP transcriptional activation observed in H vulgaris when inhibiting Wnt signaling with iCRT14 12 . This suggests a bidirectional regulatory relationship between bZIP TFs and Wnt signaling. Future research is necessary to investigate if differences in bZIP TF-mediated transcriptional activation of Wnt signaling genes contribute to the H. oligactis foot regeneration defect and how this process may be influenced by the stronger Wnt inhibitory gradient proposed for this species. Download figure Open in new tab Supplementary Figure 8. bZIP transcription factors are downregulated with ALP treatment at 3hpa. RNA expression plots show log 2 counts per million (log 2 CPM) for bZIP transcription factor genes creb, jun and fos . The blue line represents expression in DMSO-treated failed foot regenerating tissue; the green line represents expression in ALP-treated foot regenerating tissue. Download figure Open in new tab Supplementary Figure 9. ALP promotes the upregulation of foot-specific TFs at 24 hours post amputation. RNA expression plots show log 2 counts per million (log 2 CPM) for foot-specific transcription factors gata3 and nk2 . The blue line represents expression in DMSO-treated failed foot regenerating tissue; the green line represents expression in ALP-treated foot regenerating tissue. In our previous study, we found that prolonged injury-induced Wnt signaling is sufficient to induce ectopic head formation 12 . However, our current findings reveal that injury-induced Wnt signaling activation is not strictly required for head regeneration. Transient inhibition of Wnt signaling activation during the injury phase of regeneration delays, but does not block H. vulgaris head regeneration, mirroring the extended timeline we observed for H. oligactis head regeneration. Our findings suggest two potential, non-mutually exclusive mechanisms for establishing the Wnt organizer during H. oligactis head regeneration. First, the weak injury-induced expression of Wnt9/10C in H. oligactis suggests that while injury-triggered Wnt activation may contribute to head organizer formation, the associated feedforward loop could take longer to establish due to the low initial signal. Second, an unidentified mechanism may activate Wnt gene transcription later during head regeneration, either as the sole mechanism in H. oligactis or in addition to weak injury-induced Wnt activation. A similar late-acting mechanism may also function in H. vulgaris , enabling head regeneration even when injury-induced Wnt activation is inhibited ( Fig. 4G ). In contrast to head regeneration, Hydra foot regeneration requires injury-induced Wnt signaling. Our findings indicate that a key event in this process is the Wnt-dependent expression of the TF dlx2 , consistent with a prior study showing that dlx2 knockdown inhibits foot regeneration in H. vulgaris 38 . The TF gata3 has also been identified as a positive regular of basal disk fate 39 , and may be downstream of dlx2 , based on its expression dynamics in H. oligactis ALP-rescued foot regeneration. However, Hydra possess two epithelial layers, the endoderm and ectoderm, which must interact to specify the foot 40 . Interestingly, dlx2 and gata3 are both expressed in the ectoderm 29 , whereas nk2 , another foot-specific TF, is expressed in the endoderm 41 . Although nk2 has been proposed as a regulator of foot specification 41 , functional evidence remains lacking. Our study shows that dlx2 is upregulated by 12 hpa in ALP-treated foot regenerating tissue, whereas nk2 upregulation occurs at 24 hpa, suggesting that ectodermal specification may occur before endodermal specification. Future studies should investigate how Wnt signaling activates dlx2 in the ectoderm and how ectodermal and endodermal regulatory programs interact during foot regeneration. Constructing tissue-specific gene regulatory networks will be essential to understanding how Wnt signaling coordinates regeneration across both epithelial layers. Our findings suggest that foot regeneration ability is ancestral in the Hydra genus but was lost in the Oligactis group. In contrast to most Hydra species which can generate gametes continuously throughout life, species within the Oligactis group exhibit semelparity, a reproductive strategy where organisms produce many gametes before dying 42 . In H. oligactis, low temperatures induce gametogenesis, leading to somatic senescence through stem cell exhaustion 28 , 43 . One possibility is that the foot regeneration defect in H. oligactis evolved as a trade-off associated with semelparous reproduction. To explore this possibility, future research should explore the potential function of Wnt signaling in the induction of gametogenesis in H. oligactis . Similar trade-offs involving Wnt signaling may occur in other animals. For example, the semelparous planarian worm Procotyla fluviatilis cannot regenerate its head when cut near the tail due to excessive Wnt signaling activation upon injury; pharmacological downregulation of Wnt signaling rescues regeneration 44 . In addition, sexually mature male zebrafish show impaired regeneration of amputated pectoral fins due to androgen-driven inhibition of Wnt signaling, which can be reversed by pharmacological activation of Wnt signaling 45 . These examples suggest that adaptations modulating reproduction may compromise regenerative capacity, highlighting the need for further research on how Wnt signaling mediates the trade-off between reproductive strategies and regeneration in Hydra and other animals. While Wnt signaling activation during regeneration is a shared feature among distantly related animals, the gene regulatory networks linking early injury response to Wnt signaling activation are not entirely conserved across taxa 1 . Such variation limits our ability to draw broad conclusions about the evolutionary gain or loss of regenerative abilities, underscoring the need for more comparative studies. However, comparisons across long evolutionary distances can be difficult to interpret due to the distinct biological contexts in which these molecular pathways function. For this reason, studies of closely related animals with varying regenerative abilities, such as the one presented here, provide valuable insights into specific evolutionary rewiring events in the gene regulatory networks underlying regeneration. Such research can reveal the molecular changes driving the diversity of regenerative capacity observed across animals. By identifying these evolutionary changes, we may uncover the molecular requirements necessary to induce regeneration in animals with limited regenerative potential. Methods Hydra strains and culture Here we used two strains of H. oligactis . The Cold Resistant Swiss strain provided by Brigitte Galliot, was used only in regeneration experiments shown in Figure S1 43 . The second strain used for all remaining H. oligactis experiments, was collected in Innsbruck, Austria by Bert Hobmayer 12 . H. vulgaris AEP strain was used for comparative approaches throughout this work. The remaining Hydra species used for the regeneration experiments shown in Figure 1 : H. oxycnida, H. hymanae and H. viridissima, were provided by Robert E. Steele. These strains form part of a larger collection of Hydra species validated through sequencing of cytochrome c oxidase I (CO1), their collection and taxonomy have already been reported in detail 27 . All Hydra species were kept in Hydra medium (0.38mM CaCl 2 , 0.32 mM MgSO 4 X 7H2O, 0.5 mM NaHCO 3 , 0.08 mM K 2 CO 3 ), and fed three days per week with Brine Shrimp during experimentation. Phylogenetic analysis Representative validated species from each major group in the of the Hydra genus phylogeny were selected and the sequence for CO1 was used to build a maximum likelihood tree with a GTR +G +I model using MEGA 46 . Sequences of CO1 used are provided in the Source_data_1.txt file (See Source data section). Regeneration assays and foot peroxidase staining Amputations were performed under a stereoscopic microscope using a millimetric grid to precisely measure the distance from the head. Head regeneration was measured by assessing the presence of the first two tentacle buds. Foot regeneration was primarily assessed visually by identifying basal disk morphology under a stereoscopic microscope. Foot regeneration data for all species shown on Figure 1 is provided in the Source_data_2.xlsx file (See Source data section). To confirm the presence of a regenerated basal disk, foot peroxidase assays were performed in H. oligactis polyps that appeared to have regenerated their feet nine days after amputation. Animals were relaxed in 2% urethane for 5 minutes, then fixed in 4% formaldehyde at room temperature for 1 hour. Animals were then rinsed three times in phosphate buffer saline solution (PBS) + 0.25% Triton X-100 for 5 minutes each. Foot peroxidase staining was performed by incubating animals for 15 minutes in a solution of 0.02% diaminobenzidine, 0.25 % Triton X-100 and 0.003% hydrogen peroxide in PBS. Following staining, animals were rinsed in PBS + 0.25% Triton X-100 for 30 minutes, then placed in 25% glycerol for 5 minutes, followed by 50% glycerol for mounting. Lateral head organizer grafting For each time point tested, three batches of at least 10 H. vulgaris and 10 H. oligactis , were bisected. A small portion of tissue from the oral wound site was excised and set aside for grafting. Host animals were punctured at the mid-section of the body using a scalpel. The regenerating tissue from the donor oral wound site was picked up with entomological tweezers and inserted into the puncture site of the host animal. The grafted tissue was held in place against the host’s body column using tweezers and a dissection needle for 5 minutes, allowing it to adhere. Lateral head formation in the host animals was evaluated every 24 hours for 6 days, after which total number of animals with lateral heads was counted. Lateral head formation data is provided in the Source_data_9.xlsx file (See Source data section) Alsterpaullone (ALP) and iCRT14 treatments For ALP treatments, three batches of at least 20 top halves of bisected H. oligactis were incubated in either 0.05% DMSO or ALP at 2.5 µM, 5 µM or 10 µM in Hydra medium for 3 hours. Following treatment, animals were immediately washed, and foot regeneration was assessed 144 hours post-amputation. During this period, animals were fed 3 times per week. For iCRT14 treatments, 4 batches of at least 29 H. vulgaris were incubated in 5 µM iCRT14 or 0.05% DMSO for 12 hours post-bisection. After treatment, both halves of the animals were washed and monitored for head and foot regeneration every 24 hours for 96 hours. Animals were fed every 48 hours following amputation. Foot regeneration data for ALP-treated H. oligactis is provided in the Source_data_10.xlsx file (See Source data section) RNA-seq library preparation For each regeneration sample, at least 3 replicates of 30 H. oligactis were starved for 48 hours prior to bisection. Following 50% bisection, animals were incubated in either 0.05% DMSO or 5 µM ALP for 3 hours, then washed with Hydra medium. For the 0 hours post-amputation samples, animals were exposed to DMSO or 5 µM ALP for 30 seconds, then washed before RNA extraction. Regenerating animals were allowed to regenerate for 3, 12 or 48 hours, after which they were rinsed with Hydra medium and the tissue adjacent to the oral and aboral injury sites was excised for RNA extraction. The amount of tissue collected for each sample was approximately one sixth of the total body length. For ALP-treated samples, only the aboral injury site was collected. In addition, 3 replicates of 30 animals were used to collect homeostatic head tissue by decapitating just below the tentacle ring, and 3 replicates of 30 feet (peduncle + basal disk) were obtained as homeostatic foot samples. A second batch included 3 replicates of 24 hpa in DMSO and ALP, as well as 3 replicates of 0 hpa samples in DMSO and 2 replicates of homeostatic head and foot following the same conditions. Total RNA was extracted from the excised tissue fragments using Trizol TM (Thermo Fisher Scientific), followed by DNA decontamination with DNAse I (R1013, Zymo Research). A final RNA purification step was performed using the Zymo RNA Clean and Concentrator kit (R1017, Zymo Research) according to the manufacturer’s instructions. Poly(A)-enriched mRNA libraries were prepared, and 150 bp paired-end sequencing was performed on a NovaSeq 6000 by Novogene Co. RNA-seq and computational analysis Low-quality base calls and sequencing adapters were filtered out using Trimmomatic V.0.36. The reference transcriptome for mapping the trimmed reads was obtained by downloading the H. oligactis Cold Resistant (Swiss Strain) transcriptome from https://hydratlas.unige.ch/ 47 . BUSCO analysis 48 revealed a high degree of redundancy (26.3% duplicates) in this transcriptome. To reduce the redundancy, Evidentialgene tr2aacds.pl (v2017.12.21) was used to decrease redundancy 49 . BUSCO stats for both the original transcriptome and reduced transcriptome are shown in Supplementary Table 2. The resulting reduced reference (Supplementary data file 8) was then used for mapping the trimmed reads and calculating transcript counts using RSEM 50 . For annotation, predicted coding sequences were analyzed using IntersProScan 51 . Batch effects were corrected using ComBat_seq R-package 52 . Normalization of transcript counts and differential expression analysis were performed using Edge R 53 , with gene expression differences assessed using glmTreat and a false discovery rate of 0.01. Results are provided in the Source_data_5.xlsx and Source_ data_11.xlsx files. Additionally, H. vulgaris counts from Cazet et al., 2021 were reanalyzed using glmTreat for comparison. Principal Component Analysis, heatmaps, and gene expression plots were generated using in house scripts. For Orthoclust analysis, FPKMs for H. oligactis and H. vulgaris mapped reads were calculated with RSEM, and reciprocal orthology was determined using ReciprocalBlastHit.py 54 . OrthoClust was run with a p-value of 0.001, correlation threshold of 0.975, and a Kappa value of 2. Differences in gene expression patterns over time between DMSO and ALP treatments were analyzed using maSigPro, with Q set at 0.05 and R-square at 0.8. Gene Ontology enrichment analysis of maSigPro clusters was performed using FuncAssociate 3.0. RNA fluorescent in situ hybridization (FISH) FISH was performed following a previously published protocol for H. vulgaris 29 , with the only modification being the use of 250 ng of probe per sample for hybridization. Probe detection was carried out using Alexa Fluor 594 tyramide reagent (ThermoFisher). Confocal images were acquired using the Zeiss 980 LSM with Airyscan 2 confocal microscope. Z-stack images were analyzed with Fiji 55 . Statistical information Results for secondary axis formation from a regenerating graft are shown in average percentage of three different batches for each time point and species. Error bars represent the standard deviation. Three batches of at least 10 grafted animals were recorded for each time point and species of Hydra. Detailed numbers for each batch and timepoint can be found in Source_data_9.xlsx within the Source_data folder (See Source data section) Significance of the difference in secondary axis formation between species was evaluated through a two-way ANOVA, using the ‘aov’ function from the ‘stats’ package in R. This analysis showed that there is a significant difference ( p species = 2.55 X 10 -5 ) in secondary axis formation between H. oligactis and H. vulgaris . Results for foot regeneration after ALP treatment with different concentrations was shown as the mean foot regeneration percentage of three batches for each tested concentration. Error bars represent standard deviation. The difference in foot regeneration percentage between tested ALP concentrations and DMSO treated control was evaluated with a two-tailed t-test using the ‘t.test’ function from the ‘stats’ package in R. The 2.5 µM ALP showed a significant difference when compared against DMSO controls ( p = 0.03). The 5 µM ALP showed a significant difference when testes against DMSO controls ( p = 0.01). Finally, 10 µM ALP showed a significant difference when compared against DMSO controls ( p = 0.02). Detailed number for these batches of treated and control animals can be found in the Source_data_10.xlsx file within the Source data folder (See Source data section). Results for head and foot regeneration for iCRT14 treated H. vulgaris are shown in average percentage of three different batches of 30 animals for each time point and four batches of at least 29 animals in the case of DMSO treated controls. Error bars represent the standard deviation. Detailed numbers for each batch and timepoint can be found in Source_data_11.xlsx within the Source_data folder (See Source data section). Significance of the difference in foot regeneration and head regeneration percentage across time between species was evaluated through a two-way ANOVA using the ‘aov’ function from the ‘stats’ package in R. This analysis showed that there is a significant difference in foot regeneration percentage ( p treatment = 0.002824) between DMSO-treated and iCRT14-treated H. vulgaris . No statistical difference was found in head regeneration percentage between DMSO-treated and iCRT14 treated H. vulgaris . Data availability All scripts used in this study are available both as a git repository at https://github.com/cejuliano/oligactis_foot_regeneration . FASTQ files of raw RNA-seq reads and raw counts for RNA-seq are available through the Gene Expression Omnibus under the Bio Project accession number: PRJNA1231128. Source data A comprehensive list of the raw data used to produce the graphs in this study is provided here. Author information Contributions C.E.J and S.E.C conceptualized and designed the study. S.E.C, S.N., J.C., J.T., B.D.C and C.C. performed experiments. S.E.C performed data analysis. C.E.J. oversaw all the experiments. S.E.C and C.E.J. contributed to writing of the original draft and the remaining authors read and critically revised the manuscript. Ethics declarations Competing interests The authors declare no competing interests. Acknowledgements We thank Bert Hobmayer and Jack F. Cazet for their constructive comments on the manuscript and all members of Juliano Lab for discussion and suggestions during the development of this work. We also thank Bert Hobmayer for providing the Hydra oligactis Innsbruck 12 strain. This work was supported by a National Institutes of Health (NIH) grant R35 GM133689 (to C.E.J.), and a Human Frontiers Science Program Postdoctoral Fellowship (Reference number: LT000496/2020-L; to S.E.C). Footnotes https://github.com/cejuliano/oligactis_foot_regeneration References 1. ↵ Srivastava , M . Beyond Casual Resemblance: Rigorous Frameworks for Comparing Regeneration Across Species . Annu Rev Cell Dev Biol 37 , 415 – 440 ( 2021 ). OpenUrl CrossRef PubMed 2. ↵ Bely , A. E . 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