Asperous coordinates regenerative timing by regulating damage-induced WNT Signaling

Asperous coordinates regenerative timing by regulating damage-induced WNT Signaling | 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 Asperous coordinates regenerative timing by regulating damage-induced WNT Signaling View ORCID Profile Si Cave , View ORCID Profile Manashi Sonowal , View ORCID Profile Maksym Dankovskyy , View ORCID Profile Jordan Hieronymus , View ORCID Profile Chloe Van Hazel , View ORCID Profile Petra Fromme , View ORCID Profile Robin E. Harris doi: https://doi.org/10.1101/2025.06.24.661394 Si Cave 1 Arizona State University , 427 E Tyler Mall LSE 229, Tempe, AZ 85287-4501 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Si Cave Manashi Sonowal 2 Center for Applied Structural Discovery, Biodesign Institute, Arizona State University , Tempe, Arizona, 85287, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Manashi Sonowal Maksym Dankovskyy 1 Arizona State University , 427 E Tyler Mall LSE 229, Tempe, AZ 85287-4501 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maksym Dankovskyy Jordan Hieronymus 1 Arizona State University , 427 E Tyler Mall LSE 229, Tempe, AZ 85287-4501 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jordan Hieronymus Chloe Van Hazel 1 Arizona State University , 427 E Tyler Mall LSE 229, Tempe, AZ 85287-4501 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chloe Van Hazel Petra Fromme 2 Center for Applied Structural Discovery, Biodesign Institute, Arizona State University , Tempe, Arizona, 85287, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Petra Fromme Robin E. Harris 1 Arizona State University , 427 E Tyler Mall LSE 229, Tempe, AZ 85287-4501 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robin E. Harris For correspondence: Robin.Harris{at}asu.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Tissue regeneration requires precise control of signaling pathways to direct proliferation, differentiation, and patterning. While early responses to injury are well characterized, how differentiation is coordinated during later stages remains unclear. Here, we identify Asperous (Aspr), an EGF-repeat protein, as a regeneration-specific regulator in Drosophila wing discs. Aspr is dispensable for wing development but is strongly induced within 24 hours post-injury. Maintaining aspr expression inhibits differentiation and alters reparative growth, while loss impairs regeneration. Structural and expression analyses show Aspr is an extracellular protein secreted in extracellular vesicles (EVs), where it co-localizes with the WNT ligand Wingless (Wg). We find Aspr regulates post-injury but not developmental Wg signaling, potentially by influencing its secretion or availability via EVs. These findings suggest Aspr regulates WNT activity to ensure proper timing of cell fate specification during regeneration, revealing a mechanism by which signaling dynamics are temporally controlled during tissue repair. Introduction Tissue regeneration restores lost or damaged structures through coordinated reactivation of growth, differentiation, and patterning programs. Across different species and tissue types, this process frequently reuses developmental signaling pathways, including Wingless/Wnt (WNT), Bone Morphogenetic Protein (BMP), Notch, and JAK/STAT. 1 – 3 These pathways drive key regenerative processes such as stem cell activation, proliferation, morphogenesis, and repatterning. Among them, WNT signaling is particularly well studied and broadly conserved, 4 playing a pivotal role in regeneration of diverse tissues, including zebrafish fin and CNS, 5 – 9 mouse and fly intestine, 10 – 13 and planarian body axis. 14 , 15 In many contexts, WNT ligands are rapidly induced after injury, activating canonical or non-canonical pathways to promote proliferative expansion. 4 , 16 Moreover, WNT signaling is also required during the late stages of regeneration to re-establish spatial patterning cues, guiding the reconstruction of discrete tissue identities after proliferative growth. 4 , 16 However, while the presence and function of WNTs and other pathways during regeneration are well documented, how their activity is precisely timed and regulated across different phases of regeneration remains poorly understood. This becomes critical for regenerating complex tissues through successive stages, which can require events like wound epithelium formation, development of a blastema, proliferative expansion, and tissue differentiation to occur sequentially. 1 , 17 Each of these phases demand tight temporal control of signaling to maintain the correct progression of events, and understanding the mechanisms that coordinate such transitions remains an important challenge in the field. The Drosophila wing imaginal disc provides an ideal model to dissect regeneration at high resolution, owing to its well-characterized developmental programs, advanced genetic tool kit and substantial ability to regenerate following injury. 18 In addition to its historical use in studying growth and pattern formation, 19 the wing disc has also emerged as a powerful system to identify genetic factors that regulate tissue regeneration. 18 , 20 To better understand the events of injury induction and gene expression during regeneration, we previously developed a versatile genetic ablation system called DUAL Control. 21 , 22 This system enables independent induction of cell death alongside spatially and temporally restricted manipulation of gene expression in surrounding tissues, allowing us to interrogate regeneration in a controlled and reproducible manner. Using this approach, we previously identified and characterized several different genetic and epigenetic factors that are required for proper restoration of the wing disc after injury. 22 – 25 One such factor is asperous ( aspr ), a gene that encodes a putative extracellular EGF repeat–containing protein that is highly induced in early regenerating tissue. We initially identified aspr based on its proximity to a damage-responsive enhancer and its strong transcriptional upregulation following ablation, 22 although its function in regeneration remained unclear. Here we have performed a comprehensive characterization of aspr , finding that it is not expressed during normal wing disc development and is dispensable for both viability and wing formation, yet its loss impairs regeneration, suggesting a regeneration-specific role. We show that aspr is present only transiently during the early phase of regeneration, and that prolonging its expression disrupts proper regenerative growth and delays the onset of late-stage patterning. Mechanistically, we find that aspr interferes with the reactivation of several patterning genes normally induced late in regeneration downstream of Drosophila Wnt1 , wingless ( wg ), indicating aspr normally modulates WNT signaling. Further analysis reveals that the Aspr protein is secreted, and associates with extracellular vesicle-like structures, which colocalize with Wg protein explicitly during regeneration, suggesting it could potentially act as an extracellular regulator of WNT ligand distribution or availability in this context. Although vesicle-mediated transport has been proposed as a mechanism for WNT ligand trafficking during development, 26 – 29 its role in regeneration has not been previously demonstrated. Thus, our work identifies a novel, regeneration-specific modulator of WNT signaling and highlights the critical role of regulating extracellular signaling to precisely coordinate gene expression timing necessary for successful tissue regeneration. Given the conserved role of WNT signaling in regeneration across diverse species, 16 these findings have broad implications for understanding how signaling transitions are coordinated during regeneration. Results Damage-induced and developmental aspr are regulated by distinct mechanisms Our previous work to characterize epigenetic and transcriptomic changes associated with regeneration identified aspr (previously CG9572 ), 22 , 25 which RNA-seq showed to be one of the most strongly induced genes in regenerating discs. 25 Other groups have shown that aspr is damage-responsive and associated with blastema cells. 30 , 31 Notably, these studies also show aspr is absent from undamaged pouch tissue suggesting a regeneration-specific role, although this role remains unclear. To better understand its function, we visualized aspr expression after damage using HCR fluorescence in situ for transcript detection and an anti-Aspr antibody generated for this study. In undamaged early L3 discs (84 h after egg deposition, AED), aspr is expressed in the proximal notum and weakly above the dorsal hinge but is absent from the pouch ( Figure 1A ). This pattern persists in late L3 (108 h AED, Figure 1G ). Unfortunately, the anti-Aspr antibody we developed exhibits low sensitivity and only weakly detects Aspr protein (Figure S1A and S1A’). Therefore, we relied on transcript detection for subsequent analyses. Using the DUAL Control (DC) ablation system 21 to activate JNK-mediated apoptosis via activated hempiterous in the distal pouch (DC hepCA ), we found aspr is upregulated in blastema cells by 12 h after heat shock (AHS), maintained through 18-24 h, and largely absent by 36 h ( Figures 1B–E , quantified in 1F). By contrast, late L3 discs show only weak aspr upregulation following ablation ( Figure 1H ). We also tested necrotic cell death using DC gluR 1 . 23 , 24 Similar to apoptosis, necrosis triggers aspr induction around the wound in early L3, peaking at 18 h and diminishing by 36 h ( Figures 1I–J ). Notably, however, necrosis causes aspr to occur with a more punctate, potentially nuclear, appearance ( Figure 1I ), likely reflecting the distinct responses to necrotic injury we previously characterized. 23 , 24 Late L3 discs show minimal aspr expression post-necrosis ( Figure 1K ). Download figure Open in new tab Figure 1. aspr is a damage-responsive gene regulated by a DRMS enhancer ( A–E ) Time course of aspr RNA (magenta) in early L3 wing discs. Unablated control ( DC NA ) is shown in (A). Following apoptotic ablation ( DC hepCA ), aspr is strongly induced by 12 h AHS (arrowhead in B), maintained through 24 h, and reduced by 36 h (C–E). Nuclei are stained with DAPI (gray). Notum and dorsal hinge developmental expression indicated (yellow arrowheads in A), and damage-responsive expression in ablated disc (yellow arrowhead in B) ( F ) Quantification of aspr fluorescence measured in the pouch normalized to the notum. Statistics: one-way ANOVA; sample size = 10 discs per time point; ns = not significant; **** p < 0.0001. ( G–H ) aspr RNA in late L3 unablated disc (G) and ablated disc (H) showing reduced damage-induced expression in late L3. ( I–K ) Necrotic ablation ( DC gluR1 ) in early L3 induces robust aspr at 18 h (I, arrowhead) that diminishes by 36 h (J); expression is weaker following late L3 ablation (K). ( L–M ) AP-1-RFP (cyan) overlaps with damage-induced aspr in DC hepCA ablated discs (M, arrowhead) but not in unablated controls (L, open arrowhead). ( N–O ) RNA in situ for DRMS aspr -GFP (yellow) and GFP RNA (green) shows overlap in early L3 ablated discs (N, arrowhead) but weak expression in late L3 (O, open arrowhead); GFP RNA is absent from the notum (open arrowhead in N). ( P–Q ′) ptc>egr (P,Q) and ptc>hepCA (P′,Q′) ablation induce strong early L3 DRMS aspr -GFP and aspr RNA expression (P,P′) that diminishes in late L3 (Q,Q′). Dotted outlines indicate the pouch. ( R ) DC hepCA ablation in hep - /Y null hemizygous mutants shows reduced aspr in the ablated pouch (open arrowhead) but preserved developmental expression (arrowhead). ( S ) Blocking JNK in the notum with pnr>JNK DN does not suppress developmental aspr expression (arrowhead). All scale bars = 50 μm unless indicated. See also Figure S1. Full genotypes listed in Supplementary Genotypes. Beyond genetic injury, aspr is also expressed in neoplastic tumors ( sd>lglRNAi , Figure S1B–D) and soon after physical wounding (Figure S1E), supporting its role as a generalized early stress-response gene. Similarly, in other imaginal discs (leg, haltere, eye), aspr is mostly undetectable in undamaged tissue but becomes induced upon ablation (Figures S1F–H). As with many genes involved in regeneration, aspr appears to be regulated by a Damage-Responsive, Maturity-Silenced (DRMS) enhancer, a class of regulatory elements that are activated by JNK and JAK/STAT signaling but epigenetically silenced in mature discs. 22 , 25 , 32 We previously identified a DRMS enhancer upstream of aspr that becomes accessible after damage in early L3 but not late L3 discs. 22 A reporter for this region ( DRMS aspr -GFP ) contains AP-1 and Stat92E binding sites (Figure S1I), overlaps with JNK activity ( AP-1-RFP , Figures 1L–M ), and mirrors aspr expression in early L3, while being only weakly active in late L3 ( Figures 1N–O ). This behavior is consistent with the DRMS regulating aspr via JNK in the context of damage. Since regenerative ability varies across the disc, 33 we explored the regionality of DRMS aspr activity by ablating tissue along the anterior/posterior (A/P) boundary using ptc>hep CA or ptc>egr . This domain includes the notum, hinge, and pouch ( Figure 1P and Q ), all of which activate JNK after damage. 33 While only the pouch typically activates regeneration-associated genes, aspr is induced along the full stripe in early L3 discs ( Figures 1P–P ’), including non-regenerating notum tissue, although DRMS aspr -GFP is primarily activated in the pouch ( Figure 1P ). In late L3, aspr is only induced outside the pouch ( Figure 1Q–Q ’), while the DRMS aspr -GFP reporter is entirely inactive ( Figure 1Q ). Thus, damage-induced aspr is likely regulated by the DRMS aspr in the regeneration-capable pouch, while damage-responsive enhancers that are not silenced with maturity likely drive aspr expression outside of this region. Interestingly, we noted that developmental aspr expression in the notum overlaps with JNK activity that is required for thorax closure during pupariation ( Figure 1A ). 34 In hep - mutants, damage-induced aspr is diminished, while developmental expression persists ( Figure 1R ). Similarly, expression of a dominant-negative JNK in the notum ( pnr>hep DN ) fails to suppress aspr in the notum ( Figures 1S ). Since the DRMS aspr reporter is inactive in the notum of undamaged discs ( Figure 1N ), together these observations suggest that developmental and regenerative aspr are regulated by distinct mechanisms. Loss of aspr blocks regeneration but does not affect normal development of the wing disc As aspr is strongly upregulated after injury but shows limited developmental expression, we tested whether its removal affects either context. The DC system allows heat shock-induced, flip-out-mediated expression of UAS-transgenes specifically in the pouch, driven by DVE>>GAL4 during and after damage. 21 We identified an RNAi construct that strongly rescues damage induced aspr in the pouch ( Figures 2A–B ) A second RNAi line ( TRiP.HMJ22471 ) was tested, but we found it to be less effective and did not use it further. The knockdown of aspr in undamaged discs has no visible effect on wing development ( Figure 2C ), consistent with its minimal expression in normally developing discs ( Figure 1A ). However, knockdown during regeneration significantly impairs recovery in both early and late L3, shown by wing scoring ( Figure 2D–G ) and adult wing area ( Figure 2H ). The loss of aspr does not alter damage-induced JNK signaling, shown by JNK targets wg and Mmp1 , or cell death caused by DC hepCA ablation ( Figures 2I–J and M–N). Although these data do not indicate whether Aspr is a direct target of JNK signaling, or a consequence of cell death, it does support the hypothesis that Aspr is downstream of, and regulated by, a JNK-responsive DRMS enhancer ( Figure 1 ). Beyond these observations, the knockdown of aspr does not immediately indicate its function, though it is clearly essential for regeneration and mostly dispensable for normal development. Download figure Open in new tab Figure 2. aspr is required for regeneration but appears dispensable for normal development ( A–B ) aspr RNA (magenta) in early L3 discs imaged 18 h AHS after DC hepCA ablation. Control (A) shows strong damage-induced expression (arrowhead); knockdown of aspr with DVE>>aspr RNAi (B) strongly reduces damage-induced aspr (open arrowhead). Nuclei = DAPI (gray). ( C–E ) Representative adult wing outcomes: (C) knockdown of aspr with DVE>>aspr RNAi in undamaged discs (DC NA ) has no effect; (D) control ablated ( DC hepCA , DVE>>) discs have wild-type recovery; (E) knockdown of aspr with DVE>>aspr RNAi during regeneration results in impaired recovery. ( F ) Definition of regeneration scoring categories used for adult wings following ablation with DC hepCA . ( G ) Regeneration scoring plotted as proportion of wings in each category for undamaged ( DC NA ) or ablated ( DC hepCA ) discs in early or late L3, with or without aspr knockdown. aspr knockdown reduces regeneration at both stages. Sample sizes: DC NA : y RNAi n = 134, aspr RNAi n = 132; Early L3: y RNAi n = 50, aspr RNAi n = 89; Late L3: y RNAi n = 97, aspr RNAi n = 109. ( H ) Quantification of adult wing area (box plots, median and quartiles) from wings in (G). Knockdown of aspr during development ( DC NA ) does not affect adult wing size; knockdown following ablation significantly reduces wing area. Statistics: one-way ANOVA. Sample sizes: DC NA : y RNAi n = 39, aspr RNAi n = 52; Early L3: y RNAi n = 23, aspr RNAi n = 19; Late L3: y RNAi n = 25, aspr RNAi n = 18. ns = not significant; * p = 0.0266; ** p = 0.0013. ( I–J ) Ablated ( DC hepCA ) early L3 discs stained for Mmp1 (cyan) and cleaved Dcp-1 (cDcp-1, yellow). aspr knockdown does not alter Mmp1 or cDcp-1 levels relative to control. ( K–L ) Early L3 discs from aspr Mi(MIC) mutant larvae: homozygous mutant lacks developmental aspr expression in the notum (K, open arrowhead) and ablated aspr Mi(MIC) /Y discs lack both developmental and damage-induced aspr (L, open arrowheads). ( M–N ) Ablated aspr Mi(MIC ) / Y discs stained for Wg (cyan) and cDcp-1 (yellow) show that damage-induced Wg and apoptosis are unaffected in mutant discs at early (M) and late (N) L3. ( O–P ) Adult wing (O) and thorax (P) from aspr Mi(MIC) adults show no obvious developmental defects. ( Q ) Regeneration scoring for w 1118 control and aspr Mi(MIC) /+ heterozygotes following ablation ( DC hepCA ). Heterozygotes show reduced regeneration at both stages. Sample sizes: Early L3: w 1118 n = 18, aspr Mi(MIC) /+ n = 60; Late L3: w 1118 n = 41, aspr Mi(MIC) /+ n = 66. All scale bars = 50 μm unless otherwise specified. See also Figure S2. Full genotypes in Supplementary Genotypes. To confirm this, we tested the aspr MI02471 allele, a Mi[MIC] insertion that likely represents a transcriptional null. 22 HCR in situ confirms that hemizygous males and homozygous females lacked detectable aspr transcripts in the wing disc, both in undamaged and damaged conditions ( Figures 2K–L ). These mutants displayed no overt developmental phenotypes, including in wing patterning or size ( Figure 2O ). Moreover, while aspr is normally expressed in the notum, where JNK and other signals govern thoracic patterning and bristle formation, these structures were unaffected in mutants ( Figure 2P ). We did observe a modest but significant developmental delay in aspr MI02471 larvae, which is more pronounced in homozygous females (Figures S2A and C). This delay is not seen with RNAi knockdown in the wing (Figure S2B), suggesting there is either a role for aspr in developmental timing outside of the wing, or an unrelated mutation in the Mi[MIC] line. Importantly, as in the knockdown experiments, aspr MI02471 mutants showed reduced regenerative capacity in both early and late L3 discs ( Figure 2Q ). Thus, although the specific function of aspr remains unclear, these data confirm that it is required for regeneration but plays a minimal role in normal development. Maintaining aspr expression impacts repatterning during regeneration aspr is normally upregulated only during the first 24 h of repair ( Figures 1A – 1F ), and while loss-of-function analyses show it is required for regeneration, they do not clarify its role. Since aspr seems mostly dispensable for normal development but essential for early regeneration ( Figure 2 ), we investigated its function via ectopic expression. We previously used a UAS-driven multi-tagged construct ( UAS-aspr-FLAG.3xHA ) to perform preliminary investigations of its effects, 22 but this causes ectopic vein tissue in undamaged discs, a phenotype that is not seen with the untagged version we generated ( UAS-aspr ) (Figures S3A–B). Additionally, the multi-tagged construct showed inconsistent transcript levels in discs (Figure S3C). To ensure reliable findings, we generated a new single HA-tagged transgene ( UAS-aspr-HA ) and expressed it using both non-ablating (DC NA ) and ablating (DC hepCA ) versions of DUAL Control. Expression of the untagged and single tagged was confirmed by HCR and/or anti-HA staining ( Figures 3A–B ’, S3D–E). Strikingly, we found that Aspr protein occurs with two distinct appearances: “cellular Aspr,” associated with apical membranes, and “punctate Aspr,” found as discrete foci at or above the apical surface ( Figure 3A-A ’ and Figure 7G–K ). This appearance was seen when expressed in the notum, hinge and pouch regions of the disc ( Figure 3B–B ’), but was not observed with the FLAG.3xHA multiple-tagged construct (Figure S3F–F’). Despite these distinct appearances, ectopic Aspr expression during development in the pouch ( DVE>>GAL4 ), or throughout the entire anterior or posterior disc ( ci-GAL4 or hh-GAL4 ) had no visible effects on disc proliferation, signaling pathways, or gene expression ( Figures 3C–H and S6A–D), nor did it affect adult wing patterning or size ( Figures 3I-K and S3G-H). This held true for both the single HA-tagged and untagged constructs (Figure S3B), consistent with the notion that Aspr has limited developmental impact, even when ectopically expressed. Download figure Open in new tab Figure 3. Ectopic Aspr disrupts repatterning during regeneration but not normal development ( A–B ′) Aspr-HA (magenta) expressed with DVE>>GAL4 (A,A′) or ci-GAL4 (B,B′) in early L3 unablated discs ( DC NA ). Aspr-HA localizes to discrete puncta; higher magnification views (A′,B′) show focal accumulation (arrowheads). Co-stains: HA (magenta), Nubbin (nub, cyan, A), Ci (yellow, B); nuclei = DAPI (gray). ( C–F ) Dpp signaling (pMad and dpp-lacZ ) and expression of salm and nub are unchanged by aspr-HA expression in unablated ( DC NA ) discs (C–F). ( G–H ) PCNA-GFP proliferation reporter is unaltered in unablated discs ( DC NA ) expressing aspr-HA with DVE>>GAL4 . ( I–J ) Representative adult wings from undamaged discs expressing aspr-HA in the pouch (I) or anterior compartment (J) — both show normal morphology. ( K ) Quantification of wing area from experiments in (I–J); no significant change is seen with Aspr-HA (one-way ANOVA). Sample sizes: males; y RNAi n = 40, aspr-HA n = 44; females; y RNAi n = 41, aspr-HA n = 40. ( L ) Definition of regeneration scoring categories used for adult wings following ablation with DC hepCA . ( M ) Regeneration scoring of adult wings after ablation ( DC hepCA ) with pouch expression of aspr-HA using DVE>>GAL4 . Aspr-HA reduces regenerative capacity in both early and late L3. Sample sizes: Early L3; y RNAi n = 50, aspr-HA n = 81; Late L3; y RNAi n = 67, aspr-HA n = 133. ( N ) Regeneration scoring of adult wings after ablation ( DC hepCA ) with temporally restricted aspr-HA expression ( rn-GAL4, GAL80ts ): 0–24 h AHS expression has no effect; expression >24 h impairs regeneration. Sample sizes: 0–24 h n = 39; >24 h n = 30. ( O ) Wing area measurements corresponding to (M); aspr-HA reduces wing area for both sexes and stages (one-way ANOVA; **** p < 0.0001). Sample sizes: males; y RNAi n = 25, aspr-HA n = 57, females; y RNAi n = 15, aspr-HA n = 52. ( P ) Frequency of the “box wing” phenotype in regenerated adult wings from (M): early L3 aspr-HA = 38% (box wing n = 50 of 81), late L3 aspr-HA = 5% (box wing n = 7 of 133). ( Q–R ) Representative adult wings following aspr-HA expression for 0-24 h (Q) or from 24 h onward (R), as in (N). Annotations indicate longitudinal veins (LV x ) and anterior or posterior cross veins (ACV/PCV). The intervein 2 (I 2 ) region is also labeled. ( S–S ′) Example box wing phenotype, red dashed outline indicates zoomed view in (S’), highlighting defects: (1) reduced I2, (2) vein fusion, (3) bristle loss, (4) missing ACV, (5) margin loss (numbered arrowheads). All scale bars = 50 μm unless noted. See also Figure S3. Full genotypes in Supplementary Genotypes. We next examined its function during regeneration. Although endogenous aspr is normally downregulated after 24 h ( Figures 1A–F ), 31 continuous expression of aspr-HA (or untagged aspr ) throughout regeneration impaired recovery in both early and late L3 discs ( Figures 3L–M , S3I–J). To determine whether this was due to the higher level of Aspr in early regeneration or its inappropriate presence in late regeneration, we used GAL80 ts to restrict aspr-HA expression to either the first 24 h AHS, or from 24 h AHS onward. Early expression had no effect ( Figures 3N and Q ), while late expression significantly impaired regeneration ( Figures 3N and R ). This suggests that while Aspr is normally required early, and ectopic Aspr at this stage has no effect, its persistence beyond 24 h disrupts recovery. When Aspr is maintained in late regeneration, in addition to reduced regeneration, we also observed a distinct “box wing” phenotype in a subset of adults ( Figures 3O–P and S–S’): ∼38% in early L3 and 5% in late L3 ( Figure 3P ). These wings showed consistent features that predominantly affect the anterior wing ( Figures 3S-S ’), including reduced intervein 2 (I 2 ) between longitudinal veins 2 and 3 (LV 2 , LV 3 ), 2) ectopic veins along LV 2 and LV 3 , 3) loss of anterior wing margin bristles, 4) variable loss of ACV and PCV, and 5) a squared-off wing blade. These phenotypes also occurred with the untagged transgene in ablated wings (Figure S3K). However, these features were absent when aspr-HA was expressed in undamaged wings ( Figures 3I–J ) or when expression was restricted to early regeneration ( Figure 3Q ). These findings indicate that mis-patterning arises from prolonged Aspr expression. Since Aspr has only limited temporal expression during regeneration, and loss-of-function studies do not clearly reveal its specific role, we focused on the phenotypes resulting from ectopic expression to better understand the function of Aspr in regeneration. Aspr affects regrowth of the anterior compartment To better understand the "box wing" phenotype resulting from aspr expression in late regeneration, we measured the regions of the adult wing. Posterior wing area was nearly equivalent to that of ablated wild-type controls, with only a slight reduction ( Figure 4A ’), whereas the anterior was significantly reduced, accounting for the majority of loss in wing size ( Figure 4A ). This reduction reflects smaller anterior intervein regions, most significantly of the I 2 area ( Figure 4B–C ). I 3 and I 4 were only measured when the ACV was present. To confirm that Aspr affects anterior patterning and growth, we drove aspr-HA solely in the anterior or posterior compartment using ci-GAL4 or hh-GAL4 . Anterior-specific expression during regeneration produced phenotypes resembling those of whole-pouch expression: I 2 reduction, LV 2 –LV 3 fusion and thickening, ectopic vein tissue, ACV loss, and absent anterior margin bristles ( Figure 4D ). Posterior-specific expression caused only a small reduction in the I 6 area ( Figure 4E ), matching the mild posterior reduction observed in whole-pouch expression ( Figure 4A ’), but shows no other patterning defects. Expression with either driver in undamaged discs caused no wing phenotypes (Figure S3G and S3H). Thus, the overall box wing phenotype likely reflects a combination of the significant anterior and mild posterior effects ( Figure 4D and 4E ). Together, these findings strongly suggest that maintaining aspr specifically disrupts anterior growth and patterning during regeneration. Download figure Open in new tab Figure 4. Ectopic Aspr has an anterior-specific effect on regeneration ( A–A ′) Wing area measurements of adult female wings from early L3 ablated discs ( DC hepCA , DVE>>GAL4 ): (A) box-wing phenotype (green) with Aspr-HA is significantly smaller than control wings (black); (A′) anterior and posterior areas measured separately show a larger reduction in the anterior. Statistics: one-way ANOVA. Sample sizes: y RNAi n = 23, aspr-HA n = 22. **** p < 0.0001; anterior **** p >GAL4 ) expressing y RNAi (B) or aspr-HA (B′). Anterior (red) and posterior (blue) compartments shaded. Intervein regions labeled. ( C ) Quantification of anterior intervein regions I 1 –I 4 from (A′). I 2 and I 4 are significantly reduced in aspr-HA wings (one-way ANOVA). Sample sizes: I 1 ; y RNAi n = 15, aspr-HA n = 12; I 2 ; y RNAi n = 20, aspr-HA n = 18; I 3 ; y RNAi n = 17, aspr-HA n = 19; I 4 ; y RNAi n = 26, aspr-HA n = 16. I 1 , I 3 = ns; I 2 = **** p = 0.001; I4 = *** p = 0.001. ( D–E ) Anterior ( ci-GAL4 ) aspr-HA expression after ablation produces multiple anterior patterning defects (numbered arrowheads) seen in box wings; posterior ( hh-GAL4 ) aspr-HA only reduces wing area, without the anterior patterning defects (red arrowhead). ( F–G ) Regenerating disc time course ( DC hepCA , DVE>>GAL4 ) stained for HA (magenta), Nub (cyan), and Ci (yellow). Anterior pouch is consistently smaller from 12–60 h AHS with Aspr-HA (F), whereas developmental control ( DC NA , DVE>>GAL4 ) shows no change across time points with Aspr-HA (G). A/P boundary indicated by Ci. ( H–I ) Quantification of pouch compartments (anterior as % total disc in H; posterior in I) from the time course in (F) versus control. Anterior pouch is significantly reduced at 36 h, 48 h, and 60 h AHS; posterior pouch is unchanged. Sample sizes per timepoint: 24 h; y RNAi n = 20, aspr-HA n = 18; 36 h; y RNAi n = 17, aspr-HA n = 19; 48 h; y RNAi n = 26, aspr-HA n = 16; 60 h; y RNAi n = 10, aspr-HA n = 13. Anterior: 24 h = ns, 36 h * p = 0.0337, 48 h *** p = 0.0005, 60 h *** p = 0.0007. ( J–K ) aspr RNA (magenta) at 24 h AHS in unablated ( DC NA ) and ablated ( DC hepCA ) discs expressing aspr-HA ; transcript levels appear similar between conditions (J). Aspr-HA protein (K) is reduced in the anterior (open arrowhead) relative to posterior (arrowhead); Ci outlines A/P boundary. ( L ) Unablated disc with R85E08>GFP (red) shows transgene expression from the ablation domain spans both compartments similarly; Ci marks the A/P boundary. All scale bars = 50 μm unless otherwise noted. See also Figure S4. Full genotypes in Supplementary Genotypes. To examine the origin of these phenotypes, we monitored regeneration over time with aspr-HA expression and stained for nub , ci , and HA to mark the pouch, anterior compartment, and Aspr, respectively ( Figure 4F ). The anterior pouch is consistently undergrown from 12–60 h AHS, while the posterior regenerated similarly to controls. Quantification confirmed this observation, with significant anterior undergrowth at all time points except 24 h ( Figure 4H and 4I ). This aligns with the idea that persistent Aspr only disrupts late regeneration, when endogenous aspr is normally downregulated. Ectopic aspr without damage has no effect on pouch size ( Figures 4G , S4A–B), consistent with the absence of adult phenotypes ( Figure 3I ). To explore this anterior-specific effect, we examined Aspr localization. Although aspr-HA transcripts are equally expressed in both compartments ( Figure 4J–J ’), Aspr-HA protein appeared weaker in the anterior pouch of ablated discs ( Figure 4K ), which could indicate compartment-specific protein localization, stability or usage. We ruled out differences in ablation efficiency, as the salm enhancer used to induce damage drives similarly in both compartments ( Figure 4L ). These results indicate that maintaining Aspr beyond its normal early timing limits regrowth specifically in the anterior pouch. While the reason for this compartment-specificity remains unclear, these data suggest that the normal role of Aspr is to support early regenerative growth in the wing pouch. Aspr functions to regulate regeneration-specific pouch growth through vestigial To better understand the growth phenotype associated with Aspr during regeneration, we used an E2F reporter ( PCNA-GFP ) to monitor proliferation over time, with Ci staining to mark compartment boundaries. In control discs, injury triggers strong proliferation near the wound, indicative of a regeneration blastema ( Figure 5A ). This proliferation peaks over 24-48 h and returns to surrounding levels by 60 h ( Figures 5A–A ’’’ and C ). We noted that proliferation seems biased toward the anterior, consistent with previous reports showing differences in proliferative potential between compartments. 35 – 37 Ectopic aspr-HA expression reduces proliferation in both compartments, particularly in the anterior ( Figure 5B–B ’’’ and C ), matching the undergrowth seen in discs and adult wings ( Figures 4F and 4H ). aspr-HA had no effect on proliferation in undamaged discs ( Figures 3G and 3H ). We attempted to repeat these experiments with anti-PH3 and EdU incorporation, but neither was able to clearly demonstrate these differences (Figures S5A–L), likely because the E2F reporter integrates proliferative activity over time, while PH3 and EdU capture only transient mitotic or S-phase events, making subtle differences harder to detect. Download figure Open in new tab Figure 5. Aspr impairs anterior proliferation and Vestigial restoration during regeneration ( A–B L) Time course of PCNA-GFP in early L3 ablated discs ( DC hepCA , DVE>>GAL4 ): controls (A–A_) show elevated proliferation at 24–48 h with slight anterior bias that declines by 60 h; aspr-HA expressing discs (B–B_) show reduced PCNA-GFP fluorescence and loss of anterior bias. A/P boundary = Ci (yellow dotted line). ( C ) LOWESS splines of PCNA-GFP intensity across disc width for each timepoint (n = 4 discs/timepoint) illustrate loss of anterior bias in aspr-HA discs. ( D–G ′) vg QE -lacZ and vg expression dynamics: unablated discs ( DC NA , D and F) show symmetric Vg and lacZ across time points (arrowheads); ablated discs ( DC hepCA , E-E’’ and G-G’) lose Vg at 36 h (E), which is restored by 48–60 h in controls (E′-E’’). vg QE -lacZ is lost in the anterior at 36 h (G, open arrowhead), restored by 48 h (G’, arrowhead). ( H–H ′′) Ablated discs expressing aspr-HA (magenta, DC hepCA , DVE>>GAL4 ) show persistent loss of anterior Vg (cyan) from 36–60 h post-ablation (open arrowheads). Ci (yellow) indicates anterior. ( I–N ) Unablated ( DC NA , DVE>>GAL4 , I–K) and ablated ( DC hepCA , DVE>>GAL4 , L–N) discs at 36 h AHS expressing vg or vg+aspr-HA , stained for Ci (yellow) and Nub (red). Vg induces pouch overgrowth in unablated discs, which is limited by co-expression of aspr-HA in ablated discs, particularly in the anterior. ( O ) Quantification of pouch compartments for the conditions in (I–N). Anterior and posterior pouch areas (as % of total disc) are reduced by Aspr-HA, with a larger effect in the anterior. Sample sizes: anterior; y RNAi n = 10, vg n = 10, vg+aspr-HA n = 9 ; posterior; y RNAi n = 10, vg n = 10, vg+aspr-HA n = 9. Statistics: one-way ANOVA. Annotated p-values: anterior **** p < 0.0001; posterior * p = 0.0373, *** p = 0.0002, **** p >GAL4) at 36 h (Q and R) or 60 h (T and U) AHS. Following ablation, Bs is lost at 36 h (Q) and restored by 60 h in control discs (T) but remains absent in aspr-HA expressing discs (U). Ci (yellow) indicates anterior. ( V ) High magnification view of unablated disc ( DC NA ) at 60 h with I 2 intervein, longitudinal veins (LVx) labelled, indicated by Bs staining (cyan). ( W ) Adult wing with labels corresponding to disc in (V), and anterior/posterior cross veins (A/P CV). ( X-Y ) High magnification view of ablated discs ( DC hepCA , DVE>>GAL4 ) at 60 h with I 2 intervein, and longitudinal veins (LVx) labelled, indicated by Bs staining (cyan). Control discs restore anterior Bs and I2 (X), while those expressing aspr-HA discs fail to restore Bs or reestablish I 2 (Y). ( Z ) Quantification of intervein restoration across I 1 –I 7 : controls (black) show robust restoration; Aspr-HA (magenta) reduces restoration especially in anterior I 2 and I 3/4 . All scale bars = 50 μm unless otherwise indicated. See also Figure S5. Full genotypes in Supplementary Genotypes. We next screened candidate regulators of growth that might participate in regeneration, and found that vestigial (vg) , a key wing selector gene, is disrupted upon aspr misexpression. vg is normally expressed along the D/V boundary ( Figure 5D ), and is patterned by wg , notch , dpp , and autoregulation. 38 – 41 After DC hepCA ablation, Vg is reduced until 36 h AHS but restored by 48 h ( Figures 5E–E ’’). However, with sustained aspr-HA expression, Vg fails to reestablish in the anterior pouch through 60 h AHS and often remains absent until pupariation ( Figures 5H–H ’’). Interestingly, the vg quadrant enhancer (vg QE ), which initiates vg expression in response to Wg, 38 also shows anterior-specific loss after damage, typically recovering by 48 h AHS in controls ( Figures 5F–G ’ and S5M-T). Due to genetic constraints, we could not assess vg QE reporter activity in aspr-HA -expressing discs, but these results suggest that anterior vg is less robustly reinitiated after injury, and thus may be more sensitive to prolonged Aspr expression. To test whether Aspr can suppress Vg-mediated growth, we expressed vg in the presence and absence of aspr-HA in damaged and undamaged discs, and quantified pouch size. Ectopic expression of vg alone yields larger pouch size in both undamaged ( Figure 5I–J and S5U), and damaged discs ( Figure 5L–M and 5O ). However, co-expression with aspr-HA significantly suppresses this growth in damaged discs ( Figure 5N–O ), which does not occur in undamaged discs ( Figure 5K and S5U). This growth suppression primarily occurs in the anterior compartment ( Figure 5N–O ), consistent with the previous observations of compartment-specific differences in Vg restoration ( Figure 5E–H ’’). Together, these data confirm that 1) Aspr regulates Vg-mediated growth in the pouch, 2) this effect occurs preferentially in the disc anterior, and 3) that the function of Aspr is most significant in damaged discs that are undergoing regeneration. However, whether Aspr modulates Vg itself, or rather influences an intermediate factor that participates in Vg-dependent growth, is unclear (see later). Although anterior pouch tissue is reduced with ectopic Aspr, it still acquires vein/intervein identity ( Figures 3S-S ’). To assess patterning, we examined blistered (bs/DSRF) , which represses vein fate to define intervein regions ( Figure 5V–W ). 42 , 43 In undamaged discs, the vein/intervein regions are defined by bs expression at 36 h AHS and is unchanged at 60 h AHS ( Figures 5P, S and V ). After damage, bs is transiently lost but returns by 48-60 h AHS ( Figures 5Q, T and X ). aspr-HA expression has no clear effect through 36 h ( Figure 5R ), but by 60 h, I 2 fails to form and LV 2 and LV 3 are fused ( Figures 5U and Y ), mirroring adult wing defects. Since most of the pouch retains proper bs expression and vein identities despite overlapping Aspr ( Figures 5U, Y and Z ), this patterning defect possibly results indirectly from impaired growth. Alternatively, as vg has been shown to regulate bs, 39 the phenotype may stem from disrupted vg expression. Together, these findings suggest that Aspr normally promotes proportional growth during early regeneration, partly by supporting timely vg reactivation, and when mis-regulated it impairs both regenerative growth and patterning. Aspr limits expression of late-acting differentiation-associated genes specifically during regeneration Changes in Vg may account for anterior pouch size reduction and vein/intervein patterning defects in the box wing phenotype, but significant margin abnormalities and loss of bristles is also observed ( Figures 3S–S ’). To investigate this, we examined genes involved in margin cell differentiation. Bristles at the wing margin derive from sensory organ precursors specified by Notch and Wg signaling. 44 Previously, we showed that Aspr may affect expression timing of the Notch and Wg target gene cut ( ct ) at the margin specifically following injury ( Figure 6A ). 22 We revisited this by testing aspr-HA expression during regeneration. Normally, Ct is lost following ablation and restored in all discs by 60 h AHS ( Figures 6B–C ). With aspr-HA expression, Ct is fully restored in only 18% of discs (n = 50), with most showing only partial recovery ( Figure 6D ). aspr-HA had no effect on Ct in undamaged discs (Figure S6A), confirming it interferes with ct reactivation during regeneration. Furthermore, restricting expression of aspr-HA solely to the ct domain using ct-GAL4 results only in the loss of bristles component of the box wing phenotype (Figure S6F), which we attribute to Aspr’s repressive effect on Ct. This phenotype is not present when aspr-HA is driven in the ct domain in undamaged discs (Figure S6E). ct is maintained indirectly by Wg via the Notch ligands Delta and Serrate (Figure S6H). 45 Delta expression, normally restored by 60 h AHS ( Figure 6E–F ), is also suppressed by aspr-HA ( Figure 6G ), which may explain the persistent loss of Ct. As both vg and Delta (and thus indirectly ct ) are targets of Wg, we examined other differentiation-associated Wg-responsive genes. aristaless ( al ) encodes a homeobox transcription factor downstream of Wg 46 . al failed to be restored with aspr-HA by 60 h AHS ( Figure 6L–N ). Similarly, achaete ( ac ), a Wg-responsive factor that promotes neural fate 47 , 48 normally appears after 48 h time point in undamaged discs (Figure S6I), is reestablished in wild type ablated discs by 60 h AHS (83.9% of discs, Figure 6H–J ), but fails to be reestablished in most aspr-HA -expressing discs at this time point (7.9%) ( Figure 6K ). This finding, alongside the reduction in Ct ( Figure 6D ) likely underlies the reduced bristle counts observed in wings (Figure S6G). Thus, prolonged aspr disrupts the reactivation of multiple differentiation-promoting genes ( Figure 6O ), all of which are directly or indirectly downstream of Wg signaling. None of these genes were altered by aspr-HA expression in undamaged discs (Figures S6A-S6D). Many Wg targets also depend on Dpp, however, expression of a Dpp-lacZ reporter, pMad, and its target spalt ( salm ) are unaffected by aspr-HA during development or regeneration ( Figures 6L–N and S6J–O’’). Given the strong overlap between wg and aspr expression in the blastema, 31 and the fact that several affected genes lie downstream of Wg, we hypothesize that Aspr normally delays Wg-driven differentiation and sustains early regenerative growth through regulation of Vg. Download figure Open in new tab Figure 6. Aspr inhibits restoration of anterior cell-fate markers during regeneration ( A ) Unablated ( DC NA ) disc stained for Cut (Ct, cyan) showing margin expression including anterior (arrowhead). Ci (yellow, dotted line) marks the A/P boundary; nuclei = DAPI (gray). ( B–D ) Ablated discs ( DC hepCA , DVE>>GAL4 ) at 48 h (B) and 60 h (C,D) AHS stained for Ct. In GFP controls, anterior Ct is absent at 48 h (B, open arrowhead) and restored at 60 h (C, arrowhead). Ectopic Aspr-HA blocks Ct restoration at 60 h (D, open arrowhead). ( E–G ) Delta (Dl) staining: unablated (E) shows margin/vein expression; at 60 h post-ablation Dl is restored in controls (F, arrowhead) but not in aspr-HA discs (G, open arrowhead). ( H–K ) Discs as in (B–D) showing Achaete (Ac) dynamics: Ac is absent at 48 h post-ablation (I, open arrowhead) and restored by 60 h in controls (J, arrowhead); Aspr-HA prevents restoration at 60 h (K, open arrowhead). ( L–N ) Aristaless (Al) and Salm: Al is anterior-restricted and restored by 60 h in controls (M, yellow arrowhead); aspr-HA blocks Al restoration (N, open arrowhead) but does not affect Salm (N, red arrowhead). ( O ) Quantification of marker restoration at 60 h for Ct, Dl, Ac, and Al. Y-axis indicates percent discs with full (cyan), partial (turquoise), or no restoration (dark gray). Ectopic Aspr-HA reduces restoration of all anterior markers. Sample sizes: Ct: y RNAi (n = 28), aspr-HA – Full (n = 23), Partial (n = 18), None (n = 9) Dl: y RNAi – Full (n = 11), None (n = 4); aspr-HA – Full (n = 1), None (n = 16) Ac: y RNAi – Full (n = 47), None (n = 9); aspr-HA – Full (n = 3), None (n = 35) Al: y RNAi – Full (n = 9), Partial (n = 4); aspr-HA – Full (n = 1), Partial (n = 11), None (n = 3) ( P–Q ) Representative adult wings after early L3 ablation ( DC hepCA ) with Ets21C RNAi alone (P) or combined with aspr-HA (Q). Ets21C knockdown impairs regeneration; co-expression of aspr-HA rescues malformed wings. ( R ) Regeneration scoring corresponding to (P–Q). Sample sizes: Control n = 27, Ets21C RNAi n = 44, Ets21C RNAi + aspr-HA n = 88. All scale bars = 50 μm unless otherwise indicated. See also Figure S6. Full genotypes in Supplementary Genotypes. The idea that Aspr acts to delay differentiation is supported by previous work showing that aspr is downstream of the ETS transcription factor Ets21C, which promotes regeneration by supporting the expression of genes like Mmp1 , upd3 , pvf1 , and aspr , while preventing premature induction of repatterning genes like rotund and nubbin . 31 As with aspr , mutation of Ets21C causes defective, undersized wings post-ablation, but does not affect development. Knockdown of Ets21C in ablated wings shows this phenotype ( Figures 6P and R ), but this is almost entirely rescued by co-expression of aspr-HA ( Figures 6Q and R ), suggesting that a key function of Ets21C in regeneration is the activation of aspr to prevent premature differentiation and promote tissue repair. Aspr protein structure suggests a role in protein trafficking and secretion Aspr appears to regulate growth and differentiation during regeneration, but its molecular function remains unclear. To investigate this, we analyzed its predicted protein structure using AlphaFold ( Figure 7A and S7A), which reveals a solenoid-like fold with multiple epidermal growth factor (EGF)-like domains ( Figure 7B ). InterProScan and SMART predict seven EGF domains, while Prosite identifies four. These domains are typical of secreted or membrane-associated proteins involved in signaling, adhesion, and matrix organization 49 , supporting a potential extracellular role for Aspr. To further test this, we used six localization prediction tools. Four of these (SignalP, Phobius, Protter, Deeploc) predict a cleavable N-terminal signal peptide with a high-confidence cleavage site between Gly23 and Lys24 ( Figures 7C and S7B–E). TargetP and TMHMM also supported this with moderate confidence (∼0.5) ( Figures 7C and S7F–G). These tools also consistently predict Aspr to be extracellular ( Figure 7C ) reinforcing the idea it is likely secreted. Homology searches identify several structurally related extracellular proteins: human NELL2, human/mouse EDIL3, and Drosophila Dumpy ( Figure 7D ). Alignments of their signal peptides with that of Aspr revealed shared biochemical properties and α-helical structures typical of signal sequences ( Figure 7D and S7H–L), while RMSD comparisons showed strong similarity to Dumpy (0.189), NELL2 (0.248), mouse EDIL3 (0.635), and human EDIL3 (1.04) ( Figure 7E ), supporting the idea that Aspr has structural conservation with other secreted proteins. Together, these structural analyses are consistent with Aspr functioning as a secreted or membrane-associated extracellular protein. Download figure Open in new tab Figure 7. Structural and in vivo analyses show Aspr is an extracellular protein ( A ) AlphaFold predicted structure of full-length Aspr colored by pLDDT confidence: very high (>90, blue), confident (70–90, cyan), low (50–70, yellow), very low (<50, orange). ( B ) AlphaFold model highlighting seven predicted EGF-like domains (magenta) separated by non-EGF regions (cyan). Inset shows EGF domain 1 with predicted disulfide bonds (yellow) and cysteines. ( C ) Heatmap summarizing signal peptide and subcellular localization predictions from six tools (SignalP, Phobius, TargetP, Protter, TMHMM, DeepLoc); scores range 0 (low, blue) to 1 (high, yellow); grey = no data. D ) (i) Structural alignments of the Aspr signal peptide (cyan) with signal peptides from Dumpy isoform V ( Drosophila , maroon), NELL2 (human, orange), and EDIL3 (mouse, yellow; human, green). (ii) Sequence alignment showing conserved features (e.g., polarity, hydrophobicity), with residues colored by chemical class: hydrophobic (dark red), mildly hydrophobic (red), polar (blue), moderately polar (purple). ( E ) RMSD values from structural alignments in (D). Each point represents structural similarity between Aspr and comparison proteins; lower values indicate greater similarity. ( F ) Western blot of hemolymph fractions from control ( w 1118 ) and ci-GAL4>aspr-HA larvae, probed with HA and Actin antibodies. HA is detected in whole aspr-HA larvae (lane 9) and filtered hemolymph (lane 8), and in the supernatant (lane 5) but not the pellet (lane 4) of hemolymph after ultracentrifugation, consistent with secretion. No HA is detected in control larvae (lanes 2–3, 6–7). Actin is detected in whole larvae and filtrate (lanes 8–9). ( G–G’ ) Unablated ( DC NA , DVE>>GAL4 ) discs with the Crb-GFP apical marker (green), expressing aspr-HA (grey). DE-Cadherin labels cell junctions (red); DAPI (blue) labels nuclei. (G) Yellow dotted line indicates imaging planes shown in cross sections. Aspr-associated EVs are found apical to Crb-GFP, which is apical to DE-Cadherin. ( H–H’’’ ) High magnification of transverse sections in (G). Aspr-associated EVs (yellow arrowheads) occur apically to the Crb marker (H-H’) and DE-Cadherin (H’’-H’’’). Aspr also appears apically within disc cells. ( I–I’’ ) High magnification imaging of unablated (DC NA ) disc expressing aspr-HA at 24 h via ci-GAL4. (I) Surface view shows vesicles; yellow box highlights zoomed region in (I’). Open arrowhead marks area lacking vesicles beyond expression domain. (I’’) Cross-section reveals vesicles localize to the apical surface. ( J-J’’ ) High magnification imaging of ablated ( DC hepCA ) disc expressing aspr-HA at 24 h via ci-GAL4. (J) Surface view shows increased vesicles, with zoomed region in (J’). Arrowhead indicates small vesicles outside the expression domain. ( K-K’ ) Genotype as (I) imaged without permeabilization. Aspr-HA vesicles are visible (zoomed in J’) while the more diffuse intracellular cytoplasmic signal is lost, consistent with Aspr having an extracellular localization. All scale bars represent 50 μm unless otherwise indicated. See also Figure S7. Full genotypes are provided in the Supplementary Genotypes file. We experimentally tested Aspr secretion using western blots on hemolymph bled from larvae expressing aspr-HA ( ci-GAL4>UAS-aspr-HA ). HA was detected in both filtered hemolymph and larval debris ( Figure 7F , lanes 8-9), confirming previously published proteomic analysis that identified Aspr in hemolymph 50 . We further processed the filtered hemolymph by subjecting it to additional high-speed centrifugation, which separates extracellular proteins and structures like vesicles away from cells and larger debris. Repeating HA detection on this sample shows that Aspr is present in the supernatant ( Figure 7F , lane 4-5), confirming its extracellular nature. Control larvae ( w 1118 ) lacked HA signal ( Figure 7F , lanes 2-3, 6-7), while anti-Actin was used to indicate cellular presence in each sample (trace Actin in filtrates likely reflects cells such as hemocytes). Together, these findings strongly support Aspr as a secreted extracellular protein. To validate the localization of Aspr in vivo , we re-examined its distribution in the wing disc, focusing on the punctate structures previously observed ( Figures 3A–B ). High-resolution imaging of undamaged discs expressing aspr-HA revealed spherical extracellular vesicle (EV)-like structures (300–1400 nm diameter, Figure S7M–P) located above the apical surface ( Figure 7G and I –I’’), confirmed by co-staining with the apical marker Crumbs ( crb-GFP ) in transverse sections ( Figure 7H–H ’’’) and a membrane-associate GFP reporter ( CD8::GFP , Figure S7Q–R’’’). Notably, EVs do not incorporate this GFP label (Figure S7R–R’), indicating that they are unlikely to arise by simple outward budding from the labeled apical plasma membrane, but instead may originate from intracellular compartments via a secretory route, such as exosome-like vesicle release. 51 Although our anti-Aspr antibody has limited sensitivity, we successfully detected EVs using this antibody in aspr-HA -expressing discs (Figure S8A-S8B’), ruling out artifacts of HA staining. This was further validated using different anti-HA antibodies and control staining conditions (Figure S8C and S8C’). Most importantly, extracellular vesicles are also detected using the anti-Aspr antibody in ablated discs expressing only endogenous aspr , without ectopic aspr-HA (Figure S8D–S8D″). These EVs are smaller (typically <100 nm in diameter), more uniform in size, and less frequent, exhibiting features of exosome-sized vesicles rather than larger plasma membrane–derived microvesicles, 26 , 29 and suggesting that ectopic aspr-HA expression increases both EV size and abundance. These observations support the presence of Aspr-containing EVs under physiological damage conditions, but additional markers and functional assays will be required to determine their precise biogenesis, secretion route, and role in regeneration. Aspr-associated EVs appear alongside intracellular Aspr, which is enriched apically in disc cells ( Figure 7G–H ’’). An extracellular-optimized staining protocol using non-permeabilized discs eliminates the intracellular Aspr signal, allowing clearer visualization of EVs outside the cells ( Figure 7K-K ’), located between the disc proper and the overlying peripodial membrane ( Figures 7I–I ’’). These EVs form in the pouch, hinge, and notum when aspr-HA is expressed throughout the anterior compartment via ci-GAL4 , but are most frequently observed in the pouch ( Figures 7I–I ’ and 3B-B’), and can also be detected in other imaginal discs like the leg (Figure S8E). Notably, EVs are present in both undamaged and regenerating discs expressing aspr-HA ( Figure 7I-J ’). However, regenerating discs show a significantly greater number of the smaller EVs, including in regions lacking active aspr-HA expression ( Figure 7J-J ’), consistent with the possibility that extracellular Aspr may be able to disperse more broadly beyond its site of expression in regenerating discs. These EVs are distinct from the cellular debris generated by ablation (Figure S8H–J’). Overall, these structural, computational, and biochemical results demonstrate that Aspr is a secreted protein, and that it forms EVs in the wing disc, particularly during regeneration, consistent with its proposed role in extracellular signaling. Aspr co-localizes with Wg and regulates WNT signaling Maintaining aspr expression into late regeneration affects growth and patterning in ways consistent with altered Wg signaling ( Figures 5 and 6 ). Given the predicted protein structure and localization of Aspr to secreted EVs ( Figure 7 ), we tested whether Aspr influences Wg signaling via an extracellular mechanism. Wg is secreted and signals through tightly regulated processes involving chaperones, 52 lipoprotein particles, 53 cytonemes 54 , 55 juxtracrine mechanisms, 56 and via EVs such as exosomes. 26 , 29 , 57 , 58 To examine Wg regulation by Aspr, we expressed aspr-HA during development and regeneration while staining for Wg. In undamaged discs, aspr-HA has no effect on Wg levels (Figures S9A–C). In regenerating discs, however, Wg protein appears elevated from 18-48 h AHS, especially in the anterior pouch ( Figures 8A–D ). This is despite aspr-HA being driven throughout the pouch. By 60 h AHS, Wg returns to a wild type-like pattern but often appeared distorted at the A/P boundary ( Figure 8E ), which we hypothesize could be due to misaligned compartments resulting from asymmetric growth ( Figure 4 ). A similar elevation of Wg in the anterior and margin distortion occurs when aspr-HA is expressed only in the anterior (Figure S9D–H). These findings suggest that aspr-HA affects Wg during regeneration; however, Wg levels appear elevated rather than reduced, which is unexpected given the failure to reestablish multiple Wg target genes ( vg, Delta, ct, ac, al ) in the presence of Aspr-HA ( Figures 5 and 6 ). Wg signaling depends on proper epithelial polarity; one model proposes that Wg is produced at the apical surface and subsequently trafficked to the basal side, where receptor engagement activates downstream signaling. 59 , 60 Given that Aspr localizes to the apical membrane and within EVs situated above this surface ( Figure 7 and Figure S7), we hypothesize that Aspr may modulate Wg availability by sequestering the ligand apically. Such sequestration could diminish the pool of Wg accessible for signaling and/or restrict its transcytosis to the basal surface. Download figure Open in new tab Figure 8. Aspr attenuates WNT signaling during regeneration ( A–E ) Time course of Wg (cyan) in ablated discs ( DC hepCA , DVE>>GAL4 ) expressing aspr-HA (magenta), 18–60 h AHS. Ci (yellow) marks anterior and the A/P boundary (dotted line). From 18–48 h, Wg signal appears increased anteriorly; by 60 h the anterior bias is lost but Wg patterning is distorted across the margin. ( F–J ) Ablated discs ( DC hepCA , DVE>>GAL4 ) at 60 h post-ablation, stained for Achaete (Ac, cyan) and Ci (yellow). Control discs (F) restore Ac expression (arrowhead), while aspr-HA blocks Ac restoration (G, open arrowhead). Co-expression of aspr-HA with WNT pathway activators Axn RNAi (H), UAS-dsh (I), or sgg RNAi (J) restores Ac (arrowheads). ( K ) Quantification of wing phenotypes from (F-J). Ectopic aspr-HA induces a box wing phenotype in 39% of wings. This is rescued by WNT activation: 1.7% with Axn RNAi , 0% with dsh , and 9.5% with sgg RNAi . Sample sizes: DVE>>y RNAi , n=275, Axn RNAi , n=255, dsh , n=122, sgg RNAi , n=146; DVE>>aspr-HA with y RNAi , n=243, Axn RNAi , n=119, dsh , n=63, sgg RNAi , n=288. ( L ) Quantification of Ac restoration in discs from (F-J). Ectopic Aspr-HA reduces Ac restoration to 8% of discs, whereas co-activation of Wg signaling restores Ac in 81% ( Axn RNAi ) and 100% ( dsh and sgg RNAi ). Sample sizes: GFP, n=56, aspr-HA , n=38, aspr-HA + Axn RNAi , n=11, aspr-HA + dsh , n=3, aspr-HA + sgg RNAi , n=2. ( M ) Quantification of regeneration outcomes from experiments in (K), categorized by severity of tissue loss. WNT activation alone produces a distinct “ectopic WNT” phenotype (pink), shown in (O). Co-expression with aspr-HA slightly reduces this phenotype. ( N–O ) Representative adult wing phenotypes from ablated discs ( DC hepCA , DVE>>GAL4) . (N) Box wing phenotype from aspr-HA . (O) Ectopic WNT phenotype with mis-patterning and extra bristles. ( P–S ) Ablated discs ( DC hepCA , DVE>>GAL4) at 36 h post-ablation bearing vg QE -lacZ and expressing RNAi for aspr (Q), Axn (R), or sgg (S). In control discs (P), lacZ is absent in the anterior (open arrowhead). All RNAi conditions restore anterior lacZ expression (arrowheads). ( T–U ) Wg (cyan) and Aspr-HA (magenta) colocalization at 60 h AHS. In unablated discs expressing aspr-HA , ( DC NA , ci-GAL4 ) (T), Aspr-HA EVs (arrowheads) do not colocalize with Wg (open arrowheads). After damage ( DC hepCA , ci-GAL4) (U), Wg is detected at Aspr-HA vesicles (arrowheads). Yellow dotted box indicates region shown at higher magnification insets. ( V–W ) Ablated discs ( DC hepCA , DVE>>GAL4) at 24 h post-ablation stained for Wg (Cyan) and Ci (yellow), with knockdown of aspr (W), or control disc (V). Yellow dashed outline shows higher magnification inset. The loss of aspr reduces appearance of apical Wg puncta. ( X ) Unablated ( DC NA , DVE>>GAL4 ) discs with the Wls-GFP reporter (green), expressing aspr-HA (grey) and stained for Wg (red). DAPI (blue) labels nuclei. Yellow dotted line indicates imaging planes shown in cross sections. Aspr-HA overlaps Wls staining within disc cells at the apical surface. ( Y-Y’’’ ) High magnification transverse sections of disc in (X), showing the overlap of Wg with Wls (Y). and Wls with Aspr-HA (Y’-Y’’’). Yellow arrowheads indicate Aspr-associated EVs. All scale bars = 50 μm unless otherwise noted. See also Figures S8–S9. Full genotypes in Supplementary Genotypes. If this model is correct, then activating Wg signaling downstream of the receptor should rescue the reestablishment of Wg target genes caused by sustained Aspr. To test this, we activated Wg signaling downstream of the receptor using three manipulations: knockdown of Axin ( Axn ) or shaggy ( sgg ), or overexpression of disheveled ( dsh ). We assessed expression of the Wg target ac as a readout. Normally, ac is restored in nearly all regenerating discs at 60 h AHS ( Figure 8F ) but is blocked by aspr-HA ( Figure 8G ). However, the activation of Wg signaling by any of the three manipulations fully rescues ac at 60 h AHS despite persistent aspr-HA expression ( Figures 8H–J and 8L), confirming that Aspr can regulate Wg signaling at the level of the Wg ligand or receptor. Each manipulation alone also increases ac expression in both undamaged and regenerating discs (Figures S9I–N), with particularly strong effects when dsh is overexpressed (Figures S9J and M). aspr-HA mildly reduces these effects in undamaged discs (Figures S9O–R), suggesting that Aspr can temper ectopic Wg signaling to some degree even in the absence of injury. Importantly, these rescue experiments also significantly reduce the incidence of the “box wing” phenotype in regenerated wings ( Figure 8K ) and improves overall regenerative outcomes ( Figure 8M ). These manipulations, particularly the stronger Dsh overexpression and sgg knockdown, also result in a phenotype characterized by malformation and ectopic bristles within the wing blade ( Figures 8M–O and S9S–T), suggesting these manipulations can go beyond rescuing the suppressive effects of aspr-HA , resulting in an “ectopic WNT” phenotype. This phenotype also occurs in undamaged discs at an equivalent frequency (Figure S9Y–AA) and therefore is unlikely to interfere with our interpretations in the context of regeneration. These experiments are performed in the context of ectopic aspr-HA expression. To confirm that Wg signaling is regulated by Aspr endogenously, we knocked down aspr during regeneration and examined vg , a Wg responsive target gene 61 that is present at the regenerative time point when Aspr is normally expressed, and that we know is affected by Aspr; indeed, we demonstrated that ectopic Aspr not only limits vg expression after damage ( Figure 5H-H ’’), but it can also blunt the growth induced by ectopic vg during regeneration ( Figure 5M–O ). Previous work has established that Vg and Wg cooperate in a growth-promoting feedback loop during development, 41 , 62 – 66 and thus we hypothesize that if a similar dynamic occurs during regeneration, the addition of Aspr would interfere with this Vg-Wg-growth axis, resulting in the growth suppression we observe. To monitor vg we used vg QE , the vestigial quadrant enhancer that depends on Wg signaling for its activity, 38 as a transcriptional readout. Under normal conditions, vg QE is not reactivated in the anterior pouch by 36 h AHS ( Figure 8P ). However, aspr knockdown leads to premature vg QE reactivation ( Figure 8Q ), supporting the model that Aspr delays Wg-dependent re-initiation of vg . Similarly, knockdown of Axn or sgg produces the same early-activation phenotype ( Figures 8R–S ), further strengthening this hypothesis. No alterations in vg QE activity were detected with these manipulations during development (Figures S9U–S9X). Finally, we asked whether Aspr and Wg physically colocalize during regeneration. The anti-Aspr antibody was too weak to detect endogenous co-localization with Wg, and thus we examined Wg in aspr-HA expressing discs. In undamaged discs, even with optimized extracellular detection of Wg (see STAR Methods), Wg and Aspr showed no colocalization ( Figure 8T ). However, upon damage Wg now clearly associates with Aspr-positive EVs at the apical surface ( Figure 8U ). Notably, Wg localizes both on the surface and within these EVs (Figure S9AC), consistent with its presence as vesicular cargo. Supporting this interpretation, Wg staining is lost in non-permeabilized samples, indicating that its detectable epitope is internal rather than surface exposed (Figure S9AB). Moreover, aspr knockdown reduces the apical Wg-positive puncta normally observed in the tissue ( Figure 8V–W ), consistent with Aspr contributing to Wg trafficking or localization during regeneration. This idea is further supported by the colocalization of Aspr with Wntless (Wls-GFP), a well-established chaperone required for Wg secretion, 52 at the apical surface of disc cells ( Figure 8X–Y ’’’). Because Aspr is enriched apically above the Crb marker ( Figure 7G–H ’’’) and shows minimal overlap with membrane-associated GFP within EVs (Figure S7Q–R’), we hypothesize that Wg resides within Aspr-associated EVs that exhibit several hallmarks of exosome-like vesicles, although their precise identity remains to be determined. Together, these observations support a model in which Aspr modulates Wnt signaling during tissue repair by sequestering Wg within these EVs. Thus, we hypothesize that Aspr regulates the timing of Wg signaling targets by modulating Wg ligand availability ( Figure 9 ). Upon damage, aspr is initially expressed via its DRMS enhancer in blastema cells alongside wg and other secreted factors. During these early regeneration stages, Aspr may facilitate Wg trafficking to promote balanced compartment growth via vg , while restraining activation of late patterning genes such as bs and ac . After 24 h, Aspr levels decline, allowing Wg signaling to reinitiate differentiation programs to promote proper repatterning. Artificially maintaining Aspr beyond this window sequesters Wg apically in EVs, disrupting growth balance and repatterning, resulting in poor regeneration and the characteristic “box wing” phenotype. Conversely, aspr knockdown causes premature target activation, further disrupting the precise regenerative sequence required for full wing restoration. Download figure Open in new tab Figure 9. Functional model: Aspr temporally regulates WNT signaling during regeneration ( A–B ) Schematic model proposing that (A) early after injury (first ∼24 h), dying cells induce blastema formation in adjacent tissue via JNK activity, activating DRMS enhancers to drive aspr and wg expression. Aspr localizes to vesicles that sequester Wg, limiting its access to neighboring cells. (B) After ∼24 h, Aspr levels fall, permitting Wg to engage receptors and activate WNT signaling, which triggers cell-fate gene expression (e.g., vg, ac, ct, al) and re-patterning only after sufficient growth has occurred, thereby coordinating growth and patterning during regeneration. Discussion Aspr regulates regenerative WNT signaling Regeneration reactivates developmental programs, yet it often must occur within mature tissues, which poses unique challenges. Temporal coordination is particularly important, as fate specification must follow tissue regrowth to ensure proper repatterning. 1 , 67 Our findings identify aspr as a primarily regeneration-specific gene that accomplishes this by attenuating Wg signaling during early regeneration. aspr appears dispensable during development but is activated by damage-induced JNK signaling via a DRMS enhancer, 68 where it functions to properly time WNT-dependent patterning genes in regenerating tissue, potentially via sequestration of the Wg ligand in apically secreted extracellular EVs. This mechanism differs from previously described temporal regulators of regeneration, such as Ets21C and Brat, which act at the level of transcription and translation. 31 , 69 By contrast, Aspr potentially modulates ligand availability, demonstrating an alternative strategy by which the sequential steps of a regeneration program are maintained. We find that artificially maintaining Aspr disrupts this timing, delaying the restoration of WNT target genes, and leading to defective patterning and disproportional/restricted growth. These effects are rescued by pathway activation downstream of the receptor, supporting a model in which Aspr functions at or above the level of WNT ligand/receptor interaction to negatively regulate Wg signaling during early regeneration. It therefore joins factors such as Evi/Wntless, 52 Porcupine 70 , 71 , SWIM 72 , and the heparan sulfate proteoglycans Dally and Dally-like 73 , which regulate Wg signaling by controlling overall ligand availability during disc development. 57 Thus, exploring how these factors function in the context of regeneration represents an important direction for future research. Aspr is unlikely to completely block Wg signaling, but rather Aspr may fine-tune Wg signaling following injury; its ability to antagonize WNT activity when co-expressed with activated pathway components, even in undamaged discs (Figure S9P-S9R), suggests it may buffer excess signaling, which is particularly important in regenerative contexts in which signaling is inherently more variable. In regenerating wing discs, Wg is strongly induced and broadly expressed at early stages before its expression narrows and declines 69 , while in a developmental context different levels of Wg/Wnt activity are known to elicit distinct cellular responses ranging from survival to proliferation and differentiation. 74 These features are consistent with a model in which Aspr functions as a regeneration specific modulator that keeps Wg signaling within an optimal range, supporting initial proportional growth while preventing premature differentiation. Interestingly, the effects of maintaining aspr expression are more impactful in the anterior compartment. Ectopic Aspr preferentially disrupts anterior patterning, growth, and cell fate markers, and anterior proliferation is more sensitive to its expression. While Aspr is broadly induced in the pouch during wounding, this compartment-specific effect may reflect intrinsic differences in signaling dynamics, baseline proliferation rates, or the influence of specific regulators that are unique to each compartment. The observation that Aspr protein is not equally detected in the anterior versus posterior compartment of the wing disc when ectopically expressed under pouch-wide UAS control, despite transcripts being uniformly distributed, also raises the possibility that compartment-specific post-transcriptional mechanisms influence Aspr. Such asymmetries could reflect differential protein stability, localized degradation pathways, or compartment-specific modulation of secretion, trafficking, or extracellular retention. Alternatively, Aspr may require cofactors or interacting partners that are present at different levels across compartments, resulting in localized stabilization, enhanced turnover or differences in functionality. Thus, the wing disc’s intrinsic anterior–posterior heterogeneity may shape the distribution and activity of Aspr through several potential mechanisms, emphasizing the importance of understanding how tissue architecture and compartmental identity influence regenerative capacity. Although compartment-specific identity genes are thought to be largely maintained or quickly reestablished during regeneration, 69 , 75 – 77 additional studies such as those utilizing single cell sequencing, 31 may help to elucidate how compartment identity influences regenerative mechanisms and outcomes. EV-based control over regenerative timing During development of the wing disc, Wg is secreted apically and trafficked to the basolateral membrane for receptor interaction. 26 , 57 , 59 , 78 – 80 Our results suggest that in regeneration, Aspr delays this process by associating with Wg in vesicles that remain apical and separate from the signaling-competent basolateral domains. This may block WNT signaling by physically preventing ligand-receptor interaction, consistent with findings that altering Wg ligand internalization can block signaling. 57 , 81 – 83 Once aspr expression declines after 24 hours, Wg becomes available for signaling, initiating cell fate specification at the appropriate time. Alternatively, Aspr may alter the secretion or modification of Wg to alter its ability to signal in other ways, since alternative trafficking is known to alter secretion rates, location and post-translational modifications of ligands. 57 Nevertheless, this EV-based mechanism raises several questions. Is Wg encapsulation actively regulated, or a passive consequence of altered trafficking? What is the fate of Aspr vesicles? Do they carry other morphogens, such as Dpp, or regulatory factors involved in regeneration? The suppression of Dpp target al by ectopic Aspr, and the known crosstalk between Wg and Dpp, 84 – 86 suggests broader regulatory functions that remain to be explored. The localization of Wg to Aspr-containing vesicles during regeneration and not development suggests that additional injury-induced factors may mediate cargo trafficking into these EVs. Given that both Wg and Aspr are regulated by DRMS enhancers, 22 and are co-expressed in “secretory zone” cells of the blastema, 31 it is possible that other factors involved in EV formation or cargo selection are similarly regulated. Therefore, systematic identification of DRMS enhancer targets may uncover additional components or cargoes of EVs that help coordinate regeneration through this mechanism. Broader implications and future perspectives This work identifies aspr as a novel regeneration-specific gene that regulates tissue patterning by modulating WNT ligand availability via vesicle-based sequestration. These findings underscore the importance of temporal control mechanisms in regeneration and highlight vesicle trafficking as an underexplored mode of signal regulation. Beyond Drosophila , EV-mediated intercellular communication is increasingly recognized as critical in cancer and immune biology. 87 – 90 Our findings suggest that analogous mechanisms may exist in injury and regeneration contexts, and could be harnessed to enhance tissue repair. Future work should focus on the molecular determinants of Wg loading into Aspr vesicles, the identity of additional vesicle cargo, and whether other DRMS-induced genes in blastema cells are regulated via similar mechanisms. Exploring whether vertebrate functional homologs of aspr exist, and whether they influence WNT signaling during injury, may open new avenues for regenerative therapies. Ultimately, understanding how tissues control not just which genes are expressed, but also when they are activated, may be essential to unlock the full potential of regenerative biology. Limitations of the Study In this study, we identify a regeneration-specific role for Aspr, a largely uncharacterized factor induced by damage in the wing disc. Our findings suggest that Aspr may act through the formation or modulation of extracellular vesicles (EVs) that influence Wg ligand availability; however, the precise nature, origin, and functional contribution of these EVs remain incompletely understood. Additional work using high-resolution imaging and biochemical EV isolation will be required to determine how these structures affect Wg secretion or trafficking, and to characterize their endogenous formation and behavior without relying on Aspr overexpression. Furthermore, it is not yet known whether these EV-like structures contain additional molecular cargo or impact signaling pathways beyond WNT. Finally, our ability to study endogenous Aspr is limited by the low sensitivity of the antibody generated for this work. Development of an endogenously tagged Aspr allele will be critical for defining its localization, dynamics, and function during regeneration. Resource Availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Robin Harris ( robin.harris{at}asu.edu ) Materials availability Fly lines generated or used in this work are available upon request; further details regarding stock genotypes are also provided in the Supplementary Genotypes file. For more detailed protocols, please contact the first author, and any further information required to reproduce the experiments can be obtained by contacting the corresponding author. Data Availability All primary data supporting these findings are included within the manuscript or in the supplemental materials. Declaration of interests The authors declare no competing interests. Author Contributions Si Cave, Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Validation, Writing – Original Draft, Writing – Review and Editing, Visualization; Maksym Dankovsky, Investigation, Data Curation, Formal Analysis, Validation, Writing – Review and Editing, Visualization; Jordan Hieronymus, Investigation, Data Curation, Formal Analysis, Validation, Writing – Review and Editing, Visualization; Manashi Sonowal, Conceptualization, Investigation Data Curation, Formal Analysis, Validation, Writing – Original Draft, Chloe Van Hazel, Investigation Data Curation, Validation, Resources; Petra Fromme, Supervision, Conceptualization, Resources, Robin Harris, Supervision, Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Funding Acquisition, Writing – Original Draft, Writing – Review and Editing, Visualization, Project Administration. Supplemental Information Document S1. Figures S1–S9, Supplemental Genotypes document. Acknowledgements The authors would like to thank Dr. Iswar Hariharan and Dr. David Bilder of UC Berkeley, Dr. Mirka Uhlirova of the University of Cologne, Dr. Seth Bliar of the University of Wisconsin, Dr. Kirsten Guss of Dickinson College, Dr. Baotong Xie of Oregon Health & Science University, Dr. Brian Gebelein of Cincinnati’s Children’s, Dr. Sonsoles Campuzano of the Centro de Biología Molecular Severo Ochoa, Dr. Gerard Campbell of the University of Pittsburgh for stocks and reagents. Current and former members of the Harris lab for feedback, and the Bloomington Stock Center, Molecular Instruments, and Developmental Studies Hybridoma Bank for stocks and reagents. This research has received no external funding. Footnotes In revising the manuscript, we performed substantial new experimental work and textual refinements, as detailed in our point-by-point response. The key improvements include: 1. Strengthened evidence for damage-induced aspr regulation -Per reviewer request, we quantified aspr transcript levels across multiple time points using HCR-FISH with fluorescence quantification (new Fig. 1F), providing a rigorous temporal expression profile. 2. Expanded functional tests linking Aspr to Vestigial-regulated growth -We added new genetic interaction experiments showing that ectopic vg-driven overgrowth during regeneration is significantly suppressed by co-expression of aspr-HA, supporting a mechanistic connection between Aspr and WNT-dependent vg reactivation (new Fig. 5I-N, O). 3. New high-resolution imaging demonstrating extracellular vesicle localization Reviewers questioned whether Aspr puncta were genuinely extracellular. We therefore: -Used Crumbs-GFP and CD8::GFP as apical and membrane markers, -Acquired higher-magnification transverse Z-sections, and -Developed non-permeabilizing staining protocols. These clearly demonstrate that Aspr-positive structures reside apical to the epithelial surface and are distinct from the plasma membrane (new Fig. 7G-H‴, S7R-R′). 4. Evidence that Wg recruitment into vesicles depends on Aspr -We performed aspr RNAi knockdown and showed that the characteristic apical Wg-positive puncta seen after injury fail to form without Aspr (new Fig. 8V-W). 5. Tests demonstrating that Wg is encapsulated within Aspr-positive EVs -Under non-permeabilized staining, Wg signal within EVs is lost (new Fig. S9AB), -Higher-magnification imaging shows Wg localized inside, not just surrounding, Aspr vesicles (new Fig. S9AC). 6. Integration of Wntless (Wls) trafficking into the model -We added new data showing Aspr colocalizes with Wntless-GFP apically (new Fig. 8X-Y‴), supporting a model in which Aspr participates in established Wg secretory pathways. 7. 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