Tissue composition shapes differential skeletal integration strategies during axolotl limb regeneration

Axolotl, Regeneration, Skeleton, Tissue Integration, Osteoclasts, AEC. 25 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 2

26 Limb regeneration requires not only rebuilding the missing structures, but also integrating them with 27 the stump tissues. Osteoclast-mediated tissue resorption is essential for skeletal integration during 28 regeneration. However, given the cellular and structural heterogeneity along the limb skeleton, it is 29 unknown if skeletal tissue composition impacts resorption and, if so, how it is regulated. 30 Here, we show that osteoclast-mediated skeletal resorption is primarily activated in amputations 31 damaging calcified regions of the skeleton, but not in cartilaginous areas. Using a combination of 32 spatial transcriptomics and bulk RNA sequencing, we found that amputations in calcified regions 33 trigger the sustained expression of RANKL and the chemokine Loc138491483/Ccl24-like . We also 34 demonstrate that Loc138491483/Ccl24-like is sufficient to induce osteoclast presence in non-resorbing 35 amputations. Finally, our data suggests that the transcriptomic profile of the apical ectodermal cap is 36 modified according to the underlying tissue types injured by the amputation. 37 Overall, our work reveals that tissue composition at the amputation plane directs important 38 adaptations of the regenerative program to the damaged tissues, particularly regarding integration 39 strategies. These context-dependent responses will ultimately contribute to the near-seamless tissue 40 integration of the regenerating axolotl limb regardless of the amputation position. 41 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 3

42 Limb regeneration in the axolotl ( Ambystoma mexicanum ) is an important model for the study of 43 complex structure re-formation and occurs in sequential, yet largely overlapping phases 1,2. After an 44 amputation, the wound quickly heals through the establishment of a wound epithelium (WE). The 45 immune response is then established by the migration of immune cells into the stump, where they 46 release important factors and help to clear pathogens and cell debris. Meanwhile, cells in the WE 47 proliferate, forming the multilayered apical epithelial cap (AEC) 3. Together, immune cells and the 48 AEC contribute to tissue histolysis by secreting proteolytic enzymes that extensively remodel the 49 stump tissues3,4. Finally, a blastema forms under the AEC, which will then proliferate and ultimately 50 differentiate to regrow the missing tissues of the limb. 51 Although these processes are well established, the mechanisms by which newly formed tissues 52 integrate with mature tissue in the stump remain largely elusive. Remarkably, tissue integration occurs 53 irrespective of the amputation position in the axolotl limb 2,5,6, which means that regenerative 54 integration processes are equally effective regardless of the specific tissue composition affected by the 55 injury. This poses an interesting challenge especially for the regenerating skeleton, as a typical limb 56 skeletal element comprises a variety of cell types arranged in distinct conformations along its length, 57 resulting in positional differences in thickness, stiffness, and relative proportions 7. Likewise, cell and 58 tissue heterogeneity in the skeletal element can greatly differ in the span of a few micrometers, as is 59 evidenced by the diaphyseal (i.e., center) and flanking epiphyseal (i.e., proximal and distal) regions. 60 Epiphyses in the axolotl limb are mostly comprised of chondrocytes, which contribute to the growth 61 of the limb and remain cartilaginous throughout the life of the animal 7. In contrast, the diaphysis is 62 where the primary ossification center first develops, which comprises hypertrophic chondrocytes, 63 osteoblasts, and osteocytes, as well as periskeletal cells and a surrounding calcified extracellular 64 matrix (ECM)7. Moreover, skeletal integration often requires the amalgamation of nascent cartilage 65 with the calcified remains of the amputated skeletal element of the stump 6. Yet, despite these obvious 66 differences, it remains unclear whether or not the mechanisms of skeletal integration differ according 67 to the tissue composition at the amputation site. 68 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 4 Recently, we demonstrated that osteoclast-mediated tissue resorption is important for integrating the 69 regenerated radius and ulna with the previously existing skeletal elements 6. Osteoclasts are large, 70 multinucleated cells that have an immune origin 8. In mammals, these cells differentiate in a stepwise 71 manner from common myeloid progenitors (CMPs) 9,10, which also originate both macrophages and 72 dendritic cell progenitors 8. Osteoclasts resorb skeletal tissue by adhering to the bone surface and 73 degrading the calcified matrix by secreting protons (H +) and proteolytic enzymes such as Cathepsin K 74 (Ctsk) and Matrix Metalloproteinases (MMPs) 9. During axolotl limb regeneration, osteoclast-75 mediated tissue resorption occurs in a short and distinct window of time 6, which contrasts with the 76 long-lasting resorption observed in mammalian bone fractures 11. This suggests that unique underlying 77 regulatory mechanisms might be employed in regenerative integration. 78 In this work, we investigated how osteoclast-mediated skeletal resorption is regulated to promote 79 skeletal integration during limb regeneration. We found that this process is dependent on the 80 composition of the injured tissue, with resorption being triggered specifically by amputations through 81 the calcified diaphysis and primarily used for its regenerative integration. Using a combination of 82 spatial transcriptomics and bulk RNA-seq, we show that the RANK/RANKL system likely 83 orchestrates osteoclast differentiation after diaphysis amputations as early as 3 dpa. Moreover, we also 84 discovered that the previously undiscovered chemokine Loc138491483/Ccl24-like has an important 85 role in promoting osteoclast differentiation and/or recruitment. Finally, we observed that the 86 amputation site could induce significant transcriptomic differences in the AEC. 87 Altogether, our work exploring two amputation planes affecting different tissue types shows that 88 tissue composition at the injury site induces adaptations of histolysis, immune response, and in the 89 AEC, which may synergistically promote skeletal integration. This further demonstrates that early 90 regenerative mechanisms are tailored to the types of tissues affected by the amputation. These 91 adaptations ultimately make possible the successful re-formation of all missing limb tissues and their 92 integration with the mature structure regardless of the amputation position within the limb. 93 94 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 5

95 Osteoclast-mediated skeletal resorption is specifically activated after diaphysis amputations 96 To explore the mechanisms controlling tissue resorption, we chose two amputation planes that, 97 while impacting the same skeletal elements, affected regions with different cellular and ECM 98 compositions. 99 For this, we amputated through the calcified diaphysis and the cartilaginous epiphysis, and 100 compared the extent of tissue resorption after these two amputations. To accurately assess skeletal 101 tissue resorption in these two amputation planes, we utilized Sox9:Sox9-T2a-mCherrynls ( Sox9-102 mCherry) transgenic animals stained with calcein. This allowed for the clear identification of both the 103 cartilaginous skeleton, which expressed mCherry in chondrocytes, as well as the calcified diaphysis, 104 which was marked by the binding of calcein to its mineralized ECM (Fig. 1A). Using this strategy, we 105 were able to simultaneously assess 1) resorption of the remaining stump skeleton (the length of 106 mCherry signal from the elbow joint until the amputation plane); and 2) resorption specifically of the 107 calcified tissue (the length of the calcein-positive region). 108 Forelimbs of Sox9-mCherry/calcein animals were amputated and followed for 18 days. In diaphysis 109 amputated limbs, resorption of the calcified region was first observed at 7 days post-amputation (dpa) 110 in the form of gaps in the calcein staining (Fig. 1B, white arrowheads). By 9 dpa, the mineralized 111 matrix had been significantly resorbed, after which this process considerably slowed up until 18 dpa 112 (Fig. 1B, top row). In contrast, in epiphysis amputations (Fig. 1B, bottom row), both stump skeletal 113 elements and their calcified regions remained relatively unchanged. Quantification of the remaining 114 stump skeleton and respective calcified region length in diaphysis amputations revealed that, on 115 average, approximately 20% of the total length of the skeletal elements and up to 40% of the calcified 116 region of radii and ulnas were resorbed by 18 days (Fig. 1C, D). Furthermore, most of this resorption 117 occurred between 7 and 9 dpa, which agreed with previous reports6. 118 Epiphysis amputations, on the other hand, did not exhibit such extensive resorption (Fig. 1B, D). 119 However, some variations in the length of cartilage and calcified regions at 9 and 11 dpa could be 120 observed (Fig. 1C, D). To further visualize the cellular and ECM structure of tissues after diaphysis 121 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 6 and epiphysis amputations, we analyzed the histology of limb sections after these two amputations at 122 7 and 9 dpa. This showed that, in amputated epiphyses, the cartilaginous matrix of the radius and ulna 123 had undergone considerable remodeling, and that a few of the most distal chondrocytes had even 124 disappeared entirely (Fig. 1E). This explained the observed variations in the length of whole skeletal 125 elements, and matched our previous observations of cartilage undergoing histolysis12. 126 Given their roles as the main cellular effectors of skeletal tissue resorption, we analyzed osteoclast 127 prevalence in the two amputations. As it was expected, diaphysis amputations showed robust 128 osteoclast presence surrounding the calcified region of both skeletal elements at 7 and 9 dpa. In 129 contrast, only a few osteoclasts were observed asso ciated to the skeletal elements in epiphysis 130 amputations at these time points (Fig. 1E, black arrowheads, Fig. E insets). Next, we decided to 131 follow osteoclasts in vivo over time after diaphysis and epiphysis amputations. For that, we combined 132 the reporter line Sox9-mCherry with Ctsk-eGFP transgenic animals, in which eGFP is driven by the 133 promoter of the mature osteoclast marker Ctsk 6, to generate Sox9::Ctsk animals. In vivo imaging 134 confirmed the extensive presence of osteoclasts at 7 and 9 dpa in diaphysis amputations, which 135 quickly decreased at 11 dpa, and was cleared by 15 dpa (Fig. 2A, top row). In contrast, minimal to no 136 osteoclast recruitment and/or differentiation was initiated in epiphysis amputations (Fig. 2A, bottom 137 row). 138 Finally, we assessed the endogenous expression of the osteoclast marker Ctsk by Hybridization 139 Chain Reaction (HCR) to determine when osteoclast presence is first differentially established in 140 diaphysis and epiphysis amputations. While examining the Ctsk nucleotide sequence, we found that 141 another gene, Loc138578972, was present in an adjacent region and annotated as Ctsk-like (Fig. S1A). 142 The predicted nucleotide coding sequence of this gene was 73.6% identical to the one of Ctsk (Fig. 143 S1B). Moreover, the predicted protein sequence of Loc138578972 shared 73.4% and 73.7% sequence 144 identity with the axolotl Ctsk (Fig. S1C, Fig. S1F) and human CTSK peptide, respectively (Fig. S1E, 145 S1F). We thus concluded that the axolotl genome contains at least one additional Ctsk-related gene 146 and, in keeping with the most current axolotl annotation (UKY_AmexF1_1, GCF_040938575.1), the 147 gene annotated as Ctsk will continue to be referred to as “Ctsk”, whereas the gene Loc138578972 will 148 be referred to as “Ctsk-like” for the remainder of this work. 149 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 7 Analysis of both Ctsk and Ctsk-like expression showed that Ctsk-expressing cells were already 150 present at 3 dpa in diaphysis and epiphysis amputations, both in close proximity to the skeletal 151 elements and in the mesenchyme (Fig. 2B). However, large multinucleated Ctsk-positive cells, which 152 are the hallmarks of mature osteoclasts, were almost exclusively associated to diaphysis amputations 153 at 3 dpa, and were seen in even larger numbers at 5 dpa (Fig. 2B, white arrowheads, insets). Ctsk-like 154 exhibited a similar expression pattern and was largely co-expressed with Ctsk at both time points. 155 Altogether, our results show that extensive osteoclast-mediated skeletal tissue resorption is a tissue-156 dependent event, being activated by amputations directly affecting, or in close proximity to, the 157 calcified diaphyseal regions of the skeletal elements. Moreover, osteoclast recruitment and/or 158 differentiation starts early within regeneration, with Ctsk/Ctsk-like -positive multinucleated osteoclasts 159 appearing as early as 3 dpa at the injured skeletal elements in diaphysis amputations. 160 Systemic and local calcium do not significantly impact osteoclast presence in regenerating limbs 161 As one major difference between diaphysis and epiphysis amputations is the damage to the sheath of 162 calcified tissue surrounding the skeletal elements7, we explored the role of systemic and local calcium 163 in the activation of osteoclast-mediated skeletal resorption. 164 At the systemic level, blood plasma measurements in intact animals were consistent with previously 165 reported values13. While we observed some variation in calcium concentrations between intact and 166 amputated individuals especially in early time points, no significant difference between conditions 167 was observed (Fig. 3A). 168 We next investigated if, instead, local changes in calcium levels could have a role in osteoclast 169 recruitment. We thus injected amputated limbs of Sox9::Ctsk animals with either BAPTA or CaSO4 to 170 decrease or increase extracellular calcium levels in the tissue, respectively, and followed osteoclast 171 dynamics after diaphysis and epiphysis amputations (Fig. 3B). Injections of BAPTA 10mM (Fig. 3C-172 E) or CaSO4 15mM (Fig. 3F-H) had no effect on osteoclast presence in either amputation plane. 173 Hence, these results indicate that systemic calcium concentrations do not significantly change due to 174 regeneration, nor due to different amputations affecting diaphyseal or epiphyseal regions. 175 Additionally, in our experiments, changes in local calcium levels were not sufficient to trigger 176 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 8 osteoclast recruitment and/or differentiation into the damaged skeletal tissue, particularly in epiphysis 177 amputations. Overall, this suggests that neither systemic nor local changes in extracellular calcium 178 concentrations during regeneration are sufficient to induce significant osteoclast 179 recruitment/differentiation. However, we cannot rule out that other factors work together with either 180 systemic or local extracellular calcium levels to recruit or differentiate osteoclasts. 181 Spatial transcriptomics reveals differential gene expression profiles in diaphysis and epiphysis 182 amputations 183 Our data so far suggested that skeletal tissue could be involved in osteoclast recruitment and/or 184 differentiation observed in diaphysis-amputated limbs. Thus, we asked if gene expression differences 185 could inform us on regulatory factors underlying osteoclast-mediated differential resorption. To 186 address this, we used spatial transcriptomics in diaphysis- and epiphysis-amputated limbs at 3- and 5 187 dpa (Fig. 4A) – the two time points in which differential osteoclast presence first becomes evident – to 188 investigate differences in gene expression between these two conditions. 189 Clustering analysis of spatial expression dots from all samples combined revealed 19 clusters 190 representing all major tissue types contained in our tissue sections, including epidermis (clusters 5 and 191 7), muscle (clusters 4, 9, and 13), cartilage (clusters 8, and 15), periskeleton/bone (cluster 18), and 192 nerves (cluster 17). We also detected regeneration-specific clusters, particularly a blastema cluster 193 (cluster 1) enriched in the expression of Kazald214,15, two clusters associated with tissue histolysis 194 (clusters 12 and 14), and one cluster representing the AEC (cluster 16) (Fig. 4A-C; Fig. S2A-B). 195 Analysis of the expression of the resorption-associated factors Nfatc1, Ctsk, Ctsk-like, and Acp5 found 196 that these genes were highly expressed in cluster 2 (Fig. 4D, Fig. S2B). Mapping these spots back to 197 the tissue sections revealed that these genes were especially represented in diaphysis-amputated 198 skeletal elements at both 3 and 5 dpa (Fig. 4F). This, together with the fact that approximately 70% of 199 this cluster was derived from diaphysis-amputated limbs, caused us to annotate this cluster as a 200 resorption cluster (Fig. 4E inset, Fig. S2B-C). 201 As expected from having a spatial dataset with supra-cellular resolution combined with the high 202 mobility of immune cells, we could not isolate a specific immune spatial cluster. Instead, we found 203 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 9 immune markers spread across multiple clusters, including Perforin1-like (Prf1-like), Proteoglycan 3 204 (Prg3), and C-X-C motif chemokine ligand 12 (Cxcl12) in the mesenchyme (cluster 6), and two genes 205 annotated as Macrophage expressed 1-like (Mpeg1-like) in the epidermis (cluster 7) and AEC (cluster 206 16). Notably, there was an enrichment of genes related to macrophage/monocyte function like 207 Macrophage receptor with collagenous structure (Marco), a third Mpeg1-like gene, Cd68, and Cd14 208 in the resorption cluster (cluster 2). This points at the possibility that, similarly to mammals 16,17, these 209 myeloid cells could act as possible sources of osteoclast progenitors during regeneration. 210 Taken together, we show that spatial transcriptomics is able to identify clear differences in gene 211 expression between diaphysis and epiphysis amputations, and that a specific resorption cluster 212 enriched in myeloid markers is predominately present in diaphysis-amputated limbs. 213 The RANK/RANKL system likely orchestrates the activation of osteoclast-mediated skeletal resorption 214 In examined vertebrates, osteoclast progenitors differentiate into mature osteoclasts under the 215 influence of RANKL ( Tnfsf11) and RANK ( Tnfrsf11a)18–20. In our spatial dataset, we found that the 216 majority of spatial dots in the resorption cluster (cluster 2) did indeed express RANKL and RANK (Fig. 217 5A, Fig. S3A), with RANK slightly upregulated in diaphysis amputations at both time points. In 218 contrast, differences in RANKL expression were only observed at 5 dpa, being expressed in more 219 spatial dots and with overall higher expression levels in diaphysis amputations (Fig. 5B). 220 To validate our spatial data and identify the cells expressing RANK and RANKL, we performed HCR 221 for these two genes in diaphysis and epiphysis amputations at 3, 5, and 7 dpa (Fig. 5C). In epiphysis-222 amputated limbs, RANKL was observed primarily in the cartilage cells closest to the AEC at 3 dpa. Its 223 expression was decreased at 5 dpa and, by 7 dpa, only very low levels of RANKL were detected in 224 these limbs (Fig. 5C, bottom row). This contrasted with diaphysis-amputated limbs, in which RANKL 225 was robustly expressed, especially in periskeletal cells and hypertrophic chondrocytes at all analyzed 226 time points (Fig. 5C, top row). Furthermore, HCR staining for RANKL and Ctsk showed that Ctsk + 227 cells were found in close proximity with cells expressing RANKL. 228 On the other hand, RANK expression was more prevalent in diaphysis-amputated limbs at 3 and 5 229 dpa and was frequently co-expressed with Ctsk (Fig. S3B), suggesting that these cells were 230 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 10 differentiating osteoclasts. We further validated this by assessing the expression of Nfatc1 , the master 231 transcriptional factor of osteoclastogenesis 21 that promotes the expression of Ctsk and components of 232 the vacuolar V-A TPase 22. Indeed, we found that RANK-expressing cells co-expressed Nfatc1, 233 especially in diaphysis-amputated limbs (Fig. S3C). Interestingly, in these limbs, we also observed 234 cells that co-expressed RANK and Nfatc1 , but not Ctsk, suggesting that these were osteoclast 235 progenitors undergoing differentiation (Fig. S3C, white arrowheads). 236 Overall, our data indicates that RANKL expression is upregulated and sustained in periskeletal cells 237 and hypertrophic chondrocytes upon calcified diaphysis amputations, which may drive the 238 differentiation of RANK+/Nfatc1+ precursors into mature osteoclasts. In contrast, in epiphysis 239 amputations, RANKL is only expressed in distal chondrocytes, and its levels quickly decrease. This 240 suggests that, similar to mammalian osteoclastogenesis, the RANK/RANKL system has a key role in 241 osteoclast differentiation during axolotl regeneration, and that the differential activation of RANKL 242 expression after injury to the calcified diaphyseal region likely triggers tissue-dependent skeletal 243 resorption. 244 Loc138491483/Ccl24-like is sufficient to induce osteoclast presence in non-resorptive amputations 245 As osteoclast-mediated tissue resorption occurs in a relatively short and well-defined time window 246 during limb regeneration, we next searched for regeneration-specific factors that could regulate this 247 process. 248 For this, we reasoned that osteoclast progenitors and/or immature osteoclasts, identified by Nfatc1 249 expression, would be in close proximity to any putative signal factor at 3 and 5 dpa. Our analysis 250 revealed that the top 15 differentially expressed genes in Nfatc1-enriched spatial spots (Log 2 251 Expression > 2) were enriched in genes heavily involved in osteoclast function, such as V-ATPase 252 subunit genes ( Atp6v0c, Atp6v0d2, Atp6v1b2) and MMPs/ECM remodeling genes ( Ctsk, Ctsk-like, 253 Mmp9) (Fig. 6A). However, there was one gene, Loc138491483 (hereafter referred to as Loc483), that 254 was seemingly not directly related to osteoclast function while still being associated with the 255 resorption cluster (cluster 2) (Fig. 6B, C) and to the skeletal tissue in diaphysis amputations (Fig. 6D). 256 We then validated these findings by HCR at an earlier time point (1 dpa) and at 3 and 5 dpa. While no 257 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 11 differences were observed in the expression of this gene at 1 dpa, Loc483 became highly expressed in 258 diaphysis-amputated limbs both at 3 and 5 dpa when compared to epiphysis-amputated limbs (Fig. 259 6E). 260 Interestingly, Loc483 is annotated in the axolotl genome as C-C motif chemokine 24-like . A 261 phylogenetic search via the webserver aLeaves 23 revealed that its most closely related protein 262 sequence matches were Monocyte Chemoattractant Protein-1/C-C motif chemokine 2 (Mcp1/Ccl2) in 263 the caecilian Rhinatrema bivittatum and C-C motif chemokine 4 (Ccl4) in Xenopus tropicalis . 264 Additional matches could only be found in two species of sharks and in the paddlefish (Fig. S4), 265 suggesting that, despite its annotation, this gene is probably not an ortholog to the human CCL24 266 gene, and that amphibians are, to date, the only tetrapods identified to possess Loc483. 267 Nevertheless, being a chemokine made Loc483 a promising candidate as a chemoattractant for 268 macrophages and monocytes, both previously reported as sources of osteoclast progenitors 16,17,24. 269 Thus, to test if Loc483 would have the potential to promote osteoclast recruitment and/or 270 differentiation, we overexpressed this gene in Sox9::Ctsk animals. For that, we co-electroporated 271 blastemas with a construct containing the coding sequence of Loc483 under the control of the 272 ubiquitous promoter CAGGS (CAGGS-Loc483), together with a reporter plasmid containing mCherry 273 driven by the same promoter ( GAGGS-mCherry). The contralateral blastema was electroporated only 274 with the GAGGS-mCherry construct as a control. We found that, in diaphysis amputations, 275 overexpression of Loc483 had no effect on the presence of osteoclasts (Fig. 6F, G). However, Loc483 276 was able to ectopically induce the presence of osteoclasts in epiphysis-amputated limbs (Fig. 6F, H). 277 Thus, these results show that Loc483 is likely a chemokine within the amphibian immune system, 278 and that its expression is sufficient to recruit and/or differentiate osteoclasts to the amputation site. 279 Diaphysis and epiphysis amputations differentially impact other processes to related regeneration 280 To complement the spatial information of our datasets and get an overview of gene expression in 281 diaphysis- and epiphysis-amputated limbs, we performed bulk RNA sequencing (RNA-seq) using the 282 distal-most region of regenerating limbs. This found 100 and 80 differentially expressed genes 283 (DEGs) between the two amputation planes at 3 and 5 dpa, respectively. Unexpectedly, genes related 284 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 12 to bone resorption and osteoclast function such as Ctsk, Ctsk-like, Nfatc1 , and Acp5, although 285 upregulated after amputations, were not differentially expressed between diaphysis- and epiphysis-286 amputated limbs in our bulk RNA-seq dataset (Fig. S5A-E). This was especially surprising for the 287 first three of these genes, given that our spatial transcriptomics and subsequent validation by HCR 288 staining showed them as being upregulated mainly in diaphysis amputations as early as 3 dpa (Fig. 289 2B, Fig. S3B, C). The absence of differential expression in bulk RNA-seq could either be due to the 290 technical limitations of this technique, in which cellular resolution is lost and subtle changes in gene 291 expression cannot be easily detected if genes are only present in a limited number of cells, or due to 292 the expression of these genes being bolstered by their presence in processes unrelated to 293 osteoclastogenesis18,25–28. 294 Many of the detected DEGs, instead, were either uncharacterized genes (i.e., protein-coding genes 295 with no further annotation) or predicted to be non-coding RNAs (ncRNAs). Indeed, a look into the top 296 15 to 20 upregulated DEGs in diaphysis and epiphysis amputations at the two time points showed that 297 30% to 60% of these genes were computationally annotated as ncRNAs (Fig. S5A-D). Consequently, 298 we filtered out both ncRNAs and uncharacterized genes for all further downstream analyses. This 299 resulted in 60 and 30 upregulated protein-coding DEGs in epiphysis amputations at 3 and 5 dpa, 300 respectively (Fig. S5A-B), while diaphysis amputations exhibited only 10 and 8 upregulated protein-301 coding DEGs at these time points, respectively (Fig. S5C-D). 302 Gene ontology (GO) analysis for biological processes with these genes showed that upregulated 303 DEGs in 3 dpa epiphysis amputations were enriched in terms associated with response against viruses 304 and with protein folding (Fig. S5F). In contrast, upregulated DEGs in 3 dpa diaphysis amputations 305 were mostly associated with muscle tissue, as we saw Myl4, Loc138582818/Myh4-like, and 306 Loc138573356/Tpm1-like representing 3 out of the 10 upregulated DEGs. Importantly, Loc483 was 307 also significantly upregulated in diaphysis amputations at 3 dpa (Fig. S5C, G), which agreed with our 308 spatial transcriptomics datasets, as well as our previous expression and functional validations. 309 As for at 5 dpa, DEGs identified in epiphysis-amputated limbs were significantly enriched for GO 310 terms associated with epidermis and with ECM production, whereas only Cell adhesion 311 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 13 (GO:0007155) was found to be significantly associated to diaphysis-amputated limbs at the same time 312 point, with upregulation of the genes Postn, Msln, and Thbs4 (Fig. S5F). 313 Thus, these results suggest that the amputation plane, and specifically the types of tissues damaged 314 within it, might also affect other regenerative processes within the first stages of regeneration, 315 particularly ones related to muscle tissue, protein folding, and epidermis development, as well as 316 aspects of the immune response. 317 The transcriptomic profile of the AEC is impacted by tissue composition at the amputation plane 318 When we performed GO analysis for cellular component (CC) and molecular function (MF) instead, 319 we saw that the DEGs found in diaphysis-amputated limbs at 3 and 5 dpa were also significantly 320 enriched in ECM and muscle-related CC terms (Fig. 7A). Moreover, MF terms enriched for these 321 amputations at 3 dpa were generally in agreement with the CC analysis, as they were similarly 322 associated to muscle function, although no MF term was significantly enriched at 5 dpa. In contrast, 323 DEGs in 3 and 5 dpa epiphysis-amputated limbs were enriched for CC terms associated to secretion 324 or extracellular space, as well as to epidermis. Meanwhile, enriched MF terms in this amputation 325 plane at 3 dpa were associated to protein folding, acetylcholine receptor signaling, and exosome 326 function (Fig. 7A). Interestingly, by 5 dpa, these MF terms changed to being enriched in protease 327 inhibitor-related ones, such as Serine protease inhibitor (KW-0722) and Serine-type endopeptidase 328 inhibitor activity (GO:0004867). 329 Given the prevalence of GO terms related to epidermis and secretion enriched in upregulated DEGs 330 in the bulk RNA-seq from epiphysis-amputated limbs, we hypothesized that these genes were mostly 331 being expressed in the AEC. To explore this possibility, we leveraged our spatial transcriptomics 332 dataset to locate the expression of these DEGs within the context of the tissue. For that, we first took 333 all DEGs from the bulk RNA-seq previously used for GO analysis, removed genes that were not 334 expressed or were very lowly expressed in the spatial transcriptomics dataset (Log 2 Expression <1). 335 We then used Loupe Browser to display the combined average expression levels of upregulated DEGs 336 in our spatial tissue sections at 3 and 5 dpa of epiphysis (n= 43 and 36 genes, respectively) and 337 diaphysis amputations (n=8 and 7 genes, respectively). This approach revealed that, on average, 338 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 14 upregulated DEGs found by bulk RNA-seq in epiphysis amputations at 3 and 5 dpa (Fig. 7B) tended 339 to be expressed in the AEC. Indeed, visualization of representative upregulated DEGs in epiphysis-340 amputated limbs at 3 ( Psat1) and 5 dpa ( Klf17 and Csdn), found that the spatial dots with the highest 341 expression levels were localized to the WE/AEC, with variable numbers of low-expressing dots 342 scattered throughout the inner limb tissue (Fig. S6A). This was also observed when the average 343 expression of DEGs annotated as ncRNAs was analyzed (Fig. S6B). However, no such trend was 344 detected with upregulated DEGs in diaphysis limbs at either time point (Fig. 7C, Fig. S6C). The 345 combined average expression of upregulated DEGs in this amputation plane was instead found 346 throughout the limb tissue, which included the epidermis and AEC ( Msln), connective tissue ( Lum, 347 Postn), muscle (Myl4, Myh4-like, Tpm1-like), nerves (Mpz), and tendons/joints (Thbs4) (Fig. 7D). 348 Surprisingly, the combined average expression of genes in the spatial transcriptomics sections did 349 not seem to reflect the differences in expression of DEGs found by bulk RNA-seq. In particular, 350 DEGs upregulated in epiphysis amputations at 5 dpa identified by bulk RNA-seq did not appear to be 351 altered in spatial transcriptomics compared to diaphysis amputations. This could be explained as 352 either resulting from the flattening of differences between diaphysis and epiphysis amputations caused 353 by averaging the expression of multiple genes with different expression levels, by saturation of the 354 spatial dots with highly expressed genes, or simply by the fact that the analyzed tissue sections did not 355 contain the particular region of the AEC where gene expression of these DEGs was at its strongest. 356 Altogether, the transcriptomic profile differences of the AEC in diaphysis vs. epiphysis amputations 357 suggest positional adaptations of its gene expression profile during limb regeneration, particularly in 358 the secretory profile. However, more work is still needed to identify how these modifications 359 contribute to tissue regeneration and integration according to the amputation position, or what the role 360 is of the many differentially expressed ncRNAs in limb regeneration. 361

362 A crucial aspect of successful limb regeneration is the robust integration between the tissues of the 363 newly regenerated body part and the previously existing structure. Skeletal tissue integration is 364 especially complicated by its spatially and temporally dynamic tissue composition, in which a fully 365 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 15 cartilaginous skeleton at the end of development becomes progressively mineralized until achieving 366 adult patterns of ossification around sexual maturation 7. Recently, our lab showed that the tightly-367 controlled clearing of skeletal tissue by osteoclasts is essential for skeletal integration 6. However, it 368 was unknown if osteoclast-mediated resorption promoted integration in amputation planes exposing 369 different cell and matrix compositions. Moreover, it was also unclear how this process was regulated, 370 especially given its fast-acting nature during regeneration that contrasts with the slow bone 371 remodeling in fractures. 372 Here we show that skeletal integration strategies during axolotl limb regeneration are customized to 373 the tissues damaged by the amputation, and that these adaptations are initiated early on. We 374 demonstrate that osteoclast-mediated skeletal resorption is primarily activated in amputations through 375 calcified diaphyseal regions, but not through the cartilaginous epiphysis. We also demonstrate that the 376 expression of the chemokine Loc138491483/Ccl24-like is specifically sustained in diaphysis-377 amputated limbs and is sufficient to promote osteoclast presence in amputated limbs. Finally, we find 378 that the transcriptomic profile of the AEC is modified by the amputation plane, suggesting that this 379 structure may be adapted to the types of tissues injured by the amputation. 380 Previous studies found that successful tissue integration in the axolotl is impacted by factors like 381 defect size 29–31, vitamin D 32 and positional identity incompatibility 33. In this work, we set to 382 investigate the importance of the skeletal composition itself at the amputation plane. To address this, 383 we performed amputations in two sites that were closely localized within the same limb segment, and, 384 thus mainly differed on the affected skeletal tissue composition. We found that, unlike with injuries 385 affecting cartilaginous epiphyses, amputations in the calcified diaphysis undergo extensive osteoclast 386 recruitment and/or differentiation and tissue resorption. However, exactly how osteoclast-mediated 387 tissue resorption contributes to skeletal integration in diaphyseal amputations is still unknown. We 388 propose two non-mutually exclusive hypothesis, which may even act synergistically. 389 The first hypothesis is that this is a specialized additional step of histolysis that facilitates the 390 attachment of nascent cartilage cells to the skeletal stump. Tissue histolysis is a key event during 391 regeneration4,34–36 in which tissue stiffness greatly decreases 12. Furthermore, ECM remodeling has 392 been hypothesized to contribute to tissue integration in regenerating newt joints 37. It is thus likely that 393 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 16 osteoclast-mediated tissue resorption primes the mature skeleton for tissue integration by changing the 394 composition and rigidity of the calcified ECM so that differentiating chondrocytes can be correctly 395 incorporated. 396 A second hypothesis is that skeletal resorption could help release diaphyseal periskeletal cells. These 397 cells, together with dermal and interstitial fibroblasts, contribute to the regeneration of proximal 398 skeletal tissues 38–41 and are thus promising candidates to orchestrate skeletal integration. This is 399 supported by our observations that periskeletal cells are strongly influenced by the amputation plane, 400 as evidenced by their high and sustained expression of RANKL upon diaphysis amputations. The 401 activation of RANKL in periskeletal cells would 1) help coordinate skeletal tissue integration in the 402 calcified diaphysis by promoting the differentiation of myeloid immune cells into osteoclasts; and 2) 403 stimulate osteoclast maturation directly on the surface of the calcified skeletal element. 404 Our work also revealed that immune responses during limb regeneration are context-dependent and 405 adapted to promote skeletal tissue integration according to its composition. Osteoclasts derive from 406 the myeloid lineage, in which CMPs ultimately undergo terminal differentiation into fully mature 407 osteoclasts8,9. Thus, the fact that osteoclast-mediated tissue resorption is primarily triggered by 408 diaphysis amputations demonstrates that the immune response is adapted to the injured tissues. 409 Another adaptation of the immune system was the sustained expression of the previously 410 uncharacterized chemokine Loc483 in diaphysis amputations, which we demonstrated to be sufficient 411 to promote the presence of osteoclasts in regenerating limbs. The closest match to Loc483 is the 412 caecilian Mcp1/Ccl2 which, in mammals, is reported to be a potent chemotactic factor for 413 monocytes/macrophages42–44 and a promoter of osteoclast maturation 45. It is thus possible that, upon 414 calcified diaphysis injuries, Loc483 expression could have a similar role in the axolotl. Further studies 415 are needed to elucidate how Loc483 is regulated in diaphyseal amputations, as well as its role on 416 osteoclastogenesis during regeneration. 417 Finally, our study further revealed that differences in the tissues affected by the amputation plane 418 impact the transcriptomic profile of the AEC, suggesting that the AEC is a dynamic structure that can 419 likewise be adapted to specific injury contexts. This agrees with our previous study showing that the 420 AEC is important in osteoclast-mediated skeletal resorption6. However, it is still unclear how the AEC 421 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 17 impacts osteoclastogenesis, and how, in turn, the underlying tissue influences the AEC. One way by 422 which an amputation-dependent AEC could influence regeneration is through the differential secretion 423 of serine protease inhibitors, which could moderate or even terminate the histolytic process by 424 inhibiting the conversion of MMPs into their active forms 46,47. Intriguingly, we also observed many 425 genes annotated as ncRNAs being differentially expressed in the AEC in our two amputation models. 426 Given their complex biological roles 48,49, ncRNA function in the AEC could become an exciting new 427 field of study. 428 Ultimately, the fact that tissue integration mechanisms can be customized to the damaged skeletal 429 tissues highlights both the robustness and adaptability of regeneration in the axolotl. It would thus be 430 interesting to explore whether similar adaptations happen in other tissues that also display positional 431 differences in cell type and ECM composition, such as the muscle50. 432 Finally, the association between the amputation position and regenerative outcomes has been 433 reported in other models. In Xenopus laevis, regenerative potential depends on the tissues affected by 434 the amputation, and regeneration efficiency correlates inversely with the ossification status of the 435 skeletal element51,52. In neonatal mice, only amputations through the long bones in the limb, but not 436 through joints, can activate chondrocyte proliferation 53. These reports and our study thus emphasize 437 the need to, by better understanding customized regenerative responses, start challenging the 438 assumption that limb regeneration occurs using a one-size-fits-all molecular milieu. 439 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 18

440 We thank past and current members of the Sandoval-Guzmán lab for continuous support and 441 companionship during the development of this work. We are particularly grateful to Susanne Weiche 442 for their excellent technical assistance, to Maximilian Krause for his valuable comments on the 443 manuscript, and to Anja Wagner, Beate Gruhl, and Judith Konantz for their dedication to the axolotl 444 care. This work was supported by core facilities of the Technology Platform of the Center for 445 Molecular and Cellular Bioengineering (CMCB) of the TU Dresden, namely the Genome Center, the 446 Light Microscopy Facility, and the Histology Facility. 447 Funding 448 R.A. was supported by an Alexander von Humboldt-Stiftung research fellowship (PRT 1208176 449 HFST-P) and a Deutsche Forschungsgemeinschaft (DFG) Eigene Stelle Grant (AI 214/1-1, Project 450 number 523178173). S.D.K. was supported by the Dresden International Graduate School for 451 Biomedicine and Bioengineering (DIGS-BB) graduate program. M.A.C. and D.B.G. were supported 452 by ERASMUS+ traineeship mobility program. The work at the TU Dresden is co-financed with 453 tax revenues based on the budget agreed by the Saxon Landtag. 454 Conflicts of Interest 455 The authors have no conflicts of interest to declare. 456 Author Contributions 457 R.A. and T.S-G. conceived the study. R.A., C.A., and T.S-G. and acquired funding. R.A. designed 458 and performed most experiments, analyzed most data, and wrote the manuscript. S.D.K., K.B., 459 M.A.C., D.B.G and Y.S. assisted with experimental work. R.A. and S.D.K. processed and analyzed 460 bulk RNA-Seq data. R.A., U.A.F., and S.D.K.., processed and analyzed spatial transcriptomic data. 461 C.A. advised on the project. T.S-G provided supervision, critically revised and edited the manuscript. 462 All authors proofread and revised the manuscript. 463 464 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 19

465 Animal Husbandry and lines 466 Husbandry and experimental procedures were performed according to the Animal Ethics Committee 467 of the State of Saxony, Germany. Animals used were selected by their size (snout to tail = ST; snout 468 to vent = SV). All experiments in this work were performed in axolotls between 6.0 and 9.0 cm ST. 469 Axolotl husbandry was performed in the CRTD axolotl facility using methodology adapted from 54 470 and according to the European Directive 2010/63/EU, Annex III, Table 9.1. Axolotls were kept in 18-471 19°C water in a 12 h light/12 h dark cycle and a room temperature of 20-22°C. Animals were housed 472 in individual tanks categorized by a water surface (WS) area and a minimum water height (MWH). 473 Axolotls of a size up to 5 cm SV were maintained in tanks with a WS of 180 cm 2 and MWH of 4.5 474 cm. Axolotls up to 9 cm SV were maintained in tanks with a WS of 448 cm2 and MWH of 8 cm. 475 White axolotls (d/d) were used for most of the experiments. Transgenic lines used included the 476 previously published C-Ti t/+(Sox9:Sox9-T2a-mCherry)ETNKA (referred as Sox9-mCherry )55 and 477 TgTol2(Drer.Ctsk:eGFP)TSG (referred to as Ctsk-eGFP)6. 478 Animal procedures 479 Amputations were performed in the lower arm under an Olympus SZX16 stereomicroscope. For all 480 amputations, animals were anesthetized with 0.01% benzocaine (Sigma-Aldrich, #E1501) solution. 481 Diaphysis amputations were performed in the middle of the calcified diaphysis region of the radius 482 and ulna, whereas epiphysis amputations were conducted in the distal cartilaginous epiphysis of the 483 same skeletal elements. After surgical procedure, animals were returned to the benzocaine solution 484 and allowed to recover for 10 min prior to be transferred back to swimming water. 485 In vivo skeletal staining was performed using calcein (Sigma-Aldrich, #A5533) before amputations. 486 A 0.1% solution of calcein in swimming water was prepared and animals were submerged in this 487 solution for 5–10 min in the dark. After staining, axolotls were transferred to a tank with clean 488 swimming water, which was changed as many times until the water was clear, and amputated shortly 489 after. 490 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 20 Live imaging was conducted at specific time points in anesthetized d/d or transgenic animals by 491 placing them in a 100 mm petri dish, and positioning the limb accordingly. An Olympus SZX16 492 stereoscope microscope (SDF Plapo 0.75xPF) with fluorescence module was used to acquire images. 493 Length measurements of the stump skeletal elements and calcified region were performed in scaled 494 images using the line measurement tool in FIJI. 495 Tissue collection 496 Tissue collection was performed by euthanizing animals by immersion in a lethal dosage of 497 anesthesia (0.1% benzocaine). 498 For paraffin embedding and HCR, limbs were collected and fixed in 1x MEMFa (MOPS 0.1M pH 499 7.4, EGTA 2mM, MgSO4 × 7 H2O 1mM, 3.7% formaldehyde) for at least 1 overnight at 4°C. For 500 RNA-seq experiments, 1 to 1.5 mm of tissue proximal to the amputation plane was excised, flash 501 frozen in liquid nitrogen, and stored at -80°C until processed for RNA extraction. 502 For plasma measurements, blood was collected directly from the heart immediately after euthanasia 503 using heparin-coated pipette tips into heparin-coated tubes on ice. Then, samples were centrifuged 10 504 min at 3000 g, the upper phase was collected into a new heparin-coated tube, and were kept at -80°C 505 until further processing. 506 Calcium measurements in blood plasma 507 Calcium measurement from blood plasma were performed using a Dri-Chem NX600 (Fujifilm). 508 10µl of plasma from intact, diaphysis, and epiphys is amputations samples were pipetted into a Fuji 509 DRI-Chem Slide (Fujifilm, Ca-P III #2350) and calcium concentration was measured. 510 Ctsk and Ctsk-like sequence alignments 511 Sequences were aligned using EMBOSS Needle Pairwise sequence alignment56 and processed with 512 Sequence Manipulation Suite57. 513 RNA extraction, library preparation and bulk RNA sequencing 514 Sequencing was performed using 3 animals (biological replicates) per amputation site and per 515 timepoint, and each biological sample resulting from the pooling of the two amputated forelimbs of 516 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 21 each animal. RNA extraction was performed using RNAeasy Mini Plus Kit (Qiagen, #74134) 517 according to the manufacturer's instructions. Samples were disrupted and homogenized using a 518 Polytron tissue homogenizer (Kinematica, #PT1600E) in 350 µl of RLT Plus Buffer containing β -519 mercaptoethanol (Sigma, #M625). Extracted RNA was stored at -80 until processed for sequencing. 520 RNA sequencing libraries were prepared using Watchmaker mRNA Library Prep (Watchmaker 521 Genomics, #7K0078) on Biomek i7 with estimated fragment sizes of 300 - 400 bp. Poly-dT pull down 522 enrichment of mRNA was performed before sequencing 101 bp paired-end reads on an Illumina 523 NovaSeq 6000 (Illumina), generating between 50 million read pairs per sample. RNA-seq raw data 524 (fastq) has been deposited in NCBI under the Gene Expression Omnibus (GEO) accession code 525 GSEXXXXXX. 526 Bulk RNA-seq read mapping and expression analysis 527 Generated reads from diaphysis and epiphysis amputated limbs were mapped against the current 528 axolotl reference genome available from NCBI (UKY_AmexF1_1; GCF_040938575.1) using 529 HISAT2 v2.2.158. HISAT2 was run through the command line with default parameters, and a known-530 splicesite-infile created from the corresponding gtf annotation file via the 531 hisat2_extract_splice_sites.py command. StringTie (version 2.2.1 59,60) was then run through the 532 command line with standard parameters and the option of assembling novel transcripts to produce a 533 Merged Transcripts annotation file that was used for transcript quantification. Finally, normalized 534 counts per million (CPM) values for each sample were calculated using the Bioconductor package 535 edgeR (version 3.40.2 61), for R (version 4.2.2 62). Raw gene counts for mandible and limb can be 536 found in Table S2. Normalized gene counts (CPM) are provided in Table SX. 537 Gene Ontology (GO) analysis of differentially expressed genes 538 From the full list of genes found to be differentially expressed in each condition, ncRNAs and 539 uncharacterized genes were filtered. The remaining genes of interest were analyzed for significantly 540 enriched GO terms via DAVID v6.8 63 using default parameters, and are available in Tables SX-SX. 541 GO enrichment terms were considered statistically significant when p < 0.01. 542 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 22 Spatial transcriptomics sequencing and analysis 543 Spatial transcriptomics was performed using first generation Visium Spatial Gene Expression 544 System for Fresh Frozen tissue (10X Genomics) as previously described 64. Briefly, animals 8–9cm 545 snout to tail were amputated in diaphysis and epiphysis regions and allowed to regenerate until 3 or 5 546 dpa. Limbs were then harvested at the level of the upper arm, flash frozen in OCT, and then stored at 547 −80 °C. Samples were cryosectioned at -11/12 °C (chamber)/ -31°C (blade) at a thickness of 10 μ m. 548 Optimization and gene expression assays were carried out according to the manufacturer’s 549 instructions. Briefly, slides were fixed in −20 °C methanol, dried with isopropanol, and stained with 550 H&E. A tile scan of all capture areas was generated using an OlympusOVK automated slide scanner 551 system with a color camera and fluorescent module. For tissue optimization, enzymatic 552 permeabilization was conducted for 0–30 min, followed by first-strand cDNA synthesis with 553 fluorescent nucleotides. The slide was reimaged using the standard Cy3 filter cube. An optimal 554 permeabilization time of 20 min was determined by visual inspection to maximize mRNA recovery 555 while at the same time minimizing diffusion. For gene expression, the initial workflow was similar to 556 the optimization procedure and was done according to the manufacturer’s instructions. After tissue 557 lysis and RT, amplification of cDNA and library preparation – involving fragmentation, dA-Tailing, 558 adapter ligation and a 18 cycles indexing PCR under following conditions: 98 °C 45 sec, 10/14 cycles 559 [98°C 20 sec, 67°C 30 sec, 72°C 20 sec], 72°C 1 min, 4°C hold – was performed based on the 560 manufacturer’s protocol using the Library Construction kit (10X Genomics, #PN-1000190). Included 561 in this protocol was a double-sided SPRI bead (Beckman Coulter, #B23319) size selection (0.6x/0.8x) 562 purification and a final purification (0.8x). After checking the quality control and quantification with 563 Fragment Analyzer (Agilent, NGS Fragment Kit #DNF-473), the libraries were sequenced on an 564 Illumina Novaseq6000 in paired-end mode (R1/R2: 100 cycles; I1/I2: 10 cycles), generating 50-100 565 million fragment pairs for each library. 566 The raw sequencing data was then processed with the ‘count’ command of the Space Ranger 567 software (v3.1.1) provided by 10X Genomics. The Space Ranger reference for the axolotl genome 568 (UKY_AmexF1_1) was built using the Merged Transcripts gene annotation file generated from the 569 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 23 bulk RNA-seq experiments (available upon request) as input for the ‘mkref’ command of Space 570 Ranger. 571 For clustering and gene expression analysis, we used Seurat v.5.2.1 and the following analysis 572 pipeline: Barcodes (spots) are filtered by having 200 or more reads and a mitochondrial content of 573 less than 50% and features (genes) are filtered for having at least 3 cells with at least 1 read. The raw 574 data as UMI counts were regressed to remove the effect of library size using the log normalization. 575 Highly variable genes were identified using the variance-stabilizing transformation (vst), selecting the 576 top 3,000. These genes were scaled and used as input for the dimensionality reduction by PCA 577 analysis. 50 principle components were calculated and the top 20 are used for further calculations. For 578 visualization purposes, Uniform Manifold Approximation and Projection (UMAP, k nearest neighbors 579 = 30) was applied to project high-dimensional gene expression data into two dimensions. Graph-based 580 clustering (k nearest neighbors = 30, resolution = 1.0) used the Leiden algorithm 65. Marker genes that 581 were differentially expressed for each cluster were identified using the Wilcoxon rank-sum test and 582 Benjamini-Hochberg method to correct for multiple comparisons (presto v1.0.0). Markers which are 583 expressed in at least 25% of the cells in a cluster and with a Log2 fold change >2 were included. 584 RNA-seq raw data (fastq) has been deposited in NCBI under the Gene Expression Omnibus (GEO) 585 accession code GSEXXXXXX. 586 Analysis of Nfatc1 enriched spatial spots 587 Analysis of DEGs in Nfatc1 expressing spatial spots was performed in Loupe Browser v8.1.2. using 588 the Advanced Selection tool to set up a threshold of Nfatc1 fold expression (Log 2 Exp) > 2. This 589 approach selected 108 barcodes/spots. These were used to run a differential expression analysis 590 comparing the selected barcodes in the UMAP to the entire dataset. The resulting list of most 591 differentially expressed gene is supplied in Supplemental file XX. 592 Paraffin sectioning and Movat’ s pentachrome stainings 593 Histological stainings were performed in intact limbs (no amputation), and in regenerating limbs 594 after diaphysis and epiphysis amputations at 7-, and 9 dpa. 3 animals per time point were used. 595 Axolotl limbs were fixed in MEMFa and decalcified in 0.5 M EDTA for 2 weeks with daily changes 596 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 24 of the solution. Sample embedding, sectioning and staining was performed by the CMCB Histology 597 Facility, Dresden. Briefly, samples were dehydrated in a series of EtOH in RNase-free water until 598 100% EtOH, and then embedded in paraffin. Longitudinal sections of 4-5 µm were generated using a 599 microtome. Movat’s Pentachrome ( Morphisto, #12057 ) staining was performed according to the 600 manufacturer’s instructions. Imaging was performed using an Olympus OVK automated slide scanner 601 system (UPLSAPO 20x/0.75). 602 Hybridization Chain Reaction (HCR) staining 603 Probe sets for Ctsk, Ctsk-like, and Loc483 were designed using the HCR probe generator created by 604 the Monaghan Lab (https://github.com/Monaghan-Lab/probegenerator ) 66 and purchased as oligo 605 pools (oPools Oligo Pools) from Integrated DNA Technologies. Probe sequences can be found in 606 Table SX. 607 Whole mount HCR was performed according to 67 with some modifications. Briefly, limbs were 608 rehydrated through a series of MetOH in RNase-free water and washed three times in PBT (0.1% 609 Tween 20 in PBS). Tissue was then delipidated in Delipidation Solution (200mM Boric acid, 4% 610 SDS, pH 8.5 in RNAse-free water) for 2 hours at 37°C. After three washes in PBT, limbs were 611 permeabilized with Permeabilization Solution (0.3M Glycine, 2% Triton X-100, 20% DMSO in PBS) 612 for 1 hour at room temperature (RT). Limbs were washed again in PBT, incubated in pre-warmed 613 Hybridization Buffer (Molecular Instruments, #BPH01726) for 5 minutes and then pre-hybridized in 614 new Hybridization Buffer for 30 minutes at 37°C. After this, tissue was incubated overnight with 615 Hybridization Buffer containing 2 pmol per 500 µl of probe solution. The following day, limbs were 616 washed four times with agitation for 15 minutes with Wash Buffer (Molecular Instruments, 617 #BPW01726) at 37°C and two times for 5 minutes in 5× SSCT (3M NaCl, 300 mM sodium citrate, 618 0.1% Tween 20, in water) at room temperature. Pre-amplification was performed for 5 minutes at RT 619 in Amplification Buffer (Molecular Instruments, #BAM01826), followed by amplification for 16-24 620 hours at RT in Amplification buffer with 30 pmol of each hairpin. Finally, tissue was extensively 621 washed in 5× SSCT, incubated overnight with Hoechst 33258 (Abcam, #ab228550) 1:1000 in PBS, 622 and cleared in EasyIndex (LifeCanvas Technologies, #EI-500-1.52) for a minimum of one overnight. 623 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 25 Samples were mounted in a glass bottom dish in EasyIndex, and then imaged using a Zeiss LSM 980 624 inverted confocal laser scanning microscope (Plan-apochromat 10x/0.45) with 10 µm between optical 625 planes. Each HCR was performed in a minimum of 3 biological replicates. 626 Orthology inference of Loc483 with aLeaves 627 Orthology of Loc138491483 was examined using the aLeaves webserver 23 with default parameters 628 and selected databases #1 Human – Refseq, #4 Non-eutherian Mammals – Ensembl 104, #5 Non-629 mammalian Bony Vertebrates – Ensembl 104 and others, #6: Cartilaginous fish and cyclostomes, and 630 #7: All vertebrate entries except mammalians in NCBI Protein . 631 Injection of BAPTA and CaSO4 in regenerating limb blastemas 632 For the injections of BAPTA (Abcam, #ab144924), a working solution was prepared by diluting a 633 stock solution of 25mM of BAPTA (in DMSO) in APBS (80% PBS in RNAse-free water) with 1% 634 Fast Green (Sigma-Aldrich, #F7252) for easier visualization while injecting. For CaSO4, a 15mM 635 CaSO4 solution in water was directly injected in the blastemas, also with 1% Fast Green for easier 636 visualization. 637 Animals were first anesthetized and then injected using using a fine heat-pulled glass capilary to 638 inject 150 nL of solution in both limbs. Ctsk+ signal was quantified by first defining a ROI of 3.5 639 mm2, which encompassed most of the lower arm, and applying the maximum entropy threshold 640 method. The area Ctsk+ signal was then measured using the Area function in FIJI. 641 Cloning, and injection and electroporation of Loc483 in regenerating diaphysis or epiphysis limbs 642 To obtain and clone Loc483, first RNA was extracted of diaphysis amputated limbs as above. cDNA 643 was prepared using Takara PrimeScript™ 1st strand cDNA Synthesis kit (Takara Bio Inc, #6110A) 644 according the manufacturer’s instructions and using Random 6mers primers. The full coding sequence 645 of Loc483 was then amplified with Phusion® High Fidelity DNA Polymerase (New England Biolabs, 646 #M0530) from whole cDNA according to the manufacturer’s instructions and using the primers in 647 Supp file XX. The fragment contaning the coding sequence of Loc483 was then cloned into an vector 648 containing the CAGGS promoter for expression using standard cloning techniques. 649 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 26 Plasmid administration was conducted by first anesthesizing the animals and injecting a solution of 650 1.5 µg/µL of Loc483 plasmid + reporter plasmid (experimental conditions) or just the reporter 651 plasmid (control conditions) diluted in APBS (0.8X PBS) into regenerating limbs using a fine heat-652 pulled glass capilary. Both solutions contained 1% Fast Green for easier visualization while injecting 653 Animals were then immediately electroporated using a Super Electroporator NEPA21 TypeII (Nepa 654 Gene) with 2 poring pulses of 80V , 50 msec length, 50 msec interval, 9% decay rate, and 5 transfer 655 pulses of 40V , 50 msec length, 999 msec interval, 5% decay rate, using a tweezer electrode (Nepa 656 Gene, #CUY650P3). After electroporation, animals were returned to their holding tanks containing 657 swimming water, and allowed to regenerate until imaged at specific time points as before. Ctsk+ signal 658 was quantified by first defining a ROI of 3.5 mm2, applying the maximum entropy threshold method, 659 and then measured using the Area function in FIJI. 660 Image processing, analysis and quantification 661 All images were processed using Fiji 68. Processing involved selecting regions of interest, merging, 662 or splitting channels, and improving brightness and contrast levels for proper presentation in figures. 663 Maximum intensity projections were done in some confocal images, and it is stated in the respective 664 figure’s descriptions. When appropriate, stitching of tiles was done directly in the acquisition software 665 Zen (Zeiss Microscopy, Jena, Germany). 666 Data representation and statistical analysis 667 All graphs and statistical analyses were performed with GraphPad Prism 10 (GraphPad Software, 668 San Diego, CA, USA). Specific number of replicates, statistical tests and pos-hoc tests are indicated in 669 the respective figure legends. All figures were generated with Affinity Designer (Serif Europe, West 670 Bridgford, UK). 671 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 27

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9, 676–682 (2012). 845 846 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 1 2 Fig. 1 Extensive osteoclast-mediated tissue resorption is specifically triggered by amputations 3 through the calcified diaphyseal region of the radius and ulna. A. Schematic representation of 4 amputation planes through the calcified diaphysis and cartilaginous epiphysis (left) and experimental 5 set up (right). B. Time course of tissue resorption during lower arm regeneration in diaphysis and 6 epiphysis amputations at 0, 7, 9, 11, 15, and 18 dpa. Representative images of an experiment with N= 7 5 animals per condition. Scale bar: 500 µm. C. Quantification of radius and ulna length of the stump 8 after diaphysis and epiphysis amputations over time. D. Quantification of the calcified region length 9 in radii and ulnas after diaphysis and epiphysis amputations over time. E. Movat’s pentachrome 10 staining of representative longitudinal sections of intact lower arms (left) and diaphysis and epiphysis 11 limbs at 7- and 9 dpa. R, radius, U, ulna. Dashed lines represent the contours of the radius and ulna. 12 Boxes represent the inset location. Black arrowheads indicate multinucleated osteoclasts. Scale bar: 13 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 500 µm, scale bar in insets: 100 µm. For C and D, N= 5 animals. The lines show mean values over 14 time ± sd. #p<0.05, ##p<0.01, and ###p<0.001 for diaphysis vs epiphysis amputations in the ulna; 15 *p<0.05, **p<0.01 and ***p<0.001 for diaphysis vs epiphysis amputations in the radius (two-way 16 ANOVA with Tukey’s post-hoc test). 17 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 18 Fig. 2 Diaphysis amputations specifically trigger osteoclast recruitment in axolotl limb 19 regeneration. A. Time course of osteoclast presence in diaphysis and epiphysis amputations at 0, 7, 9, 20 11, 15, and 18 dpa. Chondrocytes are labelled in white, osteoclasts in green. Scale bar: 1 mm. 21 Representative images of an experiment with N= 5 animals per condition. B. Hybridization chain 22 reaction (HCR) for Ctsk (green) and Ctsk-like (magenta) at 3- and 5 dpa in representative diaphysis 23 and epiphysis amputated limbs. White arrowheads indi cate the multinucleated osteoclasts depicted in 24 corresponding insets. Scale bar: 500 µm, scale bar in insets: 50 µm. 25 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 26 27 Fig. 3 Osteoclast presence is not correlated with systemic calcium levels or impacted by local 28 calcium changes. A. Quantification of calcium levels in the serum of diaphysis and epiphysis 29 amputated animals in intact conditions, and at 12 hpa, 1-, 3-, 5-, and 9 dpa. The graph represents the 30 mean and sd of the combined results of 4 independent experiments using a minimum of 3 animals per 31 condition and per time point. B. Schematic representation of the experimental set up for the injections 32 in diaphysis and epiphysis amputated limbs. C. Time course of osteoclast presence in representative 33 diaphysis and epiphysis amputated limbs injected with DMSO (control) or 10mM BAPTA at 5-, 7-, 9- 34 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint and 11 dpa. Scale bar: 1 mm. D. Quantification of the area of Ctsk+ signal in BAPTA and DMSO 35 injected animals in diaphysis amputations. E. Quantification of the area of Ctsk+ signal in BAPTA and 36 DMSO injected animals in epiphysis amputations. F. Time course of osteoclast presence in 37 representative diaphysis and epiphysis amputated limbs injected with water (control) or CaSO4 at 5-, 38 7-, 9- and 11 dpa. Scale bar: 1 mm. G. Quantification of the area of Ctsk+ signal in water or CaSO4 39 injected animals in diaphysis amputations. H. Quantification of the area of Ctsk+ signal in water or 40 CaSO4 injected animals in epiphysis amputations. C and F show representative images of an 41 experiment with N= 4 animals per condition. For C-H, the 2 limbs of 4 animals were injected and 42 assessed per condition over time. For D, E, G and H, the lines show mean values over time. 43 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 44 45 Fig. 4 Spatial transcriptomics reveals differences in gene expression between diaphysis and 46 epiphysis amputations. A. Hematoxylin and Eosin (H&E) staining of representative longitudinal 47 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint sections of 3 and 5 dpa diaphysis and epiphysis amputated limbs used for spatial transcriptomics. 48 Scale bar: 500 µm. B. Spatial view of the 19 clusters identified by Seurat clustering analysis 49 combining spatial dots from diaphysis and epiphysis amputated limbs at 3 and 5 dpa. C. UMAP plot 50 and cluster annotation of spatial transcriptomic dots from 3 and 5 dpa diaphysis and epiphysis 51 amputated limbs. D. UMAP plots showing the expression of the resorption-associated markers Ctsk, 52 Ctsk-like, Nfatc1 and Acp5. E) UMAP plot showing the contribution of each sample to the UMAP at 3 53 dpa (left), 5 dpa (center) and both timepoints (right). F. Spatial expression profiles of Nfatc1, Ctsk and 54 Ctsk-like in representative spatial transcriptomics sections at 3- and 5 dpa. Expression levels in D and 55 F were calculated as Log2 fold expression. CT, connective tissue. 56 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 57 58 Fig. 5 The RANK/RANKL system likely orchestrates tissue-dependent osteoclast-mediated 59 skeletal resorption. A. UMAP view of the expression of RANK and RANKL. Expression levels were 60 calculated as Log2 fold expression. B. Spatial expression profiles of Tnfsf11 (RANKL) and Tnfrsf11a 61 (RANK) in representative spatial transcriptomics sections at 3 and 5 dpa. Scale bar: 500 µm. C. HCR 62 for Ctsk (green) and RANKL (magenta) at 3, 5 and 7 dpa in representative diaphysis and epiphysis 63 amputated limbs. Scale bar: 300 µm. 64 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 65 66 Fig. 6 Loc138491483/Ccl24-like is sufficient to ectopically recruit osteoclasts into epiphysis 67 amputated limbs. A. Top 15 most differentially expressed genes in Nfatc1-expressing spatial 68 transcriptomics spots. B. UMAP plots showing the expression of Loc138491483/Ccl24-like (Loc483). 69 C. Dot plot showing expression of Loc483 in the 19 annotated spatial transcriptomics clusters. D. 70 Spatial expression profiles of Loc483 representative spatial transcriptomics sections at 3 and 5 dpa. 71 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint Scale bar: 500 µm. E. HCR for Loc483 at 1-, 3- and 5 dpa in diaphysis and epiphysis amputated 72 limbs. Scale bar: 300 µm. F. Time course of osteoclast presence in representative diaphysis and 73 epiphysis amputated limbs injected with control plasmid (CAGGs-mCherry) or control plasmid + 74 CAGGS-Loc483 at 7-, 9-, and 11 dpa. Scale bar: 1 mm. G. Quantification of the area of Ctsk+ signal 75 in CAAGS-Loc483 and control (Ctrl) animals in diaphysis amputations. H. Quantification of the area 76 of Ctsk+ signal in CAAGS-Loc483 and control (Ctrl) animals in epiphysis amputations. F shows 77 representative images of an experiment with n= 5 animals per condition. For G and H, the graphs 78 represent the combined results of 2 independent experiments using a total of 5 limbs of 5 different 79 animals per condition. The lines show mean values over time ± sd. *p<0.05 for CAAGS-Loc483 vs 80 Ctrl electroporated animals (two-way ANOV A with Tukey’s post-hoc test). 81 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 82 Fig. 7 Diaphysis and epiphysis amputations induce changes in the transcriptomic profile of their 83 AEC. A. GO analysis for Cellular Component (CC) and Molecular Function (MF) terms in 84 upregulated DEGs found in bulk RNA-seq of diaphysis and epiphysis limbs at 3 and 5 dpa. B. Spatial 85 transcriptomic profile of the average expression of upregulated bulk RNA-seq DEGs in epiphysis 86 amputated limbs at 3 dpa (43 genes) and 5 dpa (36 genes). Scale bar: 500 µm. C. Spatial 87 transcriptomic profile of the average expression of upregulated bulk RNA-seq DEGs in diaphysis 88 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint amputated limbs at 3 dpa (8 genes) and 5 dpa (7 genes). In B and C, expression levels were calculated 89 as Log2 average expression. D. Dot plots showing expression of upregulated DEGs found in bulk 90 RNA-seq from diaphysis limbs in the 19 annotated spatial transcriptomics clusters at 3 and 5 dpa. 91 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint Supplementary Figures 92 93 94 Fig. S1 Ctsk-like is likely a duplication of the Ctsk gene present in the axolotl genome. A) 95 Localization of Ctsk (GCF_040938575.1) and Loc138578972 (Ctsk-like) genes in the axolotl 96 genome. B) Alignment of predicted coding sequences of Ctsk and Ctsk-like, with sequence identity 97 and similarity scores. C) Predicted protein sequence alignment of axolotl Ctsk and Ctsk-like. D) 98 Protein sequence alignment of human Ctsk (hCtsk) and axolotl Ctsk (axCtsk). E) Protein sequence 99 alignment of human Ctsk (hCtsk) and axolotl Ctsk-like (axCtsk-like). E) Table depicting calculated 100 sequence identities and sequence similarities between human hCtsk, axCtsk, and axCtsk-like (axCtsk-101 L) peptides. 102 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 103 Fig. S2 Cluster annotation and expression of known marker genes in spatial transcriptomics. A. 104 Dot plot showing the top marker genes in each of the 19 annotated clusters. B. Dot plot showing 105 expression of known marker genes for epithelia, bone, cartilage, endothelium (Endot), blood, immune 106 system, resorption and blastema (Blast) in the 19 spatial transcriptomics clusters. C. Contribution of 107 each sample to each of the 19 annotated clusters. 108 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 109 Fig. S3 RANK and RANKL system are likely involved in osteoclastogenesis during axolotl limb 110 regeneration. A. Dot plot showing the expression of Tnfsf11 (RANKL), Tnfrsf11a (RANK), and 111 Tnfrsf11b (Osteoprotegerin), in spatial clusters. B. HCR for Ctsk (green) and RANK (magenta) at 3 112 and 5 dpa in representative diaphysis and epiphysis amputated limbs. Asterisks indicate 113 autofluorescence. Scale bar: 300 µm C. Insets of B showing Ctsk (green), RANK (magenta), and 114 Nfatc1(white). Arrowheads indicate cells that are positive for RANK and Nfatc1, but not Ctsk. Scale 115 bar: 50 µm. 116 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 117 Fig. S4 Loc483 is likely not an ortholog to the human CCL24. aLeaves generated neighbor-joining 118 tree using the predicted aminoacid sequence of Loc483 gene. Bootstrap support values are shown in 119 selected nodes. Blue box contains cartilaginous fish species (Chondrichthyes), green box comprises 120 amphibian species. 121 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 122 123 Fig. S5 Bulk RNA-seq reveals that Cart and Calc amputations affected other processes than 124 osteoclastogenesis A. Top 20 most upregulated differentially expressed genes (DEGs), total number 125 of upregulated DEGs, proportion of protein-coding vs non-coding DEGs, and number of DEGs after 126 filtering used for Gene Ontology (GO) analysis in epiphysis amputations at 3 dpa. B. Top 20 most 127 upregulated DEGs, total number of upregulated DEGs, proportion of protein-coding vs non-coding 128 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint DEGs, and number of DEGs after filtering used for GO analysis in epiphysis amputations at 5 dpa. C. 129 Top 20 most upregulated DEGs, total number of upregulated DEGs, proportion of protein-coding vs 130 non-coding DEGs, and number of DEGs after filtering used for GO analysis in diaphysis amputations 131 at 3 dpa. D. Top 15 most upregulated DEGs, total number of upregulated DEGs, proportion of 132 protein-coding vs pseudo-genes vs non-coding DEGs, and number of DEGs after filtering used for 133 GO analysis in diaphysis amputations at 5 dpa. E. Gene expression levels of the osteoclast-associated 134 genes Ctsk, Ctsk-like, Nfatc1 and Acp5 in bulk RNA-seq of intact, and diaphysis and epiphysis 135 amputated limbs at 3 and 5 dpa. F. Significantly enriched biological process GO terms for diaphysis 136 and epiphysis amputated limbs at 3 and 5 dpa. G. Gene expression levels of Loc138491483/Ccl24-like 137 in bulk RNA-seq of intact, and diaphysis and epiphysis amputated limbs at 3 and 5 dpa. 138 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint 139 140 Fig. S6 AECs of diaphysis and epiphysis amputations exhibit differences in the expression of 141 non-coding genes. A. Expression of representative genes upregulated in epiphysis amputations at 3 142 dpa (Psat1) and 5 dpa (Klf17 and Csdn) in bulk RNA-seq. B. Spatial transcriptomic profile of the 143 average expression of non-coding bulk RNA-seq DEGs upregulated in epiphysis amputated limbs at 3 144 dpa (6 genes) and 5 dpa (11 genes). C. Spatial transcriptomic profile of the average expression of non-145 coding bulk RNA-seq DEGs upregulated in diaphysis amputated limbs at 3 dpa (4 genes) and 5 dpa (3 146 genes). Expression levels were calculated as Log2 average expression. In A, expression levels were 147 calculated as Log2 average expression, in B and C expression levels were calculated as average Log2 148 fold expression. Scale bar: 500 µm. 149 150 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.708175doi: bioRxiv preprint