Comparative multi-omic analysis reveals conserved and derived mechanisms of fin and limb regeneration

Domain: biology
Published: 2026-01-22
Editorial outcome: minor_revision

Abstract
Comparative studies of vertebrate appendages offer a powerful framework for uncovering shared components of an ancestral regeneration toolkit. Here, we employed a multi-omics comparative approach leveraging the regenerative capacity of the axolotl, zebrafish, and Polypterus senegalus, a fish capable of full fin regeneration. We identified conserved markers of proximal and distal blastema territories, shared activation of DNA damage repair, hif1a-mediated hypoxia response, and sequential activation of pro- and anti-inflammatory program. Apical epithelial ridge markers were expressed in both the wound epidermis and distal mesenchyme during limb and fin regeneration. Notably, hif4a-expressing erythrocytes were uniquely associated with proximal limb and fin amputations but not fin rays, while epidermal myoglobin expression was upregulated only in Polypterus and zebrafish fins. Genome-wide chromatin profiling identified candidate regeneration-responsive elements and a conserved enrichment for AP-1 transcription factor binding. Together, these findings identify shared and derived mechanisms of limb and fin regeneration.

Introduction
Among limbed vertebrates (tetrapods), salamanders are the only clade capable of regrowing severed limbs as adults. This regenerative capacity may have been inherited by tetrapods from their fish ancestors, retained in modern salamanders, partially preserved in frogs (limited to larval stages), but entirely lost in amniotes (Fig. 1a). Supporting this hypothesis, fossil evidence suggests that limb regeneration predates the origin of stem salamanders1,2. A phylogenetic survey of complete paired fin regeneration—defined as regeneration following amputation at the endoskeleton-bearing proximal fin region—demonstrated that this ability is present across species representing all major bony fish clades3,4, including zebrafish larvae5,6. Furthermore, decades of comparative studies using the zebrafish caudal fin as a model system have revealed commonalities between limb and fin ray regeneration programs7, including cell-type-specific genetic and epigenetic programs8. Altogether, these findings support a shared evolutionary origin of limb and fin regeneration, underscoring the importance of comparative studies as a powerful framework for identifying the core cellular and molecular components underlying vertebrate appendage regeneration.

While the adult zebrafish serves as a valuable model for comparative and functional studies in fin regeneration, its regenerative capacity is limited to the distal fin rays5 (Fig. 1c), a structure that lacks direct homology with limbs9 and the cellular complexity and diversity observed during limb regeneration8. Additionally, the teleost-specific whole-genome duplication (WGD) event presents challenges for genetic comparisons between zebrafish and salamanders, complicating the identification of conserved regenerative mechanisms10,11. To address these limitations, we have established the Senegal bichir, Polypterus senegalus, as a model, leveraging its remarkable regenerative abilities. Polypterus, a non-teleost ray-finned fish (actinopterygian), diverged from teleosts prior to the teleost-specific WGD (Fig. 1a), maintaining a 1:1 gene orthology with tetrapods12. Unlike zebrafish, Polypterus can readily regenerate entire fins as adults, including the proximal fin region, which contains endoskeletal elements sharing deep homology with limb bones13,14 (Fig. 1c). Additionally, the proximal fin region contains organized muscle masses, diverse connective tissue populations such as tendon and ligament fibroblasts, and fibrocartilaginous joints, contributing to its overall cellular complexity. Proximal fin amputations thus enable the assessment of regenerative processes in a cellularly intricate context, including interactions among muscle, tendon, connective tissue, cartilage precursors, vasculature, and nerves. Furthermore, our previous studies identified signaling pathways commonly upregulated in both salamanders and Polypterus4, highlighting its potential as a model for evolutionarily informed comparative studies of vertebrate appendage regeneration.

To comprehensively identify the core cellular, genetic, and gene regulatory components of Polypterus fin regeneration, we employed single-nucleus RNA-sequencing (snRNA-seq) and spatial RNA-sequencing (spatial RNA-seq). Additionally, we generated spatial RNA-seq datasets for axolotl limb regeneration and leveraged publicly available single-cell RNA-sequencing (scRNA-seq) data from axolotl limb15 and snRNA-seq data from zebrafish caudal fin8 regeneration. This approach enabled a broad comparative analysis of limb and fin regeneration during homeostasis and across successive regeneration stages. Our findings revealed conserved and derived features of limb and fin regeneration, including immune cell recruitment, infiltration of hif4a+ erythrocytes, blastema cell heterogeneity, wound epidermis and blastema gene expression profiles, proximal-distal subdivision of connective tissue cells, sequential activation of pro- and anti-inflammatory genetic programs, DNA damage response, and hypoxia-adaptive processes. Finally, using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we uncovered candidate regeneration-responsive cis-regulatory elements and identified AP-1 transcription factors as potential key conserved regulators in the early response to fin and limb amputation.

Results
The cellular landscape of Polypterus fin regeneration
To assess fin regeneration at the single-cell level, we selected timepoints representative of homeostasis (the uninjured proximal segment of the pectoral fin) and regeneration at 1, 3, and 7 days post-amputation (dpa) (Fig. 1b). Fin amputation at the level of the endoskeletal elements resulted in complete wound closure and presumptive immune cell infiltration at 1dpa (Fig. 1d). At 3dpa, the wound epidermis was multilayered, containing distinct basal and suprabasal layers, and subjacent mesenchymal cells forming the presumptive blastema. At 7dpa, the wound epidermis extended distally, forming a dorso-ventrally constricted outgrowth in which fin rays will form.

Using combinatorial barcoding technology (Parse Biosciences), we profiled almost 63,000 nuclei from fins at homeostasis and during regeneration in biological replicates. Trailmaker (Parse Biosciences, 2024) was used to process and analyze snRNA-seq data (Supplementary Fig. 1a). Unbiased clustering of all nuclei from all timepoints revealed 32 transcriptionally distinct populations (clusters) (Fig. 1e and Supplementary Fig. 1b). Cluster annotation based on transcriptional profiles identified multiple cell populations, including proliferating cells, muscle, connective tissue, glial cells, platelets, erythrocytes, endothelial cells, various immune cells and epidermal cells of multiple types (basal/intermediate/superficial epidermis, goblet cells and ionocytes) (Fig. 1e, f, Supplementary Figs. 1b and 2). We detected three populations of proliferating cells (Fig. 1e, f and Supplementary Fig. 2a, b). Proliferating cell clusters 1 and 2 presented epithelial cell features (expression of tp63 and evpl), whereas expression of col6a3 in proliferating cell 3 suggests that this cluster constitutes connective tissue proliferating cells.

To assess changes in cell populations and gene expression patterns, we evaluated cell clusters across regeneration stages. At 1dpa, the major changes observed were the increases in proliferating cells, myeloid cells, lymphoid cells, and basal and intermediate epidermal cells (Fig. 1g, h and Supplementary Fig. 2c), consistent with wound closure completion and immune cell infiltration observed via histology (Fig. 1d). At 3dpa, all immune cell types were increased relative to 1dpa, an indicative of substantial immune cell infiltration, yet the most notable change was the large increase in erythrocytes (Fig. 1g–j and Supplementary Fig. 2c). Massive immune cell infiltration, especially macrophages, and the formation of erythrocyte clumps in the limb blastema, have both been identified as features of salamander limb regeneration16,17. Finally, we observed reduction of muscle, connective tissue, and terminally differentiated epidermal cell (ionocytes, goblet cells, and superficial epidermis) clusters at 3dpa, and a reversal of this trend at 7dpa (Fig. 1g–j and Supplementary Fig. 2c).

Shared spatial gene expression domains of fin and limb regeneration
To complement our snRNA-seq dataset and enable comparisons of gene expression territories during Polypterus fin and axolotl limb regeneration, we generated high throughput, spatially resolved gene expression data using Visium spatial transcriptomics technology (10x Genomics). In both species, a regeneration series was assayed, with the goal of capturing homeostasis, wound closure, wound epidermis formation, and blastema establishment. To this end, Polypterus fins were sampled at homeostasis (proximal segment of uninjured pectoral fin), 1dpa, 3dpa, and 7dpa (Fig. 2a)—matching the stages assayed via snRNA-seq. Axolotl limb regeneration was assessed at homeostasis (uninjured forearm segment), 3dpa, 7dpa, and 14dpa (Fig. 2e)—matching stages reanalyzed from publicly available scRNA-seq data from axolotl limb15 (Supplementary Fig. 3a).

We applied k-means clustering to identify major tissue compartments across spatial transcriptomics samples. To determine the optimal number of clusters for k-means clustering, we generated elbow plots of within-cluster sum of squares (WCSS)18 across k = 2–10 (Supplementary Fig. 4). A sharp reduction in WCSS was observed from k = 2 to 4, with diminishing improvement, thereafter, indicating k = 4 as a suitable balance between cluster compactness and model complexity. Clustering identified tissue types and orthologous markers for epidermis (agr2, suprabasal layers), muscle (ckm), skeleton (col2a1), connective tissue (col6a3) and immune cells (mmp3) in the Polypterus fin (Fig. 2b) and axolotl limb (Fig. 2f). Furthermore, we assessed the expression of these markers in their corresponding cell types in our Polypterus snRNA-seq dataset and in the scRNA-seq data from axolotl limbs. Our analysis confirmed matching expression of these markers in clusters corresponding to epidermis, skeleton (a small subset of the connective tissue cluster), muscle, connective tissue and a subpopulation of myeloid cells (Fig. 2c, d, g, h), validating the combined analysis of our spatially and cellularly resolved gene expression datasets in the downstream comparisons described in subsequent sections. The combination of Polypterus snRNA-seq and spatial RNA-seq data allowed us to better refine cell cluster annotation and identify subsets of cell types within annotated clusters. This was especially relevant for the identification of the fin skeletal compartments. As described above, skeletal cells were found to correspond to a subset of connective tissue cells, as evidenced by the expression of the marker col2a1, detected via spatial RNA-seq in the Polypterus fin skeleton (Fig. 2b–d).

Distinct regeneration programs of endochondral and dermal skeleton in Polypterus
Complete fin regeneration in Polypterus requires regrowth of both the endochondral skeleton and the dermal fin rays, providing an opportunity to distinguish the cells and genetic programs that contribute to each process. To this end, we examined canonical markers of dermal and endochondral regeneration in our snRNA-seq and spatial RNA-seq datasets.

One of the best characterized markers of dermal skeleton development and regeneration is and119,20. Consistent with observations in zebrafish fin development19, we detect the onset of and1 expression in nuclei of basal epidermal cells of the Polypterus fin at 3dpa, in addition to its canonical expression in mesenchymal progenitors at 7dpa (Fig. 3a, upper panel). Conversely, col9a1—a marker of chondrocytes of endochondral growth plates in fish and mammals21,22—was expressed in a discrete subpopulation of connective tissue cells at homeostasis and throughout regeneration stages (Fig. 3a, lower panel). Spatial analysis confirmed that and1 expression marks the distal-most fin ray regeneration territory, while col9a1 was expressed throughout the cartilaginous endoskeleton and immediately subjacent to the and1 domain at 3dpa and 7dpa (Fig. 3b).

To identify gene expression signatures associated with these territories, we manually defined regions of interest at 7dpa corresponding to fin ray regeneration (and1+) and endochondral regeneration (col9a1+) (Fig. 3c). Analysis of the top 50 genes differentially expressed genes revealed distinct extracellular matrix (ECM) components enriched in each compartment (Supplementary Data 1). In our snRNA-seq dataset, structural ECM genes such as matn4 and cilp2 were specifically expressed in the connective tissue cell subpopulation corresponding to col9a1+ endochondral cells, whereas expression of vcan was also detected in other connective tissue cells and basal epidermis (Fig. 3d). In addition, the ECM remodelers ccn2a, angptl7, and emilin3 were expressed in connective tissue cells that overlapped with the col9a1+ endochondral population. Spatial RNA-seq analysis confirmed the expression of these ECM components and remodelers within and adjacent to the endochondral regeneration domain (Fig. 3e). By analyzing our spatial transcriptomics dataset of the regenerating axolotl limb at 14dpa, we showed that these genes also marked the presumptive endoskeleton regeneration front in the axolotl, immediately distal to and surrounding the endoskeleton in the stump, except for emilin3, which was expressed at low levels in scattered distal spots (Fig. 3f).

In contrast, the fin ray regeneration domain at 7dpa was characterized by expression of dermal ECM genes, including and3 and col9a1-like, a paralog of col9a1 absent from tetrapod genomes, which was expressed in subsets of basal epidermis and connective tissue cells (Fig. 3g). Spatial RNA-seq confirmed that the expression of these markers was largely restricted to the presumptive fin ray regeneration domain. This distal domain was also characterized by expression of ECM remodelers such as fkbp9, mmp11, fbln1, and emilin1. In the axolotl limb at 14dpa, fkbp9 and mmp11 were expressed in scattered distal spots, whereas fbln1 and emilin1 were enriched around the presumptive chondrogenic front. Together, our findings identified distinct connective tissue cell populations contributing to dermal versus endochondral regeneration in Polypterus and highlight conserved and divergent ECM-associated programs between fish fin rays and the tetrapod limb skeleton.

Reactivation of apical ectodermal ridge-like gene programs in the Polypterus wound epidermis
A key driver of successful epimorphic appendage regeneration is the formation of a signaling-competent wound epidermis, which subsequently matures into its specialized derivative, the apical epithelial cap (AEC). To characterize gene expression dynamics associated with wound epidermis formation during Polypterus fin regeneration, we subclustered epidermal cell populations corresponding to less differentiated layers (basal and intermediate epidermis) from our snRNA-seq dataset. These included col17a1+/tp63+ basal cells, tp63+ intermediate cells, and tp63+/mki67+ proliferative epidermal cells (Fig. 4a). At homeostasis (uninjured fin), we detected small populations of basal and proliferative cells, alongside a dominant and well-separated intermediate epidermal cluster. At 1 and 3dpa, we observed a marked expansion of basal (col17a1+) and proliferative (mki67+) populations, along with an intermediate cluster whose transcriptional profile shifted toward a basal-like state, consistent with the establishment of a wound epidermis (Fig. 4a, upper panel). Spatial transcriptomics further confirmed epithelial enrichment of the basal cell marker col17a1 and its pronounced upregulation at 3dpa (Fig. 4a, lower panel). We observed a difference in the timing of the expression peak for basal epithelial markers such as col17a1 and others (cyp27b1 and krt17–detailed below) in our snRNA-seq data compared to our spatial transcriptomics data (Fig. 4a, b). Specifically, these markers showed their highest expression levels at 1dpa in the snRNA-seq and at 3dpa in the spatial transcriptomics dataset. The intrinsic technical differences between snRNA-seq, which captures newly synthesized nuclear transcripts, and spatial transcriptomics, which primarily reflects accumulated cytoplasmic mRNAs, may explain these differences.

By combining differential gene expression analysis between regenerating and uninjured fins with evaluation of pct.1 (percentage of nuclei expressing a gene within the cluster) and pct.2 (in all other clusters), we identified a set of genes whose expression was highly enriched in the wound epidermis (Supplementary Data 2). Expression of these genes was minimal or undetected in the homeostatic epidermis but became specifically upregulated in discrete subpopulations within the regenerating epidermis, comprising no more than 40% of epidermal cells (Supplementary Fig. 5a, upper panel). A subset of these genes was also expressed in a subpopulation (≤20%) of connective tissue cells (Supplementary Fig. 5a, lower panel). The regenerating epidermis was also marked by the upregulation of several genes with a broader expression pattern (Supplementary Fig. 5b). Inspection of the scRNA-seq data from the axolotl limb regeneration and snRNA-seq data from zebrafish caudal fin regeneration (Supplementary Fig. 3a, b) revealed that several Polypterus wound epidermis-enriched genes are likewise upregulated in the regenerating epidermis of both axolotl limb and zebrafish caudal fin (Supplementary Fig. 5c–f).

Among these, krt17, cyp27b1, and mmp13 showed strong wound epidermis-restricted expression in Polypterus (Fig. 4b–d). Using hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) staining, we further validated krt17 expression predominantly at the basal wound epidermis at 3 and 7dpa (Fig. 4e). Notably, a marker of activated keratinocytes23, Krt17 has been previously reported as enriched in basal wound epidermis cells during axolotl limb regeneration24. However, the wound epidermis-restricted expression of krt17 seems to be specific to Polypterus, as in axolotl and zebrafish, expression of the krt17 orthologs was also detected in the epidermis during homeostasis (Supplementary Fig. 6a, d). Cyp27b1, which encodes a key enzyme in vitamin D metabolism25, has not previously been linked to epimorphic appendage regeneration. In Polypterus, its expression was conspicuously enriched in the wound epidermis (barely detected at homeostasis) (Fig. 4c) and may represent an early response to injury (Cyp27b1 expression peaked at 1 and 3dpa). The expression of mmp13, a gene whose axolotl ortholog has been reported as highly expressed in the limb wound epidermis26,27, was also highly enriched in the Polypterus fin regenerative epidermis but with a later temporal peak of expression compared to cyp27b1 (Fig. 4d). Both Cyp27b1 and Mmp13 displayed regeneration-enriched expression in our axolotl limb spatial transcriptomics series as well as in the axolotl scRNA-seq dataset, however expression was stronger in mesenchymal cells (Supplementary Fig. 6b, c). In zebrafish, cyp27b1 expression remained consistently low across all stages (Supplementary Fig. 6e), whereas the expression of both mmp13 paralogs was upregulated in the epidermis, alongside a connective tissue upregulation of mmp13a (Supplementary Fig. 6f, g).

Prior studies identified Frem2 as a wound epidermis marker during axolotl limb regeneration24,26. However, this previously reported Frem2 gene sequence maps to the Frem3 locus in the most recent axolotl genome release. Polypterus frem3 was the top upregulated gene in the subclustered epidermal cells at 7dpa (Supplementary Data 2). We therefore analyzed the expression of both paralogs across Polypterus, axolotl, and zebrafish datasets (Supplementary Fig. 7). In both Polypterus and axolotl, frem3/Frem3 were expressed in a distinct subset of basal epidermal cells enriched during regeneration (Supplementary Fig. 7a, c). In Polypterus, frem2 was also upregulated in the basal epidermis during regeneration (Supplementary Fig. 7b), but in axolotl, Frem2 expression was not specifically associated with regenerative stages (Supplementary Fig. 7d). In zebrafish, frem2a/b and frem3 were strongly enriched during regeneration, although their expression was more broadly distributed across basal epidermal and non-basal epidermal cell types (Supplementary Fig. 7e–g).

The establishment of an AEC is a critical step in epimorphic regeneration, as it defines a specialized signaling center that regulates blastema formation and growth. In amphibians, previous studies have shown that the gene expression program of the apical ectodermal ridge (AER)—essential for limb development—is partially reactivated during AEC formation in regenerating limbs26. In Polypterus fins at 1 and 3dpa, we identified a subset of basal epidermal cells with a high score for the expression of AER-related genes, consistent with their involvement in establishing an AEC-like structure during fin regeneration (Fig. 5a). Similar to axolotl26, part of the AEC transcriptional program in Polypterus was also expressed in connective tissue cells (Fig. 5b–i), particularly BMP-related (bambi, bmp2, bmp7) and Wnt-related (axin2, lef1) genes. Although the epidermal contribution to the AEC program appears greater in Polypterus than in axolotl (Fig. 5f–i) or zebrafish (Fig. 5j–m), the participation of connective tissue cells in AEC-associated gene expression seems to be a conserved feature across fish and amphibians.

Blastema cell heterogeneity
Fibroblast-like connective tissue cells are considered the major cellular contributors to the regenerating limb blastema24,28. Our snRNA-seq and spatial transcriptomics data identified col6a3 as a general marker for connective tissue cells (Fig. 2b–d). Connective tissue cells expressing col6a3 were present during homeostasis and across all stages of regeneration (Fig. 6a). Spatial transcriptomics data showed that during regeneration, col6a3 expression became more abundant distally, indicating migration of connective tissue cells into the region of the presumptive fin blastema at 3 and 7dpa (Fig. 2b). To confirm the location of those cells at homeostasis and during regeneration, we used HCR-FISH staining (Fig. 6b). In the uninjured fin, connective tissue cells expressing col6a3 were found in the dermis and dispersed between muscle cells. At 1dpa, clusters of col6a3-expressing cells were observed in the stump, near the amputation site. At 3dpa, col6a3-expressing cells accumulated distally in the regenerating fin and were abundant in the presumptive blastema. At 7dpa, col6a3-expressing cells were most enriched distally, and col6a3 signal intensity was higher in cells closest to the epidermis basal layer. Ultimately, col6a3 expression confirmed that connective tissue cells are major contributors to the Polypterus fin blastema.

Regeneration-enriched connective tissue states were characterized by the expansion of small, distinct subpopulations, emphasizing the heterogeneity of the connective tissue compartment during regeneration. For example, mmp19, an established marker of axolotl limb blastema cells29,30, was expressed in a subset of connective tissue cells in the Polypterus fin (Fig. 6c). We also observed the expansion of largely non-overlapping connective tissue subpopulations expressing fgf10 (Fig. 6d), an important signaling factor in limb development and regeneration31, as well as lep (Fig. 6e), whose axolotl ortholog is upregulated in the limb blastema32.

Although seemingly dispensable for axolotl limb regeneration33, Pax7+ muscle satellite cells contribute to the axolotl limb blastema34. In the uninjured Polypterus fin, pax7-expressing cells were detected in the muscle cluster. Later, at 1 and at 3dpa, pax7-expressing cells were also detected within the connective tissue clusters (Fig. 6f). Myeloid cells, particularly macrophages, infiltrate the limb blastema during regeneration. Our snRNA-seq dataset identified a substantial increase in the proportion of myeloid cells during regeneration, relative to homeostasis (Fig. 1g–j and Supplementary Fig. 2c). To assess whether these cells reached the fin blastema, we examined the expression of marco, which marks a subpopulation of myeloid cells in the axolotl limb blastema24,35. As seen in the axolotl, our snRNA-seq dataset showed marco-expressing cells in the myeloid cluster during Polypterus fin regeneration (Fig. 6g, upper panel). Spatial RNA-seq revealed expression of marco in the presumptive blastema of the Polypterus fin at 3dpa (Fig. 6g, lower panel).

Altogether, these findings suggest that, as seen in axolotls, the Polypterus fin blastema is composed of a heterogeneous cell population, with contributions from tissues such as presumptive muscle satellite cells (pax7), connective tissue cells (col6a3, mmp19, fgf10, lep), and immune cells (marco).

Emergence of proximal-distal organization within the regenerating connective tissue
While our initial analysis using K-means=4 clustering provided a consistent framework to define the major tissue compartments across all samples in our spatial transcriptomics datasets, we next asked whether finer-grained patterns could be detected within individual compartments. In particular, we noted that at later regenerative stages (Polypterus fin at 7dpa and axolotl limb at 14dpa), increasing the resolution to k=5 subdivided the connective tissue cluster into two transcriptionally and spatially distinct territories (Fig. 7a, b). One domain localized distally beneath the wound epidermis and was enriched for crabp2 and tnc, while the other occupied more proximal regions and expressed sfrp2 and dpt (Fig. 7c, d). This subdivision was observed in both Polypterus fins and axolotl limbs, suggesting that the connective tissue progressively acquires proximal-distal organization as regeneration advances.

To systematically assess the gene expression profile of these domains, we retrieved the top 100 differentially enriched genes in each compartment that could be annotated to a human ortholog (Supplementary Data 3). This orthology-based approach ensured comparability across species and highlighted conserved compartment-specific signatures. Comparison of proximal (dpt+, sfrp2+) and distal (tnc+, crabp2+) connective tissue domains revealed both shared and distinct features. Both compartments expressed markers of ECM remodeling, stress responses, and inflammation, consistent with the regenerative wound environment (Fig. 7e and Supplementary Data 3). However, proximal connective tissues showed stronger enrichment for sarcomeric and contractile genes, suggestive of myofibroblast-like states or presence of muscle tissue of the stump. In contrast, distal connective tissue was characterized by high proliferative and secretory activity, reflected in the upregulation of histone and RNA-processing genes, protein-folding machinery, and matrix-associated genes such as fibrillin-2 and thrombospondins.

The identity of these domains was further supported by scRNA-seq and snRNA-seq data (Fig. 7f–k). Subclustering analysis of connective tissue cells indicated that dpt+ and tnc+ fibroblasts define largely distinct, though partially overlapping populations, suggesting that proximal and distal connective tissue states are transcriptionally and spatially segregated during regeneration. To assess whether these connective tissue domains emerged during zebrafish fin ray regeneration, we evaluated the snRNA-seq dataset of caudal fin ray regeneration. The subclustered zebrafish connective tissue cells showed largely exclusive expression domains of dpt+ and tnca+/tncb+ cells (Fig. 7h, k), with very few connective tissue cells expressing dpt. In all three species, tnc+ distal connective tissue cells displayed elevated expression of proliferation markers such as pcna and mki67 (Fig. 7l–n), consistent with the higher proliferative activity expected near the wound site. Altogether, our results revealed the emergence of crabp2+/tnc+ proliferative fibroblasts distally and sfrp2+/dpt+ fibroblasts proximally during limb and fin regeneration.

Limb and fin regeneration followed by endoskeleton-level amputation is marked by robust expansion of hif4a+ erythrocytes
Our assessment of cellular populations emerging during Polypterus fin regeneration revealed a marked increase in erythrocytes after endoskeleton-level fin amputation (Fig. 1g–j). Spatial RNA-seq analysis of the erythrocyte-specific marker alas2 showed a broad distribution of positive spots during Polypterus fin regeneration, with alas2 expression detected near the wound site and more proximally, in the fin stump (Fig. 8a). To evaluate whether this erythrocyte expansion is a shared feature of limb and fin regeneration we evaluated Alas2 gene expression during axolotl limb regeneration as well as its zebrafish ortholog during caudal fin ray regeneration. As seen in Polypterus, Alas2-expressing erythrocytes massively expand during 3 and 7dpa, near the wound site and in the limb stump, to subside in later stages of axolotl limb regeneration stages (Fig. 8b). In contrast, erythrocyte clusters were not detected during zebrafish caudal fin ray regeneration (Fig. 8c), suggesting that this is a feature of regrowth post endoskeleton-level amputation.

In the Japanese fire-bellied newt (Cynops pyrrhogaster), erythrocyte clumps appear during limb regeneration and express genes encoding secreted growth factors and matrix metalloproteases17. In contrast, our analysis of the top 100 differentially expressed genes in the erythrocyte cluster versus all other clusters found no strong evidence supporting Polypterus or axolotl erythrocytes as a significant source of secreted growth factors or extracellular matrix remodelers (Supplementary Data 4). In adult newts, erythrocyte clumps express Newtic1, a salamander-specific gene and putative component of membrane vesicles36. However, contrary to adults, Newtic1+ erythrocytes in the regenerating limbs of larval newts do not contribute to the blastema, and Newtic1 expression is limited to gland-like structures along the skin. Likewise, our findings revealed that the axolotl Newtic1 ortholog was not expressed in the blastema but was instead confined to epidermal cells in both homeostatic and regenerating axolotl limb, as shown by in spatial RNA-seq datasets (Supplementary Fig. 8a). These findings support the hypothesis that Newtic1+ erythrocyte clumps are a feature exclusive of post-metamorphic urodele limb regeneration.

Our analysis of the top 100 upregulated genes upregulated in Polypterus and axolotl erythrocytes also revealed expression of canonical erythroid markers, such as rhag2 and hemoglobin b subunits, as well as species-specific upregulation of genes implicated in modulation of inflammatory response, such as Vipr1 in axolotl erythrocytes37, and gab3 in Polypterus erythrocytes38 (Fig. 8d and Supplementary Fig. 8b, c). Interestingly, both species showed erythrocyte upregulation of traf3ip3, a regulator of MAPK signaling and thymocyte development39, and of a paralog of the hypoxia-inducible factor 1 alpha (hif1a) gene, known for its role in translating changes of oxygen levels into cellular responses essential for wound healing and regeneration40. Phylogenetic analysis revealed that the Polypterus and axolotl hif1a-like paralogs upregulated in erythrocytes corresponded to hif4a, a homolog to the ancestral vertebrate HIFA gene, which was retained in only some ray-finned fish lineages, including zebrafish41 (Fig. 8e). In both species, upregulation of hif4a occurs almost exclusively in the erythrocyte and endothelial cell clusters (Fig. 8f, g). Finally, hif4a-expressing cells were nearly absent from the zebrafish snRNA-seq dataset (Supplementary Fig. 8d). These findings suggest that the emergence of hif4a+ erythrocytes is unique to limb and fin regeneration after endoskeleton-level amputation and the role of erythrocytes in this context may extend beyond oxygen transport.

Hypoxia response mechanisms during Polypterus fin regeneration
Evidence from various regenerative models, including mouse42, Xenopus43, axolotl44, and zebrafish45, supports a scenario where wounding drives local reactive oxygen species (ROS) production, which in turn promotes a hypoxic environment permissive to regeneration. HIF1A plays a crucial role in translating changes in oxygen levels into cellular responses essential for wound healing and regeneration40. HIF1A levels are tightly regulated by prolyl hydroxylase domain (PHD) enzymes (encoded by the EGLN1, EGLN2, and EGLN3 genes) and by the von Hippel-Lindau protein (pVHL, encoded by the VHL gene), which promote HIF1A degradation, and HIF1AN (encoded by the HIF1AN gene), which negatively regulates HIF1A transcription. Hypoxia prevents degradation of HIF1A by its negative regulators, resulting in HIF1A-induced increase in the expression of glycolytic enzymes46,47.

Recent findings demonstrated that during Xenopus and axolotl limb regeneration, the expression of HIF1A negative regulators is low, while Hif1a gene and glycolysis-related genes are highly expressed48. This suggests that these species stabilize HIF1A by maintaining low expression levels of its regulators during regeneration. We examined the expression of the Polypterus hif1a and its regulators, as well as glycolysis genes, in our spatial and snRNA-seq datasets. As seen in Xenopus and axolotl, Polypterus egln1, egln2, egln3, and hif1an expression levels remained mostly low at both uninjured and 1dpa stages, whereas hif1a expression was greatly increased (Fig. 9a, upper panel). At 3dpa, even as expression of HIF1A regulators increased, and hif1a expression decreased, hif1a remained broadly expressed in the blastema region, in contrast to its regulators (Supplementary Fig. 9a, b). Concordantly, expression dynamics of glycolysis genes largely mirrored that of hif1a (Fig. 9a, upper panel), and most were broadly expressed in the Polypterus fin at 3dpa, especially in the muscle (Supplementary Fig. 9c). We further confirmed that during axolotl limb (Fig. 9a, middle) and zebrafish caudal fin ray (Fig. 9a, lower panel) regeneration, a comparable upregulation of /HIF1a/hif1ab and glycolysis genes occurred, although in the zebrafish this program was mostly limited to the 1dpa stage. Altogether, these findings suggest an evolutionarily conserved regenerative response to hypoxia, involving HIF1A expression stabilization and activation of glycolysis.

Myoglobin (MB) is a versatile protein shown to mitigate both ROS activity and hypoxia. Specifically, high MB levels in muscle cells can increase oxygen storage capacity in marine mammals during extended dives49. Furthermore, MB attenuates oxidative stress in cardiac muscle50. Remarkably, while most vertebrates possess a single MB gene, Polypterus possesses at least 15 mb genes51, whereas frogs and salamanders have lost their MB orthologs52. We examined the expression of Polypterus mb genes in our snRNA-seq dataset and found 19 genes currently annotated as myoglobin-like genes in the Polypterus genome. Of those, LOC120537046 was markedly upregulated in the wound epidermis during fin regeneration (Fig. 9b). A previous study showed that among Polypterus myoglobin-like proteins, PseMb1—encoded by LOC120537046—exhibited the highest sequence identity to the mammalian MB gene51. Here, we refer LOC120537046 as mb1. In the uninjured and 1dpa fin tissue, mb1 was expressed in the muscle and in a few cells in the basal epidermis clusters (Fig. 9c, upper panel). At 3dpa, mb1 expression in the basal epidermis increased, and then moderately decreased at 7dpa. Spatial RNA-seq showed mb1 broadly expressed at 3dpa, with the highest levels in the muscle and wound epidermis (Fig. 9c, lower panel). Interestingly, the zebrafish mb gene was also upregulated during caudal fin ray regeneration, peaking at 4dpa (Fig. 9d), and its expression was mostly detected in basal epidermis cells at 3 and 4dpa (Fig. 9e). While the roles of the Polypterus mb1 gene in fin regeneration remain unclear, our findings suggest that mb and its paralogs may be deployed as components of a general mechanism for coping with oxygen stress during fin regeneration, providing a foundation for further investigation into their functional significance.

Activation of DNA damage and repair and switch from pro- to anti-inflammatory immune response during limb and fin regeneration
Previous studies on diverse regenerative models, including planarians53, newts54, Polypterus4, and axolotl4, have demonstrated the upregulation of DNA damage and repair genes at the onset of regeneration. Activation of DNA damage response (DDR) has been shown to be required for regeneration of both the axolotl limb55 and the zebrafish heart56. Analysis of our snRNA-seq data revealed upregulation of DDR markers during fin regeneration. For Polypterus and zebrafish, fin amputation resulted in upregulation of DNA damage sensing (atm, atr, tp53) and repair (rad51, rpa2, fen1, chaf1a/chaf1b) markers, with DNA damage sensing genes being observed in a larger number of cells when compared to DNA damage repair markers (Supplementary Fig. 10a, b). In the axolotl, DNA damage sensing and repair genes were expressed throughout regeneration in relatively similar proportions of cells (Supplementary Fig. 10c). Furthermore, in the axolotl scRNA-seq dataset, the expression of DNA damage sensing and repair genes was already detected at homeostasis. Spatial transcriptomics showed DNA damage sensing and repair gene expression becoming progressively distalized in both Polypterus fin and axolotl limb during regeneration (Supplementary Fig. 10d, e). These findings corroborate a common activation of DDR during fin and limb regeneration of the Polypterus fin and the axolotl limb.

Following axolotl limb amputation, pro- and anti-inflammatory signals are induced and sustained through regeneration16, with pro-inflammatory macrophages peaking during early to mid-blastema stages and anti-inflammatory macrophages being predominant at later stages57. Likewise, in larval zebrafish, caudal fin amputation results in accumulation of pro-inflammatory macrophages at the wound site at 1dpa, followed by an increase in anti-inflammatory macrophages58. Our analysis of the immune response landscape of Polypterus fin regeneration broadly parallels the pattern described in both the axolotl and zebrafish. Upon fin amputation, at 1dpa, we observed upregulation of il1b expression, a classic marker of pro-inflammatory macrophages59, in a subset of myeloid cells (Supplementary Fig. 11a, b). At 1dpa, the myeloid cluster also expressed high levels of spp1, a fibrogenic macrophage marker60, which was sharply downregulated at subsequent stages. Other candidate orthologs of mammalian genes associated with pro-inflammatory macrophages, such as tnfsf1561 and hsp90b162, were upregulated at 1dpa. Anti-inflammatory markers were also upregulated at 1dpa, but their expression peaked at subsequent regeneration stages (Supplementary Fig. 11a, c). tgfb1, an established immune modulator frequently presenting anti-inflammatory activity, was gradually upregulated (Supplementary Fig. 11a) and broadly expressed in cells of the myeloid cluster (Supplementary Fig. 11c) during regeneration stages. Expression of other putative orthologs of anti-inflammatory markers, hs3st1-like, clec7a-like, and mcf2l, also peaked at later stages of fin regeneration. While some overlap exists between pro- and anti-inflammatory marker expression at 1dpa, differences in the subset of cells expressing il1b/spp1 and hs3st1l1/clec7a-like within the myeloid cluster (Supplementary Fig. 11b, c) suggest that pro- and anti-inflammatory macrophages coexist in early regeneration stages. Most pro- and anti-inflammatory markers described here were expressed below the sensitivity level of spatial transcriptomics. However, we did detect expression of spp1 (Supplementary Fig. 11d) and clec7a-like (Supplementary Fig. 11e) in our Polypterus spatial RNA-seq dataset. At 1dpa, spp1 was readily detected in the wound epidermis and distal mesenchyme, whereas clec7a-like+ spots were found in the fin stump. At 3dpa, positive spots for both genes were detected distally, within and around the wound epidermis and subjacent blastema. Although detection of spp1 and clec17a-like was limited to a few dozen spots, the early detection of spp1 near the injury site at 1dpa, but not clec7a-like, is consistent with the initial wave of pro-inflammatory signals we detected in our snRNA-seq dataset. Altogether, these data support a similar dynamic of pro- and anti-inflammatory immune cell response during fin and limb regeneration.

Epigenetic analysis uncovers candidate fin regeneration-responsive elements
The identification of Tissue Regeneration Enhancer Elements (TREEs)63 active during limb and fin regeneration may ultimately reveal conserved gene regulatory networks that orchestrate a successful regenerative program. Various TREEs active during teleost fish fin ray regeneration have been identified in recent years8,64–66, yet changes to the epigenetic landscape in response to injury at the fin endoskeleton region—the homologous counterpart to the tetrapod limb—remain entirely unexplored. To address this, we used ATAC-seq to assess chromatin accessibility in the uninjured fin and at 3dpa—a stage in which a signaling-competent wound epidermis is established, and blastemal cells are readily detected. Biological triplicates of uninjured and 3dpa fin samples were used for ATAC-seq library preparation. Over 150 million paired-end reads were sequenced per sample, and the resulting libraries showed the expected fragment periodicity, separation between conditions, and clustering between replicates, along with over thirty-two and forty-seven thousand peaks called for the uninjured and 3dpa conditions, respectively (Supplementary Fig. 12a–c and Supplementary Data 5). Our analysis revealed 1,427 differentially accessible chromatin regions (adjusted p-value<0.05), or peaks, between the 3dpa and the uninjured fin tissue (Fig. 10a and Supplementary Data 6). Of those, 155 peaks mapped to unplaced DNA scaffolds. The 1272 peaks that mapped to Polypterus chromosomes were mostly (>86%) intergenic (Supplementary Fig. 12d).

Focusing on the top 100 differentially accessible peaks ranked by statistical significance, we searched for potential gene targets, reasoning that genes within their regulatory range would be differentially regulated during fin regeneration. To this end, we generated bulk RNA-seq data from uninjured and 3dpa Polypterus fins in biological triplicates. Over 40 million paired-end reads were sequenced per sample (Supplementary Data 7), and resulting libraries showed separation between conditions and clustering of replicates (Supplementary Fig. 12e). We found 4393 differentially expressed genes (Fig. 10b and Supplementary Data 8). Next, we searched for differentially expressed genes (fold change > 2, adjusted p-value < 0.05) within 600 kb up or downstream of our top 100 intergenic peaks. For peaks located in a gene desert, we expanded the search to 1.2 mb up or downstream of the peak. We found 45 genes differentially expressed at 3dpa located in the vicinity of our top 100 ATAC-seq peaks (Fig. 10b). This list included genes such as lep (Fig. 10a, b, Supplementary Fig. 12f–h), highlighted as a marker for a subpopulation of regeneration associated connective tissue cells (Fig. 6e), and bmp7 (Fig. 10a, b, Supplementary Fig. 12i–k), part of the Polypterus AER program gene set found upregulated in the regenerating epidermis and connective tissue compartments (Fig. 5b, c, e). In addition, this list included trim71, recently shown to potentiate proliferation of reprogrammed limb progenitors67 and tes, identified in our Polypterus fin snRNA-seq dataset as upregulated in the regenerating epidermis (Supplementary Data 2). Among the peaks differentially accessible in the uninjured fin, we found peak 66, located near ccn2b, a gene highly expressed at homeostasis and downregulated over 6-fold at 3dpa (Supplementary Fig. 12l–n and Supplementary Data 8). Its downregulation at 3dpa is consistent with its reported role in promoting fibrosis68.

Next, we inspected more closely three peaks ranked among the top 20 most significantly changed from the uninjured to the 3dpa fin stage: peak 5 (within dip2b gene and upstream of lima1), peak 9 (upstream of trim71), and peak 77 (upstream of tes). Peak 5, located in intron 22 of dip2b, had two genes upregulated in its vicinity: dip2b and lima1 (Fig. 10c). While further experiments will be necessary to confirm the gene under peak 5 influence, expression of lima1 seems more associated with progression of regeneration. Both genes were upregulated by just over 2-fold, bulk RNA-seq showed that lima1 expression at 3dpa is over 15 times greater than that of dip2b (Supplementary Data 8). Of note, this genomic landscape also included peak 661, located 19 kb upstream of lima1. Consistent with the bulk RNA-seq expression levels, spatial RNA-seq detected only a few positive spots of dip2b expression in the Polypterus fin stump at 3dpa (Fig. 10d). Its axolotl ortholog, Dip2b, was detected in scattered spots in the epidermis and mesenchyme (Fig. 10d). SnRNA-seq showed increased dip2b expression across all epidermal cell clusters (Fig. 10e). Unlike dip2b, lima1 and its axolotl ortholog Lima1 were expressed throughout the epidermis, but noticeably highly expressed in the wound epidermis in the spatial transcriptomics data (Fig. 10d). Concordantly, snRNA-seq showed robust lima1 expression in basal epidermis, with increasing levels of expression as regeneration progressed (Fig. 10e). Peak 9 was located 50 kb upstream of trim71 (Fig. 10f), which was upregulated at 3dpa in our bulk RNA-seq dataset. Spatial RNA-seq showed trim71 expressed in scattered spots along the proximal and distal mesenchyme, and wound epidermis of both the Polypterus fin and the axolotl limb at 3dpa (Fig. 10g). SnRNA-seq showed increased trim71 expression during fin regeneration, particularly in the connective tissue and basal epidermis clusters (Fig. 10h). Peak 77 was located approximately 13 kb upstream of tes (Fig. 10i). Spatial RNA-seq showed tes mainly expressed in the Polypterus fin wound epidermis at 3dpa and expressed in scattered spots along the wound epidermis, proximal and distal mesenchyme of the axolotl limb at 3dpa (Fig. 10j). SnRNA-seq showed expression of tes in the basal epidermis during fin regeneration (Fig. 10k).

Next, we investigated genome-wide transcription factor (TF) binding dynamics using TOBIAS69. We analyzed differential TF occupancy, or footprinting, across the 1,427 differentially accessible peaks identified between 3dpa and uninjured fin tissue. The most striking trend in our differential TF binding analysis was the enrichment of bound sites for the AP-1 family of TFs at 3dpa (Fig. 10l and Supplementary Data 9). Among AP-1 TFs, JUNB exhibited the most statistically significant differential binding at 3dpa compared to uninjured fin tissue. Consistently, the aggregated JUNB footprint plot for all 1,427 peaks displayed a well-defined JUNB footprint signal in differentially accessible genomic regions at 3dpa relative to the uninjured fin (Fig. 10m). We examined the expression of Polypterus AP-1 TFs in bulk RNA-seq data from 3dpa versus uninjured fins and found the Polypterus junb significantly upregulated at 3dpa (fold change=3.3, adjusted p-value=2.25E-12) (Supplementary Fig. 12o, Supplementary Data 9). Spatial transcriptomics data showed Polypterus junb and axolotl Junb were widely expressed in epidermal and mesenchymal cells, with Polypterus showing a more distally concentrated expression of junb (Supplementary Fig. 12p). Overall, our findings indicate that, like axolotls and zebrafish, AP-1 TFs play a crucial role in regulating the activity candidate TREEs during Polypterus fin regeneration.

Methods
Animal work
Polypterus (P. senagalus) and axolotls (Ambystoma mexicanum) specimens were maintained and used in accordance with an approved Louisiana State University (LSU) IACUC protocol IACUCAM-25-047. Juvenile fish were obtained from commercial vendors and maintained in individual tanks in a recirculating fresh water system at 27–28°C and a day-night cycle as 12h light/12h dark. Juvenile axolotls were obtained from the Ambystoma Genetic Stock Center at the University of Kentucky and maintained in individual tanks with 40% Holtfreter’s solution at 18–19°C, and a day-night cycle as 12h light/12h dark. Fish were anesthetized in 0.05% MS-222 (Sigma), diluted in fish system water, before amputations. Pectoral fins of Polypterus fish ranging from 7 to 10 cm were bilaterally amputated across the proximal endoskeleton using a sterile scalpel blade. Regenerating fins were harvested at 1, 3, and 7dpa and processed according to downstream protocols. For uninjured fin samples, a portion of the amputated fin encompassing the endoskeletal elements was sampled and processed. As we anticipate future comparisons of some of the sequence datasets described here with datasets generated from animals treated with specific protein inhibitors, regenerating fins were harvested from DMSO-treated animals (0.1% in fish system water) for bulk RNA-seq, spatial transcriptomics, and snRNA-seq experiments. For axolotl samples, animals ranging from 8 to 12 cm were anesthetized in 0.05% MS-222, diluted in 40% Holtfreter’s solution, and had their forelimbs amputated at the level of the upper arm. Regenerating limbs at 3, 7, and 14dpa were harvested and processed for spatial transcriptomics. For uninjured limb samples, a portion of the amputated limb encompassing the upper arm was sampled and processed. All animals were euthanized according to our approved IACUC protocol (0.25% MS-222 in the appropriate animal media).

Histology
Uninjured and regenerating tissues were flash-frozen using isopentane (2-methylbutane) chilled with liquid nitrogen. Isopentane was poured into a metal canister, which was placed in liquid nitrogen to equilibrate. Fresh tissues were embedded in Tissue-Tek O.C.T. compound (Sakura) in appropriate molds. Using forceps, the molds containing O.C.T. compound were placed in the equilibrated isopentane until completely frozen. Samples were stored at −80°C and later sectioned (10μm thickness) in a Leica CM1520 cryostat. Slides were stored at −80°C until subsequent use. Hematoxylin and Eosin staining was performed as previously described3. H&E slides were visualized and imaged using a Nikon Eclipse Ni microscope with a Nikon DS-Fi3 camera.

HCR-FISH. HCR-FISH was performed on 10μm fresh frozen tissue sections following the manufacturer’s protocol (Molecular Instruments). Tissue sections were stored at −80°C until use. Probes for this study were generated by Molecular Instruments and can be ordered using the following lot numbers: RTT023 (for col6a3) and SVB695 (for krt17). To begin, the sections were fixed in ice-cold 4% paraformaldehyde (PFA) (MP Biomedicals) in phosphate-buffered saline (PBS) for 15min at room temperature. After fixation, the slides for col6a3 probes were washed three times with 1X PBS for 5min each. For krt17 probe, slides were submitted to a dehydration series (5min immersion in 50%, 70%, and 2 times in 100% EtOH), followed by a PBS wash. Pre-hybridization was then performed at 37°C for 10min. Probe hybridization was carried out overnight at 37°C. Following the hybridization, the slides were washed three times with probe wash buffer at 37°C for 15min each, and then two washes were performed with 5X SSC-T buffer at room temperature for 5min each. Pre-amplification was done by applying amplification buffer to the slides for 30min at room temperature. Hairpins (H1 and H2) were denatured at 95°C for 90s and allowed to cool to room temperature for 30min in the dark. The hairpins were diluted 1:50 in amplification buffer and applied to each slide. The slides were incubated overnight at room temperature. After incubation, the slides were washed twice with 5X SSC-T buffer for 15min each (col6a3) or 4×15min washes in HCR Gold amplifier buffer (krt17). ProLong Gold with DAPI (Invitrogen, #P36925) was used as mounting media and for nuclear counterstaining. Finally, the slides were stored at 4°C in the dark until imaging.

Bulk RNA-sequencing and analysis
For RNA-sequencing, total RNA was extracted from uninjured and 3dpa pectoral fin tissue (3 biological replicates, 1 pair of fins per replicate) using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, followed by DNase I treatment and column cleanup (Qiagen RNeasy kit) to remove any residual DNA. mRNA-enriched libraries were generated and sequenced at Novogene (https://www.novogene.com/us-en). Briefly, mRNA purified using poly-T oligo-attached magnetic beads was used to generate sequencing libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina, following the manufacturer’s recommendation. Sequencing was performed on an Illumina Novaseq 6000 platform to produce 150bp paired-end reads. Sequence reads were mapped to P. senegalus Genome Assembly ASM1683550V1 using STAR V2.7.10b95. Read count extraction was performed using featureCounts v2.0.696. Read count normalization and differential gene expression analysis were performed using DEseq2 R package97. Genes with a normalized count value < 10 per row were filtered out. Differentially expressed genes were defined by a log2Fold-Change > 1.0 or <−1.0 with a False Discovery Rate (FDR) < 0.05. For subsequent comparative sequence analyses, a subset of Polypterus and axolotl genes was manually annotated according to sequence similarity and/or synteny to other vertebrate genomes (Supplementary Data 10).

Spatial transcriptomics
Cryosections (10μm thickness) from regenerating Polypterus pectoral fins (uninjured, 1, 3, and 7dpa) and regenerating axolotl forelimbs (uninjured, 3, 7, and 14dpa) were placed onto 10X Genomics Visium Spatial Gene Expression Slides. Frozen tissue blocks were prepared as described in the Histology section. Visium slide samples were processed using the 10X Genomics Spatial Gene Expression kit (#1000188) according to the instructions in the Visium Spatial Gene Expression User Guide. Tissue permeabilization was performed for 18min as previously determined using a 10X Genomics Visium Tissue Optimization Slide (#1000193). Brightfield histology images were obtained on a Leica DM6B Microscope at 10X magnification, using a Leica DFC450 color CCD camera, and stitched using the Leica Application suite X software (LASX). Generated libraries were sequenced at Novogene on an Illumina NovaSeq 6000 platform (paired-end, 150bp), with the sequencing depth of 50,000 reads per tissue covered spot. Sequence reads were mapped to P. senegalus Genome Assembly ASM1683550V1 or Ambystoma mexicanum Genome Assembly UKY_AmexF1_1. Raw sequenced data (FASTQ) and histology images were processed using SpaceRanger 3.0.1. Data visualization and generation of specific gene expression images were performed using Loupe Browser 8.0.0. Basic Seurat functions were used for generation of elbow plots in RStudio Version 2023.06.1+524.

Nuclei preparation and single-nucleus RNA-sequencing
Uninjured and regenerating pectoral fins at 1, 3, and 7dpa were dissected using sterile scalpel blades and spring scissors. For each time point, a pair of pectoral fins from two individuals was collected and pooled as one sample after nuclei isolation. A total of two biological replicates (each comprising two polled animal samples) were prepared for each time point. Pectoral fin tissue from each animal was minced first with a spring scissor and then with a razor blade in a petri dish on ice. The minced tissue was resuspended in 1 mL of 0.3X lysis buffer (lysis buffer provided in the Active Motif ATAC-seq kit, #105320, diluted 1:3 with a lysis dilution buffer: 10 mM Tris-HCl, pH: 7.5, 50 mM NaCl, 20 mM Mg2Cl, 10% BSA) and then homogenized in a pre-chilled Dounce homogenizer (12 strokes). The homogenate was filtered using a 40μm cell strainer and immediately diluted with additional 4 mL of lysis dilution buffer and then centrifuged at 400×g at 4°C for 5min in a swinging bucket rotor. The pellet was quickly washed with 1 mL of wash buffer (1X PBS/0.5% BSA), containing 40 U/μl of Protector RNAse inhibitor (Millipore-Sigma #3335399001), without resuspension, and then resuspended in 0.5 mL of wash buffer with RNAse inhibitor. At this point, samples from the two individuals were combined. Nuclei were counted using a hemocytometer, and then a volume corresponding to 106 nuclei was transferred to an Eppendorf LoBind microcentrifuge tube and centrifuged at 400×g at 4°C for 5min. Nuclei were fixed and frozen using the Evercode Nuclei Fixation Kit v3 (#NF100, #NF200) from Parse Biosciences following the manufacturer’s protocol. Fixed and frozen nuclei were then processed to generate barcoded libraries aiming for 7000 nuclei per sample using the Evercode WT v3 Kit (#WT100A-D, #WT200) as per manufacturer’s instructions. Resulting libraries were sequenced by Novogene at the depth of 20,000 reads per nuclei in an Illumina Novaseq X plus platform (paired-end, 150bp).

Single-nucleus RNA-seq analysis
Sequence reads were mapped to P. senegalus Genome Assembly ASM1683550V1. SnRNA-seq FASTQ files were processed using the TrailmakerTM pipeline module (https://app.trailmaker.parsebiosciences.com/; v1.4.0, Parse Biosciences, 2024). In this module, sequences were processed for demultiplexing, barcode correction, read alignment, and transcript quantification. Quantified transcripts were used to generate a cell-by-gene count matrix that was automatically redirected to the Insight module of Trailmaker for downstream analysis. During the Insight module processing, background, doublets, and poor-quality nuclei (high content of mitochondrial gene detection and low level of detected transcripts) were excluded using the Trailmaker default data automatic processing. For our dataset, the minimum number of transcripts per cell varied from 390 to 928, and the median number of transcripts per cell after all filters ranged from 1909 to 5199 (Trailmaker sets a minimum transcript/gene number per cell/nuclei on a per-sample basis). The maximum cutoff value for maximal percentage of mitochondrial genes was 1.21%. Doublets were filtered out based on the scDblFinder method (probability threshold from 0.60 to 0.83). Data from high-quality nuclei were submitted to data normalization, principal-component analysis, and data integration using Harmony. Clusters were identified using the Leiden method, and results were visualized by Uniform Manifold Approximation and Projection (UMAP) embedding. The presto package implementation of the Wilcoxon rank-sum test was used to define cluster-specific marker genes by comparing nuclei data of each cluster to all other nuclei. Trailmaker and Seurat v5 R package98 were used to produce heatmaps and gene expression UMAP image panels. SnRNA-seq data from zebrafish were obtained from the Gene Expression Omnibus (GEO; accession number GSE261907)8 and reprocessed using Cell Ranger. The cellranger mkref command was used to generate the genome index, and cellranger count was employed to align the reads to the reference genome. After processing, the output files (barcodes.tsv.gz, features.tsv.gz, and matrix.mtx.gz) were uploaded to Trailmaker for downstream analyses. For the axolotl dataset15, scRNA-seq data were downloaded from the NCBI Sequence Read Archive (SRA; project number PRJNA589484) and processed using the same pipeline. UMAPs presented through the manuscript were either created in Trailmaker or in RStudio using the Seurat FeaturePlot function. The analysis of geneset score for AER-related genes was performed using the Seurat AddModuleScore function in RStudio. The gene list used (axin2, bambi, bmp2a, bmp7b, cd44, dkk1, dlx2a, fgf9, fgf2, fgf8a, lef1, msx2, prdm1a, rfng, rspo2, rspo3, jag2, sp6, sp8, fn1, gja1, lgr6, notch1) was based on a previously published study26.

Bulk ATAC-seq library preparation
Uninjured and 3dpa fin tissues were collected and subsequently used for nuclei isolation and tagmentation following the ATAC-seq kit protocol (Active Motif, #105320) without modifications. For each triplicate, tissue from both left and right pectoral fins was collected and pooled to comprise one biological replica. Each library was prepared using 50,000 nuclei and amplified within ten PCR cycles. After PCR amplification, the libraries were purified and sent for sequencing. The sequencing was carried out using the NovaSeq X Plus platform, generating 150bp paired-end reads (150 million reads/sample).

ATAC-seq data analysis
The three biological replicates of each condition were processed as follows. Raw reads were trimmed using NGmerge (version 0.3)99. The trimmed reads were aligned to the P. senegalus reference genome from NCBI (ASM1683550v1)12 using Bowtie2 (version 2.2.5)100. Alignments were output as a BAM file, indexed using Samtools (version 1.18)101. Fragment size distribution was assessed using the ATACseqQC package (version 1.24.0)102 in RStudio (version 4.3.1). Trimmed reads were name-sorted using Samtools (version 1.18)101. Genrich (version 0.6.1) was used to call peaks using the parameters -j -y -r -v -m -e -f, to remove duplicate reads, multimapped reads, and reads mapped to mitochondrial chromosome. Downstream analysis of the ATAC-seq data was performed using DiffBind (version 3.10.1) to identify differential chromatin accessibility (peaks) between uninjured and regenerating fin tissues103. Peak sets generated by Genrich were associated with metadata for each sample and imported into DiffBind within RStudio (version 4.3.1). Read counts were generated using summits=75 and normalized using DESeq2 (version 1.40.2)97. Peaks that exhibited differential accessibility between the two conditions were selected based on a significance threshold of FDR < 0.05. Peaks were visualized using Integrative Genomics Viewer (IGV) (version 2.14.1). AnnotatePeaks.pl from HOMER (version 4.11) was used to identify and annotate the genes located nearest to peaks. Motif footprint enrichment was performed using TOBIAS69.

Phylogenetic analysis of HIFA homologs
The following protein sequences of HIFA homologs from representative vertebrate species were obtained from NCBI: Ambystoma mexicanum (HIF1A: XP_069495030.1, HIF2A: XP_069464544.1, HIF3A: XP_069500692.1, HIF4A: XP_069500864.1), Pleurodeles waltl (HIF1A: XP_069063495.1, HIF2A: XP_069090117.1, HIF3A: XP_069063783.1, HIF4A: XP_069063921.1), P. senegalus (HIF1A: XP_039598212.1, HIF2A: XP_039594325.1, HIF3A: XP_039624026.1, HIF4A: XP_039625223.1), Protopterus annectens (HIF1A: XP_043930427.1, HIF2A: XP_043915843.1, HIF3A: XP_043913476.1, HIF4A: XP_043931869.1), Lepisosteus oculatus (HIF1A: XP_069049245.1, HIF2A: XP_015218279.2, HIF3A: XP_069042482.1, HIF4A: XP_069036561.1), and Branchiostoma lanceolatum (HIFA: XP_066279910.1). Protein sequences were aligned using Clustal Omega v1.2.4 with default parameters, enabling MBED-like clustering for both guide tree and iteration steps. The resulting alignment in FASTA format was used as input for phylogenetic inference. Maximum-likelihood phylogenetic analysis was conducted using IQ-TREE v1.6.12 with ModelFinder to identify the best-fitting substitution model. According to the Bayesian Information Criterion (BIC), the optimal model was JTT+F+I+G4, which was subsequently used for tree reconstruction. Node support was assessed using ultrafast bootstrap with 1000 replicates. The final tree was visualized and annotated in FigTree (v1.4.4).

Statistics and reproducibility
Three biological replicates were used for both bulk RNA-seq and bulk ATAC-seq experiments on fins at homeostasis (uninjured) and at 3dpa. For snRNA-seq, two biological replicates—each consisting of pooled samples from two animals—were prepared for each time point. Spatial transcriptomics were performed on single tissue sections from each of the four time points analyzed for Polypterus fins (uninjured, 1, 3, and 7dpa) and axolotl limbs (uninjured, 3, 7, and 14dpa). Histology images from Polypterus intact and regenerating fins were selected from section series obtained from a pair of pectoral fins from two animals per stage. HCR-FISH analyses were conducted using at least two technical replicates for each of the four Polypterus fin time points (uninjured, 1, 3, and 7dpa). No power analysis was used to pre-determine sample sizes. Specific details of the statistical methods and significance values used are provided in the appropriate Methods sections describing each of the RNA/DNA sequence-based analyses and in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Sousa et al., investigated in this work whether there are shared mechanisms of regeneration between Polypterus senegalus\nfin regeneration and axolotl limb regeneration. In this study, the authors describe the following observations:\nBy using single-nucleus RNA sequencing on an early time of regeneration, the authors delineate the cell populations of\nPolypterus senegalus fin regeneration. Complemented with spatial transcriptomics of one time point in both Polypterus\nsenegalus and axolotl, the authors compared expression of genes in wound epithelium and blastema. The authors also\nobserve processes common to regeneration such as a pro to anti-inflammatory switch, DNA repair and hypoxia response.\nOverall, we are supportive of this study, since this could help to uncover the evolutionary origin of limb and fin regeneration.\nThere are several advantages of using Polypterus senegalus: their divergence from teleosts before the latter’s WGD, their\nstructural components in the fin that share characteristics with the limb bones of tetrapods, and beyond the usefulness of\nspecies comparison, the use of Polypterus senegalus can inform on ancestral vs derived mechanisms of regeneration. We\nbelieve however, that at present, many of the findings are too purely descriptive in nature, and this work could thus benefit\nfrom some additional evidence and analysis to support the claims. Particularly, how knowledge of the regenerative process\nin additional species improves our understanding of what is important for regeneration in vertebrates to occur, and how it has\nevolved over time. The details are listed below.\n1) One interesting piece of information is the description of the cell populations present at the early time points. The authors\nnoted a significant presence of erythrocytes and investigated some growth factors and ECM modulators and conclude that\nthey do not contain the secretory profile that has been described before. However, from the cellular landscape, important\ninformation about these dynamic changes could be drawn. For example, 1) what are the erythrocytes expressing besides the\nfew factors they looked at? Later in the manuscript, the expression of a HIF1a-like gene in erythrocytes seems to be unique\nof Polypterus senegalus. Characterizing this population could inform more on their function or relevance in regeneration. 2)\nwhat cell types are proliferating?\n2) The authors mention that in Japanese fire-bellied newts, erythrocyte clumps express genes of secreted growth factors and\nmatrix metalloproteases that might have a role in limb regeneration. Meanwhile, they find that this is not the case in\nPolypterus erythrocytes. However, as they have data of 3 dpa axolotl, it would be interesting for the authors to show if the\naxolotl erythrocytes are more like the newt or like the bichir. Additionally, in the mentioned newt paper\n(https://doi.org/10.1038/s41598-018-25867-x), the growth factor secreting erythrocytes largely appear around 14 dpa (stage II\nblastema) and 27 dpa (stage III blastema). Thus, could it be that the Polypterus samples in the paper are simply too early to\ndetect these erythrocyte clusters?\n3) In Figure 2, the authors mention that col17a1b was previously identified as a marker for wound epidermis, however the\nstaining shows that it is also expressed in intact epidermis and not necessarily specific of a wound epidermis. Also, col6a3\nis proposed here as a marker for blastema cells and they show HCR detection in mesenchyme, but there is not a good\noverall expression view. Since they claim the expression of col6a3 indicates that connective tissue cells are major\ncontributors to the blastema. We would suggest better images of these markers or complementing with their spatial\ntranscriptomics data.\n4) The time (3dpa) to compare both species, is chosen based on the presence of blastema cells and multilayered wound\nepidermis. This is not convincing for the following reasons: axolotls of this size, have almost no cells accumulated below the\nwound epidermis at this timepoint. The wound epithelium is still few cells in thickness compared to the histological section\nshown for Polypterus senegalus and the accumulation of cells below it. What is here marked as thickened wound epithelium\nin the axolotl appears to be the mature epidermis. The protruding bone and the normally retracting connective tissue create\nthis shape that can be confused with a blastema. This could explain for example the mismatch in gene expression in some\nof the genes of interest, because the axolotl sample corresponds to an earlier time point in Polypterus senegalus. We would\nsuggest a validation of these stages. By using either their own data and time datasets in doi: 10.1126/science.aaq0681 and\ndoi: 10.1371/journal.pcbi.100 , or those of others, the authors could provide a conclusive stage comparison.\n5) The authors expand on the species comparison of wound epithelium and blastema, however in Figure 4, they reduce their\ncomparison to a few genes. Given their data, a more comprehensive analysis could provide valuable information. For\nexample, they could provide the list of their top 50 DEG in the blastemas and wound epidermis for both species and build\nfrom there a hypothesis on the species-specific, or conserved mechanisms of regeneration. Interestingly, there are 2\nblastema clusters in Polypterus senegalus, but no mention in the text about them. There seem to be a proximal and a distal\nfield, which could be an exciting result.\n6) A strength discussed in this paper of using the Polypterus senegalus as a model of appendage regeneration, and one\nwhich we strongly agree with, is that it did not undergo the whole-genome duplication that zebrafish and other teleosts did\n(tsWGD), and thus many of its genes have 1:1 orthology with tetrapod genes. With this stated benefit, and with how many\nPolypterus senegalus genes are talked about, we wonder if the authors are missing an opportunity to formalize the gene\nnaming conventions in non-teleost actinopterygians. For example, there are cases where gene names have already been\nassigned a name in the current RefSeq annotation of Polypterus senegalus, such as pax7a and col17a1b, and the authors\nkeep these names. However, the “a” and “b” copy naming convention comes from the zebrafish, in which the ancestral gene\nwas duplicated in the tsWGD, and thus the Polypterus senegalus is not expected to possess a pax7b or col17a1a.\nMeanwhile, the authors newly annotate at least 18 genes that were previously only given LOC IDs, and for these genes they\nassign tetrapod-like gene names, i.e., no “a” or “b” versions. However, in the case of the manually annotated genes esam,\ntgfb1, fn1, fgf10, lima1, and tnc, zebrafish have “a” and “b” versions of each of them. Therefore, if the goal is to keep\nconsistency with the naming convention of already annotated genes, one of these versions would be selected and used as\nthe gene ID, e.g., esama or tgfb1b. While it is difficult to challenge already assigned gene IDs, the discussion of so many\ndifferent genes in the paper, with many being annotated for the first time, could make this a good opportunity to establish\nnames in the Polypterus senegalus that make orthology with the axolotl and other tetrapods clearer, and thus further\nhighlight the benefit of having fish models that are not teleosts.\nAdditionally, the axolotl gene nomenclature is not following the generally agreed upon style of the salamander community\n(https://doi.org/10.1002/dvdy.351), and so should be changed. The current style in the paper also makes it sometimes\nunclear if what is being discussed is a protein or a gene due to not using italics for axolotl genes.\n7) Is there a reason that the authors do not investigate the spatial location of pro-inflammatory and anti-inflammatory genes\nin their spatial transcriptomic data? They show that in the snRNA-Seq data, these genes are expressed by different\npopulations of cells, and thus it would be interesting, and potentially informative, to see if these different populations are also\nlocated in different areas of the regenerating fin.\n8) In Figure 9, it is not really clear why the genes discussed here stood out or were selected for further examination. Could\nbe better if the authors stated something like that tfpi2 was selected for study because they were interested in seeing what\ngenes were specific to Polypterus regeneration. Conversely tnfaip6 seems to have been investigated because it’s\nexpressed in the mesenchymal cells of amniotes and teleosts, demonstrating a strong conservation of its role over vertebrate\nevolution. By more explicitly stating the reasons that drove the selection of these genes, it could enable the authors to more\nclearly highlight the benefits of studying regeneration across species, and to later discuss the ways in which future\nexperiments are impacted by the knowledge of if gene roles in a particular species are derived or conserved.\nMinor comments:\n1) In Figure 1c, a color-coded correspondence of structures between species could be very helpful for people not familiar\nwith the species. For example: in the endoskeleton of the Polypterus senegalus fin what corrresponds to humerus, radius,\ncarpals, phalanges, etc. in the axolotl.\n2) There is a reference to figure (Supplementary Fig. 4c and d), however, there is no “d” panel\n3) Page 9: “Contrary to mammals, urodele amphibians such as newts can regrow severed limbs even at high environmental\noxygen concentrations.”\nThis sentence makes it sound like mammals can regrow severed limbs.\n4) Page 8: “Unlike dif1bb, lima1 and its axolotl ortholog LIMA1 were expressed… (Fig. 10d).”\ndif1bb is the wrong gene to say here. The figure states “DIP2B”, and dif1bb is not mentioned anywhere else in the paper.\n5) Page 6: “A previous study showed that among Polypterus myoglobin-like proteins, PseMb1 the product of\nLOC120537046, termed PseMb1,”\nThe phrasing of how PseMb1 was given its name should be improved.\n6) It is not clear if the authors imply that previous studies which found that Frem2 is a marker of wound epidermis cells in the\naxolotl limb have assigned the gene ID incorrectly, and that it is actually Frem3. It would be good if the authors are clearer\nabout their thoughts on axolotl Frem2/Frem3, and how its expression compares to Polypterus.\n7) A comment on comparing single-nucleus transcription (de novo trasncription) to spatial transcriptomics (whole cell\ntranscripts), would be appreciated.\n8) The authors use the word integration of data across the manuscript to refer to how they use sn-sequencing data and spatil\ntranscriptomics. This could be misleading as the data was not integerated but rather is was analysied side-by-side.", "reviewer_3": "This was an interesting paper that establishes the fish Polypterus senegalus as a new model for studying regeneration in the\nfin. Although zebrafish have been used for this purpose, only the dermal rays of the zebrafish fin can regenerate, which are\ndifferent from the endochondral bones present in limbs. Polypterus fins, on the other hand, have more extensive\nendochondral bones that are capable of regenerating, making them a better model for comparative studies of fin and limb\nregeneration. Here, the authors use a variety of multi-omics approaches, including single-nucleus RNA sequencing, spatial\ntranscriptomics, and ATAC-sequencing to identify commonalities and differences between regeneration in axolotl limbs and\nPolypterus fins.\nOverall, the contribution of a new organism for studying the process of regeneration is significant. The authors present\nseveral impressive datasets and make great strides in establishing Polypterus as a useful model. Although many of the\ngenes and pathways identified have been shown in other systems, the authors do a good job of explaining the benefits of\nstudying regeneration in Polypterus and identify some unique genes and pathways that warrant further study in the future.\nThere are no concerns about the methodology, and this is an impressive amount of data. However, the context for the\nextensive results was sometimes lacking. This made it difficult to understand the conclusions the authors were drawing and\nto assess the validity of these conclusions. Please see some minor clarifications to address below.\nMinor clarifications:\n1. Please label the figures (Fig.1, Fig. 2, etc). It was difficult to toggle back and forth between the manuscript, figure legends,\nand figures when the figures had no labels. It would also be helpful to have the lines numbered in the manuscript.\n2. An issue that was recurrent throughout the manuscript was a lack of context for the findings. Many sections in the results\nsection could use at least one clarifying sentence. Please see some select examples below.\n3. In the first results section (“the cellular landscape of Polypterus fin regeneration”) the authors mention that erythrocytes are\nnot important in Polypterus for fin regeneration while they are in salamanders. Are erythrocytes also not important in\nzebrafish fin regeneration and therefore the role of erythrocytes is limb specific? In the discussion section, the authors state\nthat this warrants further study, but more discussion about this would be useful.\n4. For the next section (“cellular contributions to the Polypterus fin wound epidermis and blastema”) there is a very nice and\nthorough description of the role of connective tissue cells in the Polypterus fin blastema, but no context provided. Is this\nsimilar in other fish and limbs? Or different?\n5. I thought that the sections about hypoxia and the DNA damage response were the clearest to understand. The authors did\na good job of describing the role of both pathways in other organisms and clearly identify Polypterus specific pathways and\ngenes that warrant more study in the future.\n6. In the section “epigenetic analysis uncovers candidate fin regeneration-responsive elements” it was unclear if the authors\nare hypothesizing that Peak 5 is driving lima1 expression instead of dip2bb. Please state this clearly if that is the case.\nFor example, for the sentence “While both genes are upregulated over 2-fold, bulk RNA-seq showed that lima1 expression at 3\ndpa is over 15 times greater than that of dip2bb” please add a sentence such as “indicating that Peak 5 is likely driving\nexpression of lima1.” If that conclusion is not correct, please state what the correct conclusion is from this section about\ndip2bb and lima1.\n7. It would be very helpful to have some sort of table identifying which genes and/or pathways are conserved and diverged\nbetween Polypterus, salamanders, and other fish. Do the authors think that regeneration is more conserved or diverged\nbetween Polypterus and salamanders? What about between Polypterus and zebrafish? Many genes and pathways were\ndiscussed, and it was difficult to get a sense of how conserved this process is between the different organisms.", "reviewer_4": "In the work from Sousa et al., the authors leverage evolutionary relationships across the vertebrate tree of life to compare\nlimb regeneration by the Urodele axolotl salamander with that of a more closely related polypterid, Polypterus senegalus.\nThe endoskeletal fin regeneration of Polypterus is a closer comparison than teleost regeneration of dermal bony fins.\nThis manuscript builds on the Schneider lab’s prior work establishing Polypterus’ regenerative ability and evolutionary\nrelationship relative to axolotl and other fish lineages (Darnet, et al., 2019, PNAS). In this manuscript, the authors perform a\nfull multi-omic comparison of axolotl limb and Polypterus fin regeneration that encompasses single nuclei RNA-seq (snRNA-\nseq), Visium spatial transcriptomics at 3 days post amputation (3dpa), and bulk Assay for Transposase-Accessible\nChromatin sequencing (ATAC-seq). The authors identify a number of similarities and differences in the transcriptional and\ngenomic regulation immediately following amputation. The rigor associated with having both limb and fin tissues subjected\nto the same experimental methodologies and analyses is a great resource for the regeneration field and represents an\nexcellent standard for future studies.\nDespite the technical standard that this work represents, I struggled to find cohesive conclusions from what seemed like\nobservational “vignettes” picked from the multi-omic datasets. I also was unclear if, or how, this work related to their prior\nanalysis of limb and fin regeneration (Darnet et al.). I don’t think this should detract from the work, but I do think there are a\nfew meaningful changes that could create a more focused and presentable manuscript.\n- My primary critique is that this work should be presented primarily as a technical resource since none of the individual\nresults sub-sections are comprehensive or mechanistic enough to make major conclusions. In that vein, I would suggest that\nthe authors put effort into making the data accessible to readers via an explorable R-Shiny site or uploading data to the\nBroad Institute Single Cell Browser (or equivalent). This would translate the technical prowess in the manuscript to a highly\nimpactful resource for the regeneration community.\n- One of the primary arguments put forth by the authors for using the Polypterus fin as a comparative model to the axolotl\nlimb, in contrast to the more ubiquitous zebrafish fin, is the presence of endoskeletal regeneration. Yet, the authors make no\nconnections between regeneration of the fin skeleton and axolotl limb bones. This seems like a large oversight, and it is not\nclear if it is because of technical limitations such as not being able to detect skeletal cell types in snRNA-seq data (no\nclusters were described). I recognize that the timepoints the authors examined are early after amputation and do not\nrepresent timepoints that skeletal patterning is happening, however a comparison of skeletal cell types and signals that\ninfluence processes like bone histolysis, and skeletal progenitor signaling could be examined even at early phases.\n- Related to the point above, the Discussion section of the manuscript is used primarily as a re-hash of result subsections\nrather than an opportunity in the manuscript to describe limitations, future outlook/directions, and postulating on a more\ncohesive overview of the work (e.g., in what ways is Polypterus regeneration more similar to teleost regeneration and what\nways is it more like limb regeneration?). I appreciate that the authors try to put some of their results in context of prior\nliterature, but it is sometimes hard to follow with non-precise reference to other papers. For example, the connection of\nKazald expression to other systems was not very clearly articulated and I’m not sure if the reference given really provides the\nevidence that the authors indicate in the discussion text.\n- As a reader, one of the most interesting and relatively untouched aspects of this work is the difference in spatial distribution\nof gene expression in the proximal-distal axis between the two organisms. The authors point this out relative to DNA\ndamage markers between Polypterus and axolotl, but this seems evident in many other gene comparisons between fin and\nlimb in the manuscript’s figures. Taking an unbiased gene set (e.g., top 100 DEGs) and determing the relative proximal-\ndistal expression zone (using some standard of normalization and distance) would be a very interesting and quantitative\ncomparison using spatial transcriptomic data. This would really leverage the author’s rigorous data in a way that is novel\nand unlikely to be represented in other manuscripts.\n- Finally, I have a hard time really agreeing on the author’s definitions of “wound epidermis” markers derived from snRNA-\nseq in Figures 1-3. The authors describe “wound epidermis” clusters, but these clusters seem to exist in the uninjured data\nset as well, just with fewer cells represented relative to post amputation. They describe col17a1b as a marker of these\nepidermal cells, but again, there are col17a1b-expressing cells before regeneration as well. It is not until Figure 5 when\nmarkers are connected between Visium and the snRNA-seq clusters do you observe marker genes that are expressed\nspecifically after amputation in wound epidermis snRNA-seq clusters. Markers like col17a1b should be clarified as\nupregulated or “amplified” during regeneration instead of markers that uniquely identify the wound epidermis. In general, I\nthink this double standard for wound epidermis markers is confusing and I would simply suggest moving the col17a1b data\nto supplemental data since the data in Figure 5 is much more convincing although it lacks high resolution HCR micrographs."}, "author_response": "Point by point response to reviewers\nWe sincerely thank the reviewers and editors for their thoughtful and constructive feedback on\nour manuscript, now entitled “Comparative multi-omic analysis reveals conserved and derived\nmechanisms of fin and limb regeneration.” We greatly appreciate the careful evaluation and insightful\nsuggestions, which have substantially improved the clarity, depth, and rigor of our study. In this revised\nversion, we have generated additional spatial transcriptomic datasets, expanded comparative analyses\nacross Polypterus, axolotl, and zebrafish, refined cell-type annotations, and clarified key aspects of our\nresults and interpretations. We have addressed all comments in detail below, with point-by-point\nresponses and corresponding changes indicated in the revised manuscript.\nReviewer Comments\nReviewer #1 (Remarks to the Author):\n1) One interesting piece of information is the description of the cell populations present at the early time\npoints. The authors noted a significant presence of erythrocytes and investigated some growth factors\nand ECM modulators and conclude that they do not contain the secretory profile that has been described\nbefore. However, from the cellular landscape, important information about these dynamic changes could\nbe drawn. For example, 1) what are the erythrocytes expressing besides the few factors they looked at?\nLater in the manuscript, the expression of a HIF1a-like gene in erythrocytes seems to be unique of\nPolypterus senegalus. Characterizing this population could inform more on their function or relevance in\nregeneration. 2) what cell types are proliferating?\nWe thank the review for prompting us to looking further into these erythrocytes. We now have analyzed\nthe recruitment of these cells in Polypterus, axolotl and zebrafish and added a results section dedicated\nto this matter, entitled: “Limb and fin regeneration followed by endoskeleton-level amputation is marked\nby robust expansion of hif4a+ erythrocytes”. (starting at line 369). We show that the emergence of these\ncells is not a hallmark of zebrafish tail fin ray regeneration. Our analysis of the top 100 differentially\nexpressed genes in erythrocytes of Polypterus and axolotl confirmed the overall lack of ECM remodeler\nor secreted morphogen expression and showed typical as well as atypical erythrocyte genes commonly\nand uniquely expressed in these species. A careful reassessment of these datasets revealed the\nerythrocyte-specific enrichment of a HIFA paralog and phylogenetic analysis supports that these genes\nencode HIF4A ortholog. The presence of Hif4a-expressing erythrocytes during regeneration in Polypterus\nand axolotl—but not zebrafish tail fin ray—possibly consists in a unique adaptation to the metabolic and\nvascular demands of regenerating an endoskeleton-bearing appendage. These analyses are shown in a\nnew figure (Fig. 8), new supplementary data (Supplementary Fig. 8) and table (Supplementary Data 8).\nIn addition, we have added a paragraph to results section entitled “The cellular landscape of Polypterus\nfin regeneration”, where we clarify that the proliferating cell 1 and 2 clusters present epithelial cell features\n(expression of tp63 and evpl), and expression of col6a3 in proliferating cell 3 cluster suggests that this\ncluster constitutes connective tissue proliferating cells (line 141).\n2) The authors mention that in Japanese fire-bellied newts, erythrocyte clumps express genes of secreted\ngrowth factors and matrix metalloproteases that might have a role in limb regeneration. Meanwhile, they\nfind that this is not the case in Polypterus erythrocytes. However, as they have data of 3 dpa axolotl, it\nwould be interesting for the authors to show if the axolotl erythrocytes are more like the newt or like the\nbichir. Additionally, in the mentioned newt paper (https://doi.org/10.1038/s41598-018-25867-x), the\ngrowth factor secreting erythrocytes largely appear around 14 dpa (stage II blastema) and 27 dpa (stage\nIII blastema). Thus, could it be that the Polypterus samples in the paper are simply too early to detect\nthese erythrocyte clusters?\nThis was a very pertinent point raised by the reviewer. Given additional comments made by this and other\nreviewers we opted to expand our single cell and spatial transcriptomics analyses by 1) generating\nadditional spatial RNA-seq datasets for Polypterus (now uninjured, 1 dpa, 3 dpa and 7 dpa) and axolotl\n(now uninjured, 3 dpa, 7 dpa, and 14 dpa); and 2) by analyzing existing axolotl single cell dataset (stages\nuninjured, 3 dpa, 7 dpa, 14 dpa, and 22 dpa). In contrast to the post-metamorphic newt, where Newtic1⁺\nerythrocyte clumps seem to express secreted factors and matrix metalloproteases during limb\nregeneration, our analyses indicate a distinct erythrocyte profile in Polypterus and axolotl. We found no\nenrichment of secreted growth factors or extracellular matrix remodelers in erythrocytes from either\nspecies (even when later stages are considered), suggesting that these cells are unlikely to contribute\ndirectly to pro-regenerative signaling. Moreover, while Newtic1 expression in adult newts is associated\nwith erythrocyte aggregates, the axolotl Newtic1 ortholog is expressed almost exclusively in epidermal\ncells in both scRNA-seq and spatial transcriptomic datasets, with no detectable expression in\nerythrocytes, which resembles what has been described for larval newts. Together, these findings support\nthe conclusion that Newtic1⁺ erythrocyte clumps are a post-metamorphic salamander feature, and that\nerythrocytes in Polypterus and axolotl instead exhibit strong Hif4a expression, consistent with a role in\nhypoxia adaptation rather than secretion of regenerative cues. These results are now described in the\nsection entitled: “Limb and fin regeneration followed by endoskeleton-level amputation is marked by\nrobust expansion of hif4a+ erythrocytes”. (starting at line 369).\n3) In Figure 2, the authors mention that col17a1b was previously identified as a marker for wound\nepidermis, however the staining shows that it is also expressed in intact epidermis and not necessarily\nspecific of a wound epidermis. Also, col6a3 is proposed here as a marker for blastema cells and they\nshow HCR detection in mesenchyme, but there is not a good overall expression view. Since they claim\nthe expression of col6a3 indicates that connective tissue cells are major contributors to the blastema. We\nwould suggest better images of these markers or complementing with their spatial transcriptomics data.\nWe thank this and other reviewers for drawing our attention to the matter of col17a1 expression in our\nstudy. This has prompted us to generate the additional spatial transcriptomics datasets for both species,\nas well as incorporating analyses of publicly available axolotl limb regeneration and zebrafish tail fin\nregeneration single cell/nucleus RNA-seq datasets, which greatly enriched our study. A more in-depth\nanalysis of wound epidermis cells and candidate markers is now reported in results section entitled\n“Reactivation of apical ectodermal ridge–like gene programs in the Polypterus wound epidermis”. (starting\nat line 225). Whereas col17a1 does become upregulated in the distal wound epidermis, it is expressed in\nhomeostasis and throughout regeneration in all species and therefore is now more accurately described\nin our manuscript as a basal epidermis marker. A refined analysis of genes differentially upregulated in\nthe presumptive wound epidermis is now provided and shows a more established marker of activated\nkeratinocytes (krt17) being conspicuously upregulated in the distal wound epidermis in both single\nnucleus RNA-seq and spatial transcriptomics datasets. HCR-FISH for krt17 was now added and largely\nmirrors the pattern we observe in our Polypterus spatial transcriptomics data. Additional markers including\ncyp27b1 and mmp13 are also shown, as are comparisons of gene expression patterns with axolotl and\nzebrafish. These data resulted in new figures and tables, specifically: Fig.4 and 5, Supplementary Figs.5,\n6 and 7, and Supplementary Data 2.\nWith regards to col6a3, we have made the following changes to accommodate this reviewer’s\nappropriate concerns: 1) we have now clarified in the results (section entitled “Blastema cell\nheterogeneity”, starting at line 299) that col6a3 is being reported not as a blastema marker but as a\ngeneral connective tissue (CT) cell marker in all species, which is expressed in all stages analyzed but\nbecomes enriched distally in the blastema region indicating migration of some CT cells to this area; 2) We\nhave added improved HCR panels to show col6a3 expression at homeostasis and during Polypterus fin\nregeneration; and 3) the addition of spatial transcriptomics datasets at later stages improved the\nvisualization of the expression dynamics of col6a3 as well as other markers of subsets of CT cells.\n4) The time (3dpa) to compare both species, is chosen based on the presence of blastema cells and\nmultilayered wound epidermis. This is not convincing for the following reasons: axolotls of this size, have\nalmost no cells accumulated below the wound epidermis at this timepoint. The wound epithelium is still\nfew cells in thickness compared to the histological section shown for Polypterus senegalus and the\naccumulation of cells below it. What is here marked as thickened wound epithelium in the axolotl appears\nto be the mature epidermis. The protruding bone and the normally retracting connective tissue create\nthis shape that can be confused with a blastema. This could explain for example the mismatch in gene\nexpression in some of the genes of interest, because the axolotl sample corresponds to an earlier time\npoint in Polypterus senegalus. We would suggest a validation of these stages. By using either their own\ndata and time datasets in doi: 10.1126/science.aaq0681 and doi: 10.1371/journal.pcbi.100 , or those of\nothers, the authors could provide a conclusive stage comparison.\nThis is a valid concern that we now address this by generating new spatial transcriptomics datasets for\nlater stages of axolotl limb regeneration (stages added: uninjured, 7 dpa and 14 dpa), as well as by\nincorporating publicly available scRNA-seq data of axolotl limb regeneration. At 7 dpa and 14 dpa the\nthickened wound epidermis is morphologically visible and shows the expression of established WE/apical\nepithelial cap markers. While interspecies stage-to-stage matching is difficult to achieve, our datasets are\nnow extended to provide the general dynamics spanning homeostasis, wound closure, AEC formation\nand blastema formation in both species.\n5) The authors expand on the species comparison of wound epithelium and blastema, however in Figure\n4, they reduce their comparison to a few genes. Given their data, a more comprehensive analysis could\nprovide valuable information. For example, they could provide the list of their top 50 DEG in the\nblastemas and wound epidermis for both species and build from there a hypothesis on the species-\nspecific, or conserved mechanisms of regeneration. Interestingly, there are 2 blastema clusters in\nPolypterus senegalus, but no mention in the text about them. There seem to be a proximal and a distal\nfield, which could be an exciting result.\nWe agree with the reviewer and have expanded our analyses on wound epidermis and the connective\ntissue cells contributing to the blastema, leveraging the newly generated spatial transcriptomics datasets\nand publicly available single cell data for axolotl and zebrafish. Differential gene expression analysis\nenabled us to 1) identify high-confidence markers for the wound epidermis; 2) identify shared and derived\npatterns of wound epidermis gene expression in Polypterus and axolotl; and 3) identify a comprehensive\nlist of genes enriched in the AEC/blastema of Polypterus, axolotl and zebrafish. These data have\nreplaced the previous Fig. 4, and are featured in section entitled “Reactivation of apical ectodermal ridge-\nlike gene programs in the Polypterus wound epidermis” (starting at line 225), in the new Figs. 4, 5 and 6,\nSupplementary Figs. 5, 6 and 7, and Supplementary Data 2.\nFurthermore, we refined our cluster analysis on spatial transcriptomics data and now provide novel\nfindings regarding the two blastema clusters, which we refer to as proximal and distal CT cell populations.\nWe had initially worked with default parameters for clustering, which led to overclustering of spatial data.\nWe corrected this now by applying within-cluster sum of squares (WCSS) as a metric of clustering\ncompactness. This resulted in fewer clusters that are more likely to be biologically meaningful. Even so,\nboth Polypterus 7 dpa and axolotl 14 dpa showed proximal and distal CT compartmentalization with k-\nmeans clusters = 5. Analysis of the top 100 genes expressed in these territories revealed clear\ndistinguishing markers and other features of these subdivided cell populations conserved between\nPolypterus and axolotl. These new analyses are described in a new section entitled “Emergence of\nproximal-distal organization within regenerating connective tissue” (starting at line 336), and featured in\nnewly produced Fig. 7, and Supplementary Data 3.\n6) A strength discussed in this paper of using the Polypterus senegalus as a model of appendage\nregeneration, and one which we strongly agree with, is that it did not undergo the whole-genome\nduplication that zebrafish and other teleosts did (tsWGD), and thus many of its genes have 1:1 orthology\nwith tetrapod genes. With this stated benefit, and with how many Polypterus senegalus genes are talked\nabout, we wonder if the authors are missing an opportunity to formalize the gene naming conventions in\nnon-teleost actinopterygians. For example, there are cases where gene names have already been\nassigned a name in the current RefSeq annotation of Polypterus senegalus, such as pax7a and\ncol17a1b, and the authors keep these names. However, the “a” and “b” copy naming convention comes\nfrom the zebrafish, in which the ancestral gene was duplicated in the tsWGD, and thus the Polypterus\nsenegalus is not expected to possess a pax7b or col17a1a. Meanwhile, the authors newly annotate at\nleast 18 genes that were previously only given LOC IDs, and for these genes they assign tetrapod-like\ngene names, i.e., no “a” or “b” versions. However, in the case of the manually annotated genes esam,\ntgfb1, fn1, fgf10, lima1, and tnc, zebrafish have “a” and “b” versions of each of them. Therefore, if the goal\nis to keep consistency with the naming convention of already annotated genes, one of these versions\nwould be selected and used as the gene ID, e.g., esama or tgfb1b. While it is difficult to challenge already\nassigned gene IDs, the discussion of so many different genes in the paper, with many being annotated for\nthe first time, could make this a good opportunity to establish names in the Polypterus senegalus that\nmake orthology with the axolotl and other tetrapods clearer, and thus further highlight the benefit of\nhaving fish models that are not teleosts.\nAdditionally, the axolotl gene nomenclature is not following the generally agreed upon style of the\nsalamander community (https://doi.org/10.1002/dvdy.351), and so should be changed. The current style\nin the paper also makes it sometimes unclear if what is being discussed is a protein or a gene due to not\nusing italics for axolotl genes.\nWe thank the reviewer for raising this point. We have now revised the manuscript by correctly naming\nPolypterus genes that do not have WGD-derived paralogs and assigning orthology/annotation to several\nLOC ID genes for both Polypterus and axolotl in the Supplementary Data 10, which features all gene\nnames and our assigned annotation. Furthermore, we thank the reviewer for suggesting the citation for\nproper salamander gene nomenclature. We have corrected axolotl gene/protein names throughout the\nmanuscript accordingly.\n7) Is there a reason that the authors do not investigate the spatial location of pro-inflammatory and anti-\ninflammatory genes in their spatial transcriptomic data? They show that in the snRNA-Seq data, these\ngenes are expressed by different populations of cells, and thus it would be interesting, and potentially\ninformative, to see if these different populations are also located in different areas of the regenerating fin.\nWe appreciate this reviewer’s question. Unfortunately, spatial transcriptomics is limited in terms of\ndetection of transcripts of moderate to low abundance or those expressed by few cells. As a result, most\ntranscripts we identified as pro- or anti-inflammatory markers of myeloid cells, while readily detected in\nsingle nucleus RNA-seq, were below detection level of 10x spatial transcriptomics. Nevertheless a few\ngenes (spp1 and clec7a-like) did show expression and are now featured in the revised Fig. 10, providing\nfurther insight into the timing and location of pro- (spp1) and anti-inflammatory (clec7a-like) responses.\n8) In Figure 9, it is not really clear why the genes discussed here stood out or were selected for further\nexamination. Could be better if the authors stated something like that tfpi2 was selected for study\nbecause they were interested in seeing what genes were specific to Polypterus regeneration. Conversely\ntnfaip6 seems to have been investigated because it’s expressed in the mesenchymal cells of amniotes\nand teleosts, demonstrating a strong conservation of its role over vertebrate evolution. By more explicitly\nstating the reasons that drove the selection of these genes, it could enable the authors to more clearly\nhighlight the benefits of studying regeneration across species, and to later discuss the ways in which\nfuture experiments are impacted by the knowledge of if gene roles in a particular species are derived or\nconserved.\nWe thank the reviewer for encouraging us to dive into a more exploratory analysis of our datasets. This\nsection was removed and expression of wound epidermis and blastema genes is more thoroughly\nexplored in the sections entitled “Reactivation of apical ectodermal ridge-like gene programs in the\nPolypterus wound epidermis” (starting at line 225), “Blastema cell heterogeneity” (starting at line 299) and\n“Emergence of proximal-distal organization within regenerating connective tissue” (starting at line 336).\nMinor comments:\n1) In Figure 1c, a color-coded correspondence of structures between species could be very helpful for\npeople not familiar with the species. For example: in the endoskeleton of the Polypterus senegalus fin\nwhat corrresponds to humerus, radius, carpals, phalanges, etc. in the axolotl.\nWe appreciate this reviewer’s suggestion. Unfortunately, direct correspondence (i.e.: bone-to-bone) of\nendoskeletal elements in ray-finned fish (Polypterus) and tetrapods is unresolved and remains an area of\nactive discussion in comparative morphology and evolutionary developmental biology. While the\nPolypterus fin contains proximal elements that are often referred to as “propterygium,” “mesopterygium,”\nand “metapterygium,” these structures do not have unequivocal homologs to specific limb bones such as\nthe humerus, radius, or ulna (see e.g. Coates, 1994 PMID: 7579518.; Boisvert et al., 2008\nhttps://doi.org/10.1038/nature07339; Yano and Tamura 2012 https://doi.org/10.1111/j.1469-\n7580.2012.01491.x). The evolutionary origin of the tetrapod limb endoskeleton likely involved elaboration\nand regionalization of the metapterygial axis, but even this interpretation is not universally accepted. If\nthis reviewer does not oppose it, we prefer to maintain the figure panel as is.\n2) There is a reference to figure (Supplementary Fig. 4c and d), however, there is no “d” panel\nWe agree with the reviewer. Issue has now been corrected – this figure has been removed.\n3) Page 9: “Contrary to mammals, urodele amphibians such as newts can regrow severed limbs even at\nhigh environmental oxygen concentrations.”\nThis sentence makes it sound like mammals can regrow severed limbs.\nWe agree with the reviewer. Issue has now been corrected. Sentence now reads: “Urodele amphibians\nsuch as newts are capable of regenerating severed limbs, even under high environmental oxygen\nconcentrations.” (line 642)\n4) Page 8: “Unlike dif1bb, lima1 and its axolotl ortholog LIMA1 were expressed… (Fig. 10d).”\ndif1bb is the wrong gene to say here. The figure states “DIP2B”, and dif1bb is not mentioned anywhere\nelse in the paper.\nThe reviewer is correct. Issue fixed. Sentence now reads: “Unlike dip2b, lima1 and its axolotl ortholog\nLima1…”. (line 547)\n5) Page 6: “A previous study showed that among Polypterus myoglobin-like proteins, PseMb1 the product\nof LOC120537046, termed PseMb1,”\nThe phrasing of how PseMb1 was given its name should be improved.\nWe agree and have revised this sentence for clarity. Sentence now reads: “A previous study showed that\namong Polypterus myoglobin-like proteins, PseMb1 — encoded by LOC120537046 — exhibited the\nhighest sequence identity to the mammalian MB gene.” (line 442).\n6) It is not clear if the authors imply that previous studies which found that Frem2 is a marker of wound\nepidermis cells in the axolotl limb have assigned the gene ID incorrectly, and that it is actually Frem3. It\nwould be good if the authors are clearer about their thoughts on axolotl Frem2/Frem3, and how its\nexpression compares to Polypterus.\nWe thank the reviewer for pointing out this issue. We have now addressed this in greater depth in the\nsection entitled “Reactivation of apical ectodermal ridge-like gene programs in the Polypterus wound\nepidermis”. (starting at line 225). Furthermore, expression dynamics of frem2/frem3 genes in Polypterus,\naxolotl and zebrafish are now shown in Supplementary Fig. 7.\n7) A comment on comparing single-nucleus transcription (de novo trasncription) to spatial transcriptomics\n(whole cell transcripts), would be appreciated.\nWe agree with the reviewer that addressing the technical differences in transcription detection between\nsnRNA-seq and spatial transcriptomics is important, as these may help explain the differences in the\ntiming of expression peaks we observed for certain genes. To address this, we added the following text\n(starting at line 236) in the section entitled “Reactivation of apical ectodermal ridge-like gene programs in\nthe Polypterus wound epidermis”: “We observed a difference in the timing of the expression peak for\nbasal epithelial markers such as col17a1 and others (cyp27b1 and krt17 – detailed below) in our snRNA-\nseq data compared to our spatial transcriptomics data (Fig. 4a and b). Specifically, these markers showed\ntheir highest expression levels at 1 dpa in the snRNA-seq and at 3 dpa in the spatial transcriptomics\ndataset. The intrinsic technical differences between snRNA-seq, which captures newly synthesized\nnuclear transcripts, and spatial transcriptomics, which primarily reflects accumulated cytoplasmic mRNAs,\nmay explain these differences.”\n8) The authors use the word integration of data across the manuscript to refer to how they use sn-\nsequencing data and spatil transcriptomics. This could be misleading as the data was not integerated but\nrather is was analysied side-by-side.\nWe agree with the reviewer and have now removed the word integration from such contexts throughout\nthe text and title.\nReviewer #3 (Remarks to the Author):\nThis was an interesting paper that establishes the fish Polypterus senegalus as a new model for studying\nregeneration in the fin. Although zebrafish have been used for this purpose, only the dermal rays of the\nzebrafish fin can regenerate, which are different from the endochondral bones present in limbs.\nPolypterus fins, on the other hand, have more extensive endochondral bones that are capable of\nregenerating, making them a better model for comparative studies of fin and limb regeneration. Here, the\nauthors use a variety of multi-omics approaches, including single-nucleus RNA sequencing, spatial\ntranscriptomics, and ATAC-sequencing to identify commonalities and differences between regeneration in\naxolotl limbs and Polypterus fins.\nOverall, the contribution of a new organism for studying the process of regeneration is significant. The\nauthors present several impressive datasets and make great strides in establishing Polypterus as a useful\nmodel. Although many of the genes and pathways identified have been shown in other systems, the\nauthors do a good job of explaining the benefits of studying regeneration in Polypterus and identify some\nunique genes and pathways that warrant further study in the future. There are no concerns about the\nmethodology, and this is an impressive amount of data. However, the context for the extensive results\nwas sometimes lacking. This made it difficult to understand the conclusions the authors were drawing and\nto assess the validity of these conclusions. Please see some minor clarifications to address below.\nMinor clarifications:\n1. Please label the figures (Fig.1, Fig. 2, etc). It was difficult to toggle back and forth between the\nmanuscript, figure legends, and figures when the figures had no labels. It would also be helpful to have\nthe lines numbered in the manuscript.\nFigures have now been labelled, and line numbers have been added.\n2. An issue that was recurrent throughout the manuscript was a lack of context for the findings. Many\nsections in the results section could use at least one clarifying sentence. Please see some select\nexamples below.\nWe thank the reviewer for bringing up this issue. We have now made a conscious effort to add concluding\nsentences to all sections.\n3. In the first results section (“the cellular landscape of Polypterus fin regeneration”) the authors mention\nthat erythrocytes are not important in Polypterus for fin regeneration while they are in salamanders. Are\nerythrocytes also not important in zebrafish fin regeneration and therefore the role of erythrocytes is limb\nspecific? In the discussion section, the authors state that this warrants further study, but more discussion\nabout this would be useful.\nWe thank this and other reviewers who raised the issue of the need for a more in-depth comparative\nanalysis of these cells across taxa. We are restating here our response to reviewer#1, which addresses\nthis matter: “We now have analyzed the recruitment of these cells in Polypterus, axolotl and zebrafish and\nadded a results section dedicated to this matter, entitled: “Limb and fin regeneration followed by\nendoskeleton-level amputation is marked by robust expansion of hif4a+ erythrocytes”. (starting at line\n369). We show that the emergence of these cells is not a hallmark of zebrafish tail fin ray regeneration.\nOur analysis of the top 100 differentially expressed genes in erythrocytes of Polypterus and axolotl\nrevealed confirmed the overall lack of ECM remodeler or secreted morphogen expression and showed\ntypical as well as atypical erythrocyte genes commonly and uniquely expressed in these species. A\ncareful reassessment of these datasets revealed the erythrocyte-specific enrichment of a HIFA paralog\nand phylogenetic analysis supports that these genes encode HIF4A ortholog. The presence of Hif4a-\nexpressing erythrocytes during regeneration in Polypterus and axolotl—but not zebrafish tail fin ray—\npossibly consists in a unique adaptation to the metabolic and vascular demands of regenerating an\nendoskeleton-bearing appendage. These analyses are shown in a new figure (Fig. 8), new\nsupplementary data (Supplementary Fig. 8) and table (Supplementary Data 8).”\n4. For the next section (“cellular contributions to the Polypterus fin wound epidermis and blastema”) there\nis a very nice and thorough description of the role of connective tissue cells in the Polypterus fin\nblastema, but no context provided. Is this similar in other fish and limbs? Or different?\nWe thank the reviewer for the constructive comment and for suggesting that we provide additional context\nto the data presented in this section. To address this and related concerns raised by other reviewers\nregarding our description of col6a3 as a marker of connective tissue contribution to the blastema, we\nhave restructured the relevant section. In the revised manuscript, we now include two new sections\nfocusing on the blastema compartment (“Blastema cell heterogeneity – line 299” and “Emergence of\nproximal-distal organization within regenerating connective tissue – line 336”).\nTo provide context, in the section “Blastema cell heterogeneity,” we have added references supporting the\nstatement that connective tissue cells are major contributors to the blastema in both axolotl and zebrafish.\nIn the following section, “Emergence of proximal-distal organization within regenerating connective tissue”\nwe combine spatial transcriptomics and sn/scRNA-seq analyses to demonstrate a proximal–distal\nsubdivision of connective tissue associated with the blastema region in both Polypterus and axolotl at\nlater stages of regeneration. Cell populations corresponding to these distinct subdomains—identified by\nexpression of specific marker genes—were also detected in a zebrafish fin regeneration snRNA-seq\ndataset. Together, these results indicate that specialization of connective tissue within the blastema\noccurs in a similar manner in regenerating fish fins and axolotl limbs.\n5. I thought that the sections about hypoxia and the DNA damage response were the clearest to\nunderstand. The authors did a good job of describing the role of both pathways in other organisms and\nclearly identify Polypterus specific pathways and genes that warrant more study in the future.\nWe thank the reviewer for appreciating how this section was presented.\n6. In the section “epigenetic analysis uncovers candidate fin regeneration-responsive elements” it was\nunclear if the authors are hypothesizing that Peak 5 is driving lima1 expression instead of dip2bb. Please\nstate this clearly if that is the case. For example, for the sentence “While both genes are upregulated over\n2-fold, bulk RNA-seq showed that lima1 expression at 3 dpa is over 15 times greater than that of dip2bb”\nplease add a sentence such as “indicating that Peak 5 is likely driving expression of lima1.” If that\nconclusion is not correct, please state what the correct conclusion is from this section about dip2bb and\nlima1.\nWe thank the reviewer for raising this issue and we have now modified this statement, which reads as\nfollows: “While further experiments will be necessary to confirm the gene under peak 5 influence,\nexpression of lima1 seems more associated with progression of regeneration. Both genes were\nupregulated by just over 2-fold, bulk RNA-seq showed that lima1 expression at 3 dpa is over 15 times\ngreater than that of dip2b” (line 539).\n7. It would be very helpful to have some sort of table identifying which genes and/or pathways are\nconserved and diverged between Polypterus, salamanders, and other fish. Do the authors think that\nregeneration is more conserved or diverged between Polypterus and salamanders? What about between\nPolypterus and zebrafish? Many genes and pathways were discussed, and it was difficult to get a sense\nof how conserved this process is between the different organisms.\nWe understand and agree with this reviewer’s request for clarification on this matter. We have now\nincluded in Fig. 11 a table that provides a schematic overview of shared and species-specific\nmechanisms and characteristics of limb and fin regeneration in axolotl, Polypterus, and zebrafish, as\nconcluded from our comparative analysis.\nReviewer #4 (Remarks to the Author):\nIn the work from Sousa et al., the authors leverage evolutionary relationships across the vertebrate tree of\nlife to compare limb regeneration by the Urodele axolotl salamander with that of a more closely related\npolypterid, Polypterus senegalus. The endoskeletal fin regeneration of Polypterus is a closer comparison\nthan teleost regeneration of dermal bony fins. This manuscript builds on the Schneider lab’s prior work\nestablishing Polypterus’ regenerative ability and evolutionary relationship relative to axolotl and other fish\nlineages (Darnet, et al., 2019, PNAS). In this manuscript, the authors perform a full multi-omic comparison\nof axolotl limb and Polypterus fin regeneration that encompasses single nuclei RNA-seq (snRNA-seq),\nVisium spatial transcriptomics at 3 days post amputation (3dpa), and bulk Assay for Transposase-\nAccessible Chromatin sequencing (ATAC-seq). The authors identify a number of similarities and\ndifferences in the transcriptional and genomic regulation immediately following amputation. The rigor\nassociated with having both limb and fin tissues subjected to the same experimental methodologies and\nanalyses is a great resource for the regeneration field and represents an excellent standard for future\nstudies.\nDespite the technical standard that this work represents, I struggled to find cohesive conclusions from\nwhat seemed like observational “vignettes” picked from the multi-omic datasets. I also was unclear if, or\nhow, this work related to their prior analysis of limb and fin regeneration (Darnet et al.). I don’t think this\nshould detract from the work, but I do think there are a few meaningful changes that could create a more\nfocused and presentable manuscript.\n- My primary critique is that this work should be presented primarily as a technical resource since none of\nthe individual results sub-sections are comprehensive or mechanistic enough to make major conclusions.\nIn that vein, I would suggest that the authors put effort into making the data accessible to readers via an\nexplorable R-Shiny site or uploading data to the Broad Institute Single Cell Browser (or equivalent). This\nwould translate the technical prowess in the manuscript to a highly impactful resource for the\nregeneration community.\nWe appreciate the reviewer’s suggestion and have now deposited and made immediately available our\nPolypterus single nucleus RNA-seq experiment in the Broad Institute Single Cell Browser.(see Data\nAvailability, line 908).\n- One of the primary arguments put forth by the authors for using the Polypterus fin as a comparative\nmodel to the axolotl limb, in contrast to the more ubiquitous zebrafish fin, is the presence of endoskeletal\nregeneration. Yet, the authors make no connections between regeneration of the fin skeleton and axolotl\nlimb bones. This seems like a large oversight, and it is not clear if it is because of technical limitations\nsuch as not being able to detect skeletal cell types in snRNA-seq data (no clusters were described). I\nrecognize that the timepoints the authors examined are early after amputation and do not represent\ntimepoints that skeletal patterning is happening, however a comparison of skeletal cell types and signals\nthat influence processes like bone histolysis, and skeletal progenitor signaling could be examined even at\nearly phases.\nWe understand and agree that this issue needs clarification. We had originally referred to our Polypterus\nexperiments as endoskeleton regeneration, while our intent was to place emphasis on the region where\nthe amputation is being made – not necessarily the endoskeletal bones. The proximal fin region bears the\nbones that are homologous to tetrapod limb bones, but it also contains the cellular complexity seen in\nlimbs. This cellular complexity (i.e.: presence of muscle, large and diverse connective tissue cell\npopulations, tendons, cartilaginous joints), is lacking in the fin ray region and hence, cannot be assess by\nstudying zebrafish fin ray regeneration. To clarify this, we changed wording in the introduction and\nthroughout the text to emphasize this point and to refer to our approach as a proximal fin amputation at\nthe level of the endoskeleton. (paragraph starting at line 100).\nNevertheless, the reviewer’s comment prompted us to examine in more detail our datasets to\nevaluate genes associated with endoskeletal or fin ray progenitors. With the addition spatial\ntranscriptomics datasets for later stages of Polypterus fin and axolotl limb regeneration, we were able to\nsingle out endochondral and fin ray regeneration regions in the Polypterus fin at 7 dpa. We have now\nadded a new figure (Fig. 3) and a differential gene expression analysis of the endochondral and fin ray\nregions (supplementary data 1) that, we believe, addresses the reviewer’s concerns. These analyses are\nnow reported in a new section entitled: “Distinct regeneration programs of endochondral and dermal\nskeleton in Polypterus.” (starting at line 185).\n- Related to the point above, the Discussion section of the manuscript is used primarily as a re-hash of\nresult subsections rather than an opportunity in the manuscript to describe limitations, future\noutlook/directions, and postulating on a more cohesive overview of the work (e.g., in what ways is\nPolypterus regeneration more similar to teleost regeneration and what ways is it more like limb\nregeneration?). I appreciate that the authors try to put some of their results in context of prior literature,\nbut it is sometimes hard to follow with non-precise reference to other papers. For example, the connection\nof Kazald expression to other systems was not very clearly articulated and I’m not sure if the reference\ngiven really provides the evidence that the authors indicate in the discussion text.\nWe thank the reviewer for bringing up this issue. We have reworked the discussion section to reduce\nrestatements of findings and to emphasize limitations, future directions, and to frame our results in the\ncontext of existing literature.\n- As a reader, one of the most interesting and relatively untouched aspects of this work is the difference in\nspatial distribution of gene expression in the proximal-distal axis between the two organisms. The authors\npoint this out relative to DNA damage markers between Polypterus and axolotl, but this seems evident in\nmany other gene comparisons between fin and limb in the manuscript’s figures. Taking an unbiased gene\nset (e.g., top 100 DEGs) and determing the relative proximal-distal expression zone (using some\nstandard of normalization and distance) would be a very interesting and quantitative comparison using\nspatial transcriptomic data. This would really leverage the author’s rigorous data in a way that is novel\nand unlikely to be represented in other manuscripts.\nWe thank this and other reviewers for raising this point and prompting us to examine this proximal-distal\naxis in more detail. We followed this and other reviewer’s suggestions and now provide evidence for this\nPD subdivision with corresponding identifying gene markers. We are restating here our response to\nreviewer#1, which addresses this matter: “we refined our cluster analysis on spatial transcriptomics data\nand now provide novel findings regarding the two blastema clusters, which we refer to as proximal and\ndistal CT cell populations. We had initially worked with default parameters for clustering, which led to\noverclustering of spatial data. We corrected this now by applying within-cluster sum of squares (WCSS)\nas a metric of clustering compactness. This resulted in fewer clusters that are more likely to be\nbiologically meaningful. Even so, both Polypterus 7 dpa and axolotl 14 dpa showed proximal and distal\nCT compartmentalization with k-means clusters = 5. Analysis of the top 100 genes expressed in these\nterritories revealed clear distinguishing markers and other features of these subdivided cell populations\nconserved between Polypterus and axolotl. These new analyses are described in a new section entitled\n“Emergence of proximal-distal organization within regenerating connective tissue” (starting at line 336),\nand featured in newly produced Fig. 7, and Supplementary Data 3.\n- Finally, I have a hard time really agreeing on the author’s definitions of “wound epidermis” markers\nderived from snRNA-seq in Figures 1-3. The authors describe “wound epidermis” clusters, but these\nclusters seem to exist in the uninjured data set as well, just with fewer cells represented relative to post\namputation. They describe col17a1b as a marker of these epidermal cells, but again, there are col17a1b-\nexpressing cells before regeneration as well. It is not until Figure 5 when markers are connected between\nVisium and the snRNA-seq clusters do you observe marker genes that are expressed specifically after\namputation in wound epidermis snRNA-seq clusters. Markers like col17a1b should be clarified as\nupregulated or “amplified” during regeneration instead of markers that uniquely identify the wound epidermis.\nIn general, I think this double standard for wound epidermis markers is confusing and I would simply suggest\nmoving the col17a1b data to supplemental data since the data in Figure 5 is much more convincing although\nit lacks high resolution HCR micrographs.\nWe thank this and other reviewers for drawing our attention to the matter of col17a1 expression in our\nstudy. This has prompted us to generate the additional spatial transcriptomics datasets for both species,\nas well as incorporating analyses of publicly available axolotl limb regeneration and zebrafish tail fin\nregeneration single cell/nucleus RNA-seq datasets, which greatly enriched our study. We restate here the\nresponse provided to reviewer #1: “A more in-depth analysis of wound epidermis cells and candidate\nmarkers is now reported in results section entitled “Reactivation of apical ectodermal ridge–like gene\nprograms in the Polypterus wound epidermis”. (starting at line 225). Whereas col17a1 does become\nupregulated in the distal wound epidermis, it is expressed in homeostasis and throughout regeneration in\nall species and therefore is now more accurately described in our manuscript as a basal epidermis\nmarker. A refined analysis of genes differentially upregulated in the presumptive wound epidermis is now\nprovided and shows a more established marker of activated keratinocytes (krt17) being conspicuously\nupregulated in the distal wound epidermis in both single nucleus RNA-seq and spatial transcriptomics\ndatasets. HCR-FISH for krt17 was now added and largely mirrors the pattern we observe in our\nPolypterus spatial transcriptomics data. Additional markers including cyp27b1 and mmp13 are also\nshown, as are comparisons of gene expression patterns with axolotl and zebrafish. These data resulted in\nnew figure and tables, specifically: Fig. 4 and 5, Supplementary Figs. 5, 6 and 7, and Supplementary\nData 2."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The corrections submitted by Sousa et al., have greatly enhanced this work. The authors have thoughtfully addressed the\ncomments and it was a pleasure to help refine it. No further comments to make.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}]

Decision letter