Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of paired appendage regeneration, regardless of the amputation level. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in polypterid fishes (Cladistia). Whether this ability evolved independently in sarcopterygians and actinopterygians or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-sequencing (RNA-seq) analysis of Polypterus and axolotl blastemas to provide support for a common origin of paired appendage regeneration in Osteichthyes (bony vertebrates). We show that, in addition to polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of 2 other nonteleost actinopterygians: the American paddlefish (Chondrostei) and the spotted gar (Holostei). Furthermore, we assessed regeneration in 4 teleost species and show that, with the exception of the blue gourami (Anabantidae), 3 species were capable of regenerating fins after endoskeleton amputation: the white convict and the oscar (Cichlidae), and the goldfish (Cyprinidae). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a shared genetic program of appendage regeneration. Comparison of RNA-seq data from early Polypterus blastema to single-cell RNA-seq data from axolotl limb bud and limb regeneration stages shows that Polypterus and axolotl share a regeneration-specific genetic program. Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in Osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration.

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema—a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of complete limb and paired fin regeneration, respectively. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in Polypterid fishes (Cladistia). Whether complete appendage regeneration in sarcopterygians and actinopterygians evolved independently or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-seq analysis to provide support for a common origin of a paired appendage regeneration in osteichthyes (bony vertebrates).We show that, in addition to Polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of all major actinopterygian clades: the American paddlefish, (Chondrostei), the spotted gar (Holostei), as well as in two cichlid species, the white convict and the oscar (Teleostei). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a pan-osteichthyes genetic program of appendage regeneration.Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration. Δ ΔCT method. methods 25, 402-408 (2001).

BackgroundConvergent evolution has been a challenging topic for decades, being cetaceans, pinnipeds and sirenians textbook examples of three independent origins of equivalent phenotypes. These mammalian lineages acquired similar anatomical features correlated to an aquatic life, and remarkably differ from their terrestrial counterparts. Whether their molecular evolutionary history also involved similar genetic mechanisms underlying such morphological convergence nevertheless remained unknown. To test for the existence of convergent molecular signatures, we studied the molecular evolution of Hox genes in these three aquatic mammalian lineages, comparing their patterns to terrestrial mammals. Hox genes are transcription factors that play a pivotal role in specifying embryonic regional identity of nearly any bilateral animal, and are recognized major agents for diversification of body plans.ResultsWe detected few signatures of positive selection on Hox genes across the three aquatic mammalian lineages and verified that purifying selection prevails in these sequences, as expected for pleiotropic genes. Genes found as being positively selected differ across the aquatic mammalian lineages, but we identified a substantial overlap of their developmental functions. Such pattern likely resides on the duplication history of Hox genes, which probably provided different possible evolutionary routes for achieving the same phenotypic solution.ConclusionsOur results indicate that convergence occurred at a functional level of Hox genes along three independent origins of aquatic mammals. This conclusion reinforces the idea that different changes in developmental genes may lead to similar phenotypes, probably due to the redundancy provided by the participation of Hox paralogous genes in several developmental functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0682-4) contains supplementary material, which is available to authorized users.

Salamanders, frog tadpoles and diverse lizards have the remarkable ability to regenerate tails. Palaeontological data suggest that this capacity is plesiomorphic, yet when the developmental and genetic architecture of tail regeneration arose is poorly understood. Here, we show morphological and molecular hallmarks of tetrapod tail regeneration in the West African lungfish Protopterus annectens , a living representative of the sister group of tetrapods. As in salamanders, lungfish tail regeneration occurs via the formation of a proliferative blastema and restores original structures, including muscle, skeleton and spinal cord. In contrast with lizards and similar to salamanders and frogs, lungfish regenerate spinal cord neurons and reconstitute dorsoventral patterning of the tail. Similar to salamander and frog tadpoles, Shh is required for lungfish tail regeneration. Through RNA-seq analysis of uninjured and regenerating tail blastema, we show that the genetic programme deployed during lungfish tail regeneration maintains extensive overlap with that of tetrapods, with the upregulation of genes and signalling pathways previously implicated in amphibian and lizard tail regeneration. Furthermore, the lungfish tail blastema showed marked upregulation of genes encoding post-transcriptional RNA processing components and transposon-derived genes. Our results show that the developmental processes and genetic programme of tetrapod tail regeneration were present at least near the base of the sarcopterygian clade and establish the lungfish as a valuable research system for regenerative biology.

Salamanders, frog tadpoles, and lizards possess the remarkable ability to regenerate tails. The fossil record suggests that this capacity is an ancestral tetrapod trait, yet its evolutionary history remains unclear. Here we examine tail regeneration in a living representative of the sister group of tetrapods, the West African lungfish Protopterus annectens. We show that, as seen in salamanders, lungfish tail regeneration occurs via formation of a proliferative blastema and restores original structures including muscle, skeleton and spinal cord. Contrary to lizards and similar to salamanders, lungfish regenerate spinal cord neurons and reconstitute dorsoventral patterning of the tail.Similar to salamander and frog tadpoles, we show that Shh is required for lungfish tail regeneration. Through RNA-seq analysis of uninjured and regenerating tail blastema we show that lungfish deploy a genetic program comparable to that of tetrapods, showing upregulation of genes and signaling pathways previously implicated in amphibian and lizard tail regeneration. Furthermore, the tail blastema showed marked upregulation of genes encoding post-transcriptional RNA processing components and transposon-derived genes. Collectively, our study establishes the lungfish as a valuable research system for regenerative biology and provides insights into the evolution of cellular and molecular processes underlying vertebrate tail regeneration.

20Regenerative ability varies tremendously across species. A common feature of regeneration of 21 appendages such as limbs, fins, antlers, and tails is the formation of a blastema--a transient 22 structure that houses a pool of progenitor cells that regenerate the missing tissue. We have 23 identified the expression of von Willebrand Factor D and EGF Domains (vwde) as a common 24 feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative 25 species. Further, vwde expression is tightly linked to the ability to regenerate appendages. 26 Functional experiments demonstrate a requirement for vwde in regeneration and indicate that 27 Vwde is a potent mitogen in the blastema. These data identify a key role for vwde in regenerating 28 blastemas and underscore the power of an evolutionarily-informed approach for identifying 29 conserved genetic components of regeneration. 30 The underlying reasons why some animals have the ability to regenerate complex 32 structures, while others cannot, remains an important and open question. This knowledge gap has 33 led to intense study of how regeneration-competent species are able to perform complex multi-34 tissue regeneration, with a particular focus on the ability to regenerate paired appendages, such 35 as limbs and fins. However, this has long been a pursuit without an understanding of whether this 36 ability was present when paired appendages first evolved or was acquired by certain 37 phylogenetic lineages (e.g. urodele amphibians). 38Recent work regarding the evolutionary origins of regenerative capacity has indicated 39 that the ability to regenerate paired appendages is an inherited feature of the fin-to-limb 40 transition [1][2][3][4]. Evidence found in the fossil record [3,4], functional studies across species [2], 41 and comparisons of gene expression profiles of regenerating tissue [1,2] support the notion that 42 paired appendage regeneration is a feature lost by certain lineages and was not a newly derived 43 capacity in highly regenerative lineages. This indicates that the amniote lineage (which includes 44 humans) has lost regenerative tendencies in appendages over evolutionary time. Therefore, the 45 ability to stimulate regeneration in non-regenerative species, potentially in a therapeutic context, 46 may require the re-initiation of a core, evolutionary conserved program. 47All species that are able to regenerate appendages share a conserved trait: the ability to 48 form a blastema. The blastema is the morphological structure that forms at the amputation plane 49 and houses the progenitor cells responsible for regeneration. Recent efforts have focused on 50 elucidating the molecular definition of the blastema, with many of these efforts aimed at the 51 axolotl limb blastema due to the ease of tissue acquisition and the ability to perform 52 experimentation in the lab [5][6][7][8][9][10][11][12][13]. These studies provide a wealth of information about 53 transcriptomic changes over time, cell types, and blastema-e...

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