Deep evolutionary origin of limb and fin regeneration

Abstract

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.

von Willebrand Factor D and EGF Domains is an evolutionarily conserved and required feature of blastemas capable of multi-tissue appendage regeneration

“…We explored the possibility that vwde could be a common feature of blastemas responsible for regenerating paired fins, which share a deep homology with limbs [15], and likely share an inherited gene regulatory program for regeneration [1,2]. We chose two highly regenerative, but distantly related, fish species to determine if vwde expression was a conserved feature of blastemas capable of regenerating paired appendages.…”

“…We first inspected publicly available transcriptome datasets of lungfish and Polypterus regenerating fins for the vwde orthologs we previously identified (Figure 1F). The lungfish LG29893_g1_i1 contig was upregulated in blastemas 21 days post-amputation (dpa) relative to uninjured fins [1], and the Polypterus PS64836c0_g1_i1 contig was upregulated in 9 dpa blastemas relative to uninjured fins [2]. Assessment of expression levels via qPCR at various regeneration stages showed an upregulation of vwde coinciding with blastema formation during lungfish fin regeneration (Supplemental Figure 2A).…”

“…Recent work, most notably next generation sequencing, has led to a plethora of information about the genes and cells that define the blastema [1,2,5–13]. However, it is difficult to determine which genes may have functional relevance based purely on their expression.…”

“…Recent work regarding the evolutionary origins of regenerative capacity has indicated that the ability to regenerate paired appendages is an inherited feature of the fin-to-limb transition [1–4]. Evidence found in the fossil record [3,4], functional studies across species [2], and comparisons of gene expression profiles of regenerating tissue [1,2] support the notion that paired appendage regeneration is a feature lost by certain lineages and was not a newly derived capacity in highly regenerative lineages. This indicates that the amniote lineage (which includes humans) has lost regenerative tendencies in appendages over evolutionary time.…”

“…These studies provide a wealth of information about transcriptomic changes over time, cell types, and blastema-enriched genes. More recently, sequencing efforts of non-model species have allowed for comparisons to the axolotl limb blastema and indicate a core molecular signature that is shared between the blastemas of distantly related species [1,2]. Due to these similarities and the common evolutionary origin of limb regeneration capacity, we can use an evolutionarily-informed approach to understand what constitutes a blastema and for identifying core features required for regeneration.…”