Changes in regeneration-responsive enhancers shape regenerative capacities in vertebrates

Regulatory elements of fish regeneration Some animals regenerate extensively, whereas others, such as mammals, do not. The reason behind this difference is not clear. If the genetic mechanisms driving regeneration are evolutionarily conserved, the study of distantly related species that are subjected to different selective pressures could identify distinguishing species-specific and conserved regeneration-responsive mechanisms. Zebrafish and the short-lived African killifish are separated by ∼230 million years of evolutionary distance and, as such, provide a biological context to elucidate molecular mechanisms. Wang et al. identify both species-specific and evolutionarily conserved regeneration programs in these fish. They also provide evidence that elements of this program are subjected to evolutionary changes in vertebrate species with limited or no regenerative capacities. Science, this issue p. eaaz3090 Comparison of two related but evolutionarily distant fish species reveals regulatory elements involved in tissue regeneration. INTRODUCTION The ability to regenerate tissues lost to damage or disease is widely but nonuniformly distributed in vertebrates. Some animals such as teleost fishes can regenerate a variety of organs, including amputated appendages, heart ventricles, and the spinal cord, whereas others such as mammals cannot. Even though regeneration has been the subject of extensive phylogenetic, developmental, cellular, and molecular studies, the mechanisms underlying the broad disparity of regenerative capacities in animals remain elusive. Changes in cis-regulatory elements have been shown to be a major source of morphological diversity. Emerging evidence indicates that injury-dependent gene expression may be controlled by injury-responsive enhancer elements. However, ablations of these previously characterized elements from the zebrafish (Danio rerio) and Drosophila have shown that they are generally dispensable for regeneration. Therefore, whether conserved regeneration-responsive, rather than injury-responsive, elements exist in vertebrate genomes and how they evolved remain to be conclusively demonstrated. RATIONALE Identification of conserved regeneration-responsive enhancers (RREs) requires two related but evolutionarily distant species that are capable of regeneration. The dramatic differences in life history and the ~230 million years of evolutionary distance between the zebrafish and the African killifish Nothobranchius furzeri provide a unique biological context in which to distinguish between species-specific and conserved RREs. We reasoned that applying histone H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq, a marker for active enhancers), bulk RNA sequencing (RNA-seq), and single-cell RNA-seq (scRNA-seq) would identify RREs activated by amputation and help to determine their target gene expression at the single-cell level. Furthermore, we took advantage of the fast sexual maturation of African killifish to rapidly generate transgenic reporter assays to validate predicted RREs and to facilitate their functional testing in adult regeneration. RESULTS We uncovered both large differences in the genomic responses to amputation in killifish and zebrafish and an evolutionarily conserved teleost regeneration response program (RRP), which is mainly deployed by regeneration-specific blastema cells. Bioinformatic analyses revealed that activation of the RRP, which includes known effectors of regeneration in zebrafish such as inhibin beta A (inhba), was differentially activated in mammals that are robust (Acomys cahirinus) and weak regenerators (Mus musculus). Functional testing by systematic transgenic reporter assays of the conserved inhba RRE from killifish, zebrafish, and humans identified species-specific variations. Deletion of the killifish inhba RRE significantly perturbed caudal fin regeneration and abrogated cardiac regeneration. Furthermore, inhba RRE activity required the presence of predicted binding motifs for the activator protein 1 (AP-1) complex. Lastly, AP-1–binding motifs can be identified in the conserved and nonconserved teleost RREs reported in this study, indicating that AP-1 may be required for both injury and regeneration responses. CONCLUSION We propose an RRE-based model for the loss of regenerative capacities during evolution. In our model, the ancestral function for AP-1–enriched RREs was to activate a regenerative response that included both injury and regeneration. Through the course of evolution and speciation, regeneration and injury responses became dissociated from each other in some but not all enhancers. In extant species, regeneration-competent animals maintain the ancestral enhancer activities to activate both injury and regeneration responses, whereas in regeneration-incompetent animals, repurposing of ancestral enhancers may have led to the retention of injury response activities but to the loss of the regeneration response. RREs and vertebrate regeneration. Comparative H3K27ac ChIP-seq, bulk RNA-seq, and scRNA-seq of two distantly related teleost species (African killifish and zebrafish) during the early stages of regeneration helped to identify evolutionarily conserved RREs active in blastemal cells. Systematic transgenic reporter assays validated the putative RREs and helped to identify species-specific variations of an RRE essential for killifish regeneration. Our study provides a testable hypothesis based on enhancer repurposing to explain the uneven distribution of regenerative capacities in vertebrates. Vertebrates vary in their ability to regenerate, and the genetic mechanisms underlying such disparity remain elusive. Comparative epigenomic profiling and single-cell sequencing of two related teleost fish uncovered species-specific and evolutionarily conserved genomic responses to regeneration. The conserved response revealed several regeneration-responsive enhancers (RREs), including an element upstream to inhibin beta A (inhba), a known effector of vertebrate regeneration. This element activated expression in regenerating transgenic fish, and its genomic deletion perturbed caudal fin regeneration and abrogated cardiac regeneration altogether. The enhancer is present in mammals, shares functionally essential activator protein 1 (AP-1)–binding motifs, and responds to injury, but it cannot rescue regeneration in fish. This work suggests that changes in AP-1–enriched RREs are likely a crucial source of loss of regenerative capacities in vertebrates.