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.

中文翻译：

---

再生反应增强子的变化塑造了脊椎动物的再生能力

鱼类再生的调控要素 一些动物广泛再生，而其他动物，如哺乳动物，则不会。这种差异背后的原因尚不清楚。如果驱动再生的遗传机制在进化上是保守的，那么对受到不同选择压力的远缘物种的研究可以确定区分物种特异性和保守的再生响应机制。斑马鱼和短命的非洲鳉鱼相距约 2.3 亿年的进化距离，因此为阐明分子机制提供了生物学背景。王等人。确定这些鱼的物种特异性和进化上保守的再生计划。他们还提供证据表明，该计划的元素受到脊椎动物物种的进化变化的影响，再生能力有限或没有再生能力。科学，本期第 3 页。eaaz3090 对两种相关但进化距离较远的鱼类进行比较，揭示了参与组织再生的调控元件。引言 再生因损伤或疾病而丧失的组织的能力广泛但不均匀地分布在脊椎动物中。硬骨鱼等一些动物可以再生多种器官，包括截肢的附肢、心室和脊髓，而哺乳动物等其他动物则不能。尽管再生一直是广泛的系统发育、发育、细胞和分子研究的主题，但动物再生能力广泛差异背后的机制仍然难以捉摸。顺式调节元件的变化已被证明是形态多样性的主要来源。新出现的证据表明，损伤依赖性基因表达可能受损伤反应增强子元件的控制。然而，对斑马鱼 (Danio rerio) 和果蝇的这些先前特征元素的消融表明，它们通常对于再生是可有可无的。因此，脊椎动物基因组中是否存在保守的再生反应元件而不是损伤反应元件以及它们如何进化仍有待最终证明。基本原理 鉴定保守的再生响应增强子 (RRE) 需要两个相关但进化上遥远的能够再生的物种。生活史的巨大差异以及斑马鱼和非洲鳉鱼 Nothobranchius furzeri 之间约 2.3 亿年的进化距离为区分物种特异性和保守的 RRE 提供了独特的生物学背景。我们推断，应用组蛋白 H3K27ac 染色质免疫沉淀测序（ChIP-seq，活性增强子的标记）、批量 RNA 测序（RNA-seq）和单细胞 RNA-seq（scRNA-seq）将识别截肢激活的 RRE，并帮助在单细胞水平确定它们的靶基因表达。此外，我们利用非洲鳉鱼的快速性成熟来快速生成转基因报告分析，以验证预测的 RRE 并促进它们在成虫再生中的功能测试。结果 我们发现了鳉鱼和斑马鱼截肢的基因组反应的巨大差异以及进化上保守的硬骨鱼再生反应程序（RRP），该程序主要由再生特异性胚泡细胞部署。生物信息学分析表明，RRP 的激活，包括已知的斑马鱼再生效应器，如抑制素 beta A (inhba)，在强壮的哺乳动物 (Acomys cahirinus) 和弱再生器 (Mus musculus) 中被不同地激活。通过对来自鳉鱼、斑马鱼和人类的保守的 inhba RRE 进行系统的转基因报告分析的功能测试，确定了物种特异性变异。删除鳉鱼 inhba RRE 显着扰乱尾鳍再生和废除心脏再生。此外，inhba RRE 活性需要存在预测的激活蛋白 1 (AP-1) 复合物的结合基序。最后，AP-1 结合基序可以在本研究报告的保守和非保守硬骨鱼 RRE 中鉴定，表明 AP-1 可能是损伤和再生反应所必需的。结论 我们提出了一种基于 RRE 的模型，用于在进化过程中丧失再生能力。在我们的模型中，富含 AP-1 的 RRE 的祖先功能是激活包括损伤和再生在内的再生反应。通过进化和物种形成的过程，再生和损伤反应在一些但不是所有增强子中彼此分离。在现存物种中，具有再生能力的动物维持祖先增强剂的活性以激活损伤和再生反应，而在再生能力不足的动物中，祖先增强剂的重新利用可能导致损伤反应活动的保留，但导致再生反应的丧失。RREs和脊椎动物再生。在再生的早期阶段，比较两种远缘硬骨鱼物种（非洲鳉鱼和斑马鱼）的 H3K27ac ChIP-seq、bulk RNA-seq 和 scRNA-seq，有助于识别在胚细胞中活跃的进化上保守的 RRE。系统的转基因报告基因检测验证了假定的 RRE，并帮助确定了鳉鱼再生所必需的 RRE 的物种特异性变异。我们的研究提供了一个基于增强子再利用的可检验假设，以解释脊椎动物再生能力的不均匀分布。脊椎动物的再生能力各不相同，这种差异背后的遗传机制仍然难以捉摸。两种相关硬骨鱼的比较表观基因组分析和单细胞测序揭示了物种特异性和进化上保守的基因组对再生的反应。保守的反应揭示了几种再生反应增强剂 (RRE)，包括一种已知的脊椎动物再生效应器抑制素 beta A (inhba) 上游的元素。该元素激活了再生转基因鱼的表达，其基因组缺失扰乱了尾鳍再生并完全消除了心脏再生。增强剂存在于哺乳动物中，具有功能必需的激活蛋白 1 (AP-1) 结合基序，并对损伤作出反应，但它不能挽救鱼类的再生。