Comparative Genomic Studies on the Evolution of Morphological Complexity

非对称动物物种的基因组测序为分子创新提供了宝贵的见解，这些创新推动了动物早期进化中多样性和复杂性的爆发。刺胞动物是统一在一个门中的生物体，它们利用刺细胞（刺细胞）来捕获猎物和防御捕食者，并且作为两侧对称动物的姐妹类群，它们在系统发育中占据着关键的地位。就基因内容和结构而言，刺胞动物基因组与人类基因组非常相似，而这些生物体之所以特别具有研究吸引力，是因为我们观察到，与经典无脊椎动物模型相比，刺胞动物的基因组编码了更多与人类疾病基因的同源物 (1)。我们正在领导努力，将选定的刺胞动物建立为新的模式生物，这些生物有可能为人类生物学和人类健康的重要问题提供信息，为专注于特定人类疾病的转化研究奠定基础。 我们对两种水螅属物种的基因组进行了测序和注释：H. echinata 和 H. symbiolongicarpus（手稿正在准备中）。这些简单的生物体特别适合作为模型系统，因为它们拥有一种特定类型的多能性间质细胞（i-细胞），为组织再生提供基础，表达已知其双边同源物参与干细胞生物学的基因。水螅也是殖民性的，拥有同种异体识别系统，可以为与宿主移植物排斥相关的重要问题提供见解。我们的测序方法涉及执行 PacBio 长读长测序和 Dovetail 长程支架，对两个基因组产生非常高的覆盖率；组装的 H. symbiolongicarpus 基因组的 N50 值使其成为迄今为止测序的最连续的无脊椎动物基因组之一。在这些组装体中可以轻松识别一组进化上保守的单拷贝直向同源物的绝大多数，并且对这些全基因组测序数据的分析已经为染色质压缩 (2) 和动物神经发生 (3) 的进化提供了重要的见解。 同种异体识别。水螅只是已鉴定出同种异体识别 (Alr) 基因的三种无脊椎动物之一，我们的水螅序列数据的可用性使我们能够更详细地描述同种异体识别复合物 (ARC) 的基因组结构，揭示其固有的复杂性。我们的工作揭示了一个由 41 个基因座组成的令人惊讶的大家族，这些基因座编码 Ig 结构域，打破了许多传统上用于定义 Ig 结构域的“规则”，这表明 Ig 超家族的广度比之前想象的要大。这项工作还表明，V-set Ig 结构域存在于刺胞动物和人类的最后一个共同祖先中，其出现的时间比之前推测的要早得多。几个 Alr 基因被证明具有 ITAM 和 ITIM，这表明 ITAM/ITIM 介导的信号传导可能在无脊椎动物同种异体识别中发挥作用——这一发现具有潜在意义，因为 ITAM/ITIM 介导的信号传导对于无脊椎动物免疫系统中的自我/非自我识别至关重要。这一发现表明无脊椎动物和脊椎动物识别系统之间存在深度同源性 (4)。 神经发生。哺乳动物中新神经元的产生主要发生在胚胎和胎儿发育期间。在成年生活中，神经发生相当有限，导致这些动物的再生能力较差。相比之下，一些无脊椎动物在一生中保持产生所有神经元亚型的能力，其再生能力优于哺乳动物神经系统。 Hydractinia 适合神经发生的研究，因为它在组织稳态过程中不断产生新的神经元来替代老化的神经元。使用干细胞和神经细胞的转基因报告动物、体内成像、遗传干扰以及细胞类型特异性和单细胞转录组学，我们对水螅发育、组织稳态和再生中的神经发生进行了分子和细胞分析。人们发现 SoxB 基因在神经干细胞中按顺序起作用；此外，表达Piwi1和Soxb1的干细胞具有广泛的发育潜力，在分化为成熟神经细胞之前会成为表达Soxb2的神经祖细胞。 SoxB 基因的敲低也被证明会导致胚胎神经发生的复杂缺陷。这些数据让我们深入了解 SoxB 基因的进化及其在后生动物神经发生中的功能 (5)。 性别决定。性别决定发生在各种动物物种中，但我们关于性别决定机制的大部分知识仅来自少数两侧对称动物类群，这限制了我们推断动物性别决定进化历史的能力。我们生成了 H. symbiolongicarpus 基因组的连锁图谱，并使用该图谱确定该物种具有 XX/XY 性别决定系统。这项工作描绘了 Y 染色体的假常染色体和非重组区域，表明后者编码许多具有男性性腺特异性表达的基因。这些发现将Hydractinia确立为一种用于性别决定研究的易处理的非两侧对称动物模型系统，为了解整个动物界的性别进化奠定了基础，并可能揭示6亿多年前真后生动物祖先中存在的途径(6)。 九头蛇基因组计划。鉴于刺胞动物作为两侧对称动物的姐妹类群在进化上的重要地位，我们的小组参与了水螅基因组测序项目，这项工作为普通水螅菌株 AEP（最常见的实验室水螅菌株）产生了第一个染色体水平基因组组装 (7)。这种组装使得系统发育足迹能够揭示保守的顺式调控元件和功能性转录结合基序的预测。 Hi-C 实验提供了染色质中可能影响基因表达的局部接触域的证据；这些与两侧对称动物中发现的拓扑相关结构域具有不同的特征，为动物转录调控的进化提供了线索。最后，单细胞分析确定了转录因子，它们是该生物体细胞命运的关键调节因子。 数据共享：Hydractinia 和 Hydra 基因组项目门户。我们开发了 Hydractinia 基因组项目门户网站，位于 https://research.nhgri.nih.gov/Hydractinia。通过该门户获得的数据范围远远超出了通过 GenBank 获得的序列数据，它提供了额外的生物信息，旨在提高我们小组生成的测序数据的实用性。它还提供定制的交互式 JBrowse 前端，用于可视化两个物种的组装、基因预测、组装、转录本、预测功能域、非编码 RNA 序列和甲基化数据。我们使用相同的方法构建和维护一个联合公共门户，用于研究再生刺胞动物九头蛇的研究人员生成的基因组数据，位于 https://research.nhgri.nih.gov/Hydra (7)。 (1) 麦克斯韦，E.K.等人。 BMC 进化生物学 14: 212, 2014 (2) Torok, A. 等人，表观遗传学与染色质 9: 36，2016 (3) Gahan, J.M. 等人，Dev.生物。 428：224-231，2017 (4) Huene, A.L. 等人，Proc。国家。阿卡德。科学。美国，正在出版。 (5) Chrysostomou, E. 等人，eLife 11：e78793，2022。 (6) Chen, R. 等人，BioRxiv，doi.org/10.1101/2022.03.22.485406，2022。 (7) Cazet, J.F. 等人，BioRxiv，doi.org/10.1101/2022.06.21.496857，2022。

Genomic sequencing of non-bilaterian animal species has provided invaluable insight into the molecular innovations that have fueled the outbreak of diversity and complexity in the early evolution of animals. The cnidarians are organisms unified in a single phylum based on their use of cnidocytes (stinging cells) for capturing prey and defense from predators, and they occupy a key phylogenetic position as the sister group to bilaterian animals. Cnidarian genomes are remarkably similar to the human genome in terms of gene content and structure, and what makes these organisms particularly attractive for study is our observation that the genomes of cnidarians encode more homologs to human disease genes than do classic invertebrate models (1). We are leading efforts to establish selected cnidarians as new model organisms that have the potential to inform important questions in human biology and human health, laying the groundwork for translational studies focused on specific human diseases. We have sequenced and annotated the genomes of two Hydractinia species: H. echinata and H. symbiolongicarpus (manuscript in preparation). What makes these simple organisms particularly well-suited as a model system lies in the fact that they possess a specific type of interstitial cell (an i-cell) that is pluripotent and provides the basis for tissue regeneration, expressing genes whose bilateral homologs are known to be involved in stem cell biology. Hydractinia is also colonial, possessing an allorecognition system that may provide insights into important questions related to host-graft rejection. Our sequencing approach involved performing both PacBio long-read sequencing and Dovetail long-range scaffolding, yielding very high coverage for both genomes; the N50 value of the assembled H. symbiolongicarpus genome make it one of the most contiguous invertebrate genomes sequenced to date. The vast majority of a set of evolutionarily conserved single-copy orthologs can be easily identified in these assemblies, and analyses of these whole-genome sequencing data have already provided important insights into the evolution of chromatin compaction (2) and animal neurogenesis (3). Allorecognition. Hydractinia is only one of three invertebrates in which allorecognition (Alr) genes have been identified, and the availability of our Hydractinia sequence data has enabled us to characterize the genomic structure of the allorecognition complex (ARC) in greater detail, revealing its inherent complexity. Our work has revealed a surprisingly large family of 41 loci that encode Ig domains that break many of the 'rules' traditionally used to define an Ig domain, suggesting that the breadth of the Ig superfamily is larger than previously thought. This work also indicates that V-set Ig domains existed in the last common ancestor of cnidarians and humans, arising far earlier in evolution than previously surmised. Several Alr genes were shown to have ITAMs and ITIMs, suggesting that ITAM/ITIM-mediated signaling could play a role in inverebrate allorecognition - a finding of potential significance, as ITAM/ITIM-mediated signaling is essential in self/non-self recognition in the invertebrate immune system. This finding suggests that deep homologies exist between invertebrate and vertebrate recognition systems (4). Neurogenesis. Generation of new neurons in mammals occurs mainly during embryonic and fetal development. In adult life, neurogenesis is rather limited, resulting in poor regenerative capabilities in these animals. By contrast, some invertebrates maintain the abilities to generate all neuronal subtypes throughout life, outperforming the mammalian nervous system in their ability to regenerate. Hydractinia lends itself to the study of neurogenesis, as it continuously generates new neurons during tissue homeostasis to replace aged neurons. Using transgenic reporter animals for stem cells and neural cells, in vivo imaging, genetic interference, and cell type-specific and single-cell transcriptomics, we have conducted a molecular and cellular analysis of neurogenesis in Hydractinia development, tissue homeostasis, and regeneration. SoxB genes were found to act sequentially in neural stem cells; further, stem cells expressing Piwi1 and Soxb1, which have broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes were also shown to result in complex defects in embryonic neurogenesis. These data provide insight into the evolution of SoxB genes and their function in neurogenesis across the Metazoa (5). Sex Determination. Sex determination occurs across animal species, but most of our knowledge about the mechanisms of sex determination comes from only a handful of bilaterian taxa, limiting our ability to infer the evolutionary history of sex determination in animals. We have generated a linkage map of the genome of the H. symbiolongicarpus and used this map to determine that this species has an XX/XY sex determination system. This work delineated the pseudoautosomal and non-recombining regions of the Y chromosome, showing that the latter encodes a number of genes with male gonad-specific expression. These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination, providing a foundation for understanding how sex has evolved across the animal kingdom and possibly revealing pathways that were present in the eumetazoan ancestor over 600 million years ago (6). Hydra Genome Project. Given the evolutionarily important position of cnidarians as the sister group to the bilaterians, our group has been involved in the Hydra genome sequencing project, an effort that has produced the first chromosome-level genome assembly for H. vulgaris strain AEP, the most common laboratory Hydra strain (7). This assembly has enabled phylogenetic footprinting to reveal conserved cis-regulatory elements and the prediction of functional transcriptional binding motifs. Hi-C experiments have provided evidence of localized contact domains in chromatin that likely influence gene expression; these have different features than the topologically associated domains identified in bilaterians, providing clues as to the evolution of transcriptional regulation in animals. Finally, single-cell analyses identified transcriptional factors that are key regulators of cell fate in this organism. Data Sharing: The Hydractinia and Hydra Genome Project Portals. We have developed the Hydractinia Genome Project Portal, located at https://research.nhgri.nih.gov/hydractinia. The scope of data available through the Portal goes well-beyond the sequence data available through GenBank, providing additional biological information intended to increase the utility of the sequencing data generated by our group. It also provides a customized, interactive JBrowse front-end for visualizing assemblies, gene predictions, assembled, transcripts, predicted functional domains, non-coding RNA sequences, and methylation data from both species. We have used the same approach in building and maintaining an allied public portal for genomic data being generated by investigators studying the regenerative cnidarian Hydra, located at https://research.nhgri.nih.gov/hydra (7). (1) Maxwell, E.K. et al. BMC Evolutionary Biology 14: 212, 2014 (2) Torok, A. et al., Epigenetics & Chromatin 9: 36, 2016 (3) Gahan, J.M. et al., Dev. Biol. 428: 224-231, 2017 (4) Huene, A.L. et al., Proc. Natl. Acad. Sci. USA, in press. (5) Chrysostomou, E. et al., eLife 11: e78793, 2022. (6) Chen, R. et al., BioRxiv, doi.org/10.1101/2022.03.22.485406, 2022. (7) Cazet, J.F. et al., BioRxiv, doi.org/10.1101/2022.06.21.496857, 2022.