Phylogenomic Studies on the Evolution of Morphological Complexity

形态复杂性演化的系统基因组学研究

• 批准号: 10267085
• 负责人: Andreas Baxevanis
• 金额: $ 57.01万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10267085
• 关键词: Adolescent; Affinity Chromatography; Alleles; Animal Model; Animals; Architecture; Bilateral; Biological; Biological Assay; Biological Models; Biological Process; Biology; Biomedical Research; CRISPR/Cas technology; Cells; Chromatin; Cnidaria; Collaborations; Communities; Complex; Copy Number Polymorphism; Custom; Data; Databases; Development; Developmental Biology; Disease Outbreaks; Distant; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Environment; Epigenetic Process; Epitopes; Evolution; Exhibits; Extracellular Domain; Genbank; Gene Duplication; Gene Expression Profile; Generations; Genes; Genetic Polymorphism; Genome; Genomic approach; Genomics; Germ; Germ Cells; Gonadal structure; Graft Rejection; Health; Homologous Transplantation; Human; Human Biology; Intuition; Invertebrates; Ireland; Manuscripts; Mediating; Messenger RNA; Methods; Methylation; Mnemiopsis; Modeling; Molecular; Morphology; Names; National Institute of Child Health and Human Development; Natural Killer Cells; Natural regeneration; Organism; Orthologous Gene; Pattern Formation; Phenotype; Phylogenetic Analysis; Play; Positioning Attribute; Preparation; Process; Protein Family; Proteins; RNA Sequences; Receptor Cell; Research; Resources; RiboTag; Ribosomes; Role; Science; Signal Transduction; Sister; Somatic Cell; Study models; System; Tissues; Transcript; Transgenic Organisms; Translating; Trees; Universities; Untranslated RNA; Zebrafish; adult stem cell; animal cloning; base; cell type; comparative genomics; data sharing; database structure; design; embryo tissue; enhancer-binding protein AP-2; gain of function; genome sequencing; genome-wide; genomic data; gonad development; human disease; in vivo; innovation; insight; interstitial cell; mutant; neurogenesis; novel; pluripotency; promoter; stem cell biology; tissue regeneration; tool; transcriptome sequencing; translational study; usability; whole genome

The study of our most distant animal relatives through the use of phylogenetic and comparative genomic approaches has significantly advanced our understanding of the relationship between genomic and morphological complexity, the evolution of multicellularity, and the emergence of novel cell types. These findings are leading to the establishment of 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. The cnidarians, organisms unified in a single phylum based on their use of cnidocytes to capture prey and for defense from predators, occupy a key phylogenetic position as the sister group to the bilaterians. Previous phylogenomic analyses performed by our group have revealed that the genomes of cnidarians encode more homologs to human disease genes than do classic invertebrate models (1), strongly positioning the cnidarians as powerful model systems for the study of biological processes such as pluripotency, regeneration, lineage commitment, and allorecognition. Given their experimental tractability, including the ability to perform CRISPR/Cas9-mediated gene knock-ins (2), we are actively sequencing and annotating the genomes of two Hydractinia species: H. echinata and H. symbiolongicarpus. 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 (or 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. Using PacBio, Illumina, and Dovetail-based strategies, high-coverage sequencing data indicate an estimated genome size of 774 Mb for H. echinata (84x coverage) and 514 Mb for H. symbiolongicarpus (94x coverage); these genomes are AT-rich (65%) and highly repetitive (47-51%). The N50 for the H. symbiolongicarpus genome exceeds 2.2 MB, making this one of the most contiguous animal 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 (3) and metazoan neurogenesis (4). Allorecognition. The analysis of these Hydractinia genomes has also revealed a heretofore unappreciated complexity of the mechanisms underlying allorecognition. Previously, it was thought that two genes (named Alr1 and Alr2) found within the allorecognition complex (ARC) controlled the ability of colonies to distinguish self from non-self through potential signal transduction motifs in their extracellular domains. Analysis of our highly contiguous whole-genome sequence data has revealed there are 10 putative Alr genes located within the 12 Mb allorecognition complex, with fusion assays indicating that Alr4 is a putative third allodeterminant (manuscript in preparation). The genomic architecture of the ARC is similar to that of mammalian natural killer cell receptors that also exhibit high levels of allelic polymorphism, gene duplication, and copy number variation, suggesting common mechanisms of genomic evolution in both systems. Germ Cell Induction. Clonal animals such as Hydractinia do not sequester a germline during embryogenesis, instead producing gametes from adult stem cells that can also contribute to somatic tissues. However, how germ fate is induced in these animals and whether this process is related to bilateral embryonic germline induction remains an open question. Along with our collaborators at the University of Ireland-Galway, we have shown that transcription factor AP2 (Tfap2), a major regulator of mammalian germline induction, acts as a molecular switch that commits i-cells to germ fate in Hydractinia. Tfap2 mutants were shown to lack germ cells, developing only rudimentary gonads, while transplanted allogenic wild-type cells rescued gonad development but not germ cell induction in Tfap2 mutants. Further, forced expression of Tfap2 in i-cells converted them to germ cells ectopically in non-gonadal tissues of embryos and juveniles, but Tfap2 expression produced no discernible phenotype in somatic cells. These data show that Tfap2 acts cell-autonomously and is essential and sufficient to induce germ cell fate in i-cells, also acting non-cell-autonomously downstream of germ cell induction to promote gonad development. Therefore, Tfap2 is a conserved regulator of germ cell commitment across germline-sequestering and germline-non-sequestering animals (5). Data Sharing. Given the increased emphasis on the development of new animal models for the study of human health, it is extremely important that genomic data generated using these emerging research organisms be disseminated to the biomedical research community in as accessible a fashion as possible. To facilitate access to and use of the data generated during the course of our sequencing, assembly, and annotation efforts, 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. The structure of the database parallels that of the Mnemiopsis Genome Project Portal (6), a research organism database that we originally developed in 2013 (and significantly expanded in 2020) as a straightforward model whose design places a premium on usability, intuitive navigation, and clarity, as well as providing easy access to value-added data that is not available elsewhere. Finally, in a collaboration with Brant Weinstein (NICHD/DIR), we profiled the gene expression patterns of undisturbed endothelial cells in living animals using a novel AngioTag zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment (7). This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope-tagged Rpl10a 60S ribosomal subunit progein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. TRAP-RNAseq on AngioTag animals indicated strong enrichment of endothelial-specific genes and uncovered novel endothelially expressed genes. Additionally, UAS:RiboTag transgenic lines were generated to allow for the study of a wider array of zebrafish cell and tissue types using TRAP-RNAseq methods. This new tool offers an unparalleled resource to study cause and effect relationships in the context of gene loss or gain of function in vivo. (1) Maxwell, E.K. et al. BMC Evolutionary Biology 14: 212, 2014 (2) Sanders, S.M. et al. BMC Genomics 19: 649, 2018 (3) Trk, A. et al., Epigenetics & Chromatin 9: 36, 2016 (4) Gahan, J.M. et al., Dev. Biol. 428: 224-231, 2017 (5) DuBuc, T.Q. et al., Science 367: 757-762, 2020 (6) Moreland, R.T. et al., Database (Oxford) 1-9 (doi:10.1093/database/baaa029), 2020 (7) Miller, M. et al., BioRxiv (doi:10.1101/815696), 2019

通过使用系统发育和比较基因组方法对我们最遥远的动物亲戚的研究显着提高了我们对基因组和形态复杂性之间关系的理解，多细胞性的演变以及新型细胞类型的出现。这些发现导致建立了新的模型生物体，这些生物有可能为人类生物学和人类健康中的重要问题提供信息，从而为转化研究奠定了基础，该研究的重点是特定人类疾病。 Cnidarians，根据使用Cnidocytes捕获猎物和防御捕食者的捕食者，在单个门中统一的生物体占据了捕食者的关键系统发育位置，作为双层人群的姊妹组。我们小组进行的先前的系统基因组学分析表明，与经典无脊椎动物模型（1）相比，CNIDARIANS的基因组编码更多的人类疾病基因同源物，将Cnidarians作为强大的模型系统，用于研究生物学过程，例如多能，再生，再生，线条承诺和Allorercognite，以及。鉴于它们的实验性障碍性，包括执行CRISPR/CAS9介导的基因敲击的能力（2），我们正在积极地测序和注释两种水分种类的基因组：H。echinata和H. symbiolongicarpus。这些简单的生物特别适合作为模型系统的原因在于它们具有多能具有多能的特定类型的间质细胞（或I细胞），并为组织再生提供了基础，表达了其双侧同源物与干细胞生物学有关的基因。 Hydractinia也是殖民地，具有同种异体认知系统，该系统可能会洞悉与宿主 - 移植拒绝相关的重要问题。 使用PACBIO，ILLUMINA和基于燕尾的策略，高覆盖测序数据表明，H. echinata（84倍覆盖范围）的估计基因组大小为774 MB，H。symbiolongicarpus（94倍覆盖范围）的基因组大小为774 Mb；这些基因组是丰富的（65％）和高度重复的（47-51％）。 H. symbiolongicarpus基因组的N50超过2.2 Mb，这使得这是迄今为止测序的最连续动物基因组之一。在这些组件中很容易识别一组进化保守的单拷贝直系同源物的绝大多数，并且对这些全基因组测序数据的分析已经为染色质压实（3）和后生神经发生的演变提供了重要的见解（4）。 同种识别。对这些Hydractinia基因组的分析也揭示了源自同种识别的机制的不受欢迎的复杂性。以前，人们认为在同种异体认知复合物（ARC）中发现的两个基因（命名为ALR1和ALR2）控制着菌落通过其细胞外域中的潜在信号转导基序区分自我与非自我的能力。对我们高度连续的全基因组序列数据的分析表明，在12 MB同种异体识别复合物中，有10个假定的ALR基因，融合分析表明ALR4是推定的第三个同层同层（制备中的手稿）。 ARC的基因组结构与哺乳动物天然杀伤细胞受体的基因组结构相似，这些细胞受体也表现出高水平的等位基因多态性，基因重复和拷贝数变化，这表明了这两个系统中基因组进化的常见机制。 生殖细胞诱导。克隆动物（例如Hydractinia）在胚胎发生过程中不会隔离种系，而是从成年干细胞中产生配子，这也可以有助于体细胞组织。但是，这些动物如何诱导细菌命运，以及该过程是否与双侧胚胎种系诱导有关仍然是一个悬而未决的问题。与我们在爱尔兰大学 - 加alway大学的合作者一起，我们已经表明，哺乳动物种系诱导的主要调节剂AP2（TFAP2）是一种分子开关，在Hydractinia中将I-Cells赋予胚芽命运。 TFAP2突变体被证明缺乏生殖细胞，仅发育基本的性腺，而移植的同种异性野生型细胞挽救了性腺发育，但在TFAP2突变体中没有生殖细胞诱导。此外，在I细胞中，TFAP2强迫表达在胚胎和少年的非辅助组织中将其转化为生殖细胞，但是TFAP2表达在体细胞中没有明显的表型。这些数据表明，TFAP2在I-Cells中诱导生殖细胞命运至关重要，并且足以诱导生殖细胞诱导的下游，以促进性腺发育。因此，TFAP2是跨种系序列和种系的非序列动物的生殖细胞承诺的保守调节剂（5）。 数据共享。鉴于人们越来越重视开发新的动物模型以进行人类健康研究，因此，使用这些新兴研究生物生成的基因组数据以尽可能易于访问的方式传播到生物医学研究界。为了促进在我们的测序，组装和注释工作过程中访问和使用生成的数据，我们开发了位于https://research.nhgri.nih.gov/hydractinia的Hydractinia Genome Project Portal。通过门户网站获得的数据范围可以通过GenBank良好的序列数据，提供了其他生物学信息，旨在增加我们小组生成的测序数据的实用性。它还提供了一种自定义的交互式JBROWSE前端，用于可视化组件，基因预测，组装，转录本，预测的功能域，非编码RNA序列和来自这两个物种的甲基化数据。数据库的结构与Mnemiopsis Genome Project Portal（6）的结构相似，这是我们最初于2013年开发的研究生物数据库（并在2020年显着扩展），作为一种直接的模型，其设计具有优质的可用性，直观的导航和清晰性，并且可以轻松地访问价值添加的数据，该数据不可用其他地方可用。 最后，在与Brant Weinstein（NICHD/DIR）的合作中，我们使用了一种新型的Angiotag斑马鱼转基因系介绍了活动物中不受干扰的内皮细胞的基因表达模式，该系列允许在本机环境中隔离从内皮细胞中主动翻译MRNA（7）。该转基因线使用内皮细胞特异性的KDRL启动子来驱动表位标记的RPL10A 60S核糖体亚基前尿素的表达，从而使核糖体亲和力纯化（TRAP）活跃地翻译内皮细胞mRNA。 Angiotag动物上的陷阱 - rnaseq表明内皮特异性基因的强烈富集和发现的新型内皮表达基因。此外，生成了UAS：核糖转基因线，以研究使用陷阱 - rnaseq方法研究更广泛的斑马鱼细胞和组织类型。该新工具提供了一个无与伦比的资源，可以在基因丧失或体内功能增益的背景下研究因果关系。 （1）E.K。Maxwell等。 BMC进化生物学14：212，2014 （2）Sanders，S.M。等。 BMC基因组学19：649，2018 （3）Trk，A。等，表观遗传学和染色质9：36，2016 （4）Gahan，J.M。等，Dev。生物。 428：224-231，2017 （5）Dubuc，T.Q。等，科学367：757-762，2020 （6）R.T. Moreland等，数据库（牛津）1-9（doi：10.1093/database/baaa029），2020年 （7）Miller，M。等，Biorxiv（doi：10.1101/815696），2019年