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: 10020053
• 负责人: Andreas Baxevanis
• 金额: $ 55.57万
• 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/10020053
• 关键词: Adolescent; Alleles; Animal Model; Animals; Architecture; Bilateral; Binding; Biological Assay; Biological Models; Biological Process; Biology; CRISPR/Cas technology; Cells; Chromatin; Chromosomes; Cnidaria; Code; Collaborations; Complex; Consensus Sequence; Copy Number Polymorphism; Cytoplasmic Tail; DNA; Data; Development; Developmental Biology; Disease Outbreaks; Distant; Elements; Embryo; Embryonic Development; Eukaryota; Evolution; Exhibits; Extracellular Domain; Female; Future; Gene Duplication; Gene Expression Regulation; Generations; Genes; Genetic Polymorphism; Genetic Recombination; Genome; Genomic Segment; Genomic approach; Genomics; Germ; Germ Cells; Gonadal structure; Graft Rejection; Haploidy; Health; Homologous Transplantation; Human; Human Biology; Invertebrates; Ireland; Knock-in; Link; Maintenance; Manuscripts; Maps; Mediating; Modeling; Molecular; Morphology; Names; Natural Killer Cells; Natural regeneration; Nature; Organism; Orthologous Gene; Pattern Formation; Phenotype; Phylogenetic Analysis; Play; Positioning Attribute; Preparation; Process; Protein Family; Proteins; Quantitative Trait Loci; Receptor Cell; Recombinant DNA; Reproduction; Ribosomes; Role; Signal Transduction; Sister; Somatic Cell; Specificity; System; Time; Tissues; Transcription Factor AP-2 Alpha; Trees; Universities; Work; adult stem cell; animal cloning; base; cell type; comparative genomics; embryo tissue; enhancer-binding protein AP-2; genetic sex determination; genome integrity; genome sequencing; genome-wide; gonad development; human disease; innovation; insight; interstitial cell; male; mutant; neurogenesis; novel; offspring; pluripotency; promoter; reference genome; sex; sex determination; stem cell biology; tissue regeneration; translational study; 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-in (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 and metazoan neurogenesis. With respect to the highly repetitive nature of these genomes, repetitive DNA has been implicated in chromatin organization, regulation of gene expression, and the maintenance of genome integrity, but large repeats are often not found in reference genomes due to inherent technical challenges related to sequencing and assembly. Despite these challenges, we have been able to determine the consensus sequence and genomic architecture of rDNA repeats in Hydractinia. Compared to human, Hydractinia has four times as many rDNA repeats in its genome and, while the coding sequences for each ribosomal component are similarly organized and roughly the same size, its intergenic spacers (IGSs) are 100 times shorter than those seen in humans. Given their significantly shorter size, we are exploring whether control elements found in human and vertebrate IGSs are located outside the Hydractinia rDNA cluster. If so, the entire rDNA array may be under the control of a single promoter, enabling it to meet the high demand for ribosomes during regeneration. 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 candidate Alr genes located within the 12 Mb allorecognition complex, with fusion assays indicating that either Alr3 or 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. Future work is focused on the extensive sequence diversity seen in the cytoplasmic tails of these Alr proteins and whether recombination in this genomic region has the potential to create novel binding specificities. While sexual reproduction is ubiquitous among eukaryotes, with sex determination strategies varying greatly among different species, little is known about the mechanisms that determine sex in non-bilaterians. In collaboration with our colleagues at the University of Pittsburgh, we were able to identify a 3 Mb genomic region that co-segregates with sex in Hydractinia. The strategy used to identify this region was based on the construction of a linkage map and subsequent analysis for QTLs linked to sex. 15 linkage groups were recovered in both the male and female maps, corresponding to the haploid chromosome number for Hydractinia. These maps were then used to perform quantitative trait locus analysis to identify markers linked to sex. No sex-linked QTLs were identified on the female map, but a single region on one chromosome of the male map was found to be significantly correlated with sex (LOD > 18, p < 0.001). This region includes four SNPs spanning 3 Mb that co-segregated with sex in 84 of the 87 offspring. These results are consistent with a genetic sex determination system in which males are the heterogametic sex (XX/XY; manuscript in preparation). Finally, 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 (manuscript under review). (1) Maxwell, E.K. et al. BMC Evolutionary Biology 14: 212, 2014. (2) Sanders, S.M. et al. BMC Genomics 19: 649, 2018.

通过使用系统发育和比较基因组方法对我们最远的动物亲戚进行研究，极大地增进了我们对基因组和形态复杂性、多细胞进化以及新细胞类型出现之间关系的理解。这些发现导致了新模式生物的建立，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，为专注于特定人类疾病的转化研究奠定基础。 刺胞动物是基于利用刺胞细胞捕获猎物和防御捕食者而统一在一个门中的生物体，作为两侧对称动物的姐妹类群，占据着重要的系统发育地位。我们小组之前进行的系统发育分析表明，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)，这有力地将刺胞动物定位为研究多能性、再生、谱系定型和同种异体识别等生物过程的强大模型系统。鉴于它们的实验易处理性，包括执行 CRISPR/Cas9 介导的基因敲入的能力 (2)，我们正在积极对两种水螅属物种的基因组进行测序和注释：H. echinata 和 H. symbiolongicarpus。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（或 i-cell），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。 使用基于 PacBio、Illumina 和 Dovetail 的策略，高覆盖率测序数据表明 H. echinata 的估计基因组大小为 774 Mb（84 倍覆盖率），H. symbiolongicarpus 为 514 Mb（94 倍覆盖率）；这些基因组富含 AT (65%) 并且高度重复 (47-51%)。 H. symbiolongicarpus 基因组的 N50 超过 2.2 MB，使其成为迄今为止测序的最连续的动物基因组之一。在这些组件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和后生动物神经发生的进化提供了重要的见解。 鉴于这些基因组的高度重复性质，重复DNA与染色质组织、基因表达调控和基因组完整性的维持有关，但由于与测序和组装相关的固有技术挑战，在参考基因组中通常找不到大量重复。尽管存在这些挑战，我们仍然能够确定海螅中 rDNA 重复序列的共有序列和基因组结构。与人类相比，Hydractinia 的基因组中的 rDNA 重复次数是人类的四倍，虽然每个核糖体成分的编码序列的组织相似且大小大致相同，但其基因间间隔区 (IGS) 比人类中的间隔区短 100 倍。鉴于其尺寸显着缩短，我们正在探索人类和脊椎动物 IGS 中发现的控制元件是否位于水螅 rDNA 簇之外。如果是这样，整个 rDNA 阵列可能处于单个启动子的控制之下，使其能够满足再生过程中对核糖体的高需求。 对这些水螅基因组的分析还揭示了同种异体识别机制的复杂性，这一点迄今为止未被认识到。此前，人们认为同种异体识别复合体 (ARC) 中发现的两个基因（名为 Alr1 和 Alr2）控制着集落通过其胞外域中潜在的信号转导基序区分自身和非自身的能力。对我们高度连续的全基因组序列数据的分析表明，有 10 个候选 Alr 基因位于 12 Mb 同种异体识别复合体中，融合分析表明 Alr3 或 Alr4 是假定的第三个同种异体决定簇（手稿正在准备中）。 ARC 的基因组结构与哺乳动物自然杀伤细胞受体的基因组结构相似，也表现出高水平的等位基因多态性、基因重复和拷贝数变异，表明两个系统中基因组进化的共同机制。未来的工作重点是在这些 Alr 蛋白的细胞质尾部中观察到的广泛序列多样性，以及该基因组区域中的重组是否有可能产生新的结合特异性。 虽然有性生殖在真核生物中普遍存在，不同物种之间的性别决定策略差异很大，但人们对非对称动物性别决定机制知之甚少。通过与匹兹堡大学的同事合作，我们能够鉴定出 Hydractinia 中与性别共分离的 3 Mb 基因组区域。用于识别该区域的策略基于连锁图谱的构建以及随后对与性别相关的QTL的分析。在雄性和雌性图谱中均发现了 15 个连锁群，对应于水螅的单倍体染色体数目。然后使用这些图谱进行数量性状基因座分析，以识别与性别相关的标记。在雌性图谱上没有发现与性别相关的 QTL，但在雄性图谱的一条染色体上的单个区域被发现与性别显着相关（LOD > 18，p < 0.001）。该区域包括 4 个跨度为 3 Mb 的 SNP，在 87 个后代中的 84 个中与性别共分离。这些结果与遗传性别决定系统一致，其中男性是异配性别（XX/XY；手稿正在准备中）。 最后，像水螅这样的克隆动物在胚胎发生过程中不会隔离生殖系，而是从成体干细胞中产生配子，这些配子也可以形成体细胞组织。然而，如何在这些动物中诱导生殖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。我们与爱尔兰大学戈尔韦分校的合作者一起证明，转录因子 AP2 (Tfap2)（哺乳动物种系诱导的主要调节因子）在水螅中充当分子开关，使 i 细胞承担生殖命运。 Tfap2突变体被证明缺乏生殖细胞，仅发育出初级性腺，而移植的同种异体野生型细胞挽救了Tfap2突变体的性腺发育，但不能诱导生殖细胞。此外，在胚胎和幼体的非性腺组织中，i细胞中Tfap2的强制表达将它们异位转化为生殖细胞，但Tfap2表达在体细胞中没有产生可辨别的表型。这些数据表明，Tfap2 细胞自主地发挥作用，并且对于诱导 i-cell 中的生殖细胞命运至关重要且足够，还在生殖细胞诱导的下游非细胞自主地发挥作用，以促进性腺发育。因此，Tfap2 是种系隔离和种系非隔离动物中生殖细胞定型的保守调节因子（手稿正在审查中）。 (1) 麦克斯韦，E.K.等人。 BMC 进化生物学 14: 212, 2014。 (2) 桑德斯，S.M.等人。 BMC 基因组学 19：649，2018。