Comparative Genomic Studies on the Evolution of Morphological Complexity

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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 submitted). 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. 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. The gene family is surprisingly large, containing a total of 41 loci with all but two being located within one of three Alr clusters. Structural studies using Colabfold indicate that the N-terminal domains of these Alr proteins are Ig domains. This is the first time that V-set Ig domains have been identified outside of the Bilateria, indicating that this domain was present in the last common ancestor of cnidarians and humans, over 700 million years ago. Several of these genes also encode for ITAMs and ITIMs, providing evidence that ITAM-mediated signaling may play a key role in invertebrate allorecognition (4). The Hydra Genome. 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 (5). 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 novel candidate regulators of cell-type-specific transcription that have likely been conserved across two cnidarian clades that diverged over 200 million years ago. Senescence. Cellular senescence has long been thought to be related to aging and disease. During our studies of Hydractinia, we noted that small pieces of tissues from the animals head can regenerate a fully functional individual even though the head does not contain any stem cells that are normally required for regeneration in this animal. Using a variety of techniques, we were able to determine that amputation injury induces senescence in adjacent cells, with these senescent cells emitting a signal that causes the surrounding cells to reprogram to pluripotency. Senescent cells are then expelled from the tissue. Inhibition of senescence prevented cellular reprogramming and regeneration, while ectopic induction of senescence induced stem cells, leading to faster regeneration (6). Additional studies published during the reporting period include the determination that Hydractinia uses an XY sex determination system, the first time that the basis of sex determination has been identified unambiguously in any cnidarian or non-bilaterian species (7). Also, a study aimed at understanding the downstream effects of adenosine methylation showed that randomly incorporated 6mA delays zygotic transcription initiation in Hydractinia and that the dioxygenase Alkbh1 acts as a 6mA 'cleaner', facilitating timely zygotic genome activation (8). 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. The structure of the database parallels that of our Mnemiopsis Genome Project Portal (9), providing access to value-added data that is not available elsewhere, and we have used the same approach in building and maintaining an allied public portal for genomic data from our Hydra genome project studies, located at https://research.nhgri.nih.gov/hydra (5). Identification of Conserved Non-Coding Elements Across the Metazoa. Previous studies of conserved non-coding elements (CNEs) have been rather limited in scope, focusing on comparisons between a small number of species given the sheer amount of computational power needed to perform these analyses at scale. We were able to overcome this computational barrier and systematically explored patterns of CNE conservation across the animal kingdom (manuscript submitted). In addition to the large-scale identification of thousands of CNEs in over 50 genomes, we find that CNEs from cnidarians, molluscs, and arthropods have evolved repeatedly and cluster around the same genes, particularly homeodomain genes and those involved in neural development. We also have shown that transposable elements are a major source of CNEs, strongly suggesting that these elements play a critical role in the evolution of regulatory potential within genomes. (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 et al., Proc. Natl. Acad. Sci. USA 119: e2207374119, 2022 (5) Cazet et al., Genome Res. 33: 283-293, 2023 (6) Salinas-Saavedra et al., Cell Rep. 112687, 2023 (7) Chen et al., BMC Biology 21: 32, 2023 (8) Febrimarsa et al., EMBO J. e112934, 2023 (9) Moreland et al., Database 1-9, 2020