Comparative Genomic Studies on the Evolution of Morphological Complexity

National Human Genome Research Institute

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Abstract

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.