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 also 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, lineage commitment, and allorecognition. Given this observation and their experimental tractability, we are actively sequencing and annotating the genomes of two Hydractinia species: H. echinata and H. symbiolongicarpus. What makes these simple organisms particularly attractive for study is that they possess a specific type of interstitial cell (or 'i-cell') that is pluripotent, expressing genes whose bilateral homologs are known to be involved in stem cell biology. Hydractinia is also colonial, with a complex allorecognition system that lends itself to the study of host-graft rejection. Using PacBio, Illumina, and Dovetail-based strategies, sequencing at high coverage (84x for H. echinata, 94x for H. symbiolongicarpus) has been completed, with the N50 for the H. symbiolongicarpus genome exceeding 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 easily be identified in these assemblies, and analyses of these whole-genome sequencing data have already provided important insights into the evolution of chromatin compaction, with Hydractinia using a set of novel H2B histone variants rather than protamines to compact its sperm DNA; nuclease accessibility assays confirm that the chromatin is less accessible in sperm than in somatic cells, presumably due to the N-terminal tails of these H2B variants binding to linker DNA. Current work on the ectopic expression of these H2B variants in combination with canonical H2B translation-block morpholino co-injection appears to result in a reduced cell proliferation rate and premature death during embryogenesis (2). 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. However, we have now identified a 11.5 Mb repeat-rich region containing at least 30 Alr-like sequences (including Alr1 and Alr2), with all of Hydractinia's Alr-like genes being located within the ARC and not elsewhere in the genome. 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. Continuing studies are focused on the underlying complexity of the ARC, 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. The experimental utility of Hydractinia in experimental settings has been greatly augmented with our successful application of CRISPR/Cas9 genome editing approaches to generate targeted genomic insertions (knock-ins) in H. symbiolongicarpus (3). CRISPR/Cas9 was used to knock fluorescent reporters and small affinity tags into the endogenous eukaryotic elongation factor 1 alpha (Eef1a) locus. Transgenes were expressed ubiquitously and were stable over two generations of breeding. Further, CRISPR/Cas9 genome editing was also used to mark endogenous proteins with two different affinity tags, enabling in vivo and ex vivo protein studies. This work represents the first reported case of successful CRISPR/Cas9-mediated knock-ins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9-inserted transgene in a cnidarian species. Ongoing work in the Computational Genomics Unit includes the characterization of the transcriptional profiles of i-cells at different stages of neurogenesis in adult feeding polyps. Data from FACS-sorted i-cells from transgenic animals expressing reporters for key markers of specific stages in the neurogenic pathway are being used to perform RNA-seq differential expression analyses in order to identify new cell type-specific markers, as well as candidate genes and signaling pathways involved in cell fate determination. We have also identified a complete ribosomal gene consensus sequence in Hydractinia and have determined the genomic architecture of its rDNA repeats. A comprehensive protein domain structural analysis indicates that Hydractinia does not possess the canonical UBF protein, which is required for the recruitment of the Pol I transcription machinery during ribosome biogenesis. This opens the possibility that Hydractinia might employ a different mechanism for regulating transcription of rDNA genes and nucleolar formation than that used by higher eukaryotes. Continuing comparative genomics studies are focused on the comparison of these rDNA repeats and transcription factors in regenerative vs. non-regenerative organisms, with the goal of revealing key mechanisms that underlie regenerative capacity. (1) Maxwell, E.K. et al. Evolutionary Profiling Reveals the Heterogeneous Origins of Classes of Human Disease Genes: Implications for Modeling Disease Genetics in Animals. BMC Evolutionary Biology 14: 212, 2014. (2) Torok, A. et al. The Role of Novel SPKK Repeat-Rich H2B Variants in the Sperm of Hydractinia. European Society for Evolutionary Developmental Biology Conference, Galway, Ireland, 2018. (3) Sanders, S.M. et al. CRISPR/Cas9-Mediated Gene Knock-In in the Hydroid Hydractinia symbiolongicarpus. BMC Genomics, in press. Preprint available through BioRxiv, at www.biorxiv.org/content/early/2018/06/08/342592.
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