Many metazoan genomes are characterized by highly conserved chromosomal homologies that predate the ancient origin of this clade. This conservation has been tested by expansions of selfish DNA elements, in particular transposable elements (TEs). While comparative genomics studies have highlighted their diversity across animal genomes, common principles underlying their evolution along deeply conserved chromosomes have been elusive. A detailed mechanistic understanding from phylogenetically key and early branching animal species has been lacking. We present a comprehensive stem-cell resolved genomic and transcriptomic study of the freshwater cnidarian Hydra, an animal characterized by its high regenerative capacity, the ability to propagate clonally, and an apparent lack of aging. Using single-haplotype telomere-to-telomere genome assemblies of two recently diverged strains and utilizing unique features of hydra biology allowed us to sequence and compare the individual genomes of hydra’s three stem cell lineages. We show that distinct TE families are active at both transcriptional and genomic levels via non-random insertions in each of these lineages. We show that the core set of these active TE families, primarily composed of DNA elements, is evolutionarily deeply conserved and contributes to consistent genomic expansions in metazoan lineages. These anciently active TEs differentially contribute to structural variants around loci associated with cell proliferation and long-range topological contacts. This is in strong contrast to the frequently observed and highly varied substantial genome expansions that often happen via retroelements. Our study suggests an ancient and conserved role for these core TEs as self-renewing components of animal chromosomes.

The regenerative potential of animals varies widely, even among closely related species. In a comparative study of regeneration across the Hydra genus, we found that while most species exhibit robust whole-body regeneration, Hydra oligactis and other members of the Oligactis clade display substantially reduced rates of foot regeneration. To investigate the mechanisms underlying this deficiency, we analyzed transcriptional responses during head and foot regeneration in H. oligactis. Our analysis revealed that the general injury response in H. oligactis lacks strong activation of Wnt signaling, a pathway essential for Hydra vulgaris foot regeneration. Notably, transient treatment with a Wnt agonist in H. oligactis triggered a foot-specific transcriptional program, significantly increasing rates of foot regeneration. Our transcriptional profiling also revealed dlx2 as a likely high-level regulator of foot regeneration, dependent on Wnt signaling activation. Our study establishes a comparative framework for understanding the molecular basis of regeneration in Hydra and provides a new platform for investigating the evolution of regenerative mechanisms.

Hydra , a simple freshwater cnidarian, occurs in both moving and still bodies of water. Flow and corresponding forces are ubiquitous factors in Hydra ’s environment. Even in still water, brief exposure to a burst of flow (e.g., due to wind) can influence the surface attachment and dispersal of polyps. Additionally, alignment with flow may play a crucial role in minimizing forces and regulating feeding behaviors in Hydra . However, the response to flow (particularly in the presence of gravity) has remained underexplored. Using vertically oriented microfluidic chambers, we investigated the biomechanical response of Hydra vulgaris and two additional Hydra species to fluid flow. For Hydra vulgaris , strong surface attachment was observed for all flow rates (0 - 100 mL/hr, corresponding to average velocities of 0 - 2.2 mm/s for the chambers). The experiments indicated alignment of the body column with the flow direction at high flow rates (≥ 50 mL/hr). Alignment with flow was examined by quantifying the angle between the organisms (using a vector connecting head-to-foot) and the flow direction for multiple individuals (N = 9). Most individuals of H. vulgaris exhibited alignment under high flow. While preliminary, comparisons with H. hymanae and H. oxycnida suggested species-specific differences H. hymanae showed tentacle deformation but lacked clear alignment. H. oxycnida did not remain attached under flow. Additional experiments explored the combined effects of flow and osmolarity. These results support the use of microfluidic tools to examine flow-related behaviors and highlight the potential for comparative biomechanics across Hydra species. ### Competing Interest Statement The authors have declared no competing interest.

The Hydra nervous system is the paradigm of a ‘simple nerve net’. Nerve cells in Hydra , as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of the Hydra nerve net by immunostaining fixed polyps with a novel antibody that stains all nerve cells in Hydra . Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution TEM (transmission electron microscopy) and serial block face SEM (scanning electron microscopy) show that the nerve nets consist of bundles of parallel overlapping neurites. Results from transgenic lines show that neurite bundles include different neural circuits and hence that neurites in bundles require circuit-specific recognition. Nerve cell-specific innexins indicate that gap junctions can provide this specificity. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells.

Hydractinia symbiolongicarpus is a pioneering model organism for stem cell biology, being one of only a few animals with adult pluripotent stem cells (known as i-cells). However, the unavailability of a chromosome-level genome assembly has hindered a comprehensive understanding of global gene regulatory mechanisms underlying the function and evolution of i-cells. Here, we report the first chromosome-level genome assembly of H. symbiolongicarpus (HSymV2.0) using PacBio HiFi long-read sequencing and Hi-C scaffolding. The final assembly is 483 Mb in total length with 15 chromosomes representing 99.8% of the assembly. Repetitive sequences were found to account for 296 Mb (61%) of the total genome; we provide evidence for at least two periods of repeat expansion in the past. A total of 25,825 protein-coding genes were predicted in this assembly, which include 93.1% of the metazoan Benchmarking Universal Single-Copy Orthologs (BUSCO) gene set. 92.8% (23,971 genes) of the predicted proteins were functionally annotated. The H. symbiolongicarpus genome showed a high degree of macrosynteny conservation with the Hydra vulgaris genome. This chromosome-level genome assembly of H. symbiolongicarpus will be an invaluable resource for the research community that enhances broad biological studies on this unique model organism.

Cnidarians display a wide diversity of life cycles. Among the main cnidarian clades, only Medusozoa possesses a swimming life cycle stage called the medusa, alternating with a benthic polyp stage. The medusa stage was repeatedly lost during medusozoan evolution, notably in the most diverse medusozoan class, Hydrozoa. Here, we show that the presence of the homeobox gene Tlx in Cnidaria is correlated with the presence of the medusa stage, the gene having been lost in clades that ancestrally lack a medusa (anthozoans, endocnidozoans) and in medusozoans that secondarily lost the medusa stage. Our characterization of Tlx expression indicate an upregulation of Tlx during medusa development in three distantly related medusozoans, and spatially restricted expression patterns in developing medusae in two distantly related species, the hydrozoan Podocoryna carnea and the scyphozoan Pelagia noctiluca. These results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage in the evolution of Hydrozoa.

Selective plane illumination microscopy (SPIM), or light sheet microscopy, is a powerful imaging approach. However, access to and interfacing microscopes with microfluidics have remained challenging. Complex interfacing with microfluidics has limited the SPIM's utility for studying the hydrodynamics of freely moving multicellular organisms. We developed SPIM-Flow, an inexpensive light sheet platform that enables easy integration with microfluidics. We used SPIM-Flow to investigate the hydrodynamics of a freely moving Hydra polyp via particle tracking in millimeter-sized chambers. Initial experiments across multiple animals, feeding on a chip (Artemia franciscana nauplii used as food), and baseline behaviors (tentacle swaying, elongation, and bending) indicated the organisms' health inside the system. Fluidics were used to investigate Hydra's response to flow. The results suggested that the animals responded to an established flow by bending and swaying their tentacles in the flow direction. Finally, using SPIM-Flow in a proof-of-concept experiment, the shear stress required to detach an animal from a surface was demonstrated. Our results demonstrated SPIM-Flow's utility for investigating the hydrodynamics of freely moving animals.

We constructed the first chromosome-level genome assembly of Hydractinia symbiolongicarpus (the HSymV2.0 assembly) using PacBio HiFi long-read sequencing and Hi-C scaffolding. The HSymV2.0 assembly (NCBI accession number: JARBIS000000000) is 483 Mb in total length with 15 chromosomes, which represent 99.8% of the assembly. We also provide its genome annotation with 25,959 protein-coding genes as well as cDNA/CDS/Protein sequences. Index for cellranger is also provided for single-cell RNA-seq analysis. The HSymV2.0 assembly will be an invaluable resource for the research community performing a wide range of studies with H. symbiolongicarpus.

Animal genomes show networks of deeply conserved gene linkages whose phylogenetic scope and chromosomal context remain unclear. Here, we report chromosome-scale conservation of synteny among bilaterians, cnidarians, and sponges and use comparative analysis to reconstruct ancestral chromosomes across major animal groups. Comparisons among diverse metazoans reveal the processes of chromosome evolution that produced contemporary karyotypes from their Precambrian progenitors. On the basis of these findings, we introduce a simple algebraic representation of chromosomal change and use it to establish a unified systematic framework for metazoan chromosome evolution. We find that fusion-with-mixing, a previously unappreciated mode of chromosome change, has played a central role. We find that relicts of several metazoan chromosomal units are preserved in unicellular eukaryotes. These conserved pre-metazoan linkages include the chromosomal unit that encodes the most diverse set of metazoan homeobox genes, suggesting a candidate genomic context for the early diversification of this key gene family.

The evolutionary origin of nutrient trafficking, a key development in metazoans, has been relatively unexplored. A new study in a model sea anemone exploits click chemistry and gene editing to provide insight into how nutrient trafficking may have arisen.

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The transcriptional target of the Hippo pathway, the transcriptional coactivator YAP, inhibits the initiation of budding in Hydra and is regulated by Hydra LATS. In addition, we show functions of the Hippo pathway in regulation of actin organization and cell proliferation in Hydra . We hypothesize that the Hippo pathway served as a link between continuous cell division, cell density, and axis formation early in metazoan evolution.

SummaryHow did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The target of Hippo pathway, the transcriptional co-activator YAP, inhibits the initiation of budding in Hydra, and is regulated by Hydra LATS. In addition, we show functions of Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that Hippo pathway served as a link between continuous cell division, cell density and axis formation early in metazoan evolution.

We present a new transgenic Hydra vulgaris line expressing a distinct fluorescent protein in each of the three cell lineages of the adult polyp. Plasmid microinjection was used to generate a novel transgenic Hydra line expressing the yellow fluorescent protein YPet in the ectodermal epithelial cell lineage. Tissue grafting was then used to combine a YPet animal with a line that expresses DsRed2 in the endodermal epithelial lineage and eGFP in the interstitial cell (i-cell) lineage. The resulting triple-labeled ("tricolored") transgenic line provides, for the first time, a Hydra in which all three cell lineages can be imaged simultaneously in vivo. We show example confocal images of whole animals and individual cells to illustrate the imaging capabilities that this new line makes possible. We also used this line to carry out new studies of cell fate in the tentacles. Specifically, we evaluated the well-accepted notion that all tentacle cells are terminally differentiated and are displaced or migrate exclusively towards the distal end of the tentacle. We found that ectodermal and endodermal epithelial cells are displaced distally, as expected. In contrast, members of the i-cell lineage, which resembled neuronal precursors, could migrate out of a tentacle into the body column. This example illustrates how this tricolored transgenic line enables new in vivo studies of cell behaviors in Hydra.

Cephalopods fascinate us but have been out of the reach of experimental manipulations at the genetic level. A new study describes editing of a gene in a squid using CRISPR.

How an animal establishes its body axis is a fundamental question in developmental biology. The freshwater cnidarian Hydra is an attractive model for studying axis formation because it is radially symmetric, with a single oral-aboral axis. It was recently proposed that the orientation of the new body axis in a regenerating Hydra polyp is determined by the oral-aboral orientation of the actin-myosin contractile processes (myonemes) in the animal's outer epithelial layer. However, it remained unclear how the oral-aboral polarity of the body axis would be defined. As Wnt signaling is known to control axis polarity in Hydra and bilaterians, we hypothesized that it plays a role in axis formation during regeneration of Hydra tissue pieces. We tested this hypothesis using pharmacological perturbations and novel grafting experiments to set Wnt signaling and myoneme orientation perpendicular to each other to determine which controls axis formation. Our results demonstrate that Wnt signaling is the dominant encoder of axis orientation and polarity, in line with its conserved role in axial patterning.

Fat, Fat-like, and Dachsous family cadherins are giant proteins that regulate planar cell polarity (PCP) and cell adhesion in bilaterians. Their evolutionary origin can be traced back to prebilaterian species, but their ancestral function(s) are unknown. We identified Fat-like and Dachsous cadherins in Hydra, a member of phylum Cnidaria a sister group of bilaterian. We found Hydra does not possess a true Fat homolog, but has homologs of Fat-like (HyFatl) and Dachsous (HyDs) that localize at the apical membrane of ectodermal epithelial cells and are planar polarized perpendicular to the oral-aboral axis of the animal. Using a knockdown approach we found that HyFatl is involved in local cell alignment and cell-cell adhesion, and that reduction of HyFatl leads to defects in tissue organization in the body column. Overexpression and knockdown experiments indicate that the intracellular domain (ICD) of HyFatl affects actin organization through proline-rich repeats. Thus, planar polarization of Fat-like and Dachsous cadherins has ancient, prebilaterian origins, and Fat-like cadherins have ancient roles in cell adhesion, spindle orientation, and tissue organization.

Despite the fact that Hydra has been studied for more than 200 years, we know surprisingly little about its life history. We show that Hydra vulgaris embryos hatch sporadically over a period ranging from a few days to nine months. We also report, for what seems to be the first time, the presence of Hydra in a vernal pool. Phylogenetic analysis and sexual crossing show that this Hydra is a member of the cosmopolitan Vulgaris clade and is not reproductively isolated from other members of the clade. Our findings lead us to hypothesize that Hydra evolved in an unstable freshwater habitat in which survival required that its life cycle include the use of a bet-hedging reproductive strategy and the formation of an embryo that is desiccation resistant and that can remain dormant for long periods of time.

Fat family cadherins are enormous proteins that regulate planar cell polarity (PCP) and cell adhesion in bilaterian animals. Their evolutionary origin can be traced back to prebilaterian species, but their ancestral function(s) are unknown. We identified Fat-like and Dachsous cadherins in Hydra , a member of the early-diverging metazoan phylum Cnidaria. Hydra has a simple body plan with only two epithelial layers and radial symmetry. We find that Hydra homologues of Fat-like (HyFat) and Dachsous (HyDs) co-localize at the apico-lateral membrane of ectodermal epithelial cells. Remarkably, HyFat is planar polarized perpendicular to the oral-aboral axis of the animal. Using knockdown approaches we found that HyFat is involved in the regulation of local cell alignment, but is dispensable for the global alignment of ectodermal myonemes along the oral-aboral axis. The intracellular domain (ICD) of HyFat is involved in the morphogenesis of ectodermal myonemes. Thus, Fat family cadherins have ancient, prebilaterian functions in cell adhesion, tissue organization and planar polarity.