A new look at the architecture and dynamics of the Hydra nerve net.

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.

Differentiation trajectories of the Hydra nervous system reveal transcriptional regulators of neuronal fate.

The small freshwater cnidarian polyp Hydra vulgaris uses adult stem cells (interstitial stem cells) to continually replace neurons throughout its life. This feature, combined with the ability to image the entire nervous system (Badhiwala et al., 2021; Dupre & Yuste, 2017) and availability of gene knockdown techniques (Juliano, Reich, et al., 2014; Lohmann et al., 1999; Vogg et al., 2022), makes Hydra a tractable model for studying nervous system development and regeneration at the whole-organism level. In this study, we use single-cell RNA sequencing and trajectory inference to provide a comprehensive molecular description of the adult nervous system. This includes the most detailed transcriptional characterization of the adult Hydra nervous system to date. We identified eleven unique neuron subtypes together with the transcriptional changes that occur as the interstitial stem cells differentiate into each subtype. Towards the goal of building gene regulatory networks to describe Hydra neuron differentiation, we identified 48 transcription factors expressed specifically in the Hydra nervous system, including many that are conserved regulators of neurogenesis in bilaterians. We also performed ATAC-seq on sorted neurons to uncover previously unidentified putative regulatory regions near neuron-specific genes. Finally, we provide evidence to support the existence of transdifferentiation between mature neuron subtypes and we identify previously unknown transition states in these pathways. All together, we provide a comprehensive transcriptional description of an entire adult nervous system, including differentiation and transdifferentiation pathways, which provides a significant advance towards understanding mechanisms that underlie nervous system regeneration.

Deeply conserved synteny and the evolution of metazoan chromosomes.

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.

Expansion of a single transposable element family is associated with genome-size increase and radiation in the genus Hydra.

Transposable elements are one of the major contributors to genome-size differences in metazoans. Despite this, relatively little is known about the evolutionary patterns of element expansions and the element families involved. Here we report a broad genomic sampling within the genus Hydra, a freshwater cnidarian at the focal point of diverse research in regeneration, symbiosis, biogeography, and aging. We find that the genome of Hydra is the result of an expansion event involving long interspersed nuclear elements and in particular a single family of the chicken repeat 1 (CR1) class. This expansion is unique to a subgroup of the genus Hydra, the brown hydras, and is absent in the green hydra, which has a repeat landscape similar to that of other cnidarians. These features of the genome make Hydra attractive for studies of transposon-driven genome expansions and speciation.

Regionalized nervous system in Hydra and the mechanism of its development.

The last common ancestor of Bilateria and Cnidaria is considered to develop a nervous system over 500 million years ago. Despite the long course of evolution, many of the neuron-related genes, which are active in Bilateria, are also found in the cnidarian Hydra. Thus, Hydra is a good model to study the putative primitive nervous system in the last common ancestor that had the great potential to evolve to a more advanced one. Regionalization of the nervous system is one of the advanced features of bilaterian nervous system. Although a regionalized nervous system is already known to be present in Hydra, its developmental mechanisms are poorly understood. In this study we show how it is formed and maintained, focusing on the neuropeptide Hym-176 gene and its paralogs. First, we demonstrate that four axially localized neuron subsets that express different combination of the neuropeptide Hym-176 gene and its paralogs cover almost an entire body, forming a regionalized nervous system in Hydra. Second, we show that positional information governed by the Wnt signaling pathway plays a key role in determining the regional specificity of the neuron subsets as is the case in bilaterians. Finally, we demonstrated two basic mechanisms, regionally restricted new differentiation and phenotypic conversion, both of which are in part conserved in bilaterians, are involved in maintaining boundaries between the neuron subsets. Therefore, this study is the first comprehensive analysis of the anatomy and developmental regulation of the divergently evolved and axially regionalized peptidergic nervous system in Hydra, implicating an ancestral origin of neural regionalization.

Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps.

Nerve cells and spontaneous coordinated behavior first appeared near the base of animal evolution in the common ancestor of cnidarians and bilaterians. Experiments on the cnidarian Hydra have demonstrated that nerve cells are essential for this behavior, although nerve cells in Hydra are organized in a diffuse network and do not form ganglia. Here we show that the gap junction protein innexin-2 is expressed in a small group of nerve cells in the lower body column of Hydra and that an anti-innexin-2 antibody binds to gap junctions in the same region. Treatment of live animals with innexin-2 antibody eliminates gap junction staining and reduces spontaneous body column contractions. We conclude that a small subset of nerve cells, connected by gap junctions and capable of synchronous firing, act as a pacemaker to coordinate the contraction of the body column in the absence of ganglia.

Interstitial stem cells in Hydra: multipotency and decision-making.

Interstitial stem cells in Hydra constitute a population of multipotent cells, which continuously give rise to differentiated products during the growth and budding of Hydra polyps. They also give rise to germ cells in animals undergoing sexual differentiation. Cloning experiments have shown that interstitial stem cells are multipotent. In vivo tracing of stem cell lineages has revealed that stem cells divide symmetrically to yield two stem cells or asymmetrically to yield one stem cell daughter and one daughter cell which initiates nerve or nematocyte differentiation. Following commitment, some nerve cell precursors migrate from the body column into the head or foot region, thus giving rise to the high density of nerve cells observed in these regions. Stem cell proliferation is regulated by changes in the self-renewal probability and is controlled by stem cell density. Nerve cell commitment is controlled by several peptides including the Head Activator. Factors affecting nematocyte commitment are not known, but wnt and notch signaling are both required for differentiation of committed precursors.

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem ß-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

A genomic view of 500 million years of cnidarian evolution.

Cnidarians (corals, anemones, jellyfish and hydras) are a diverse group of animals of interest to evolutionary biologists, ecologists and developmental biologists. With the publication of the genome sequences of Hydra and Nematostella, whose last common ancestor was the stem cnidarian, researchers are beginning to see the genomic underpinnings of cnidarian biology. Cnidarians are known for the remarkable plasticity of their morphology and life cycles. This plasticity is reflected in the Hydra and Nematostella genomes, which differ to an exceptional degree in size, base composition, transposable element content and gene conservation. It is now known what cnidarian genomes, given 500 million years, are capable of; as we discuss here, the next challenge is to understand how this genomic history has led to the striking diversity seen in this group.

Nematogalectin, a nematocyst protein with GlyXY and galectin domains, demonstrates nematocyte-specific alternative splicing in Hydra.

Taxonomically restricted genes or lineage-specific genes contribute to morphological diversification in metazoans and provide unique functions for particular taxa in adapting to specific environments. To understand how such genes arise and participate in morphological evolution, we have investigated a gene called nematogalectin in Hydra, which has a structural role in the formation of nematocysts, stinging organelles that are unique to the phylum Cnidaria. Nematogalectin is a 28-kDa protein with an N-terminal GlyXY domain (glycine followed by two hydrophobic amino acids), which can form a collagen triple helix, followed by a galactose-binding lectin domain. Alternative splicing of the nematogalectin transcript allows the gene to encode two proteins, nematogalectin A and nematogalectin B. We demonstrate that expression of nematogalectin A and B is mutually exclusive in different nematocyst types: Desmonemes express nematogalectin B, whereas stenoteles and isorhizas express nematogalectin B early in differentiation, followed by nematogalectin A. Like Hydra, the marine hydrozoan Clytia also has two nematogalectin transcripts, which are expressed in different nematocyte types. By comparison, anthozoans have only one nematogalectin gene. Gene phylogeny indicates that tandem duplication of nematogalectin B exons gave rise to nematogalectin A before the divergence of Anthozoa and Medusozoa and that nematogalectin A was subsequently lost in Anthozoa. The emergence of nematogalectin A may have played a role in the morphological diversification of nematocysts in the medusozoan lineage.

The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins.

The fresh water polyp Hydra belongs to the phylum Cnidaria, which diverged from the metazoan lineage before the appearance of bilaterians. In order to understand the evolution of apoptosis in metazoans, we have begun to elucidate the molecular cell death machinery in this model organism. Based on ESTs and the whole Hydra genome assembly, we have identified 15 caspases. We show that one is activated during apoptosis, four have characteristics of initiator caspases with N-terminal DED, CARD or DD domain and two undergo autoprocessing in vitro. In addition, we describe seven Bcl-2-like and two Bak-like proteins. For most of the Bcl-2 family proteins, we have observed mitochondrial localization. When expressed in mammalian cells, HyBak-like 1 and 2 strongly induced apoptosis. Six of the Bcl-2 family members inhibited apoptosis induced by camptothecin in mammalian cells with HyBcl-2-like 4 showing an especially strong protective effect. This protein also interacted with HyBak-like 1 in a yeast two-hybrid assay. Mutation of the conserved leucine in its BH3 domain abolished both the interaction with HyBak-like 1 and the anti-apoptotic effect. Moreover, we describe novel Hydra BH-3-only proteins. One of these interacted with Bcl-2-like 4 and induced apoptosis in mammalian cells. Our data indicate that the evolution of a complex network for cell death regulation arose at the earliest and simplest level of multicellular organization, where it exhibited a substantially higher level of complexity than in the protostome model organisms Caenorhabditis and Drosophila.

Apoptosis in pre-Bilaterians: Hydra as a model.

Hydra is a member of the ancient metazoan phylum Cnidaria and is an especially well investigated model organism for questions of the evolutionary origin of metazoan processes. Apoptosis in Hydra is important for the regulation of cellular homeostasis under different conditions of nutrient supply. The molecular mechanisms leading to apoptosis in Hydra are surprisingly extensive and comparable to those in mammals. Genome wide sequence analysis has revealed the presence of large caspase and Bcl-2 families, the apoptotic protease activating factor (APAF-1), inhibitors of apoptotic proteases (IAPs) and components of a putative death receptor pathway. Regulation of apoptosis in Hydra may involve BH-3 only proteins and survival pathways, possibly including insulin signalling.

The BCL-2-like protein CED-9 of C. elegans promotes FZO-1/Mfn1,2- and EAT-3/Opa1-dependent mitochondrial fusion.

The mammalian dynamin-related guanosine triphosphatases Mfn1,2 and Opa1 are required for mitochondrial fusion. However, how their activities are controlled and coordinated is largely unknown. We present data that implicate the BCL-2-like protein CED-9 in the control of mitochondrial fusion in Caenorhabditis elegans. We demonstrate that CED-9 can promote complete mitochondrial fusion of both the outer and inner mitochondrial membrane. We also show that this fusion is dependent on the C. elegans Mfn1,2 homologue FZO-1 and the C. elegans Opa1 homologue EAT-3. Furthermore, we show that CED-9 physically interacts with FZO-1 in vivo and that the ability of CED-9 to interact with FZO-1 is important for its ability to cause mitochondrial fusion. CED-9-induced mitochondrial fusion is not required for the maintenance of mitochondrial morphology during embryogenesis or in muscle cells, at least under normal conditions and in the absence of stress. Therefore, we propose that the BCL-2-like CED-9 acts through FZO-1/Mfn1,2 and EAT-3/Opa1 to promote mitochondrial fusion in response to specific cellular signals.

Further characterization of the PW peptide family that inhibits neuron differentiation in Hydra.

From an evolutionary point of view, Hydra has one of the most primitive nervous systems among metazoans. Two different groups of peptides that affect neuron differentiation were identified in a systematic screening of peptide signaling molecules in Hydra. Within the first group of peptides, a neuropeptide, Hym-355, was previously shown to positively regulate neuron differentiation. The second group of peptides encompasses the PW family of peptides that negatively regulate neuron differentiation. In this study, we identified the gene encoding PW peptide preprohormone. Moreover, we made the antibody that specifically recognizes LPW. In situ hybridization and immunohistochemical analyses showed that the PW peptides and the gene encoding them were expressed in ectodermal epithelial cells throughout the body except for the basal disk. The PW peptides are produced by epithelial cells and are therefore termed "epitheliopeptides." Together with Hym-355, the PW family peptides mediate communication between neurons and epithelial cells and thereby maintain a specific density of neurons in Hydra.

Evolution of complex structures: minicollagens shape the cnidarian nematocyst.

The generation of biological complexity by the acquisition of novel modular units is an emerging concept in evolutionary dynamics. Here, we review the coordinate evolution of cnidarian nematocysts, secretory organelles used for capture of prey, and of minicollagens, proteins constituting the nematocyst capsule. Within the Cnidaria there is an increase in nematocyst complexity from Anthozoa to Medusozoa and a parallel increase in the number and complexity of minicollagen proteins. This complexity is primarily manifest in a diversification of N- and C-terminal cysteine-rich domains (CRDs) involved in minicollagen polymerization. We hypothesize that novel CRD motifs alter minicollagen networks, leading to novel capsule structures and nematocyst types.

Cilium evolution: identification of a novel protein, nematocilin, in the mechanosensory cilium of Hydra nematocytes.

The cnidocil at the apical end of Hydra nematocytes is a mechanosensory cilium, which acts as a "trigger" for discharge of the nematocyst capsule. The cnidocil protrudes from the center of the cnidocil apparatus and is composed of singlet and doublet microtubules surrounding an electron-dense central filament. In this paper, we identify a novel protein, nematocilin, which is localized in the central filament. Immunofluorescence staining and immunogold electron microscopy show that nematocilin forms filaments in the central core of the cnidocil. Nematocilin represents a new member of the intermediate filament superfamily. Two paralogous sequences of nematocilin are present in the Hydra genome and appear to be the result of recent gene duplication. Comparison of the exon-intron structure suggests that the nematocilin genes evolved from the nuclear lamin gene by conserving exons encoding the coiled-coil domains and replacing the C-terminal lamin domains. Molecular phylogenetic analyses also support the hypothesis of a common ancestor between lamin and nematocilin. Comparison of cnidocil structures in different cnidarians indicates that a central filament is present in the cnidocils of several hydrozoan and a cubozoan species but is absent in the cnidocils of anthozoans. A nematocilin homolog is absent in the recently completed genome of the anthozoan Nematostella. Thus, the evolution of a novel ciliary structure, which provides mechanical rigidity to the sensory cilium during the process of mechanoreception, is associated with the evolution of a novel protein.

The evolutionary emergence of cell type-specific genes inferred from the gene expression analysis of Hydra.

Cell lineages of cnidarians including Hydra represent the fundamental cell types of metazoans and provides us a unique opportunity to study the evolutionary diversification of cell type in the animal kingdom. Hydra contains epithelial cells as well as a multipotent interstitial cell (I-cell) that gives rise to nematocytes, nerve cells, gland cells, and germ-line cells. We used cDNA microarrays to identify cell type-specific genes by comparing gene expression in normal Hydra with animals lacking the I-cell lineage, so-called epithelial Hydra. We then performed in situ hybridization to localize expression to specific cell types. Eighty-six genes were shown to be expressed in specific cell types of the I-cell lineage. An additional 29 genes were expressed in epithelial cells and were down-regulated in epithelial animals lacking I-cells. Based on the above information, we constructed a database (http://hydra.lab.nig.ac.jp/hydra/), which describes the expression patterns of cell type-specific genes in Hydra. Most genes expressed specifically in either I-cells or epithelial cells have homologues in higher metazoans. By comparison, most nematocyte-specific genes and approximately half of the gland cell- and nerve cell-specific genes are unique to the cnidarian lineage. Because nematocytes, gland cells, and nerve cells appeared along with the emergence of cnidarians, this suggests that lineage-specific genes arose in cnidarians in conjunction with the evolution of new cell types required by the cnidarians.

Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling.

BACKGROUND: 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. RESULTS: We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. CONCLUSION: Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra.

Continuous molecular evolution of protein-domain structures by single amino acid changes.

Protein structures cluster into families of folds that can result from extremely different amino acid sequences [1]. Because the enormous amount of genetic information generates a limited number of protein folds [2], a particular domain structure often assumes numerous functions. How new protein structures and new functions evolve under these limitations remains elusive. Molecular evolution may be driven by the ability of biomacromolecules to adopt multiple conformations as a bridge between different folds [3-6]. This could allow proteins to explore new structures and new tasks while part of the structural ensemble retains the initial conformation and function as a safeguard [7]. Here we show that a global structural switch can arise from single amino acid changes in cysteine-rich domains (CRD) of cnidarian nematocyst proteins. The ability of these CRDs to form two structures with different disulfide patterns from an identical cysteine pattern is distinctive [8]. By applying a structure-based mutagenesis approach, we demonstrate that a cysteine-rich domain can interconvert between two natively occurring domain structures via a bridge state containing both structures. Comparing cnidarian CRD sequences leads us to believe that the mutations we introduced to stabilize each structure reflect the birth of new protein folds in evolution.