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Neuropeptides are evolutionarily ancient peptide hormones of the nervous and neuroendocrine systems, and are thought to have regulated metamorphosis in early animal ancestors. In particular, the deeply conserved Wamide family of neuropeptides—shared across Bilateria (e.g. insects and worms) and its sister group Cnidaria (e.g. jellyfishes and corals...
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... 121 comparison of the N. vectensis genome and cDNA sequences instead shows that the GLWamide 122 gene consists of two exons (Figure 1-figure supplement 1A). The analysis of putative 123 endoproteolytic cleavage sites (i.e. ...
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... and basic amino acid residues; (Darmer et al., 1991;124 Leviev and Grimmelikhuijzen, 1995) and C-terminal amidation sites (i.e. C-terminal Gly 125 residue) in the predicted precursor protein sequence suggests that the GLWamide gene encodes 126 different copy numbers of nine distinct GLWamide peptides that vary in N-terminal sequence 127 ( Figure 1-figure supplement 1B, C). We note that previously documented NvLWamide-like gene 128 (Nv242283; (Havrilak et al., 2017) is predicted to generate and release QCPPGLWGC peptides, 129 but not GLWamides, because of the lack of the canonical amidation signal, glycine, at the C- 130 terminus (see Figure 5-figure supplement 1 for experimental validation). ...
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... precursor transcripts and GLWamide peptides, respectively. Consistent with 158 previously published gene/peptide expression data ( Nakanishi et al., 2012;Watanabe et al., 159 2014), GLWamide precursor transcripts were first detected in ectodermal sensory cells in the 160 outer epithelium and in the pharynx during early-mid planula development (arrowheads in 161 oriented perpendicular to the oral-aboral axis (inset in Fig.1D; (Watanabe et al., 2014)). ...
Citations
... In addition to knockouts, it may be possible to knock in a target sequence to determine whether a candidate locus is sufficient to change heat tolerance. Although this technology is not currently available in reef-building corals, CRISPR/Cas9-based genomic knock-in has been successful in several cnidarians, including an anemone (Nematostella vectensis) [82][83][84], a hydroid (Hydractinia symbiolongicarpus) [85], and a non-reef-building temperate coral (Astrangia poculata) [86]. Therefore, it should be possible to extend CRISPR/Cas9 methods to allow for knock-ins and allelic replacements in reef-building corals [77]. ...
... 29,54 N. vectensis is a powerful developmental model that can easily produce thousands of embryos on demand with simple light and temperature cues. Many techniques for manipulating gene expression are already well established in N. vectensis, including CRISPR/Cas9 genome editing, [55][56][57][58] stable and transient cell-type-specific transgenesis, [59][60][61][62] and various forms of gene knockdown techniques (e.g., antisense morpholinos, RNAi, shRNA, dominant-negative approaches). 60,63,64 Together, these attributes make N. vectensis well-suited for investigating gene function during biomineralization. ...
Coral conservation requires a mechanistic understanding of how environmental stresses disrupt biomineralization, but progress has been slow, primarily because corals are not easily amenable to laboratory research. Here, we highlight how the starlet sea anemone, Nematostella vectensis, can serve as a model to interrogate the cellular mechanisms of coral biomineralization. We have developed transgenic constructs using biomineralizing genes that can be injected into Nematostella zygotes and designed such that translated proteins may be purified for physicochemical characterization. Using fluorescent tags, we confirm the ectopic expression of the coral biomineralizing protein, SpCARP1, in Nematostella. We demonstrate via calcein staining that SpCARP1 concentrates calcium ions in Nematostella, likely initiating the formation of mineral precursors, consistent with its suspected role in corals. These results lay a fundamental groundwork for establishing Nematostella as an in vivo system to explore the evolutionary and cellular mechanisms of coral biomineralization, improve coral conservation efforts, and even develop novel biomaterials.
... Being composed of two cellular layers, the ectoderm and the endoderm, Nematostella have a simple body plan and tissue organization [11][12][13][14]. Additionally, Nematostella possess a nervous system that takes the form of a nerve net, composed of sensory cells, glandular cells, multipolar ganglion cells and cnidocytes (stinging cells) [4,15, 16]. ...
Background: The starlet sea anemone, Nematostella vectensis, is an emerging model organism with a high regenerative capacity, which was recently found to possess an orthologue to the human LRRK2 gene (nvLRRK2). The leucine rich repeat kinase 2 (LRRK2) gene, when mutated, is the most common cause of inherited Parkinson’s Disease (PD). Its protein product (LRRK2) has implications in a variety of cellular processes, however, the full function of LRRK2 is not well established. Current research is focusing on understanding the function of LRRK2, including both its physiological role as well as its pathobiological underpinnings.
Methods: We used bioinformatics to determine the cross-species conservation of LRRK2, then applied drugs targeting the kinase activity of LRRK2 to examine its function in development, homeostasis and regeneration in Nematostella vectensis.
Results: An in-silico characterization and phylogenetic analysis of nvLRRK2 comparing it to human LRRK2 highlighted key conserved motifs and residues. In vivo analyses inhibiting the kinase function of this enzyme demonstrated a role of nvLRRK2 in development and regeneration of N. vectensis. These findings implicate a developmental role of LRRK2 in Nematostella, adding to the expanding knowledge of its physiological function.
Conclusions: Our work introduces a new model organism with which to study LRRK biology. We show a necessity for LRRK2 in development and regeneration. Given the short generation time, genetic trackability and in vivo imaging capabilities, this work introduces Nematostella vectensis as a new model in which to study genes linked to neurodegenerative diseases such as Parkinson’s.
... Finally, our work also opens up new avenues in experimental neuroscience in cnidarians (Bosch et al., 2017 ). With readily available genetic manipulation techniques in N. vectensis and other species (Ikmi et al., 2014 ;Nakanishi and Martindale, 2018 ;Paix et al., 2023 ) (Artigas et al., 2020 ;Sanders et al., 2018 ;Wittlieb et al., 2006 ), the identified neuropeptide-receptor interactions will enable genetic manipulations of both ligand and receptor(s), to reveal the biological functions of peptidergic signaling. ...
Neuropeptides are ancient signaling molecules in animals but only few peptide receptors are known outside bilaterians. Cnidarians possess a large number of G protein-coupled receptors (GPCRs) – the most common receptors of bilaterian neuropeptides – but most of these remain orphan with no known ligands. We searched for neuropeptides in the sea anemone Nematostella vectensis and created a library of 64 peptides derived from 33 precursors. In a large-scale pharmacological screen with these peptides and 161 N. vectensis GPCRs, we identified 31 receptors specifically activated by one of 14 peptides. Mapping GPCR and neuropeptide expression to single-cell sequencing data revealed how cnidarian tissues are extensively wired by multilayer peptidergic networks. Phylogenetic analysis identified no direct orthology to bilaterian peptidergic systems and supports the independent expansion of neuropeptide signaling in cnidarians from a few ancestral peptide-receptor pairs.
... Finally, our work also opens up new avenues in experimental neuroscience in cnidarians (Bosch et al., 2017). With readily available genetic manipulation techniques in N. vectensis and other species (Ikmi et al., 2014;Nakanishi and Martindale, 2018;Paix et al., 2023) (Artigas et al., 2020;Sanders et al., 2018;Wittlieb et al., 2006), the identified neuropeptide-receptor interactions will enable genetic manipulations of both ligand and receptor(s), to reveal the biological functions of peptidergic signaling. Overall, we identified receptors for peptides from nearly half of the Nematostella neuropeptide precursors, including the receptors of the ancient PRXamide and QGRFamide peptides. ...
Neuropeptides are ancient signaling molecules in animals but only few peptide receptors are known outside bilaterians. Cnidarians possess a large number of G protein-coupled receptors (GPCRs), the most common receptors of bilaterian neuropeptides, but most of these remain orphan with no known ligands. We searched for neuropeptides in the sea anemone Nematostella vectensis and created a library of 64 peptides derived from 33 precursors. In a large-scale pharmacological screen with these peptides and 161 N. vectensis GPCRs, we identified 31 receptors specifically activated by one of 14 peptides. Mapping GPCR and neuropeptide expression to single-cell sequencing data revealed how cnidarian tissues are extensively wired by multilayer peptidergic networks. Phylogenetic analysis identified no direct orthology to bilaterian peptidergic systems and supports the independent expansion of neuropeptide signaling in cnidarians from a few ancestral peptide-receptor pairs.
... Improvements in husbandry and long-term culture will facilitate generation of animal lines in Hydroides, enabling Hydroides to keep pace with advancements in other marine invertebrate larvae. [51][52][53][54][55][56] In parallel, emerging synthetic biology tools in established model microbes also hold promise for the manipulation of more diverse marine bacteria that promote Hydroides development. 57 These new tools compliment powerful classical bacterial genetics approaches which have already helped reveal key aspects of Hydroides interaction with stimulatory microbes. ...
Background
The biofouling marine tube worm, Hydroides elegans, is an indirect developing polychaete with significance as a model organism for questions in developmental biology and the evolution of host‐microbe interactions. However, a complete description of the life cycle from fertilization through sexual maturity remains scattered in the literature, and lacks standardization.
Results and discussion
Here, we present a unified staging scheme synthesizing the major morphological changes that occur during the entire life cycle of the animal. These data represent a complete record of the life cycle, and serve as a foundation for connecting molecular changes with morphology.
Conclusions
The present synthesis and associated staging scheme are especially timely as this system gains traction within research communities. Characterizing the Hydroides life cycle is essential for investigating the molecular mechanisms that drive major developmental transitions, like metamorphosis, in response to bacteria.
... In neural organisms, the neuro-endocrine system is the main mediator between the external and internal world, modulating development, sensation, memory, metabolism, and behavior De Marco et al., 2016;Jennings & de Lecea, 2020;Nakanishi & Martindale, 2018a;Seehausen et al., 2008;Soares et al., 2010). To generate a functional neuroendocrine system, several cell types must be differentiated and maintained. ...
Cnidaria (i.e., sea anemones, jellyfish, corals) and Bilateria (i.e., vertebrates, sea stars, fruit flies), are sister groups that diverged around 600 million years ago. Despite the long evolutionary time, many cellular differentiation mechanisms, cell types, tissues and behaviors are conserved. Such as neurons, mechanosensory hair cells, feeding behaviors, peristaltic movements, and sleep. Recent advances in genomics, molecular biology and microscopy have fueled an increased interest in understanding cnidarian nervous and neuroendocrine systems. Understanding the developmental mechanisms and the mode of operation of Cnidarian nervous systems helps to reconstruct the ancestral nervous system of the last common ancestor of Cnidaria and Bilateria. Thus, also shedding light in fundamental aspects of Bilaterian nervous systems. Here, the 'starlet sea anemone' Nematostella vectensis, a powerful cnidarian model organism was used to address the gene expression pattern of Pit1, a conserved gene shared between Cnidaria and Bilateria. In Chapter 1, a method to extract DNA and genotype embryos of Nematostella without sacrificing the animal was established, with possible application to other non-sea anemone cnidarians. Early genotyping is fundamental for addressing phenotypes during development, thus opening the door to study the function of any gene of interest during larval pre-metamorphic stages. In Chapter 2, the expression pattern of Pit1 and detailed cellular morphology of Pit1-positive cells was characterized for the first time in Nematostella. Complex neuronal networks and diverse sensory cells were found. Furthermore, the foundation for future functional studies of Pit1 was laid by establishing stable CRISPR-Cas9 knockout and transgenic reporter lines.
... GLWamide, one of the most studied neuropeptides, is responsive in tentacle regeneration of E. pallida. GLWamide was previously revealed to be involved in the process of muscle contraction in the sea anemone genus Anthopleura and the coral Euphyllia ancora (Takahashi & Hatta, 2011;Shikina et al., 2020), metamorphosis in H. magnipapillata (Takahashi & Hatta, 2011), settlement in the coral Acropora palmata (Erwin & Szmant 2010;Takahashi & Hatta, 2011), and transition timing modulation of N. vectensis from planula larvae to polyps (Nakanishi & Martindale, 2018). Given its participation in cnidarian tissue regeneration, this study has now revealed that the level of pleiotropy of GLWamide is higher than previously thought. ...
Cnidarians including sea anemones, corals, hydra, and jellyfishes are a group of animals well known for their regeneration capacity. However, how non-coding RNAs such as microRNAs (also known as miRNAs) contribute to cnidarian tissue regeneration is poorly understood. Here, we sequenced and assembled the genome of the sea anemone Exaiptasia pallida collected in Hong Kong waters. The assembled genome size of E. pallida is 229.21 Mb with a scaffold N50 of 10.58 Mb and BUSCO completeness of 91.1%, representing a significantly improved genome assembly of this species. The organization of ANTP-class homeobox genes in this anthozoan further supported the previous findings in jellyfishes, where most of these genes are mainly located on three scaffolds. Tentacles of E. pallida were excised, and both mRNA and miRNA were sequenced at 9 time points (0 h, 6 h, 12 h, 18 h, 1 day, 2, 3, 6, and 8 days) from regenerating tentacles. In addition to the Wnt signaling pathway and homeobox genes that are shown to be likely involved in tissue regeneration as in other cnidarians, we have shown that GLWamide neuropeptides, and for the first time sesquiterpenoid pathway genes could potentially be involved in the late phase of cnidarian tissue regeneration. The established sea anemone model will be useful for further investigation of biology and evolution in, and the effect of climate change on this important group of animals.
... In this report, we use the starlet sea anemone N. vectensis to investigate the role of POU-IV in the development of cnidarian hair cells. N. vectensis is a convenient model for studies of mechanisms of cnidarian development because of the availability of the genome sequence (Putnam et al., 2007) and a wide range of powerful molecular genetic tools including CRISPR-Cas9 genome editing (Ikmi et al., 2014;Nakanishi and Martindale, 2018). During embryogenesis, N. vectensis gastrulates by invagination to form an embryo consisting of ectoderm and endoderm separated by the extracellular matrix known as the mesoglea (Kraus and Technau, 2006;Magie et al., 2007). ...
... F1 pou-iv +/-heterozygotes were subsequently crossed with each other to produce F2 offspring, a quarter of which, on average, were pou-iv -/-mutants. pou-iv -/-mutants were identified by PCR-based genotyping methods ( Figure 3B and C) using genomic DNA extracted from polyp tentacles (Ikmi et al., 2014) or from pieces of tissue isolated from early embryos (Nakanishi and Martindale, 2018;Silva and Nakanishi, 2019). Western blotting with the anti-POU-IV has confirmed that pou-iv -/-polyps express mutant POU-IV lacking DNA-binding domains (18.7 kDa), but not wildtype POU-IV (35.2 kDa) ( Figure 3D), validating the specificity of the antibody. ...
Although specialized mechanosensory cells are found across animal phylogeny, early evolutionary histories of mechanoreceptor development remain enigmatic. Cnidaria (e.g. sea anemones and jellyfishes) is the sister group to well-studied Bilateria (e.g. flies and vertebrates), and has two mechanosensory cell types - a lineage-specific sensory-effector known as the cnidocyte, and a classical mechanosensory neuron referred to as the hair cell. While developmental genetics of cnidocytes is increasingly understood, genes essential for cnidarian hair cell development are unknown. Here we show that the class IV POU homeodomain transcription factor (POU-IV) - an indispensable regulator of mechanosensory cell differentiation in Bilateria and cnidocyte differentiation in Cnidaria - controls hair cell development in the sea anemone cnidarian Nematostella vectensis. N. vectensis POU-IV is postmitotically expressed in tentacular hair cells, and is necessary for development of the apical mechanosensory apparatus, but not of neurites, in hair cells. Moreover, it binds to deeply conserved DNA recognition elements, and turns on a unique set of effector genes - including the transmembrane-receptor-encoding gene polycystin 1 - specifically in hair cells. Our results suggest that POU-IV directs differentiation of cnidarian hair cells and cnidocytes via distinct gene regulatory mechanisms, and support an evolutionarily ancient role for POU-IV in defining the mature state of mechanosensory neurons.
... 192 In this report, we use the starlet sea anemone N. vectensis to investigate the role of IV in the development of cnidarian hair cells. N. vectensis is a convenient model for studies of 194 mechanisms of cnidarian development because of the availability of the genome sequence 195 (Putnam et al., 2007) and a wide range of powerful molecular genetic tools including Cas9 genome editing (Ikmi et al., 2014, Nakanishi andMartindale, 2018). During embryogenesis,197 N. vectensis gastrulates by invagination to form an embryo consisting of ectoderm and endoderm 198 separated by the extracellular matrix known as the mesoglea Technau, 2006, Magie 199 et al., 2007). ...
Although specialized mechanosensory cells are found across animal phylogeny, early evolutionary histories of mechanoreceptor development remain enigmatic. Cnidaria (e.g. sea anemones and jellyfishes) is the sister group to well-studied Bilateria (e.g. flies and vertebrates), and has two mechanosensory cell types – a lineage-specific sensory-effector known as the cnidocyte, and a classical mechanosensory neuron referred to as the hair cell. While developmental genetics of cnidocytes is increasingly understood, genes essential for hair cell development are unknown. Here we show that the class IV POU homeodomain transcription factor (POU-IV) – an indispensable regulator of mechanosensory cell differentiation in Bilateria and cnidocyte differentiation in Cnidaria – controls hair cell development in the sea anemone cnidarian Nematostella vectensis. N. vectensis POU-IV is postmitotically expressed in tentacular hair cells, and is necessary for development of the apical mechanosensory apparatus, but not of neurites, in hair cells. Moreover, it binds to deeply conserved DNA recognition elements, and turns on a unique set of effector genes – including the transmembrane-receptor-encoding gene polycystin 1 – specifically in hair cells. Our results suggest that POU-IV directs differentiation of cnidarian hair cells and cnidocytes via distinct gene regulatory mechanisms, and support an evolutionarily ancient role for POU-IV in defining the mature state of mechanosensory neurons.
