Distinct gene expression dynamics in developing and regenerating crustacean limbs.

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

Epigenetic machinery is functionally conserved in cephalopods.

BACKGROUND: Epigenetic regulatory mechanisms are divergent across the animal kingdom, yet these mechanisms are not well studied in non-model organisms. Unique features of cephalopods make them attractive for investigating behavioral, sensory, developmental, and regenerative processes, and recent studies have elucidated novel features of genome organization and gene and transposon regulation in these animals. However, it is not known how epigenetics regulates these interesting cephalopod features. We combined bioinformatic and molecular analysis of Octopus bimaculoides to investigate the presence and pattern of DNA methylation and examined the presence of DNA methylation and 3 histone post-translational modifications across tissues of three cephalopod species. RESULTS: We report a dynamic expression profile of the genes encoding conserved epigenetic regulators, including DNA methylation maintenance factors in octopus tissues. Levels of 5-methyl-cytosine in multiple tissues of octopus, squid, and bobtail squid were lower compared to vertebrates. Whole genome bisulfite sequencing of two regions of the brain and reduced representation bisulfite sequencing from a hatchling of O. bimaculoides revealed that less than 10% of CpGs are methylated in all samples, with a distinct pattern of 5-methyl-cytosine genome distribution characterized by enrichment in the bodies of a subset of 14,000 genes and absence from transposons. Hypermethylated genes have distinct functions and, strikingly, many showed similar expression levels across tissues while hypomethylated genes were silenced or expressed at low levels. Histone marks H3K27me3, H3K9me3, and H3K4me3 were detected at different levels across tissues of all species. CONCLUSIONS: Our results show that the DNA methylation and histone modification epigenetic machinery is conserved in cephalopods, and that, in octopus, 5-methyl-cytosine does not decorate transposable elements, but is enriched on the gene bodies of highly expressed genes and could cooperate with the histone code to regulate tissue-specific gene expression.

The Lottia gigantea shell matrix proteome: re-analysis including MaxQuant iBAQ quantitation and phosphoproteome analysis.

BACKGROUND: Although the importance of proteins of the biomineral organic matrix and their posttranslational modifications for biomineralization is generally recognized, the number of published matrix proteomes is still small. This is mostly due to the lack of comprehensive sequence databases, usually derived from genomic sequencing projects. However, in-depth mass spectrometry-based proteomic analysis, which critically depends on high-quality sequence databases, is a very fast tool to identify candidates for functional biomineral matrix proteins and their posttranslational modifications. Identification of such candidate proteins is facilitated by at least approximate quantitation of the identified proteins, because the most abundant ones may also be the most interesting candidates for further functional analysis. RESULTS: Re-quantification of previously identified Lottia shell matrix proteins using the intensity-based absolute quantification (iBAQ) method as implemented in the MaxQuant identification and quantitation software showed that only 57 of the 382 accepted identifications constituted 98% of the total identified matrix proteome. This group of proteins did not contain obvious intracellular proteins, such as cytoskeletal components or ribosomal proteins, invariably identified as minor components of high-throughput biomineral matrix proteomes. Fourteen of these major proteins were phosphorylated to a variable extent. All together we identified 52 phospho sites in 20 of the 382 accepted proteins with high confidence. CONCLUSIONS: We show that iBAQ quantitation may be a useful tool to narrow down the group of functional biomineral matrix protein candidates for further research in cell biology, genetics or materials research. Knowledge of posttranslational modifications in these major proteins could be a valuable addition to previously published proteomes. This is true especially for phosphorylation, because this modification was already shown to modify mineralization processes in some instances.

The Genome Sequences of 118 Taxonomically Diverse Eukaryotes of the Salish Sea.

We present the complete genome sequences of 118 taxonomically diverse eukaryotes from the Salish Sea. Illumina sequencing was performed on genetic material from wild-collected individuals. The reads were assembled using a de novo method followed by a finishing step. The raw and assembled data are publicly available via Genbank.

Ocean to Tree: Leveraging Single-Molecule RNA-Seq to Repair Genome Gene Models and Improve Phylogenomic Analysis of Gene and Species Evolution.

Understanding gene evolution across genomes and organisms, including ctenophores, can provide unexpected biological insights. It enables powerful integrative approaches that leverage sequence diversity to advance biomedicine. Sequencing and bioinformatic tools can be inexpensive and user-friendly, but numerous options and coding can intimidate new users. Distinct challenges exist in working with data from diverse species but may go unrecognized by researchers accustomed to gold-standard genomes. Here, we provide a high-level workflow and detailed pipeline to enable animal collection, single-molecule sequencing, and phylogenomic analysis of gene and species evolution. As a demonstration, we focus on (1) PacBio RNA-seq of the genome-sequenced ctenophore Mnemiopsis leidyi, (2) diversity and evolution of the mechanosensitive ion channel Piezo in genetic models and basal-branching animals, and (3) associated challenges and solutions to working with diverse species and genomes, including gene model updating and repair using single-molecule RNA-seq. We provide a Python Jupyter Notebook version of our pipeline (GitHub Repository: Ctenophore-Ocean-To-Tree-2023 https://github.com/000generic/Ctenophore-Ocean-To-Tree-2023 ) that can be run for free in the Google Colab cloud to replicate our findings or modified for specific or greater use. Our protocol enables users to design new sequencing projects in ctenophores, marine invertebrates, or other novel organisms. It provides a simple, comprehensive platform that can ease new user entry into running their evolutionary sequence analyses.

Epithelial wound healing in Clytia hemisphaerica provides insights into extracellular ATP signaling mechanisms and P2XR evolution.

Epithelial wound healing involves the collective responses of many cells, including those at the wound margin (marginal cells) and those that lack direct contact with the wound (submarginal cells). How these responses are induced and coordinated to produce rapid, efficient wound healing remains poorly understood. Extracellular ATP (eATP) is implicated as a signal in epithelial wound healing in vertebrates. However, the role of eATP in wound healing in vivo and the cellular responses to eATP are unclear. Almost nothing is known about eATP signaling in non-bilaterian metazoans (Cnidaria, Ctenophora, Placozoa, and Porifera). Here, we show that eATP promotes closure of epithelial wounds in vivo in the cnidarian Clytia hemisphaerica (Clytia) indicating that eATP signaling is an evolutionarily ancient strategy in wound healing. Furthermore, eATP increases F-actin accumulation at the edges of submarginal cells. In Clytia, this indicates eATP is involved in coordinating cellular responses during wound healing, acting in part by promoting actin remodeling in cells at a distance from the wound. We also present evidence that eATP activates a cation channel in Clytia epithelial cells. This implies that the eATP signal is transduced through a P2X receptor (P2XR). Phylogenetic analyses identified four Clytia P2XR homologs and revealed two deeply divergent major branches in P2XR evolution, necessitating revision of current models. Interestingly, simple organisms such as cellular slime mold appear exclusively on one branch, bilaterians are found exclusively on the other, and many non-bilaterian metazoans, including Clytia, have P2XR sequences from both branches. Together, these results re-draw the P2XR evolutionary tree, provide new insights into the origin of eATP signaling in wound healing, and demonstrate that the cytoskeleton of submarginal cells is a target of eATP signaling.

Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels.

Ultrasound has been used to non-invasively manipulate neuronal functions in humans and other animals. However, this approach is limited as it has been challenging to target specific cells within the brain or body. Here, we identify human Transient Receptor Potential A1 (hsTRPA1) as a candidate that confers ultrasound sensitivity to mammalian cells. Ultrasound-evoked gating of hsTRPA1 specifically requires its N-terminal tip region and cholesterol interactions; and target cells with an intact actin cytoskeleton, revealing elements of the sonogenetic mechanism. Next, we use calcium imaging and electrophysiology to show that hsTRPA1 potentiates ultrasound-evoked responses in primary neurons. Furthermore, unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to c-fos expression and contralateral limb responses in response to ultrasound delivered through an intact skull. Collectively, we demonstrate that hsTRPA1-based sonogenetics can effectively manipulate neurons within the intact mammalian brain, a method that could be used across species.

Social tolerance in Octopus laqueus-A maximum entropy model.

Octopus laqueus is a small tropical octopus found in Okinawa, Japan and the greater Indo-Pacific. Octopus are often viewed as solitary animals but O. laqueus live in close proximity in the wild, and will potentially encounter one another on a regular basis, raising the possibility of social tolerance. Adopting shared den occupancy in aquaria as a potential measure of social tolerance in O. laqueus, we studied the animals' preference for shared dens over solitude. We characterized dependence of sharing preference on sex, den availability and den occupancy density. We designed two simple social tolerance assays in aquaria with a total of 45 daily measurements: (i) Pots Equal, with equal numbers of octopuses and dens and (ii) Pots Limited, with a 3:1 ratio of octopuses to dens. We found that O. laqueus will socially tolerate other individuals by sharing tanks and dens and with typically no loss to cannibalism or escape. However, animals also exhibit significant levels of social repulsion, and individuals often chose a solitary den when given the option. The patterns of den occupancy are observed to be consistent with a maximum entropy model that balances seeking shelter against avoiding other animals. The model accurately captures and predicts the data and can be generalized to other organisms and their social interactions. Overall, in O. laqueus the preference for a den is stronger than the preference to be solitary. The animals are tolerant of others with a mixture of sizes in the tank and even in a den, a reported first for octopuses outside mating. The relaxed disposition and social tolerance of O. laqueus make it a promising species to work with in the lab to explore social and potentially other behaviors in octopuses.

A Conserved Role for Serotonergic Neurotransmission in Mediating Social Behavior in Octopus.

Human and octopus lineages are separated by over 500 million years of evolution [1, 2] and show divergent anatomical patterns of brain organization [3, 4]. Despite these differences, growing evidence suggests that ancient neurotransmitter systems are shared across vertebrate and invertebrate species and in many cases enable overlapping functions [5]. Sociality is widespread across the animal kingdom, with numerous examples in both invertebrate (e.g., bees, ants, termites, and shrimps) and vertebrate (e.g., fishes, birds, rodents, and primates) lineages [6]. Serotonin is an evolutionarily ancient molecule [7] that has been implicated in regulating both invertebrate [8] and vertebrate [9] social behaviors, raising the possibility that this neurotransmitter's prosocial functions may be conserved across evolution. Members of the order Octopoda are predominantly asocial and solitary [10]. Although at this time it is unknown whether serotonergic signaling systems are functionally conserved in octopuses, ethological studies indicate that agonistic behaviors are suspended during mating [11-13], suggesting that neural mechanisms subserving social behaviors exist in octopuses but are suppressed outside the reproductive period. Here we provide evidence that, as in humans, the phenethylamine (+/-)-3,4-methylendioxymethamphetamine (MDMA) enhances acute prosocial behaviors in Octopus bimaculoides. This finding is paralleled by the evolutionary conservation of the serotonin transporter (SERT, encoded by the Slc6A4 gene) binding site of MDMA in the O. bimaculoides genome. Taken together, these data provide evidence that the neural mechanisms subserving social behaviors exist in O. bimaculoides and indicate that the role of serotonergic neurotransmission in regulating social behaviors is evolutionarily conserved.

Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours.

Imaging living organisms at high spatial resolution requires effective and innocuous immobilization. Long-term imaging places further demands on sample mounting with minimal perturbation of the organism. Here we present a simple, inexpensive method for rapid encapsulation of small animals of any developmental stage within a photo-crosslinked polyethylene glycol (PEG) hydrogel, gently restricting movement within their confined spaces. Immobilized animals maintain their original morphology in a hydrated environment compatible with chemical treatment, optical stimulation, and light-sheet microscopy. We demonstrate prolonged three-dimensional imaging of neural responses in the nematode Caenorhabditis elegans, recovery of viable organisms after 24 h, and imaging of larger squid hatchlings. We characterize a range of hydrogel and illumination conditions for immobilization quality, and identify paralytic-free conditions suitable for high-resolution single-cell imaging. Overall, PEG hydrogel encapsulation provides fast, versatile, and gentle mounting of small living organisms, from yeast to zebrafish, for continuous observation over hours.

Structural and Functional Components of the Skate Sensory Organ Ampullae of Lorenzini.

The skate, a cartilaginous fish related to sharks and rays, possesses a unique electrosensitive sensory organ known as the ampullae of Lorenzini (AoL). This organ is responsible for the detection of weak electric field changes caused by the muscle contractions of their prey. While keratan sulfate (KS) is believed to be a component of a jelly that fills this sensory organ and has been credited with its high proton conductivity, modern analytical methods have not been applied to its characterization. Surprisingly, total glycosaminoglycan (GAG) analysis demonstrates that the KS from skate jelly is extraordinarily pure, containing no other GAGs. This KS had a molecular weight of 20 to 30 kDa, consisting primarily of N-linked KS comprised mostly of a monosulfated disaccharide repeating unit, →3) Gal (1→4) GlcNAc6S (1→. Proteomic analysis of AoL jelly suggests that transferrin, keratin, and mucin serve as KS core proteins. Actin and tropomyosin are responsible for assembling the macrostructure of the jelly, and parvalbumin α-like protein and calreticulin regulate calcium and potassium channels involved in the transduction of the electrical signal, once conducted down the AoL by the jelly, serving as the molecular basis for electroreception.