Gene Expression Patterns in the Ctenophore Pleurobrachia bachei: In Situ Hybridization.

In situ hybridization is a powerful and precise tool for revealing cell- and tissue-specific gene expression and a critical approach to validating single-cell RNA-seq (scRNA-seq). However, applying it to highly fragile animals such as ctenophores is challenging. Here, we present an in situ hybridization protocol for adult Pleurobrachia bachei (Cydippida)-a notable reference species representing the earliest-branching metazoan lineage, Ctenophora, sister to the rest of Metazoa. We provided expression patterns for several markers of cell phenotypes, as illustrated examples. The list includes predicted small secretory molecules/neuropeptides, WntX, genes encoding RNA-binding proteins (Musashi, Elav, Dicer, Argonaut), Neuroglobin, and selected transcription factors such as BarX. Both cell- and organ-specific expression of these genes further support the convergent evolution of many ctenophore innovations, which are remarkably distinct from tissue and organ specification in other basal metazoan lineages.

DNA Isolation Long-Read Genomic Sequencing in Ctenophores.

Long-read sequencing has proven the necessity for high-quality genomic assemblies of reference species, including enigmatic ctenophores. Obtaining high-molecular-weight genomic DNA is pivotal to this process and has proven highly problematic for many species. Here, we discuss different methodologies for gDNA isolation and present a protocol for isolating gDNA for several members of the phylum Ctenophora. Specifically, we describe a Pacific Biosciences library construction method used in conjunction with gDNA isolation methods that have proven successful in obtaining high-quality genomic assemblies in ctenophores.

RNA Isolation from Ctenophores.

RNA-seq or transcriptome analysis of individual cells and small cell populations is essential for virtually any biomedical field. Here, we examine and discuss the different methods of RNA isolation specific to ctenophores. We present a convenient, inexpensive, and reproducible protocol for RNA-seq libraries that are designed for low quantities of samples. We demonstrated these methods on early (one, two, four, eight cells) embryonic and developmental stages, tissues, and even a single aboral organ from the ctenophore Pleurobrachia bachei and other ctenophore species (e.g., Mnemiopsis, Bolinopsis, and Beroe).

DNA Methylation in Basal Metazoans: Insights from Ctenophores.

Epigenetic modifications control gene expression without altering the primary DNA sequence. However, little is known about DNA methylation in invertebrates and its evolution. Here, we characterize two types of genomic DNA methylation in ctenophores, 5-methyl cytosine (5-mC) and the unconventional form of methylation 6-methyl adenine (6-mA). Using both bisulfite sequencing and an ELISA-based colorimetric assay, we experimentally confirmed the presence of 5-mC DNA methylation in ctenophores. In contrast to other invertebrates studied, Mnemiopsis leidyi has lower levels of genome-wide 5-mC methylation, but higher levels of 5-mC methylation in promoters when compared with gene bodies. Phylogenetic analysis showed that ctenophores have distinct forms of DNA methyltransferase 1 (DNMT1); the zf-CXXC domain type, which localized DNMT1 to CpG sites, and is a metazoan specific innovation. We also show that ctenophores encode the full repertoire of putative enzymes for 6-mA DNA methylation, and these genes are expressed in the aboral organ of Mnemiopsis. Using an ELISA-based colorimetric assay, we experimentally confirmed the presence of 6-mA methylation in the genomes of three different species of ctenophores, M. leidyi, Beroe abyssicola, and Pleurobrachia bachei. The functional role of this novel epigenomic mark is currently unknown. In summary, despite their compact genomes, there is a wide variety of epigenomic mechanisms employed by basal metazoans that provide novel insights into the evolutionary origins of biological novelties.

The ctenophore genome and the evolutionary origins of neural systems.

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of 'classical' neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

Distinct expression patterns of glycoprotein hormone subunits in the lophotrochozoan Aplysia: implications for the evolution of neuroendocrine systems in animals.

Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific ß subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHß5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHß subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHß5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHß, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHß confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHß5 subunit the seatbelt structure is absent in AcaGPHß. We also found that AcaGPHα and AcaGPHß subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands.

Rapid evolution of the compact and unusual mitochondrial genome in the ctenophore, Pleurobrachia bachei.

Ctenophores are one of the most basally branching lineages of metazoans with the largest mitochondrial organelles in the animal kingdom. We sequenced the mitochondrial (mtDNA) genome from the Pacific cidipid ctenophore, Pleurobrachia bachei. The circular mitochondrial genome is 11,016 nts, with only 12 genes, and one of the smallest metazoan mtDNA genomes recorded. The protein coding genes are intronless cox1-3, cob, nad1, 3, 4, 4L and 5. The nad2 and 6 genes are represented as short fragments whereas the atp6 gene was found in the nuclear genome. Only the large ribosomal RNA subunit and two tRNAs were present with possibly the small subunit unidentifiable due to extensive fragmentation. The observed unique features of this mitochondrial genome suggest that nuclear and mitochondrial genomes have evolved at very different rates. This reduced mtDNA genome sharply contrasts with the very large sizes of mtDNA found in other basal metazoans including Porifera (sponges), and Placozoa (Trichoplax).

Molecular characterization of NMDA-like receptors in Aplysia and Lymnaea: relevance to memory mechanisms.

The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic glutamate receptors and has been implicated in synaptic plasticity, memory acquisition, and learning in both vertebrates and invertebrates, including molluscs. However, the molecular identity of NMDA-type receptors in molluscs remains unknown. Here, we cloned two NMDA-type receptors from the sea slug Aplysia californica, AcNR1-1 and AcNR1-2, as well as their homologs from the freshwater pulmonate snail Lymnaea stagnalis, LsNR1-1 and LsNR1-2. The cloned receptors contain a signal peptide, two extracellular segments with predicted binding sites for glycine and glutamate, three recognized transmembrane regions, and a fourth hydrophobic domain that makes a hairpin turn to form a pore-like structure. Phylogenetic analysis suggests that both the AcNR1s and LsNR1s belong to the NR1 subgroup of ionotrophic glutamate receptors. Our in situ hybridization data indicate highly abundant, but predominantly neuron-specific expression of molluscan NR1-type receptors in all central ganglia, including identified motor neurons in the buccal and abdominal ganglia as well as groups of mechanosensory cells. AcNR1 transcripts were detected extrasynaptically in the neurites of metacerebral cells of Aplysia. The widespread distribution of AcNR1 and LsNR1 transcripts also implies diverse functions, including their involvement in the organization of feeding, locomotory, and defensive behaviors.