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).

Gap Junctions in Ctenophora.

Gap junction proteins form specialized intercellular communication channels, including electrical synapses, that regulate cellular metabolism and signaling. We present a molecular inventory of the gap junction proteins-innexins (INX-like) in ctenophores, focusing on two reference species, Pleurobrachia bachei and Mnemiopsis leidyi. Innexins were identified in more than 15 ctenophore species, including such genera as Euplokamis, Pukia, Hormiphora, Bolinopsis, Cestum, Ocyropsis, Dryodora, Beroe, benthic ctenophores, Coeloplana and Vallicula, and undescribed species of Mertensiidae. The observed diversity of innexins resulted from the independent expansion of this family from the common ancestor of ctenophores. Innexins show the conserved topology with four transmembrane domains connected by two extracellular loops, which bridge intracellular gaps. However, INX-like genes have highly diverse exon organization and low percentage identity for their amino acid sequences within the same species and between ctenophore species. Such a broad scope of molecular diversity differs from innexins in other phyla. We predicted posttranslational modifications in innexins: 249 and 188 for M. leidyi and P. bachei, respectively. Neither their number nor their locations were conserved within or between species. When the number of posttranslational modifications is factored into the innexins' radiation, the potential for molecular and physiological diversity within gap junctions of ctenophores is almost unfathomable. RNA-seq and in situ hybridization data revealed that innexins are expressed across embryogenesis, including early cleavage stages and gastrulation. They are abundant in all adult tissues, with the highest expression level in the aboral organ (the major integrative center and the gravity sensor in ctenophores), followed by tentacles and comb plates. Nevertheless, each organ and tissue has a unique combination of innexins, suggesting their involvement in complex integrative functions and behaviors of ctenophores.

DNA Methylation in Ctenophores.

Epigenomic regulation and dynamic DNA methylation, in particular, are widespread mechanisms orchestrating the genome operation across time and species. Whole-genome bisulfite sequencing (WGBS) is currently the only method for unbiasedly capturing the presence of 5-methylcytosine (5-mC) DNA methylation patterns across an entire genome with single-nucleotide resolution. Bisulfite treatment converts unmethylated cytosines to uracils but leaves methylated cytosines intact, thereby creating a map of all methylated cytosines across a genome also known as a methylome. These epigenomic patterns of DNA methylation have been found to regulate gene expression and influence gene evolution rates between species. While protocols have been optimized for vertebrate methylome production, little adaptation has been done for invertebrates. Creating a methylome reference allows comparisons to be made between rates of transcription and epigenomic patterning in animals. Here we present a method of library construction for bisulfite sequencing optimized for non-bilateral metazoans such as the ctenophore, Mnemiopsis leidyi. We have improved upon our previously published method by including spike-in genomic DNA controls to measure methylation conversion rates. By pooling two bisulfite conversion reactions from the same individual, we also produced sequencing libraries that yielded a higher percentage of sequenced reads uniquely mapping to the reference genome. We successfully detected 5-mC in whole-animal methylomes at CpG, CHG, and CHH sites and visualized datasets using circos diagrams. The proof-of-concept tests were performed both under control conditions and following injury tests with changes in methylation patterns of genes encoding innexins, toxins and neuropeptides. Our approach can be easily adapted to produce epigenomes from other fragile marine animals.

Impact of the COVID-19 Pandemic Regulations on the Health Status and Medical Care of Children with Trisomy 21.

BACKGROUND: While children are considered at low risk for COVID-19, little is known about the impact of SARS-CoV-2 on paediatric risk patients like children with Trisomy 21 (T21). As these children often need regular therapy and various medical appointments, this study aimed to investigate the possible impact of the COVID-19 pandemic on children with T21. PATIENTS AND METHODS: Parents of children with T21 in the age of 0-12 years in Saxony-Anhalt were interviewed via phone in June 2021 regarding the health status and medical care of their children during the past 15 months of pandemic. RESULTS: 37 children with mean age of 6.1 years (min 0; max 12) were included in the study. The majority did not have any additional congenital anomalies. Surveyed parents hardly reported adverse changes of health status during the pandemic, but rather improvements, such as decreased number of respiratory infections and more time spend with their children. Outpatient appointments and therapy were cancelled or postponed at the onset of the pandemic, but parents reported low impact on their child's health and development. The main concern seemed to be lack of childcare during school and day-care closures and uncertainty concerning possible health impacts of an infection on their children. CONCLUSION: There was low impact of the COVID-19 pandemic on health and medical care of children with T21 in our study population. Further research is needed to help weigh the child's individual risk of infection against the need for medical treatment and therapy when dealing with paediatric risk patients.

Cross-sectional study to assess awareness of cytomegalovirus infection among pregnant women in Germany.

BACKGROUND: Study aimed to assess awareness of congenital cytomegalovirus (CMV) infection and its determinants in pregnancy. METHODS: Cross-sectional survey was conducted in five hospital-based maternity units in Germany. Pregnant women attending the maternity departments completed interviewer/self-administered survey questionnaire. High-risk group was defined according to contact with children under five years of age (at home or at work). Quantitative analyses using multivariable logistic regression were performed. RESULTS: One thousand two hundred thirty-three pregnant women were included. 48.5% (n = 598) of women reported any knowledge about risk of CMV infection during pregnancy. CMV infection was less known than other infections or diseases (education about toxoplasmosis 95.5% (n = 1,177), listeriosis 60.5% (n = 746). 38% (n = 468) of participants received education about CMV. CMV awareness was associated with the level of education and employment in childcare or medical care. Only 32% (n = 394) of the women made use of serological screening for CMV during pregnancy (individual health service). 40.8% (n = 503) of pregnant women were classified as high-risk group. They had significantly higher knowledge and education about CMV, and msignificantlycant more often use of the serological screening. CONCLUSIONS: Less than half of pregnant women surveyed were aware of potential risk associated with CMV infection during pregnancy. In our study,one-third third of pregnant women made use of the serological screening for CMV. Regarding the lack of current consensus on the role of serological CMV screening for pregnant women, hygiene preventive measures are the only evidence-based recommendation for pregnant women and knowledge increase could potentially have major public health impact.

A single-cell transcriptomic atlas of complete insect nervous systems across multiple life stages.

Molecular profiles of neurons influence neural development and function but bridging the gap between genes, circuits, and behavior has been very difficult. Here we used single cell RNAseq to generate a complete gene expression atlas of the Drosophila larval central nervous system composed of 131,077 single cells across three developmental stages (1 h, 24 h and 48 h after hatching). We identify 67 distinct cell clusters based on the patterns of gene expression. These include 31 functional mature larval neuron clusters, 1 ring gland cluster, 8 glial clusters, 6 neural precursor clusters, and 13 developing immature adult neuron clusters. Some clusters are present across all stages of larval development, while others are stage specific (such as developing adult neurons). We identify genes that are differentially expressed in each cluster, as well as genes that are differentially expressed at distinct stages of larval life. These differentially expressed genes provide promising candidates for regulating the function of specific neuronal and glial types in the larval nervous system, or the specification and differentiation of adult neurons. The cell transcriptome Atlas of the Drosophila larval nervous system is a valuable resource for developmental biology and systems neuroscience and provides a basis for elucidating how genes regulate neural development and function.

Single cell RNA-seq analysis reveals temporally-regulated and quiescence-regulated gene expression in Drosophila larval neuroblasts.

The mechanisms that generate neural diversity during development remains largely unknown. Here, we use scRNA-seq methodology to discover new features of the Drosophila larval CNS across several key developmental timepoints. We identify multiple progenitor subtypes - both stem cell-like neuroblasts and intermediate progenitors - that change gene expression across larval development, and report on new candidate markers for each class of progenitors. We identify a pool of quiescent neuroblasts in newly hatched larvae and show that they are transcriptionally primed to respond to the insulin signaling pathway to exit from quiescence, including relevant pathway components in the adjacent glial signaling cell type. We identify candidate "temporal transcription factors" (TTFs) that are expressed at different times in progenitor lineages. Our work identifies many cell type specific genes that are candidates for functional roles, and generates new insight into the differentiation trajectory of larval neurons.

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution.

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide­sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.

Evolution of glutamatergic signaling and synapses.

Glutamate (Glu) is the primary excitatory transmitter in the mammalian brain. But, we know little about the evolutionary history of this adaptation, including the selection of l-glutamate as a signaling molecule in the first place. Here, we used comparative metabolomics and genomic data to reconstruct the genealogy of glutamatergic signaling. The origin of Glu-mediated communications might be traced to primordial nitrogen and carbon metabolic pathways. The versatile chemistry of L-Glu placed this molecule at the crossroad of cellular biochemistry as one of the most abundant metabolites. From there, innovations multiplied. Many stress factors or injuries could increase extracellular glutamate concentration, which led to the development of modular molecular systems for its rapid sensing in bacteria and archaea. More than 20 evolutionarily distinct families of ionotropic glutamate receptors (iGluRs) have been identified in eukaryotes. The domain compositions of iGluRs correlate with the origins of multicellularity in eukaryotes. Although L-Glu was recruited as a neuro-muscular transmitter in the early-branching metazoans, it was predominantly a non-neuronal messenger, with a possibility that glutamatergic synapses evolved more than once. Furthermore, the molecular secretory complexity of glutamatergic synapses in invertebrates (e.g., Aplysia) can exceed their vertebrate counterparts. Comparative genomics also revealed 15+ subfamilies of iGluRs across Metazoa. However, most of this ancestral diversity had been lost in the vertebrate lineage, preserving AMPA, Kainate, Delta, and NMDA receptors. The widespread expansion of glutamate synapses in the cortical areas might be associated with the enhanced metabolic demands of the complex brain and compartmentalization of Glu signaling within modular neuronal ensembles.

The American lobster genome reveals insights on longevity, neural, and immune adaptations.

The American lobster, Homarus americanus, is integral to marine ecosystems and supports an important commercial fishery. This iconic species also serves as a valuable model for deciphering neural networks controlling rhythmic motor patterns and olfaction. Here, we report a high-quality draft assembly of the H. americanus genome with 25,284 predicted gene models. Analysis of the neural gene complement revealed extraordinary development of the chemosensory machinery, including a profound diversification of ligand-gated ion channels and secretory molecules. The discovery of a novel class of chimeric receptors coupling pattern recognition and neurotransmitter binding suggests a deep integration between the neural and immune systems. A robust repertoire of genes involved in innate immunity, genome stability, cell survival, chemical defense, and cuticle formation represents a diversity of defense mechanisms essential to thrive in the benthic marine environment. Together, these unique evolutionary adaptations contribute to the longevity and ecological success of this long-lived benthic predator.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

ATP signaling in the integrative neural center of Aplysia californica.

ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica-a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

Neural versus alternative integrative systems: molecular insights into origins of neurotransmitters.

Transmitter signalling is the universal chemical language of any nervous system, but little is known about its early evolution. Here, we summarize data about the distribution and functions of neurotransmitter systems in basal metazoans as well as outline hypotheses of their origins. We explore the scenario that neurons arose from genetically different populations of secretory cells capable of volume chemical transmission and integration of behaviours without canonical synapses. The closest representation of this primordial organization is currently found in Placozoa, disk-like animals with the simplest known cell composition but complex behaviours. We propose that injury-related signalling was the evolutionary predecessor for integrative functions of early transmitters such as nitric oxide, ATP, protons, glutamate and small peptides. By contrast, acetylcholine, dopamine, noradrenaline, octopamine, serotonin and histamine were recruited as canonical neurotransmitters relatively later in animal evolution, only in bilaterians. Ligand-gated ion channels often preceded the establishment of novel neurotransmitter systems. Moreover, lineage-specific diversification of neurotransmitter receptors occurred in parallel within Cnidaria and several bilaterian lineages, including acoels. In summary, ancestral diversification of secretory signal molecules provides unique chemical microenvironments for behaviour-driven innovations that pave the way to complex brain functions and elementary cognition. This article is part of the theme issue 'Basal cognition: multicellularity, neurons and the cognitive lens'.

The diversification and lineage-specific expansion of nitric oxide signaling in Placozoa: insights in the evolution of gaseous transmission.

Nitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans-one of the early-branching animal lineages. In contrast to other invertebrates studied, Trichoplax and Hoilungia have three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes in Trichoplax. Using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax and Hoilungia exemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa and the preservation of conservative NOS architecture from prokaryotic ancestors.

Sodium action potentials in placozoa: Insights into behavioral integration and evolution of nerveless animals.

Placozoa are small disc-shaped animals, representing the simplest known, possibly ancestral, organization of free-living animals. With only six morphological distinct cell types, without any recognized neurons or muscle, placozoans exhibit fast effector reactions and complex behaviors. However, little is known about electrogenic mechanisms in these animals. Here, we showed the presence of rapid action potentials in four species of placozoans (Trichoplax adhaerens [H1 haplotype], Trichoplax sp.[H2], Hoilungia hongkongensis [H13], and Hoilungia sp. [H4]). These action potentials are sodium-dependent and can be inducible. The molecular analysis suggests the presence of 5-7 different types of voltage-gated sodium channels, which showed substantial evolutionary radiation compared to many other metazoans. Such unexpected diversity of sodium channels in early-branched metazoan lineages reflect both duplication events and parallel evolution of unique behavioral integration in these nerveless animals.

Periconceptional folic acid supplement use among women of reproductive age and its determinants in central rural Germany: Results from a cross sectional study.

BACKGROUND: Prevalence of neural tube defects (NTD) has not decreased in Germany despite longstanding recommendations for folic acid supplementation. To examine the prevalence of periconceptional folic acid supplement use and associated factors among German women of reproductive age. METHODS: Cross-sectional survey was conducted in hospital-based maternity units in rural Germany. A sample of 1,004 women of reproductive age, either pregnant or in their early postpartum period, took interviewer/self-administered paper-based survey questionnaire. Prevalence of periconceptional folic acid supplement use was assessed, where periconception was defined as 1 month prior to and 3 months post-conception. Prevalence odds ratios (POR) and 95% confidence intervals (CI) using crude and adjusted logistic regression analysis were estimated to examine determinants of folic acid supplement use. RESULTS: Prevalence of folic acid supplement use was 41.5% (95% CI: 37.7%, 45.7%). Multivariable analysis showed lack of educational qualifications, unplanned pregnancy, later diagnosis of pregnancy, increased parity, and not having an awareness of importance of folic acid for optimal pregnancy outcomes were associated with not taking periconceptional folic acid supplements. Books, doctors, friends, media, were sources of information. CONCLUSION: Periconceptional folic acid is sub-optimal in rural Germany and thus failing to prevent NTDs. Targeted promotion of folic acid supplement use should be conducted periodically by gynecologists and primary care physicians during annual medical screenings. Mandatory folic acid fortification of staple foods is a complementary approach to overcome limitations of individual behaviors of folic acid supplement intake, and should be considered as it has been proven effective in multiple countries.

Microchemical identification of enantiomers in early-branching animals: Lineage-specific diversification in the usage of D-glutamate and D-aspartate.

D-amino acids are unique and essential signaling molecules in neural, hormonal, and immune systems. However, the presence of D-amino acids and their recruitment in early animals is mostly unknown due to limited information about prebilaterian metazoans. Here, we performed the comparative survey of L-/D-aspartate and L-/D-glutamate in representatives of four phyla of early-branching Metazoa: cnidarians (Aglantha); placozoans (Trichoplax), sponges (Sycon) and ctenophores (Pleurobrachia, Mnemiopsis, Bolinopsis, and Beroe), which are descendants of ancestral animal lineages distinct from Bilateria. Specifically, we used high-performance capillary electrophoresis for microchemical assays and quantification of the enantiomers. L-glutamate and L-aspartate were abundant analytes in all species studied. However, we showed that the placozoans, cnidarians, and sponges had high micromolar concentrations of D-aspartate, whereas D-glutamate was not detectable in our assays. In contrast, we found that in ctenophores, D-glutamate was the dominant enantiomer with no or trace amounts of D-aspartate. This situation illuminates prominent lineage-specific diversifications in the recruitment of D-amino acids and suggests distinct signaling functions of these molecules early in the animal evolution. We also hypothesize that a deep ancestry of such recruitment events might provide some constraints underlying the evolution of neural and other signaling systems in Metazoa.