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.

Hyperpolarization-activated, cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning.

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K(+) and Na(+) ions, and is selectively blocked by Cs(+) and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 µM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.

Single-cell semiconductor sequencing.

RNA-seq or transcriptome analysis of individual cells and small-cell populations is essential for virtually any biomedical field. It is especially critical for developmental, aging, and cancer biology as well as neuroscience where the enormous heterogeneity of cells present a significant methodological and conceptual challenge. Here we present two methods that allow for fast and cost-efficient transcriptome sequencing from ultra-small amounts of tissue or even from individual cells using semiconductor sequencing technology (Ion Torrent, Life Technologies). The first method is a reduced representation sequencing which maximizes capture of RNAs and preserves transcripts' directionality. The second, a template-switch protocol, is designed for small mammalian neurons. Both protocols, from cell/tissue isolation to final sequence data, take up to 4 days. The efficiency of these protocols has been validated with single hippocampal neurons and various invertebrate tissues including individually identified neurons within a simpler memory-forming circuit of Aplysia californica and early (1-, 2-, 4-, 8-cells) embryonic and developmental stages from basal metazoans.

Single-neuron transcriptome and methylome sequencing for epigenomic analysis of aging.

Enormous heterogeneity in transcription and signaling is the feature that slows down progress in our understanding of the mechanisms of normal aging and age-related diseases. This is critical for neurobiology of aging where the enormous diversity of neuronal populations presents a significant challenge in experimental design. Here, we introduce Aplysia as a model for genomic analysis of aging at the single-cell level and provide protocols for integrated transcriptome and methylome profiling of individually identified neurons during the aging process. These single-cell RNA-seq and DNA methylation assays (methyl-capture/methyl enrichment) are compatible with all major next generation sequencing platforms (we used Roche/454 and SOLiD/Life Technologies as illustrative examples) and can be used to integrate an epigenetic signature with transcriptional output. The described sequencing library construction protocol provides both quantitative and directional information from transcriptional profiling of individual cells. Our results also confirm that different copies of DNA in polyploid Aplysia neurons behave similarly with respect to their DNA methylation.

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.

Phylogenomics reveals deep molluscan relationships.

Evolutionary relationships among the eight major lineages of Mollusca have remained unresolved despite their diversity and importance. Previous investigations of molluscan phylogeny, based primarily on nuclear ribosomal gene sequences or morphological data, have been unsuccessful at elucidating these relationships. Recently, phylogenomic studies using dozens to hundreds of genes have greatly improved our understanding of deep animal relationships. However, limited genomic resources spanning molluscan diversity has prevented use of a phylogenomic approach. Here we use transcriptome and genome data from all major lineages (except Monoplacophora) and recover a well-supported topology for Mollusca. Our results strongly support the Aculifera hypothesis placing Polyplacophora (chitons) in a clade with a monophyletic Aplacophora (worm-like molluscs). Additionally, within Conchifera, a sister-taxon relationship between Gastropoda and Bivalvia is supported. This grouping has received little consideration and contains most (>95%) molluscan species. Thus we propose the node-based name Pleistomollusca. In light of these results, we examined the evolution of morphological characters and found support for advanced cephalization and shells as possibly having multiple origins within Mollusca.

Creation by mutagenesis of a mammalian Ca(2+) channel beta subunit that confers praziquantel sensitivity to a mammalian Ca(2+) channel.

Voltage-gated Ca(2+) channel beta subunits are important modulators of the pore-forming alpha(1) subunit. We have cloned two schistosome beta subunits that confer sensitivity to the antischistosomal drug praziquantel (PZQ) to an otherwise insensitive mammalian alpha(1) subunit. The primary site of beta subunit interaction with alpha(1) subunits is the beta interaction domain (BID). The BID contains two conserved serines (225, 235 in rat beta2a) that constitute consensus sites for protein kinase C phosphorylation. However, these serines are absent in these schistosome beta subunits. Here we show that the capability to confer PZQ sensitivity can be created in the rat beta2a subunit by eliminating both serines in the BID. These results are consistent with, and should help our understanding of, the selective toxicity of PZQ.