Andrea B. Kohn

Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida.

Deuterostomes comprise vertebrates, the related invertebrate chordates (tunicates and cephalochordates) and three other invertebrate taxa: hemichordates, echinoderms and Xenoturbella. The relationships between invertebrate and vertebrate deuterostomes are clearly important for understanding our own distant origins. Recent phylogenetic studies of chordate classes and a sea urchin have indicated that urochordates might be the closest invertebrate sister group of vertebrates, rather than cephalochordates, as traditionally believed. More remarkable is the suggestion that cephalochordates are closer to echinoderms than to vertebrates and urochordates, meaning that chordates are paraphyletic. To study the relationships among all deuterostome groups, we have assembled an alignment of more than 35,000 homologous amino acids, including new data from a hemichordate, starfish and Xenoturbella. We have also sequenced the mitochondrial genome of Xenoturbella. We support the clades Olfactores (urochordates and vertebrates) and Ambulacraria (hemichordates and echinoderms). Analyses using our new data, however, do not support a cephalochordate and echinoderm grouping and we conclude that chordates are monophyletic. Finally, nuclear and mitochondrial data place Xenoturbella as the sister group of the two ambulacrarian phyla. As such, Xenoturbella is shown to be an independent phylum, Xenoturbellida, bringing the number of living deuterostome phyla to four.

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.

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.

Neuronal Transcriptome of Aplysia: Neuronal Compartments and Circuitry

Molecular analyses of Aplysia, a well-established model organism for cellular and systems neural science, have been seriously handicapped by a lack of adequate genomic information. By sequencing cDNA libraries from the central nervous system (CNS), we have identified over 175,000 expressed sequence tags (ESTs), of which 19,814 are unique neuronal gene products and represent 50%-70% of the total Aplysia neuronal transcriptome. We have characterized the transcriptome at three levels: (1) the central nervous system, (2) the elementary components of a simple behavior: the gill-withdrawal reflex-by analyzing sensory, motor, and serotonergic modulatory neurons, and (3) processes of individual neurons. In addition to increasing the amount of available gene sequences of Aplysia by two orders of magnitude, this collection represents the largest database available for any member of the Lophotrochozoa and therefore provides additional insights into evolutionary strategies used by this highly successful diversified lineage, one of the three proposed superclades of bilateral animals.

Specific sites in the Beta Interaction Domain of a schistosome Ca2+ channel β subunit are key to its role in sensitivity to the anti-schistosomal drug praziquantel

Praziquantel, the drug of choice against schistosomiasis, disrupts calcium (Ca2+) homeostasis in schistosomes via an unknown mechanism. Voltage-gated Ca2+ channels are heteromultimeric transmembrane protein complexes that contribute to impulse propagation and also regulate intracellular Ca2+ levels. Beta subunits modulate the properties of the pore-forming alpha1 subunit of high voltage-activated Ca2+ channels. Unlike other Ca2+ channel beta subunits, which have current stimulatory effects, a beta subunit subtype found in S. mansoni (SmbetaA) and S. japonicum (Sjbeta) dramatically reduces current levels when co-expressed with Ca2+ channel alpha1 subunits in Xenopus oocytes. It also confers praziquantel sensitivity to the mammalian Cav2.3 alpha1 subunit. The Beta Interaction Domains (BIDs) of SmbetaA and Sjbeta lack 2 conserved serines that each constitute a consensus site for protein kinase C (PKC) phosphorylation. Here, we use site-directed mutagenesis of schistosome beta subunits to show that these unique functional properties are correlated with the absence of these consensus PKC sites in the BID. Furthermore, a second schistosome beta subunit subtype contains both serines in the BID, enhances currents through alpha1 subunits, and does not confer praziquantel sensitivity. Thus, phosphorylation sites in the BID may play important roles in defining the modulatory properties and pharmacological sensitivities of schistosome Ca2+ channel beta subunits.

Regulation of neuronal excitability by interaction of Fragile X Mental Retardation Protein with Slack potassium channels

Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na+-activated K+ channels (KNa), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel–FMRP interactions may link changes in neuronal firing to changes in protein translation.

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.

Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family

SUMMARY Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1) uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA, but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B0 type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

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).

Do Different Neurons Age Differently? Direct Genome-Wide Analysis of Aging in Single Identified Cholinergic Neurons

Aplysia californica is a powerful experimental system to study the entire scope of genomic and epigenomic regulation at the resolution of single functionally characterized neurons and is an emerging model in the neurobiology of aging. First, we have identified and cloned a number of evolutionarily conserved genes that are age-related, including components of apoptosis and chromatin remodeling. Second, we performed gene expression profiling of different identified cholinergic neurons between young and aged animals. Our initial analysis indicates that two cholinergic neurons (R2 and LPl1) revealed highly differential genome-wide changes following aging suggesting that on the molecular scale different neurons indeed age differently. Each of the neurons tested has a unique subset of genes differentially expressed in older animals, and the majority of differently expressed genes (including those related to apoptosis and Alzheimers disease) are found in aging neurons of one but not another type. The performed analysis allows us to implicate (i) cell specific changes in histones, (ii) DNA methylation and (iii) regional relocation of RNAs as key processes underlying age-related changes in neuronal functions and synaptic plasticity. These mechanisms can fine-tune the dynamics of long-term chromatin remodeling, or control weakening and the loss of synaptic connections in aging. At the same time our genomic tests revealed evolutionarily conserved gene clusters associated with aging (e.g., apoptosis-, telomere- and redox-dependent processes, insulin and estrogen signaling and water channels).