Cristina Grande

Receptor subtypes involved in the presynaptic and postsynaptic actions of dopamine on striatal interneurons

By stimulating distinct receptor subtypes, dopamine (DA) exerts presynaptic and postsynaptic actions on both large aspiny (LA) cholinergic and fast-spiking (FS) parvalbumin-positive interneurons of the striatum. Lack of receptor- and isoform-specific pharmacological agents, however, has hampered the progress toward a detailed identification of the specific DA receptors involved in these actions. To overcome this issue, in the present study we used four different mutant mice in which the expression of specific DA receptors was ablated. In D1 receptor null mice, D1R-/-, DA dose-dependently depolarized both LA and FS interneurons. Interestingly, SCH 233390 (10 μm), a D1-like (D1 and D5) receptor antagonist, but not l-sulpiride (3–10 μm), a D2-like (D2, D3, D4) receptor blocker, prevented this effect, implying D5 receptors in this action. Accordingly, immunohistochemical analyses in both wild-type and D1R-/- mice confirmed the expression of D5 receptors in both cholinergic and parvalbumin-positive interneurons of the striatum. In mice lacking D2 receptors, D2R-/-, the DA-dependent inhibition of GABA transmission was lost in both interneuron populations. Both isoforms of D2 receptor, D2L and D2S, were very likely involved in this inhibitory action, as revealed by the electrophysiological analysis of the effect of the DA D2-like receptor agonist quinpirole in two distinct mutants lacking D2L receptors and expressing variable contents of D2S receptors. The identification of the receptor subtypes involved in the actions of DA on different populations of striatal cells is essential to understand the circuitry of the basal ganglia and to develop pharmacological strategies able to interfere selectively with specific neuronal functions.

Differential population structuring of two closely related fish species, the mackerel (Scomber scombrus) and the chub mackerel (Scomber japonicus), in the Mediterranean Sea

Population genetic structures of the mackerel (Scomber scombrus) and chub mackerel (Scomber japonicus) were studied in the Mediterranean Sea. Fragments of 272 bp (S. scomber) and 387 bp (S. japonicus) of the 5′‐end of the mitochondrial control region were sequenced from spawning individuals collected off the coasts of Greece, Italy, Spain, and Portugal. High levels of mitochondrial control region haplotypic diversity (> 0.98) were found for both Scomber species. Nucleotide diversity was higher in the mackerel (0.022) than in the chub mackerel (0.017). Global FST values were also higher and significant in the mackerel (0.024, P < 0.0001) as opposed to the chub mackerel (0.003, P > 0.05). Molecular variance analyses showed differential genetic structuring for these two closely related species. There is extensive gene flow between Mediterranean Sea and Atlantic Ocean populations of chub mackerel, which are organized into a larger panmictic unit. In contrast, Mediterranean Sea populations of mackerel show some degree of genetic differentiation and are structured along an east–west axis. The analysed eastern Mediterranean Sea mackerel populations (Greece, Italy) are clearly separated from that of the western Mediterranean Sea (Barcelona), which forms a panmictic unit with eastern Atlantic Ocean populations. The genetic structures of both species showed asymmetric migration patterns and indicated population expansion.

Evolution of gastropod mitochondrial genome arrangements

BackgroundGastropod mitochondrial genomes exhibit an unusually great variety of gene orders compared to other metazoan mitochondrial genome such as e.g those of vertebrates. Hence, gastropod mitochondrial genomes constitute a good model system to study patterns, rates, and mechanisms of mitochondrial genome rearrangement. However, this kind of evolutionary comparative analysis requires a robust phylogenetic framework of the group under study, which has been elusive so far for gastropods in spite of the efforts carried out during the last two decades. Here, we report the complete nucleotide sequence of five mitochondrial genomes of gastropods (Pyramidella dolabrata, Ascobulla fragilis, Siphonaria pectinata, Onchidella celtica, and Myosotella myosotis), and we analyze them together with another ten complete mitochondrial genomes of gastropods currently available in molecular databases in order to reconstruct the phylogenetic relationships among the main lineages of gastropods.ResultsComparative analyses with other mollusk mitochondrial genomes allowed us to describe molecular features and general trends in the evolution of mitochondrial genome organization in gastropods. Phylogenetic reconstruction with commonly used methods of phylogenetic inference (ME, MP, ML, BI) arrived at a single topology, which was used to reconstruct the evolution of mitochondrial gene rearrangements in the group.ConclusionFour main lineages were identified within gastropods: Caenogastropoda, Vetigastropoda, Patellogastropoda, and Heterobranchia. Caenogastropoda and Vetigastropoda are sister taxa, as well as, Patellogastropoda and Heterobranchia. This result rejects the validity of the derived clade Apogastropoda (Caenogastropoda + Heterobranchia). The position of Patellogastropoda remains unclear likely due to long-branch attraction biases. Within Heterobranchia, the most heterogeneous group of gastropods, neither Euthyneura (because of the inclusion of P. dolabrata) nor Pulmonata (polyphyletic) nor Opisthobranchia (because of the inclusion S. pectinata) were recovered as monophyletic groups. The gene order of the Vetigastropoda might represent the ancestral mitochondrial gene order for Gastropoda and we propose that at least three major rearrangements have taken place in the evolution of gastropods: one in the ancestor of Caenogastropoda, another in the ancestor of Patellogastropoda, and one more in the ancestor of Heterobranchia.

Neogastropod phylogenetic relationships based on entire mitochondrial genomes

BackgroundThe Neogastropoda is a highly diversified group of predatory marine snails (Gastropoda: Caenogastropoda). Traditionally, its monophyly has been widely accepted based on several morphological synapomorphies mostly related with the digestive system. However, recent molecular phylogenetic studies challenged the monophyly of Neogastropoda due to the inclusion of representatives of other caenogastropod lineages (e.g. Littorinimorpha) within the group. Neogastropoda has been classified into up to six superfamilies including Buccinoidea, Muricoidea, Olivoidea, Pseudolivoidea, Conoidea, and Cancellarioidea. Phylogenetic relationships among neogastropod superfamilies remain unresolved.ResultsThe complete mitochondrial (mt) genomes of seven Neogastropoda (Bolinus brandaris, Cancellaria cancellata, Conus borgesi, Cymbium olla, Fusiturris similis, Nassarius reticulatus, and Terebra dimidiata) and of the tonnoidean Cymatium parthenopeum (Littorinimorpha), a putative sister group to Neogastropoda, were sequenced. In addition, the partial sequence of the mitochondrial genome of the calyptraeoidean Calyptraea chinensis (Littorinimorpha) was also determined. All sequenced neogastropod mt genomes shared a highly conserved gene order with only two instances of tRNA gene translocation. Phylogenetic relationships of Neogastropoda were inferred based on the 13 mt protein coding genes (both at the amino acid and nucleotide level) of all available caenogastropod mitochondrial genomes. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses failed to recover the monophyly of Neogastropoda due to the inclusion of the tonnoidean Cymatium parthenopeum within the group. At the superfamily level, all phylogenetic analyses questioned the taxonomic validity of Muricoidea, whereas the monophyly of Conoidea was supported by most phylogenetic analyses, albeit weakly. All analyzed families were recovered as monophyletic except Turridae due to the inclusion of Terebridae. Further phylogenetic analyses based on either a four mt gene data set including two additional Littorinimorpha or combining mt and nuclear sequence data also rejected the monophyly of Neogastropoda but rendered rather unresolved topologies. The phylogenetic performance of each mt gene was evaluated under ML. The total number of resolved internal branches of the reference (whole-mt genome) topology was not recovered in any of the individual gene phylogenetic analysis. The cox2 gene recovered the highest number of congruent internal branches with the reference topology, whereas the combined tRNA genes, cox1, and atp8 showed the lowest phylogenetic performance.ConclusionPhylogenetic analyses based on complete mt genome data resolved a higher number of internal branches of the caenogastropod tree than individual mt genes. All performed phylogenetic analyses agreed in rejecting the monophyly of the Neogastropoda due to the inclusion of Littorinimorpha lineages within the group. This result challenges morphological evidence, and prompts for further re-evaluation of neogastropod morphological synapomorphies. The important increase in number of analyzed positions with respect to previous studies was not enough to achieve conclusive results regarding phylogenetic relationships within Neogastropoda. In this regard, sequencing of complete mtDNAs from all closely related caenogastropod lineages is needed. Nevertheless, the rapid radiation at the origin of Neogastropoda may not allow full resolution of this phylogeny based only on mt data, and in parallel more nuclear sequence data will also need to be incorporated into the phylogenetic analyses.

The complete mitochondrial genome of Scutopus ventrolineatus (Mollusca : Chaetodermomorpha) supports the Aculifera hypothesis

BackgroundWith more than 100000 living species, mollusks are the second most diverse metazoan phylum. The current taxonomic classification of mollusks recognizes eight classes (Neomeniomorpha, Chaetodermomorpha, Polyplacophora, Monoplacophora, Cephalopoda, Gastropoda, Bivalvia, and Scaphopoda) that exhibit very distinct body plans. In the past, phylogenetic relationships among mollusk classes have been contentious due to the lack of indisputable morphological synapomorphies. Fortunately, recent phylogenetic analyses based on multi-gene data sets are rendering promising results. In this regard, mitochondrial genomes have been widely used to reconstruct deep phylogenies. For mollusks, complete mitochondrial genomes are mostly available for gastropods, bivalves, and cephalopods, whereas other less-diverse lineages have few or none reported.ResultsThe complete DNA sequence (14662 bp) of the mitochondrial genome of the chaetodermomorph Scutopus ventrolineatus Salvini-Plawen, 1968 was determined. Compared with other mollusks, the relative position of protein-coding genes in the mitochondrial genome of S. ventrolineatus is very similar to those reported for Polyplacophora, Cephalopoda and early-diverging lineages of Bivalvia and Gastropoda (Vetigastropoda and Neritimorpha; but not Patellogastropoda). The reconstructed phylogenetic tree based on combined mitochondrial and nuclear sequence data recovered monophyletic Aplacophora, Aculifera, and Conchifera. Within the latter, Cephalopoda was the sister group of Gastropoda and Bivalvia + Scaphopoda.ConclusionsPhylogenetic analyses of mitochondrial sequences showed strong among-lineage rate heterogeneity that produced long-branch attraction biases. Removal of long branches (namely those of bivalves and patellogastropods) ameliorated but not fully resolved the problem. Best results in terms of statistical support were achieved when mitochondrial and nuclear sequence data were concatenated.

Molecular dissection of dopamine receptor signaling

The use of genetically engineered mice has provided substantial new insights into the functional organization of the striatum. Increasing evidence suggests that specific genes expressed within the striatum contribute to its functional activity. We studied the dopamine (DA) D1 receptor gene and one of its downstream targets, the transcription factor c-Fos. We have evaluated the functional interaction between the D1 and D2 DA receptor subtypes at the cellular and behavioral levels. Our results show that haloperidol, a DA D2-class receptor antagonist, activates c-Fos predominantly in enkephalin-positive striatal neurons, which project to the globus pallidus and are thought to mediate motor inhibition. Deletion of the DA D1 receptor increased the responsiveness of enkephalin neurons to haloperidol, in that haloperidol-induced increases in c-Fos and catalepsy were enhanced in D1 receptor knockout mice. These results suggest a functionally opposing role of the D1 receptor against the D2 DA-class receptors in the striatum.

Deployment of regulatory genes during gastrulation and germ layer specification in a model spiralian mollusc Crepidula

Background: During gastrulation, endoderm and mesoderm are specified from a bipotential precursor (endomesoderm) that is argued to be homologous across bilaterians. Spiralians also generate mesoderm from ectodermal precursors (ectomesoderm), which arises near the blastopore. While a conserved gene regulatory network controls specification of endomesoderm in deuterostomes and ecdysozoans, little is known about genes controlling specification or behavior of either source of spiralian mesoderm or the digestive tract. Results: Using the mollusc Crepidula, we examined conserved regulatory factors and compared their expression to fate maps to score expression in the germ layers, blastopore lip, and digestive tract. Many genes were expressed in both ecto‐ and endomesoderm, but only five were expressed in ectomesoderm exclusively. The latter may contribute to epithelial‐to‐mesenchymal transition seen in ectomesoderm. Conclusions: We present the first comparison of genes expressed during spiralian gastrulation in the context of high‐resolution fate maps. We found variation of genes expressed in the blastopore lip, mouth, and cells that will form the anus. Shared expression of many genes in both mesodermal sources suggests that components of the conserved endomesoderm program were either co‐opted for ectomesoderm formation or that ecto‐ and endomesoderm are derived from a common mesodermal precursor that became subdivided into distinct domains during evolution. Developmental Dynamics 244:1215–1248, 2015.

Evolution, divergence and loss of the Nodal signalling pathway: new data and a synthesis across the Bilateria

Since the discovery that the TGF-β signalling molecule Nodal and its downstream effector Pitx have a parallel role in establishing asymmetry between molluscs and deuterostomes the debate over the degree to which this signalling pathway is conserved across the Bilateria as a whole has been ongoing. Further taxon sampling is critical to understand the evolution and divergence of this signalling pathway in animals. Using genome and transcriptome mining we confirmed the presence of nodal and Pitx in a range of additional animal taxa for which their presence has not yet been described. In situ hybridization was used to show the embryonic expression of these genes in brachiopods and planarians. We show that both nodal and Pitx genes are broadly conserved across the Spiralia, and nodal likely appeared in the Bilaterian stem lineage after the divergence of the Acoelomorpha. Furthermore, both nodal and Pitx mRNA appears to be expressed in an asymmetric fashion in the brachiopod Terebratalia transversa. No evidence for the presence of a Lefty ortholog could be found in the non-deuterostome genomic resources examined. Nodal expression is asymmetric in a number of spiralian lineages, indicating a possible ancestral role of the Nodal/Pitx cascade in the establishment of asymmetries across the Bilateria.

Characterization of the complete mitochondrial genomes from Polycladida (Platyhelminthes) using next-generation sequencing.

The complete mitochondrial genomes of three polycladids, the acotylean Hoploplana elisabelloi and the cotyleans Enchiridium sp. and Prosthiostomum siphunculus have been assembled with high coverage from Illumina sequencing data. The mt genomes contain 36 genes including 12 of the 13 protein-coding genes characteristic for metazoan mitochondrial genomes, two ribosomal RNA genes, and 22 transfer RNA genes. Gene annotation, gene order, genetic code, start and stop codons and codon bias have been identified. In comparison with the well investigated parasitic Neodermata, our analysis reveals a great diversity of gene orders within Polycladida and Platyhelminthes in general. By analyzing representative genomes of the main groups of Platyhelminthes we explored the phylogenetic relationships of this group. The phylogenetic analyses strongly supported the monophyly of Polycladida, and based on a small taxon sampling suggest the monophyly of Acotylea and Cotylea.

Morphology and DNA barcodes reveal the presence of the non-native land planarian Obama marmorata (Platyhelminthes : Geoplanidae) in Europe

Abstract. We report for the first time the occurrence of Obama marmorata in the Iberian Peninsula as an introduced species from the Neotropical Region. The species is also reported for the first time in Argentina. The identification was made on the basis of morphological evidence. The divergence levels of a fragment of the mitochondrial cytochrome c oxidase I gene from Argentinean and Iberian samples were studied. The morphology of samples from the two regions was the same and matched the original description of the species. A DNA barcoding matrix was constructed using new sequences from O. marmorata and sequences of related species taken from GenBank. Among the Geoplanidae, interspecific divergence ranged between 3.3 and 14.4%, while intraspecific divergence percentages were 0–1.2%, signalling the presence of a DNA barcoding gap. All O. marmorata sequences, irrespective of their geographic origin, form a well supported clade with an intraspecific divergence of 0–0.9 (average = 0.4%). These results indicate the utility of DNA barcoding to discover allochthonous species in this group of organisms.