Genetic strategies to analyze primary TRP channel-expressing cells in mice

Transient receptor potential (TRP) ion channels regulate fundamental biological processes throughout the body. TRP channel dysfunction has been causally linked to a number of disease states and thus establishes these channels as promising therapeutic targets. In order to dissect the physiological role of individual TRP channels in specific tissues, a detailed understanding of the expression pattern of the different TRP channels throughout the organism is essential. We provide an overview of recent efforts to generate novel TRP channel reporter mouse strains for all 28 TRP channels encoded in the mouse genome to understand expression of these channels with a single-cell resolution in an organism-wide manner. The reporter mice will enable both the visualization and manipulation of all primary TRP channel-expressing cells allowing an unprecedented wealth in variety to investigate TRP channel function in vivo. As proof of principle, we provide preliminary results documenting TRPM5 expression throughout the entire body of juvenile and adult mice.     

New insight into the evolution of aquaporins from flowering plants and vertebrates: orthologous identification and functional transfer is possible

Aquaporins (AQPs) represent a family of channel proteins that transport water and/or small solutes across cell membranes in the three domains of life. In all previous phylogenetic analysis of aquaporin, trees constructed using proteins with very low amino acid identity (<15%) were incongruent with rRNA data. In this work, restricting the evolutionary study of aquaporins to proteins with high amino acid identity (>25%), we showed congruence between AQPs and organismal trees. On the basis of this analysis, we defined 19 orthologous gene clusters in flowering plant species (3 PIP-like, 7 TIP-like, 6 NIP-like and 3 SIP-like). We described specific conserved motifs for each subfamily and each cluster, which were used to develop a method for automatic classification. Analysis of amino acid identity between orthologous monocotyledon and dicotyledon AQPs from each cluster, suggested that PIPs are under high evolutionary constraint. The phylogenetic analysis allowed us the assignment of orthologous aquaporins for very distant animal lineages (tetrapods-fishes). We also demonstrated that the location of all vertebrate AQPs in the ortholog clusters could be predicted by comparing their amino acid identity with human AQPs. We defined four AQP subfamilies in animals: AQP1-like, AQP8-like, AQP3-like and AQP11-like. Phylogenetic analysis showed that the four animal AQPs subfamilies are related with PIP-like, TIP-like, NIP-like and SIP-like subfamilies, respectively. Thus, this analysis would allow the prediction of individual AQPs function on the basis of orthologous genes from Arabidopsis thaliana and Homo sapiens.     

PHYLOGENY OF THE HYDRODICTYACEAE (CHLOROPHYCEAE): INFERENCES FROM rDNA DATA1

The hydrodictyacean green algal lineage has been the focus of much research due to the fossil record of at least some members, their ornamented cell walls, and their distinctive reproductive strategies. The phylogeny of the family was, until recently, exclusively morphology based. This investigation examines hydrodictyacean isolates from several culture collections, focusing on sequences from ribosomal data: 18S rDNA, 26S rDNA (partial), and internal transcribed spacer (ITS)‐2 data. Results from phylogenetic analyses of independent and combined data matrices support the Hydrodictyaceae as a monophyletic lineage that includes isolates of Chlorotetraedron, Hydrodictyon, Pediastrum, Sorastrum, and Tetraedron. Phylogenetic analyses of rDNA data indicate that the three‐dimensional coenobium of Hydrodictyon is evolutionarily distinct from the three‐dimensional coenobium of Sorastrum. The more robust aspects of the ITS‐2 data corroborate the 18S+26S rDNA topology and provide a structural autapomorphy for the Hydrodictyaceae and Neochloridaceae, that is, an abridgment of helix IV in the secondary structure. The rDNA data do not support monophyly of Pediastrum but rather suggest the existence of four additional hydrodictyacean genera: Monactinus, Parapediastrum, Pseudopediastrum, and Stauridium.     

Parendozoicomonas haliclonae gen. nov. sp. nov. isolated from a marine sponge of the genus Haliclona and description of the family Endozoicomonadaceae fam. nov. comprising the genera Endozoicomonas,Parendozoicomonas, and Kistimonas

Two Gram-stain-negative, facultative anaerobic, motile, rod-shaped strains, S-B4-1U^T and JOB-63a, forming small whitish transparent colonies on marine agar, were isolated from a sponge of the genus Haliclona. The strains shared 99.7% 16S rRNA gene sequence identity and a DNA-DNA hybridization value of 100%, but were differentiated by genomic fingerprinting using rep-PCRs. 16S rRNA gene sequence phylogeny placed the strains as a sister branch to the monophyletic genus Endozoicomonas (Oceanospirillales; Gammaproteobacteria) with 92.3–94.3% 16S rRNA gene sequence similarity to Endozoicomonas spp., 91.9 and 92.1% to Candidatus Endonucleobacter bathymodiolin, and 91.9 to 92.1% to the type strains of Kistimonas spp. Core genome based phylogeny of strain S-B4-1U^T confirmed the phylogenetic placement. Major fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) and 8 (C_18:1 ω7c/C_18:1 ω6c) followed by C_10:0 3-OH, C_16:0, and C_18:0. The G + C content was 50.1–51.4 mol%. The peptidoglycan diamino acid of strain S-B4-1U^T was meso-diaminopimelic acid, the predominant polyamine spermidine, the major respiratory quinone ubiquinone Q-9; phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine were major polar lipids. Based on the clear phylogenetic distinction, the genus Parendozoicomonas gen. nov. is proposed, with Parendozoicomonas haliclonae sp. nov. as type species and strain S-B4-1U^T (= CCM 8713^T = DSM 103671^T = LMG 29769^T) as type strain and JOB-63a as a second strain of the species. Based on the 16S rRNA gene sequence phylogeny of the Oceanospirillales within the Gammaproteobacteria, the Endozoicomonaceae fam. nov. is proposed including the genera Endozoicomonas, Parendozoicomonas, and Kistimonas as well as the Candidatus genus Endonucleobacter.     

A new regulatory principle for in vivo biochemistry: Pleiotropic low affinity regulation by the adenine nucleotides – Illustrated for the glycolytic enzymes of Saccharomyces cerevisiae

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme’s catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology.     

FISH mapping of 35S and 5S rRNA genes in Artemisia subgenus Dracunculus (Asteraceae): changes in number of loci during polyploid evolution and their systematic implications

Fluorescence in situ hybridization (FISH) with 35S and 5S rDNA probes was used to characterize cytogenetically representatives of Artemisia subgenus Dracunculus and allied species and to explore their evolution following polyploidization. At the diploid level two rDNA loci were observed in most species belonging to the A. dracunculus complex, a pattern considered to be the ancestral state for diploid Artemisia. In contrast, representative species from the Eurasian grade which belong to the other major lineage of the subgenus had more heterogeneous rDNA profiles, with three to five loci at the diploid level. Divergent patterns of locus evolution were also detected in polyploids, with the number and distribution of rDNA loci broadly fitting the two main diversification lineages in the subgenus. In the polyploid complex of A. dracunculus, the number of rDNA loci was almost proportional to ploidy, although monoploid genome size was shown to decrease with increasing ploidy. However, in polyploids from the Eurasian grade we found a remarkable reduction in the number of rDNA sites, suggesting that these species might have experienced either a complete loss of loci or a significant reduction in the number of repeats following polyploid formation. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013 , 171 , 655–666.     

SNX26, a GTPase-activating Protein for Cdc42, Interacts with PSD-95 Protein and Is Involved in Activity-dependent Dendritic Spine Formation in Mature Neurons

SNX26, a brain-enriched RhoGAP, plays a key role in dendritic arborization during early neuronal development in the neocortex. In mature neurons, it is localized to dendritic spines, but little is known about its role in later stages of development. Our results show that SNX26 interacts with PSD-95 in dendritic spines of cultured hippocampal neurons, and as a GTPase-activating protein for Cdc42, it decreased the F-actin content in COS-7 cells and in dendritic spines of neurons. Overexpression of SNX26 resulted in a GTPase-activating protein activity-dependent decrease in total protrusions and spine density together with dramatic inhibition of filopodia-to-spine transformations. Such effects of SNX26 were largely rescued by a constitutively active mutant of Cdc42. Consistently, an shRNA-mediated knockdown of SNX26 significantly increased total protrusions and spine density, resulting in an increase in thin or stubby type spines at the expense of the mushroom spine type. Moreover, endogenous expression of SNX26 was shown to be bi-directionally modulated by neuronal activity. Therefore, we propose that in addition to its key role in neuronal development, SNX26 also has a role in the activity-dependent structural change of dendritic spines in mature neurons.     

Novel ROSA26 Cre-reporter Knock-in C57BL/6N Mice Exhibiting Green Emission before and Red Emission after Cre-mediated Recombination

The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F₁ mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F₁ mice and R26GRR /Ins1-Cre F₁ mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).