Mammalian cerebellar astrocytes critically regulate the differentiation and maturation of neuronal Purkinje cells and granule precursors. The G protein‐coupled receptor 37‐like 1 (Gpr37l1) is ...expressed by Bergmann astrocytes and interacts with patched 1 (Ptch1) at peri‐ciliary membranes. Cerebellar primary astrocyte cultures from wild‐type and Gpr37l1 null mutant mouse pups were established and studied. Primary cilia were produced by cultures of both genotypes, as well as Ptch1 and smoothened (Smo) components of the sonic hedgehog (Shh) mitogenic pathway. Compared to wild‐type cells, Gpr37l1−/− astrocytes displayed striking increases in proliferative activity, Ptch1 protein expression and internalization, intracellular cholesterol content, ciliary localization of Smo, as well as a marked production of active Shh. Similar effects were reproduced by treating wild‐type astrocytes with a putative prosaptide ligand of Gpr37l1. These findings indicate that Gpr37l1–Ptch1 interactions specifically regulate Ptch1 internalization and trafficking, with consequent stimulation of Shh production and activation of proliferative signaling.
Genetic ablation of G protein‐coupled receptor 37‐like 1 results in increased patched 1 expression and internalization. This leads to the intracellular accumulation of cholesterol, with concomitant stimulation of sonic hedgehog (Shh) production and activation of Shh‐induced proliferative signaling.
BACKGROUND: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the ...International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms. RESULTS: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems. CONCLUSIONS: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.
ABSTRACT
The mammalian G‐protein‐coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located ...within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37‐null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5‐fold increase in Gpr37‐/‐ P21 pups) and their increased apoptosis (e.g., 1.8‐fold and 3.5‐fold increase in Gpr3T‐/‐ P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ±16 mg; P< 0.05) and epididymal sperm count and motility (e.g., 1.5‐fold and 1.45‐fold decrease in Gpr37‐/‐ mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3‐fold increase in Gpr37‐/‐ P10 and P21 pups; Gli2, 1.4‐fold and 1.6‐fold increase in Gpr37‐/‐ P10 and P21 pups, respectively) including patched homolog 1 (1.3‐fold increase in Gpr3T‐/‐ P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation.—La Sala, G., Marazziti, D., Di Pietro, C., Golini, E., Mateoni, R., Tocchini‐Valentini, G. P. Modulation of Dhh signaling and altered Sertoli cell function in mice lacking the GPR37‐prosaposin receptor. FASEB J. 29, 2059‐2069 (2015). www.fasebj.org
The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research ...findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We have characterized a homodimeric tRNA endonuclease from the euryarchaeota
(FERAC), a facultative anaerobe which can grow at temperatures ranging from 35 to 42 °C. This enzyme, contrary to the ...eukaryal tRNA endonucleases and the homotetrameric
(METJA) homologs, is able to cleave minimal BHB (bulge-helix-bulge) substrates at 30 °C. The expression of this enzyme in
(SCHPO) enables the use of its properties as effectors by inserting BHB motif introns into hairpin loops normally seen in mRNA transcripts. In addition, the FERAC endonuclease can create proteins with new functionalities through the recombination of protein domains.
In the developing cerebellum, the proliferation and differentiation of glial and neuronal cell types depend on the modulation of the sonic hedgehog (Shh) signaling pathway. The vertebrate ...G-protein-coupled receptor 37-like 1 (GPR37L1) gene encodes a putative G-protein–coupled receptor that is expressed in newborn and adult cerebellar Bergmann glia astrocytes. This study shows that the ablation of the murine Gpr37l1 gene results in premature down-regulation of proliferation of granule neuron precursors and precocious maturation of Bergmann glia and Purkinje neurons. These alterations are accompanied by improved adult motor learning and coordination. Gpr37l1 ⁻/⁻ mice also exhibit specific modifications of the Shh signaling cascade. Specific assays show that in Bergmann glia cells Gpr37l1 is associated with primary cilium membranes and it specifically interacts and colocalizes with the Shh primary receptor, patched 1. These findings indicate that the patched 1–associated Gpr37l1 receptor participates in the regulation of postnatal cerebellum development by modulating the Shh pathway.
The G-protein coupled receptor 37-like 1 (Gpr37l1) is specifically expressed in most astrocytic glial cells, including cerebellar Bergmann astrocytes and interacts with patched 1 (Ptch1), a ...co-receptor of the sonic hedgehog (Shh)-smoothened (Smo) signaling complex. Gpr37l1 null mutant mice exhibit precocious post-natal cerebellar development, with altered Shh-Smo mitogenic cascade and premature down-regulation of granule cell precursor (GCP) proliferation. Gpr37l1 expression is downregulated in medulloblastoma (MB) and upregulated in glioma and glioblastoma tumors.
Shh-associated MBs originate postnatally, from dysregulated hyperproliferation of GCPs in developing cerebellum's external granular layer (EGL), as shown in heterozygous Ptch1+/− knock-out mouse strains that model human MB occurrence and progression.
This study investigates cerebellar MB phenotypes in newly produced Gpr37l1, Ptch1 double mutant mice. Natural history analysis shows that Gpr37l1 genetic ablation, in Ptch1+/− model animals, results in marked deferment of post-natal tumor occurrence and decreased incidence of more aggressive tumor types. It is also associated with the delayed and diminished presence of more severe types of hyperplastic lesions in Ptch1+/− mice. Consistently, during early post-natal development Gpr37l1−/−;Ptch1+/− pups exhibit reduction in cerebellar GCP proliferation and EGL thickness and a precocious, sustained expression of wingless-type MMTV integration site member 3 (Wnt3), a specific inhibitor of Shh-induced neuronal mitogenesis, in comparison with Ptch1+/− heterozygous single mutants. These findings highlight the specific involvement of Gpr37l1 in modulating postnatal cerebellar Shh-Ptch1-Smo mitogenic signaling in both normal and pathological conditions. The novel Gpr37l1−/−;Ptch1+/− mouse models may thus be instrumental in the detailed characterization of the initial phases of Shh-associated MB insurgence and development.
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•A novel Gpr37l1−/-Ptch1+/− mouse strain has been produced and studied.•Medulloblastoma occurrence is delayed in Gpr37l1−/-Ptch1+/− mice.•Aggressive tumor types are less frequent in Gpr37l1−/-Ptch1+/− mice.•Postnatal cerebellum development is altered in Gpr37l1−/-Ptch1+/− mice.
In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) ...with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit
Tsen54
gene. Our analysis determined that the embryonic development in
Tsen54
null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects.
Evolution of introns in the archaeal world Tocchini-Valentini, Giuseppe D; Fruscoloni, Paolo; Tocchini-Valentini, Glauco P
Proceedings of the National Academy of Sciences - PNAS,
03/2011, Letnik:
108, Številka:
12
Journal Article
Recenzirano
Odprti dostop
The self-splicing group I introns are removed by an autocatalytic mechanism that involves a series of transesterification reactions. They require RNA binding proteins to act as chaperones to ...correctly fold the RNA into an active intermediate structure in vivo. Pre-tRNA introns in Bacteria and in higher eukaryote plastids are typical examples of self-splicing group I introns. By contrast, two striking features characterize RNA splicing in the archaeal world. First, self-splicing group I introns cannot be found, to this date, in that kingdom. Second, the RNA splicing scenario in Archaea is uniform: All introns, whether in pre-tRNA or elsewhere, are removed by tRNA splicing endonucleases. We suggest that in Archaea, the protein recruited for splicing is the preexisting tRNA splicing endonuclease and that this enzyme, together with the ligase, takes over the task of intron removal in a more efficient fashion than the ribozyme. The extinction of group I introns in Archaea would then be a consequence of recruitment of the tRNA splicing endonuclease. We deal here with comparative genome analysis, focusing specifically on the integration of introns into genes coding for 23S rRNA molecules, and how this newly acquired intron has to be removed to regenerate a functional RNA molecule. We show that all known oligomeric structures of the endonuclease can recognize and cleave a ribosomal intron, even when the endonuclease derives from a strain lacking rRNA introns. The persistance of group I introns in mitochondria and chloroplasts would be explained by the inaccessibility of these introns to the endonuclease.
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