Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis

Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-1, inhibin beta A, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 mu M) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 mu M can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

Differential Shh, Bmp and Wnt gene expressions during craniofacial development in mice

In this study, Bmp-4, Wnt-5a and Shh gene expressions were compared during early craniofacial development in mice by comparative non-isotopic in situ hybridization. Wild-type C57BL/6J mice were studied at various stages of embryonic development (from 8.5- to 13.5-day-old embryos - E8.5-13.5). During early odontogenesis, transcripts for Bmp-4, Shh and Wnt-5a were co-localised at the tooth initiation stage. At E8.5, Shh mRNA expression was restricted to diencephalon and pharyngeal endoderm. Before maxillae and mandible ossification, Bmp-4 and Wnt-5a signals were detected in the mesenchymal cells and around Meckel`s cartilage. During palatogenesis, Shh was expressed only in the epithelium and Wnt-5a only in the mesenchyme of the elevating palatal shelves. During tongue development, Shh expression was found in mesenchyme, probably contributing to tongue miogenesis, while Wnt-5a signal was in the epithelium, possibly during placode development and papillae formation. Taken together, these findings suggest that Bmp-4, Shh and Wnt-5a gene expressions may act together on the epithelial mesenchymal interactions occurring in several aspects of the early mouse craniofacial development, such as odontogenesis, neuronal development, maxillae and mandible ossification, palatogenesis and tongue formation. (C) 2009 Elsevier GmbH. All rights reserved.

SOX18 and the transcriptional regulation of blood vessel development

SOX18 is a transcription factor that is transiently expressed in nascent endothelial cells during embryonic development and adult neovascularization. This protein belongs to the SOX family of transcription factors, ih,which are proving to be some of the key regulators of cell-type specification in the vertebrate embryo. Natural mutations in the Sox18 gene have been shown to result to cardiovascular dysfunction, in some cases leading to death. Available evidence thus implicates Sox18 as an important regulator of vascular development, most likely playing a key role in endothelial cell specification. However; the genetic knockout of Sox18 in mice has produced a confounding result that complicates our understanding of the molecular mode of action of the SOX18 protein. We speculate that Sox18 inky act in a redundant fashion with closely related genes such as Sox7 and/or Sox17. (C) 2001, Elsevier Science Inc.

Sox18 expression in blood vessels and feather buds during chicken embryogenesis

Sox18 encodes a transcription factor known to be important for the development of blood vessels and hair follicles in mice. In order to study the functional conservation of this gene through evolution, we have isolated and characterized Sox18 in chickens. cSox18 shows a high degree of sequence homology to both the mouse and human orthologues, particularly in the high mobility group DNA-binding domain and to a lesser extent in the transcriptional activation domain. A region of unusually high sequence conservation at the C-terminus may represent a further, previously unrecognized functional domain. Both the chicken and human proteins appear to be truncated at the N-terminus relative to mouse SOX18. In situ hybridization analyses showed expression in the developing vasculature and feather follicles, consistent with reported expression in the mouse embryo. In addition, cSox18 mRNA was observed in the retina and claw beds. (C) 2001 Elsevier Science B.V. All rights reserved.

Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells

Sonic Hedgehog is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. Disruption of the Hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. The identification of genes regulated by Hedgehog is crucial to understanding how disruption of this pathway leads to neoplastic transformation. We have used a Sonic Hedgehog (Shh) responsive mouse cell line, C3H/10T1/2, to provide a model system for hedgehog target gene discovery. Following activation of cell cultures with Shh, RNA was used to interrogate microarrays to investigate downstream transcriptional consequences of hedgehog stimulation. As a result 11 target genes have been identified, seven of which are induced (Thrombomodulin, GILZ, BF-2, Nr4a1, IGF2, PMP22, LASP1) and four of which are repressed (SFRP-1, SFRP-2, Mip1-gamma, Amh) by Shh. These targets have a diverse range of putative functions and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery.

The receptor tyrosine kinase regulator sprouty1 is a target of the tumor suppressor WT1 and important for kidney development

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

Bacterial cellulose-lactoferrin as an antimicrobial edible packaging

Bacterial cellulose (BC) films from two distinct sources (obtained by static culture with Gluconacetobacter xylinus ATCC 53582 (BC1) and from a commercial source (BC2)) were modified by bovine lactoferrin (bLF) adsorption. The functionalized films (BC+bLF) were assessed as edible antimicrobial packaging, for use in direct contact with highly perishable foods, specifically fresh sausage as a model of meat products. BC+bLF films and sausage casings were characterized regarding their water vapour permeability (WVP), mechanical properties, and bactericidal efficiency against two food pathogens, Escherichia coli and Staphylococcus aureus. Considering their edibility, an in vitro gastrointestinal tract model was used to study the changes occurring in the BC films during passage through the gastrointestinal tract. Moreover, the cytotoxicity of the BC films against 3T3 mouse embryo fibroblasts was evaluated. BC1 and BC2 showed equivalent density, WVP and maximum tensile strength. The percentage of bactericidal efficiency of BC1 and BC2 with adsorbed bLF (BC1+bLF and BC2+bLF, respectively) in the standalone films and in inoculated fresh sausages, was similar against E. coli (mean reduction 69 % in the films per se versus 94 % in the sausages) and S. aureus (mean reduction 97 % in the films per se versus 36 % in the case sausages). Moreover, the BC1+bLF and BC2+bLF films significantly hindered the specific growth rate of both bacteria. Finally, no relevant cytotoxicity against 3T3 fibroblasts was found for the films before and after the simulated digestion. BC films with adsorbed bLF may constitute an approach in the development of bio-based edible antimicrobial packaging systems.

The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome.

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.

In vivo fate mapping with SCL regulatory elements identifies progenitors for primitive and definitive hematopoiesis in mice.

One of the principal issues facing biomedical research is to elucidate developmental pathways and to establish the fate of stem and progenitor cells in vivo. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for investigating this process. Here, we employ transcriptional regulatory elements from the stem cell leukemia (SCL) gene to selectively label primitive and definitive hematopoiesis. We report that SCL-labelled cells arising in the mid to late streak embryo give rise to primitive red blood cells but fail to contribute to the vascular system of the developing embryo. Restricting SCL-marking to different stages of foetal development, we identify a second population of multilineage progenitors, proficient in contributing to adult erythroid, myeloid and lymphoid cells. The distinct lineage-restricted potential of SCL-labelled early progenitors demonstrates that primitive erythroid cell fate specification is initiated during mid gastrulation. Our data also suggest that the transition from a hemangioblastic precursors with endothelial and blood forming potential to a committed hematopoietic progenitor must have occurred prior to SCL-marking of definitive multilineage blood precursors.

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein beta

Background. Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. Methods. Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. Results. C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ¿ (IFN¿). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN¿ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFN¿-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia.

Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1.

Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

Modulation of murine blastocyst hatching in vitro by glutamine and tryptophan

Enrichment of culture media with amino acids improves embryo development. However, little is known about the specific action of each amino acid during embryogenesis. The present study was undertaken to examine the effect of L-glutamine (Gln) and tryptophan (Trp) on mouse embryo hatching, expansion and viability in vitro. Blastocysts were collected from 6- to 8-week-old female BALB/c mice (N = 30) and cultured in M2 medium containing either 0.125, 0.25 or 0.5 mM Trp, 1 mM Gln, or M2 alone. Gln significantly increased (100%; P < 0.05) blastocyst hatching at 24 h compared to M2 alone or Trp; moreover, Trp inhibited blastocyst hatching when compared to M2 alone (P < 0.05) at 72 h. In contrast, the percentage of embryos reaching the state of expanded blastocyst at 48 h was significantly higher in medium with 1 mM Gln (66.6%; P < 0.05) or with 0.125 mM Trp (61.1%; P < 0.05). Unexpectedly, Trp increased the percentage of degenerated blastocysts after 48 h (67.7%; P < 0.05), while Gln preserved blastocyst viability. These results suggest that Gln may enhance blastocyst hatching, expansion and viability in vitro.

Untersuchungen zur Rolle von Isoformen katalytischer Untereinheiten der PKA

ZUSAMMENFASSUNG: Proteinkinasen übernehmen zentrale Aufgaben in der Signaltransduktion höherer Zellen. Dabei ist die cAMP-abhängige Proteinkinase (PKA) bezüglich ihrer Struktur und Funktion eine der am besten charakterisierten Proteinkinasen. Trotzdem ist wenig über direkte Interaktionspartner der katalytischen Untereinheiten (PKA-C) bekannt. In einem Split-Ubiquitin basiertem Yeast Two Hybrid- (Y2H-)System wurden potenzielle Interaktionspartner der PKA-C identifiziert. Als Bait wurden sowohl die humane Hauptisoform Cα (hCα) als auch die Proteinkinase X (PrKX) eingesetzt. Nach der Bestätigung der Funktionalität der PKA-C-Baitproteine, dem Nachweis der Expression und der Interaktion mit dem bekannten Interaktionspartner PKI wurde ein Y2H-Screen gegen eine Mausembryo-cDNA-Expressionsbibliothek durchgeführt. Von 2*10^6 Klonen wurden 76 Kolonien isoliert, die ein mit PrKX interagierendes Preyprotein exprimierten. Über die Sequenzierung der enthaltenen Prey-Vektoren wurden 25 unterschiedliche, potenzielle Interaktionspartner identifiziert. Für hCα wurden über 2*10^6 S. cerevisiae-Kolonien untersucht, von denen 1.959 positiv waren (1.663 unter erhöhter Stringenz). Über die Sequenzierung von ca. 10% der Klone (168) konnten Sequenzen für 67 verschiedene, potenzielle Interaktionspartner der hCα identifiziert werden. 15 der Preyproteine wurden in beiden Screens identifiziert. Die PKA-C-spezifische Wechselwirkung der insgesamt 77 Preyproteine wurde im Bait Dependency Test gegen largeT, ein Protein ohne Bezug zum PKA-System, untersucht. Aus den PKA-C-spezifischen Bindern wurden die löslichen Preyproteine AMY-1, Bax72-192, Fabp3, Gng11, MiF, Nm23-M1, Nm23-M2, Sssca1 und VASP256-375 für die weitere in vitro-Validierung ausgewählt. Die Interaktion von FLAG-Strep-Strep-hCα (FSS-hCα) mit den über Strep-Tactin aus der rekombinanten Expression in E. coli gereinigten One-STrEP-HA-Proteinen (SSHA-Proteine) wurde über Koimmunpräzipitation für SSHA-Fabp3, -Nm23-M1, -Nm23-M2, -Sssca1 und -VASP256-375 bestätigt. In SPR-Untersuchungen, für die hCα kovalent an die Oberfläche eines CM5-Sensorchips gekoppelt wurde, wurden die ATP/Mg2+-Abhängigkeit der Bindungen sowie differentielle Effekte der ATP-kompetitiven Inhibitoren H89 und HA-1077 untersucht. Freie hCα, die vor der Injektion zu den SSHA-Proteinen gegeben wurde, kompetierte im Gegensatz zu FSS-PrKX die Bindung an die hCα-Oberfläche. Erste kinetische Analysen lieferten Gleichgewichtsdissoziationskonstanten im µM- (SSHA-Fabp3, -Sssca1), nM- (SSHA-Nm23-M1, –M2) bzw. pM- (SSHA-VASP256-375) Bereich. In funktionellen Analysen konnte eine Phosphorylierung von SSHA-Sssca1 und VASP256-375 durch hCα und FSS-PrKX im Autoradiogramm nachgewiesen werden. SSHA-VASP256-375 zeigte zudem eine starke Inhibition von hCα im Mobility Shift-Assay. Dieser inhibitorische Effekt sowie die hohe Affinität konnten jedoch auf eine Kombination aus der Linkersequenz des Vektors und dem N-Terminus von VASP256-375 zurückgeführt werden. Über die Wechselwirkungen der hier identifizierten Interaktionspartner Fabp3, Nm23-M1 und Nm23-M2 mit hCα können in Folgeuntersuchungen neue PKA-Funktionen insbesondere im Herzen sowie während der Zellmigration aufgedeckt werden. Sssca1 stellt dagegen ein neues, näher zu charakterisierendes PKA-Substrat dar.