No impact of malaria infection or immunisation on the expression of the immune GTPase GIMAP1 at the protein or mRNA levels (Article no. 53)

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish scyliorhinus canicula defines ancient and derived roles for fox genes in vertebrate development

In the human genome, members of the FoxC, FoxF, FoxL1, and FoxQ1 gene families are found in two paralagous clusters. Here we characterize all four gene families in the dogfish Seyliorhinus canicula, a member of the cartilaginous fish lineage that diverged before the radiation of osteichthyan vertebrates. We identify two FoxC genes, two FoxF genes, and single FoxQ1 and FoxL1 genes, demonstrating cluster duplication preceded the radiation of gnathostomes. The expression of all six genes was analyzed by in situ hybridization. The results show conserved expression of FoxL1, FoxF, and FoxC genes in different compartments of the mesoderm and of FoxQ1 in pharyngeal endoderm and its derivatives, confirming these as ancient sites of Fox gene expression, and also illustrate multiple cases of lineage-specific expression domains. Comparison to invertebrate chordates shows that the majority of conserved vertebrate expression domains mark tissues that are part of the primitive chordate body plan.

Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of rnRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells

The aims were to examine ovarian expression of bone morphogenetic protein (BMP) ligands/receptor mRNAs in the chicken and to test the hypothesis that theca-derived BMP(s) modulates granulosa cell function in a paracrine manner. RT-PCR revealed expression of multiple BMPs in granulosa and theca cells from prehierarchical and preovulatory follicles with greater expression in theca cells; both cell types expressed BMP receptors-1A, -1B and -II consistent with tissue responsiveness. Preovulatory granulosa cells F1, F2 and F3/4) were cultured with BMP-6 (expressed by theca but not granulosa) in the presence/absence of LH, FSH or 8-Br-cAMP. RMP-6 increased 'basal' and gonadotrophin-induced inhibin-A and progesterone secretion by each cell type but did not enhance the effect of 8-Br-cAMP. This indicates that the observed synergism between BMP-6 and gonadotrophin might involve BMP-induced up-regulation of gonadotrophin receptors. In support of this, BMP-6 alone increased LH-receptor (LHR) mRNA in F1 cells and FSH-receptor (FSHR) mRNA in F1, F2 and F3/4 cells. RMP-6 also enhanced LH/FSH-induced LHR transcript amount in each cell type but did not raise FSHR transcript amounts above those induced by BMP-6 alone. To further explore BMP6 action on inhibin-A secretion, we quantified inhibin/activin subunits (alpha, beta(A), beta(B)) mRNAs. Consistent with its effect on inhibin-A secretion, BMP-6 enhanced 'basal' expression of alpha- and beta(A)-Subunit mRNA in F1, F2 and F3/4 cells, and beta(B)-subunit mRNA in F3/4 cells. BMP-6 markedly enhanced FSH/LH-induced expression of alpha-subunit in all follicles and FSH-induced beta(A)-subunit in F2 and F3/4 follicles but not in F1 follicles. Neither BMP-6 alone, nor FSH/LH alone, affected 'basal' OB mRNA abundance. However, co-treatment with gonadotrophin and BMP-6 greatly increased beta(B)-subunit expression, the response being lowest in F1 follicles and greatest in F3/4 follicles. Collectively, these results support the hypothesis that intra-ovarian OMPs of thecal origin have a paracrine role in modulating granulosa cell function in the chicken in a preovulatory stage-dependent manner.

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

Objectives: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. Methods: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We. determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. Results: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. Conclusions: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

Expression of metallothionein genes I and II in bank vole clethrionomys glareolus populations chronically exposed in situ to heavy metals

The expression of two metallothionein genes (Mt-I and Mt-II) in the liver, kidney, and gonad of bank voles collected at four metal-contaminated sites (Cd, Zn, Pb, and Fe) were measured using the quantitative real-time PCR method (QPCR). Relative Mt gene expression was calculated by applying a normalization factor (NF) using the expression of two housekeeping genes, ribosomal 18S and beta-actin. Relative Mt expression in tissues of animals from contaminated sites was up to 54.8-fold higher than those from the reference site for Mt-I and up to 91.6-fold higher for Mt-II. Mt-II gene expression in the livers of bank voles from contaminated sites was higher than Mt-I gene expression. Inversely, Mt-II expression in the kidneys of voles was lower than Mt-I expression. Positive correlations between cadmium levels in the tissues and Mt-I were obtained in all studied tissues. Zinc, which undergoes homeostatic regulation, correlated positively with both Mt-I and Mt-II gene expression only in the kidney. Results showed that animals living in chronically contaminated environments intensively activate detoxifying mechanisms such as metallothionein expression. This is the first time that QPCR techniques to measure MT gene expression have been applied to assess the impact of environmental metal pollution on field collected bank voles.

Expression of target and reference genes in Daphnia magna exposed to ibuprofen

Background: Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR). The accurate measurement of target gene expression requires normalisation to an invariant internal control e. g., total RNA or reference genes. Reference genes are preferable, as they control for variation inherent in the cDNA synthesis and PCR. However, reference gene expression can vary between tissues and experimental conditions, which makes it crucial to validate them prior to application. Results: We evaluated 10 candidate reference genes for QPCR in Daphnia magna following a 24 h exposure to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) at 0, 20, 40 and 80 mg IB l(-1). Six of the 10 candidates appeared suitable for use as reference genes. As a robust approach, we used a combination normalisation factor (NF), calculated using the geNorm application, based on the geometric mean of three selected reference genes: glyceraldehyde-3-phosphate dehydrogenase, ubiquitin conjugating enzyme and actin. The effects of normalisation are illustrated using as target gene leukotriene B4 12-hydroxydehydrogenase (Ltb4dh), which was upregulated following 24 h exposure to 63-81 mg IB l(-1). Conclusions: As anticipated, use of the NF clarified the response of Ltb4dh in daphnids exposed to sublethal levels of ibuprofen. Our findings emphasise the importance in toxicogenomics of finding and applying invariant internal QPCR control(s) relevant to the study conditions.

Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H2O2) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

Forced expression of the cyclin B1-CDC2 complex induces proliferation in adult rat cardiomyocytes

Repair of the mature mammalian myocardium following injury is impaired by the inability of the majority of cardiomyocytes to undergo cell division. We show that overexpression of the cyclin B1-CDC2 (cell division cycle 2 kinase) complex re-initiates cell division in adult cardiomyocytes. Thus strategies targeting the cyclin B1-CDC2 complex might re-initiate cell division in mature cardiomyocytes in vivo and facilitate myocardial regeneration following injury.

Influence of mutations affecting gonadotropin production or responsiveness on expression of inhibin subunit mRNA and protein in the mouse ovary

Measurement of inhibins A and B in the serum of normal cyclic rodents has implicated FSH in the regulation of these peptides within the ovary. To extend these observations we have used a panel of mutant mice carrying mutations which affect either the production of, or the ability to respond to, FSH and LH. As a consequence, the females are infertile and show different degrees of follicular development. The aim of this study was to measure inhibin gene transcription in the ovaries of these mutant females together with inhibin protein levels in ovaries and serum and to relate these to follicular development within the ovary. Comparison was made with a pool of normal/heterozygous females. In hpg females where lack of GnRH production results in the absence of gonadotropin synthesis, in FSHbeta knockout (FSHbetaKO) females where disruption of the gene encoding FSHbeta results in the absence of FSH production, and in FSH receptor knockout (FSHRKO) females which are unable to respond to circulating FSH, follicular development remains at the pre-antral stage in these three mutants. Only in the hpg females were common inhibin alpha subunit mRNA levels significantly lower than normal. In these three mutants, however, mRNA levels for both the betaA and betaB subunits were extremely low compared with normal mice. At the protein level, neither inhibin A nor B was detected in the serum of these three mutants; however inhibin B, albeit at very low levels, was detectable within the ovaries. These observations confirm a major role for FSH in the control of transcription of the RA and betaB genes but suggest that the constitutive transcription of the alpha subunit is less dependent on FSH. In contrast, in LH receptor knockout (LuRKO) female mice inhibin betaA subunit mRNA levels were similar to those measured in normal/heterozygous females but levels of inhibin alpha and betaB subunit mRNAs were significantly higher than in the normal group. This was reflected in significantly higher inhibin B protein levels in ovaries and serum. An inability to respond to LH combined with high circulating levels of FSH leads to a high proportion of antral follicles in LuRKO females, with granulosa cells constituting the major cell type within the ovary. The high percentage of antral granulosa cells is likely to account for the significantly higher levels of inhibin B production in these ovaries.

Expression of AmphiCoe, an amphioxus COE/EBF gene, in the developing central nervous system and epidermal sensory neurons

The COE/EBF gene family marks a subset of prospective neurons in the vertebrate central and peripheral. nervous system; including neurons deriving from some ectodermal placodes. Since placodes are often considered unique to vertebrates, we have characterised an amphioxus COE/EBF gene with the aim of using it as a marker to examine the timing and location of peripheral neuron differentiation. A single COE/EBF family member, AmphiCoe, was isolated from the amphioxus Branchiostoma floridae: AmphiCoe lies basal to the vertebrate COE/EBF genes in molecular phylogenetic analysis, suggesting that the duplications that formed the vertebrate COE/EBF family were specific to the vertebrate lineage. AmphiCoe is expressed in the central nervous system and in a small number of scattered ectodermal cells on the flanks of neurulae stage embryos. These cells become at least largely recessed beneath the ectoderm. Scanning electron microscopy was used to examine embryos in which the ectoderm had been partially peeled away. This revealed that these cells have neuronal morphology, and we infer that they are the precursors of epidermal primary sensory neurons. These characters lead us to suggest that differentiation of some ectodermal cells into sensory neurons with a tendency to sink beneath the embryonic surface represents a primitive feature that has become incorporated into placodes during vertebrate evolution. (C) 2004 Wiley-Liss, Inc.