Thermal evolution of gene expression profiles in Drosophila subobscura

Background: Despite its pervasiveness, the genetic basis of adaptation resulting in variation directly or indirectly related to temperature (climatic) gradients is poorly understood. By using 3-fold replicated laboratory thermal stocks covering much of the physiologically tolerable temperature range for the temperate (i.e., cold tolerant) species Drosophila subobscura we have assessed whole-genome transcriptional responses after three years of thermal adaptation, when the populations had already diverged for inversion frequencies, pre-adult life history components, and morphological traits. Total mRNA from each population was compared to a reference pool mRNA in a standard, highly replicated two-colour competitive hybridization experiment using cDNA microarrays.Results: A total of 306 (6.6%) cDNA clones were identified as 'differentially expressed' (following a false discovery rate correction) after contrasting the two furthest apart thermal selection regimes (i.e., 13°C vs . 22°C), also including four previously reported candidate genes for thermotolerance in Drosophila (Hsp26, Hsp68, Fst, and Treh). On the other hand, correlated patterns of gene expression were similar in cold- and warm-adapted populations. Analysis of functional categories defined by the Gene Ontology project point to an overrepresentation of genes involved in carbohydrate metabolism, nucleic acids metabolism and regulation of transcription among other categories. Although the location of differently expressed genes was approximately at random with respect to chromosomes, a physical mapping of 88 probes to the polytene chromosomes of D. subobscura has shown that a larger than expected number mapped inside inverted chromosomal segments.Conclusion: Our data suggest that a sizeable number of genes appear to be involved in thermal adaptation in Drosophila, with a substantial fraction implicated in metabolism. This apparently illustrates the formidable challenge to understanding the adaptive evolution of complex trait variation. Furthermore, some clustering of genes within inverted chromosomal sections was detected. Disentangling the effects of inversions will be obviously required in any future approach if we want to identify the relevant candidate genes.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Analysis of gene expression patterns in animals

During my PhD, my aim was to provide new tools to increase our capacity to analyse gene expression patterns, and to study on a large-scale basis the evolution of gene expression in animals. Gene expression patterns (when and where a gene is expressed) are a key feature in understanding gene function, notably in development. It appears clear now that the evolution of developmental processes and of phenotypes is shaped both by evolution at the coding sequence level, and at the gene expression level.Studying gene expression evolution in animals, with complex expression patterns over tissues and developmental time, is still challenging. No tools are available to routinely compare expression patterns between different species, with precision, and on a large-scale basis. Studies on gene expression evolution are therefore performed only on small genes datasets, or using imprecise descriptions of expression patterns.The aim of my PhD was thus to develop and use novel bioinformatics resources, to study the evolution of gene expression. To this end, I developed the database Bgee (Base for Gene Expression Evolution). The approach of Bgee is to transform heterogeneous expression data (ESTs, microarrays, and in-situ hybridizations) into present/absent calls, and to annotate them to standard representations of anatomy and development of different species (anatomical ontologies). An extensive mapping between anatomies of species is then developed based on hypothesis of homology. These precise annotations to anatomies, and this extensive mapping between species, are the major assets of Bgee, and have required the involvement of many co-workers over the years. My main personal contribution is the development and the management of both the Bgee database and the web-application.Bgee is now on its ninth release, and includes an important gene expression dataset for 5 species (human, mouse, drosophila, zebrafish, Xenopus), with the most data from mouse, human and zebrafish. Using these three species, I have conducted an analysis of gene expression evolution after duplication in vertebrates.Gene duplication is thought to be a major source of novelty in evolution, and to participate to speciation. It has been suggested that the evolution of gene expression patterns might participate in the retention of duplicate genes. I performed a large-scale comparison of expression patterns of hundreds of duplicated genes to their singleton ortholog in an outgroup, including both small and large-scale duplicates, in three vertebrate species (human, mouse and zebrafish), and using highly accurate descriptions of expression patterns. My results showed unexpectedly high rates of de novo acquisition of expression domains after duplication (neofunctionalization), at least as high or higher than rates of partitioning of expression domains (subfunctionalization). I found differences in the evolution of expression of small- and large-scale duplicates, with small-scale duplicates more prone to neofunctionalization. Duplicates with neofunctionalization seemed to evolve under more relaxed selective pressure on the coding sequence. Finally, even with abundant and precise expression data, the majority fate I recovered was neither neo- nor subfunctionalization of expression domains, suggesting a major role for other mechanisms in duplicate gene retention.

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression.

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection.

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.

Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain.

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.

A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin κ C as a compatible prognostic marker in human solid tumors.

PURPOSE: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. EXPERIMENTAL DESIGN: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell-derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non-small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. RESULTS: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. CONCLUSION: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining.

Differential regulation of Na-K-ATPase isoform gene expression by T3 during rat brain development.

A fetal rat telencephalon organotypic cell culture system was found to reproduce the developmental pattern of Na-K-adenosinetriphosphatase (ATPase) gene expression observed in vivo [Am. J. Physiol. 258 (Cell Physiol. 27): C1062-C1069, 1990]. We have used this culture system to study the effects of triiodothyronine (T3; 0.003-30 nM) on mRNA abundance and basal transcription rates of Na-K-ATPase isoforms. Steady-state mRNA levels were low at culture day 6 (corresponding to the day of birth) but distinct for each isoform alpha 3 much greater than beta 1 = beta 2 greater than alpha 2 greater than alpha 1. At culture day 6, T3 did not modify mRNA abundance of any isoform. At culture day 12 (corresponding to day 7 postnatal), T3 increased the mRNA level of alpha 2 (4- to 7-fold), beta 2 (4- to 5-fold), alpha 1 (3- to 6-fold), and beta 1 (1.5-fold), whereas alpha 3 mRNA levels remained unchanged. Interestingly, the basal transcription rate for each isoform differed strikingly (alpha 2 greater than alpha 1 much greater than beta 1 = beta 2 greater than alpha 3) but remained stable throughout 12 days of culture and was not regulated by T3. Thus we observed an inverse relationship between rate of transcription and rate of mRNA accumulation for each alpha-isoform, suggesting that alpha 1- and alpha 2-mRNA are turning over rapidly whereas alpha 3-mRNA is turning over slowly. Our data indicate that one of the mechanisms by which T3 selectively controls Na-K-ATPase gene expression during brain development in vitro occurs at the posttranscriptional level.

Comparative modular analysis of gene expression in vertebrate development

The focus of my PhD research was the concept of modularity. In the last 15 years, modularity has become a classic term in different fields of biology. On the conceptual level, a module is a set of interacting elements that remain mostly independent from the elements outside of the module. I used modular analysis techniques to study gene expression evolution in vertebrates. In particular, I identified ``natural'' modules of gene expression in mouse and human, and I showed that expression of organ-specific and system-specific genes tends to be conserved between such distance vertebrates as mammals and fishes. Also with a modular approach, I studied patterns of developmental constraints on transcriptome evolution. I showed that none of the two commonly accepted models of the evolution of embryonic development (``evo-devo'') are exclusively valid. In particular, I found that the conservation of the sequences of regulatory regions is highest during mid-development of zebrafish, and thus it supports the ``hourglass model''. In contrast, events of gene duplication and new gene introduction are most rare in early development, which supports the ``early conservation model''. In addition to the biological insights on transcriptome evolution, I have also discussed in detail the advantages of modular approaches in large-scale data analysis. Moreover, I re-analyzed several studies (published in high-ranking journals), and showed that their conclusions do not hold out under a detailed analysis. This demonstrates that complex analysis of high-throughput data requires a co-operation between biologists, bioinformaticians, and statisticians.

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation.

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma.

Background: Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells. Results: The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance. Conclusions: Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-enviroment in testicular cancer.

An allele-specific, stochastic gene expression process controls the expression of multiple Ly49 family genes and generates a diverse, MHC-specific NK cell receptor repertoire.

Mouse NK cells express MHC class I-specific inhibitory Ly49 receptors. Since these receptors display distinct ligand specificities and are clonally distributed, their expression generates a diverse NK cell receptor repertoire specific for MHC class I molecules. We have previously found that the Dd (or Dk)-specific Ly49A receptor is usually expressed from a single allele. However, a small fraction of short-term NK cell clones expressed both Ly49A alleles, suggesting that the two Ly49A alleles are independently and randomly expressed. Here we show that the genes for two additional Ly49 receptors (Ly49C and Ly49G2) are also expressed in a (predominantly) mono-allelic fashion. Since single NK cells can co-express multiple Ly49 receptors, we also investigated whether mono-allelic expression from within the tightly linked Ly49 gene cluster is coordinate or independent. Our clonal analysis suggests that the expression of alleles of distinct Ly49 genes is not coordinate. Thus Ly49 alleles are apparently independently and randomly chosen for stable expression, a process that directly restricts the number of Ly49 receptors expressed per single NK cell. We propose that the Ly49 receptor repertoire specific for MHC class I is generated by an allele-specific, stochastic gene expression process that acts on the entire Ly49 gene cluster.

Cardiac raptor deletion impairs adaptive hypertrophy, alters metabolic gene expression and causes heart failure in mice

Background: Mammalian target of rapamycin (mTOR), a central regulator of cell growth, is found in two structurally and functionally distinct multiprotein complexes called mTOR complex (mTORC)1 and mTORC2. The specific roles of each of these branches of mTOR signaling have not been dissected in the adult heart. In the present study, we aimed to bring new insights into the function of cardiac mTORC1-mediated signaling in physiological as well as pathological situations.Methods: We generated mice homozygous for loxP-flanked raptor and positive for the tamoxifen-inducible Cre recombinase (MerCreMer) under control of the α- myosin heavy chain promoter. The raptor gene encodes an essential component of mTORC1. Gene ablation was induced at the age of 10-12 weeks, and two weeks later the raptor cardiac-knockout (raptor-cKO) mice started voluntary cagewheel exercise or were subjected to transverse aortic constriction (TAC) to induce pressure overload.Results: In sedentary raptor-cKO mice, ejection fractions gradually decreased, resulting in significantly reduced values at 38 days (P < 0.001). Raptor-cKO mice started to die during the fifth week after the last tamoxifen injection. At that time, the mortality rate was 36% in sedentary (n = 11) and 64% in exercising (n = 14) mice. TAC-induced pressure overload resulted in severe cardiac dysfunction already at earlier timepoints. Thus, at 7-9 days after surgery, ejection fraction and fractional shortening values were 22.3% vs 43.5% and 10.2% vs 21.5% in raptor-cKO vs wild-type mice, respectively. This was accompanied by significant reductions of ventricular wall and septal thickness as well as an increase in left ventricular internal diameter. Moreover, ventricular weight to tibial length ratios were increased in wild-type, but not in the raptor-cKO TAC mice. Together, this shows that raptor-cKO mice rapidly developed dilated cardiomyopathy without going through a phase of adaptive hypertrophy. Expression of ANP and β-MHC was induced in all raptor-cKO mice irrespective of the cardiac load conditions. Consistent with reduced mTORC1 activity, phosphorylation of ribosomal S6 kinase and 4E-BP1 was blunted, indicating reduced protein synthesis. Moreover, expression of multiple genes involved in the regulation of energy metabolism was altered, and followed by a shift from fatty acid to glucose oxidation.Conclusion: Our study suggests that mTORC1 coordinates protein and energy metabolic pathways in the heart. Moreover, we demonstrate that raptor is essential for the cardiac adaptation to increased workload and importantly, also for normal physiological cardiac function.

IFN Stimulated Gene Expression in the Liver is a Better Predictor of Treatment Response in Chronic Hepatitis C than the IL28B (IFN lambda 3) Genotype

BACKGROUND: Therapy of chronic hepatitis C (CHC) with pegIFNα/ribavirin achieves a sustained virologic response (SVR) in ∼55%. Pre-activation of the endogenous interferon system in the liver is associated with non-response (NR). Recently, genome-wide association studies described associations of allelic variants near the IL28B (IFNλ3) gene with treatment response and with spontaneous clearance of the virus. We investigated if the IL28B genotype determines the constitutive expression of IFN stimulated genes (ISGs) in the liver of patients with CHC. METHODS: We genotyped 93 patients with CHC for 3 IL28B single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, rs12980275), extracted RNA from their liver biopsies and quantified the expression of IL28B and of 8 previously identified classifier genes which discriminate between SVR and NR (IFI44L, RSAD2, ISG15, IFI22, LAMP3, OAS3, LGALS3BP and HTATIP2). Decision tree ensembles in the form of a random forest classifier were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS: The minor IL28B allele (bad risk for treatment response) was significantly associated with increased expression of ISGs, and, unexpectedly, with decreased expression of IL28B. Stratification of the patients into SVR and NR revealed that ISG expression was conditionally independent from the IL28B genotype, i.e. there was an increased expression of ISGs in NR compared to SVR irrespective of the IL28B genotype. The random forest feature score (RFFS) identified IFI27 (RFFS = 2.93), RSAD2 (1.88) and HTATIP2 (1.50) expression and the HCV genotype (1.62) as the strongest predictors of treatment response. ROC curves of the IL28B SNPs showed an AUC of 0.66 with an error rate (ERR) of 0.38. A classifier with the 3 best classifying genes showed an excellent test performance with an AUC of 0.94 and ERR of 0.15. The addition of IL28B genotype information did not improve the predictive power of the 3-gene classifier. CONCLUSIONS: IL28B genotype and hepatic ISG expression are conditionally independent predictors of treatment response in CHC. There is no direct link between altered IFNλ3 expression and pre-activation of the endogenous system in the liver. Hepatic ISG expression is by far the better predictor for treatment response than IL28B genotype.