Alexandre S. Cristino

Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR

For obtaining accurate and reliable gene expression results it is essential that quantitative realtime RT-PCR (qRT-PCR) data are normalized with appropriate reference genes. The current exponential increase in postgenomic studies on the honey bee, Apis mellifera, makes the standardization of qRT-PCR results an important task for ongoing community efforts. For this aim we selected four candidate reference genes (actin, ribosomal protein 49, elongation factor 1-alpha, tbp-association factor) and used three software-based approaches (geNorm, BestKeeper and NormFinder) to evaluate the suitability of these genes as endogenous controls. Their expression was examined during honey bee development, in different tissues, and after juvenile hormone exposure. Furthermore, the importance of choosing an appropriate reference gene was investigated for two developmentally regulated target genes. The results led us to consider all four candidate genes as suitable genes for normalization in A. mellifera. However, each condition evaluated in this study revealed a specific set of genes as the most appropriated ones.ZusammenfassungDie quantitative real-time RT-PCR (qRT-PCR) Methode enwickelt sich zu einer der am häufigsten benutzten Methode zur Quantifizierung von mRNAs. Für exakte und zuverlässig reproduzierbare Ergebnisse ist es allerdings notwendig, dass die jeweiligen qRT-PCR Werte mittels eines geeigneten Referenzgens normalisiert werden. Trotz der Verfügbarkeit von Referenzgen-Studien bei verschiedenen Organismen ist die Bewertung geeigneter Referenzgene für den jeweiligen Organismus, in diesem Fall die Honigbiene Apis mellifera, erforderlich. Genau dies war das Ziel der vorliegenden Untersuchung, und dafür wählten wir folgende Gene als Kandidaten aus: actin, rp49, ef1-alpha and tbp-af (Tab. I). Diese wurden mittels der Computerprogramme geNorm, BestKeeper und Normfinder auf ihre Eignung und Eigenschaften als Referenzgene getestet. Wir untersuchten dies in verschiedenen biologischen Zusammenhängen: in verschiedenen Phasen der postembryonalen Entwicklung, in verschiedenen Geweben und Organen und nach Behandlung mit Juvenilhormon (JH-III).Für Studien zur Postembryonalentwicklung erwiesen sich bei Verwendung von geNorm actin und tbp-af und bei Verwendung von BestKeeper und NormFinder rp49 als die jeweils stabilsten Gene (Tab. II und III). Trotz dieser Unterschiede in der Rangfolge der Eignung zeigten alle vier Referenzgene für das Entwicklungsstadium F3 die höchsten mRNA-Werte an (Abb. 1). Dies weist darauf hin, dass die Regulierung der Genexpression in der Entwicklung in weitem Maße korreliert ist, und dass sie in der Larval- und Pupalentwicklung wahrscheinlich stark von physiologischen Faktoren beeinflusst wird. Ähnliche Unterschiede in der Eignung als Referenzgene waren auch bei den Gewebestudien und nach JH-III-Behandlung zu sehen. Während geNorm in diesen Situationen actin und ef1-alpha als die am besten geeigneten Gene auswies (Tab. II und III), waren dies rp49 und ef1-alpha, wenn BestKeeper und NormFinder als Bewertungsprogramme eingesetzt wurden (Tab. II und III). Obwohl bei den Vergleichen verschiedener Gewebe und Organe die Expressionswerte aller vier Gene signifikant schwankten (Abb. 2), wurden sie bei den BestKeeper- und geNorm-Analysen als stabile Referenz-Gene bewertet. Für Untersuchungen zu Behandlungen mit JH-III waren die stabilsten Gene ef1-alpha und tbp-af (geNorm), tbp-af (Best-Keeper) und actin (NormFinder) (Tab. II and III). Da wir für die Kontrollen (Aceton behandelteArbeiterinnen) und die JH-III behandelten Arbeiterinnen keine Unterschiede in der Expression dieser Gene fanden (Abb. 3), zeigt dies, dass bei solchen Versuchen alle vier Gene zur Normalisierung eingesetzt werden können.In einem weiteren Versuchsansatz untersuchten wir, welche Auswirkungen die Auswahl von Referenzgenen auf die jeweiligen Zielgene hat. Für die Zielgene jhe-like und proPO fanden wir, dass die Normalisierung mittels eines weniger geeigneten Referenzgens eine deutliche Auswirkung auf die Berechnung der relativen Genexpressionswwerte hat, insbesondere, wenn die Proben niedrige mRNA-Werte haben (Abb. 4).Unsere Untersuchung zeigt, dass alle vier Gene im Prinzip als Referenzgene für quantitative Genexpressionsstudien bei A. mellifera eingesetzt werden können. Da jedoch jeder biologische Kontext eine jeweils andere Kombination von Referenzgenen als am besten geeignet auswies, schlagen wir vor, dass jeweils immer zwei dieser Gene für die Normalisierung verwendet werden.

Metabolic enzymes associated with xenobiotic and chemosensory responses in Nasonia vitripennis.

The numbers of glutathione S‐transferase, cytochrome P450 and esterase genes in the genome of the hymenopteran parasitoid Nasonia vitripennis are about twice those found in the genome of another hymenopteran, the honeybee Apis mellifera. Some of the difference is associated with clades of these families implicated in xenobiotic resistance in other insects and some is in clades implicated in hormone and pheromone metabolism. The data support the hypothesis that the eusocial behaviour of the honeybee and the concomitant homeostasis of the nest environment may obviate the need for as many gene/enzyme systems associated with xenobiotic metabolism as are found in other species, including N. vitripennis, that are thought to encounter a wider range of potentially toxic xenobiotics in their diet and habitat.

Neurodevelopmental and neuropsychiatric disorders represent an interconnected molecular system

Many putative genetic factors that confer risk to neurodevelopmental disorders such as autism spectrum disorders (ASDs) and X-linked intellectual disability (XLID), and to neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD) and schizophrenia (SZ) have been identified in individuals from diverse human populations. Although there is significant aetiological heterogeneity within and between these conditions, recent data show that genetic factors contribute to their comorbidity. Many studies have identified candidate gene associations for these mental health disorders, albeit this is often done in a piecemeal fashion with little regard to the inherent molecular complexity. Here, we sought to abstract relationships from our knowledge of systems level biology to help understand the unique and common genetic drivers of these conditions. We undertook a global and systematic approach to build and integrate available data in gene networks associated with ASDs, XLID, ADHD and SZ. Complex network concepts and computational methods were used to investigate whether candidate genes associated with these conditions were related through mechanisms of gene regulation, functional protein–protein interactions, transcription factor (TF) and microRNA (miRNA) binding sites. Although our analyses show that genetic variations associated with the four disorders can occur in the same molecular pathways and functional domains, including synaptic transmission, there are patterns of variation that define significant differences between disorders. Of particular interest is DNA variations located in intergenic regions that comprise regulatory sites for TFs or miRNA. Our approach provides a hypothetical framework, which will help discovery and analysis of candidate genes associated with neurodevelopmental and neuropsychiatric disorders.

Deep Sequencing of Organ- and Stage-Specific microRNAs in the Evolutionarily Basal Insect Blattella germanica (L.) (Dictyoptera, Blattellidae)

Background microRNAs (miRNAs) have been reported as key regulators at post-transcriptional level in eukaryotic cells. In insects, most of the studies have focused in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphon pisum) have had their miRNAs identified. Therefore, the study of the miRNAs of the evolutionarily basal hemimetabolan Blattella germanica may provide valuable insights on the structural and functional evolution of miRNAs. Methodology/Principal Findings Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 70 miRNA candidates conserved in other insects and 170 candidates specific to B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that number of reads can be used as a quantitative approach. Five novel miRNA precursors were identified and validated by PCR and sequencing. Known miRNAs and novel candidates were also validated by decreasing levels of their expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates. Conclusions/Significance Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and dicer-1 RNAi assay is shown to be a reliable method for validation of novel miRNAs.

Caste development and reproduction: a genome-wide analysis of hallmarks of insect eusociality

The honey bee queen and worker castes are a model system for developmental plasticity. We used established expressed sequence tag information for a Gene Ontology based annotation of genes that are differentially expressed during caste development. Metabolic regulation emerged as a major theme, with a caste‐specific difference in the expression of oxidoreductases vs. hydrolases. Motif searches in upstream regions revealed group‐specific motifs, providing an entry point to cis‐regulatory network studies on caste genes. For genes putatively involved in reproduction, meiosis‐associated factors came out as highly conserved, whereas some determinants of embryonic axes either do not have clear orthologs (bag of marbles, gurken, torso), or appear to be lacking (trunk) in the bee genome. Our results are the outcome of a first genome‐based initiative to provide an annotated framework for trends in gene regulation during female caste differentiation (representing developmental plasticity) and reproduction.

Complex networks: the key to systems biology

Though introduced recently, complex networks research has grown steadily because of its potential to represent, characterize and model a wide range of intricate natural systems and phenomena. Because of the intrinsic complexity and systemic organization of life, complex networks provide a specially promising framework for systems biology investigation. The current article is an up-to-date review of the major developments related to the application of complex networks in biology, with special attention focused on the more recent literature. The main concepts and models of complex networks are presented and illustrated in an accessible fashion. Three main types of networks are covered: transcriptional regulatory networks, protein-protein interaction networks and metabolic networks. The key role of complex networks for systems biology is extensively illustrated by several of the papers reviewed.

Odor memories regulate olfactory receptor expression in the sensory periphery.

Odor learning induces structural and functional modifications throughout the olfactory system, but it is currently unknown whether this plasticity extends to the olfactory receptors (Or) in the sensory periphery. Here, we demonstrate that odor learning induces plasticity in olfactory receptor expression in the honeybee, Apis mellifera. Using quantitative RT‐PCR analysis, we show that six putative floral scent receptors were differentially expressed in the bee antennae depending on the scent environment that the bees experienced. Or151, which we characterized using an in vitro cell expression system as a broadly tuned receptor binding floral odorants such as linalool, and Or11, the specific receptor for the queen pheromone 9‐oxo‐decenoic acid, were significantly down‐regulated after honeybees were conditioned with the respective odorants in an olfactory learning paradigm. Electroantennogram recordings showed that the neural response of the antenna was similarly reduced after odor learning. Long‐term odor memory was essential for inducing these changes, suggesting that the molecular mechanisms involved in olfactory memory also regulate olfactory receptor expression. Our study demonstrates for the first time that olfactory receptor expression is experience‐dependent and modulated by scent conditioning, providing novel insight into how molecular regulation at the periphery contributes to plasticity in the olfactory system.

Neuroligin-associated microRNA-932 targets actin and regulates memory in the honeybee

Increasing evidence suggests small non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) control levels of mRNA expression during experience-related remodelling of the brain. Here we use an associative olfactory learning paradigm in the honeybee Apis mellifera to examine gene expression changes in the brain during memory formation. Brain transcriptome analysis reveals a general downregulation of protein-coding genes, including asparagine synthetase and actin, and upregulation of ncRNAs. miRNA-mRNA network predictions together with PCR validation suggest miRNAs including miR-210 and miR-932 target the downregulated protein-coding genes. Feeding cholesterol-conjugated antisense RNA to bees results in the inhibition of miR-210 and of miR-932. Loss of miR-932 impairs long-term memory formation, but not memory acquisition. Functional analyses show that miR-932 interacts with Act5C, providing evidence for direct regulation of actin expression by an miRNA. An activity-dependent increase in miR-932 expression may therefore control actin-related plasticity mechanisms and affect memory formation in the brain.

Towards a molecular characterization of autism spectrum disorders: an exome sequencing and systems approach

The hypothetical ‘AXAS’ gene network model that profiles functional patterns of heterogeneous DNA variants overrepresented in autism spectrum disorder (ASD), X-linked intellectual disability, attention deficit and hyperactivity disorder and schizophrenia was used in this current study to analyze whole exome sequencing data from an Australian ASD cohort. An optimized DNA variant filtering pipeline was used to identify loss-of-function DNA variations. Inherited variants from parents with a broader autism phenotype and de novo variants were found to be significantly associated with ASD. Gene ontology analysis revealed that putative rare causal variants cluster in key neurobiological processes and are overrepresented in functions involving neuronal development, signal transduction and synapse development including the neurexin trans-synaptic complex. We also show how a complex gene network model can be used to fine map combinations of inherited and de novo variations in families with ASD that converge in the L1CAM pathway. Our results provide an important step forward in the molecular characterization of ASD with potential for developing a tool to analyze the pathogenesis of individual affected families.

Rapid regulation of microRNA following induction of long-term potentiation in vivo.

Coordinated regulation of gene expression is essential for consolidation of the memory mechanism, long-term potentiation (LTP). Triggering of LTP by N-methyl-D-aspartate receptor (NMDAR) activation rapidly activates constitutive and inducible transcription factors, which promote expression of genes responsible for LTP maintenance. As microRNA (miRNA) coordinate expression of genes related through seed sites, we hypothesize that miRNA contribute to the regulation of the LTP-induced gene response. MiRNA function primarily as negative regulators of gene expression. As LTP induction promotes a generalized rapid up-regulation of gene expression, we predicted a complementary rapid down-regulation of miRNA levels. Accordingly, we carried out global miRNA expression profiling in the rat dentate gyrus 20 min post-LTP induction in vivo. Consistent with our hypothesis, we found a large number of differentially expressed miRNA, the majority down-regulated. Detailed analysis of miR-34a-5p and miR-132-3p revealed this down-regulation was transient and NMDAR-dependent, whereby block of NMDARs released an activity-associated inhibitory mechanism. Furthermore, down-regulation of mature miR-34a-5p and miR-132-3p occurred solely by post-transcriptional mechanisms, occurring despite an associated up-regulation of the pri-miR-132 transcript. To understand how down-regulation of miR-34a-5p and miR-132-3p intersects with the molecular events occurring following LTP, we used bioinformatics to identify potential targets. Previously validated targets included the key LTP-regulated genes Arc and glutamate receptor subunits. Predicted targets included the LTP-linked kinase, Mapk1, and neuropil-associated transcripts Hn1 and Klhl11, which were validated using luciferase reporter assays. Furthermore, we found that the level of p42-Mapk1, the protein encoded by the Mapk1 transcript, was up-regulated following LTP. Together, these data support the interpretation that miRNA, in particular miR-34a-5p and miR-132-3p, make a surprisingly rapid contribution to synaptic plasticity via dis-inhibition of translation of key plasticity-related molecules.