Circadian Rhythm and Sleep Analyses in a Fruit Fly Model of Fragile X Syndrome Using a Video-Based Automated Behavioral Research System.

Fragile X syndrome (FXS) is caused by the full mutation in the FMR1 gene on the Xq27.3 chromosome region. It is the most common monogenic cause of autism spectrum disorder (ASD) and inherited intellectual disability (ID). Besides ASD and ID and other symptoms, individuals with FXS may exhibit sleep problems and impairment of circadian rhythm (CR). The Drosophila melanogaster models of FXS, such as dFMR1B55, represent excellent models for research in the FXS field. During this study, sleep patterns and CR in dFMR1B55 mutants were analyzed, using a new platform based on continuous high-resolution videography integrated with a highly-customized version of an open-source software. This methodology provides more sensitive results, which could be crucial for all further research in this model of fruit flies. The study revealed that dFMR1B55 male mutants sleep more and can be considered weak rhythmic flies rather than totally arrhythmic and present a good alternative animal model of genetic disorder, which includes impairment of CR and sleep behavior. The combination of affordable videography and software used in the current study is a significant improvement over previous methods and will enable broader adaptation of such high-resolution behavior monitoring methods.

A novel microduplication in INPP5A segregates with schizophrenia spectrum disorder in the family of a patient with both childhood onset schizophrenia and autism spectrum disorder.

Childhood-Onset Schizophrenia (COS) is a very rare and severe psychiatric disorder defined by adult schizophrenia symptoms occurring before the age of 13. We report a microduplication in the 10q26.3 region including part of the Inositol Polyphosphate-5-Phosphatase A (INPP5A) gene that segregates with Schizophrenia Spectrum Disorders (SSDs) in the family of a female patient affected by both COS and Autism Spectrum Disorder (ASD). Phenotyping and genotyping (including CGH-array) were performed for mother, healthy sister, and affected child according to the GenAuDiss protocol (NCT02565524). The duplication size is 324 kb and is present in a patient with COS and in her mother with SSD, but not in the patient's healthy sister. INPP5A encodes a membrane-associated 43 kDa type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase. This protein is found both in mouse and human brains and we found that its Drosophila homologue 5PtaseI is specifically expressed in the central nervous system. Hydrolyzed products from InsP3 5-phosphatases mobilize intracellular calcium, which is relevant for dendritic spine morphogenesis in neurons and altered in both schizophrenia and ASD. These may constitute arguments in favor of this gene alteration in the pathophysiology of COS.

Using a Combination of Novel Research Tools to Understand Social Interaction in the Drosophila melanogaster Model for Fragile X Syndrome.

Fragile X syndrome (FXS), the most common monogenic cause of inherited intellectual disability and autism spectrum disorder, is caused by a full mutation (>200 CGG repeats) in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene. Individuals with FXS experience various challenges related to social interaction (SI). Animal models, such as the Drosophila melanogaster model for FXS where the only ortholog of human FMR1 (dFMR1) is mutated, have played a crucial role in the understanding of FXS. The aim of this study was to investigate SI in the dFMR1B55 mutants (the groups of flies of both sexes simultaneously) using the novel Drosophila Shallow Chamber and a Python data processing pipeline based on social network analysis (SNA). In comparison with wild-type flies (w1118), SNA analysis in dFMR1B55 mutants revealed hypoactivity, fewer connections in their networks, longer interaction duration, a lower ability to transmit information efficiently, fewer alternative pathways for information transmission, a higher variability in the number of interactions they achieved, and flies tended to stay near the boundaries of the testing chamber. These observed alterations indicate the presence of characteristic strain-dependent social networks in dFMR1B55 flies, commonly referred to as the group phenotype. Finally, combining novel research tools is a valuable method for SI research in fruit flies.

Transactivation in Drosophila of human enhancers by human transcription factors involved in congenital heart diseases.

BACKGROUND: The human transcription factors (TFs) GATA4, NKX2.5 and TBX5 form part of the core network necessary to build a human heart and are involved in Congenital Heart Diseases (CHDs). The human natriuretic peptide precursor A (NPPA) and α-myosin heavy chain 6 (MYH6) genes are downstream effectors involved in cardiogenesis that have been demonstrated to be in vitro targets of such TFs. RESULTS: To study the interactions between these human TFs and their target enhancers in vivo, we overexpressed them in the whole Drosophila cardiac tube using the UAS/GAL4 system. We observed that all three TFs up-regulate their natural target enhancers in Drosophila and cause developmental defects when overexpressed in eyes and wings. CONCLUSIONS: A strong potential of the present model might be the development of combinatorial and mutational assays to study the interactions between human TFs and their natural target promoters, which are not easily undertaken in tissue culture cells because of the variability in transfection efficiency, especially when multiple constructs are used. Thus, this novel system could be used to determine in vivo the genetic nature of the human mutant forms of these TFs, setting up a powerful tool to unravel the molecular genetic mechanisms that lead to CHDs.

A Pilot Study on Early-Onset Schizophrenia Reveals the Implication of Wnt, Cadherin and Cholecystokinin Receptor Signaling in Its Pathophysiology.

Early-Onset Schizophrenia (EOS) is a very rare mental disorder that is a form of schizophrenia occurring before the age of 18. EOS is a brain disease marked by an early onset of positive and negative symptoms of psychosis that impact development and cognitive functioning. Clinical manifestations commonly include premorbid features of Autism Spectrum Disorder (ASD), attention deficits, Intellectual Disability (ID), neurodevelopmental delay, and behavioral disturbances. After the onset of psychotic symptoms, other neuropsychiatric comorbidities are also common, including obsessive-compulsive disorder, major depressive disorder, expressive and receptive language disorders, auditory processing, and executive functioning deficits. With the purpose to better gain insight into the genetic bases of this disorder, we developed a pilot project performing whole exome sequencing of nine trios affected by EOS, ASD, and mild ID. We carried out gene prioritization by combining multiple bioinformatic tools allowing us to identify the main pathways that could underpin the neurodevelopmental phenotypes of these patients. We identified the presence of variants in genes belonging to the Wnt, cadherin and cholecystokinin receptor signaling pathways.

The hexapeptide and linker regions of the AbdA Hox protein regulate its activating and repressive functions.

The Hox family transcription factors control diversified morphogenesis during development and evolution. They function in concert with Pbc cofactor proteins. Pbc proteins bind the Hox hexapeptide (HX) motif and are thereby thought to confer DNA binding specificity. Here we report that mutation of the AbdA HX motif does not alter its binding site selection but does modify its transregulatory properties in a gene-specific manner in vivo. We also show that a short, evolutionarily conserved motif, PFER, in the homeodomain-HX linker region acts together with the HX to control an AbdA activation/repression switch. Our in vivo data thus reveal functions not previously anticipated from in vitro analyses for the hexapeptide motif in the regulation of Hox activity.

Childhood-Onset Schizophrenia: A Systematic Overview of Its Genetic Heterogeneity From Classical Studies to the Genomic Era.

Childhood-onset schizophrenia (COS), a very rare and severe chronic psychiatric condition, is defined by an onset of positive symptoms (delusions, hallucinations and disorganized speech or behavior) before the age of 13. COS is associated with other neurodevelopmental disorders such as autism spectrum disorder (ASD) and attention deficit and hyperactivity disorder. Copy number variations (CNVs) represent well documented neurodevelopmental disorder risk factors and, recently, de novo single nucleotide variations (SNVs) in genes involved in brain development have also been implicated in the complex genetic architecture of COS. Here, we aim to review the genetic changes (CNVs and SNVs) reported for COS, going from previous studies to the whole genome sequencing era. We carried out a systematic review search in PubMed using the keywords "childhood(early)-onset schizophrenia(psychosis)" and "genetic(s) or gene(s) or genomic(s)" without language and date limitations. The main inclusion criteria are COS (onset before 13 years old) and all changes/variations at the DNA level (CNVs or SNVs). Thirty-six studies out of 205 met the inclusion criteria. Cytogenetic abnormalities (n = 72, including 66 CNVs) were identified in 16 autosomes and 2 sex chromosomes (X, Y), some with a higher frequency and clinical significance than others (e.g., 2p16.3, 3q29, 15q13.3, 22q11.21 deletions; 2p25.3, 3p25.3 and 16p11.2 duplications). Thirty-one single nucleotide mutations in genes principally involved in brain development and/or function have been found in 12 autosomes and one sex chromosome (X). We also describe five SNVs in X-linked genes inherited from a healthy mother, arguing for the X-linked recessive inheritance hypothesis. Moreover, ATP1A3 (19q13.2) is the only gene carrying more than one SNV in more than one patient, making it a strong candidate for COS. Mutations were distributed in various chromosomes illustrating the genetic heterogeneity of COS. More than 90% of CNVs involved in COS are also involved in ASD, supporting the idea that there may be genetic overlap between these disorders. Different mutations associated with COS are probably still unknown, and pathogenesis might also be explained by the association of different genetic variations (two or more CNVs or CNVs and SNVs) as well as association with early acquired brain lesions such as infection, hypoxia, or early childhood trauma.

Reduction of Fmr1 mRNA Levels Rescues Pathological Features in Cortical Neurons in a Model of FXTAS.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a rare disorder associated to the presence of the fragile X premutation, a 55-200 CGG repeat expansion in the 5' UTR of the FMR1 gene. Two main neurological phenotypes have been described in carriers of the CGG premutation: (1) neurodevelopmental disorders characterized by anxiety, attention deficit hyperactivity disorder (ADHD), social deficits, or autism spectrum disorder (ASD); and (2) after 50 years old, the FXTAS phenotype. This neurodegenerative disorder is characterized by ataxia and a form of parkinsonism. The molecular pathology of this disorder is characterized by the presence of elevated levels of Fragile X Mental Retardation 1 (FMR1) mRNA, presence of a repeat-associated non-AUG (RAN) translated peptide, and FMR1 mRNA-containing nuclear inclusions. Whereas in the past FXTAS was mainly considered as a late-onset disorder, some phenotypes of patients and altered learning and memory behavior of a mouse model of FXTAS suggested that this disorder involves neurodevelopment. To better understand the physiopathological role of the increased levels of Fmr1 mRNA during neuronal differentiation, we used a small interfering RNA (siRNA) approach to reduce the abundance of this mRNA in cultured cortical neurons from the FXTAS mouse model. Morphological alterations of neurons were rescued by this approach. This cellular phenotype is associated to differentially expressed proteins that we identified by mass spectrometry analysis. Interestingly, phenotype rescue is also associated to the rescue of the abundance of 29 proteins that are involved in various pathways, which represent putative targets for early therapeutic approaches.

Modeling Fragile X Syndrome in Drosophila.

Intellectual disability (ID) and autism are hallmarks of Fragile X Syndrome (FXS), a hereditary neurodevelopmental disorder. The gene responsible for FXS is Fragile X Mental Retardation gene 1 (FMR1) encoding the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in RNA metabolism and modulating the expression level of many targets. Most cases of FXS are caused by silencing of FMR1 due to CGG expansions in the 5'-UTR of the gene. Humans also carry the FXR1 and FXR2 paralogs of FMR1 while flies have only one FMR1 gene, here called dFMR1, sharing the same level of sequence homology with all three human genes, but functionally most similar to FMR1. This enables a much easier approach for FMR1 genetic studies. Drosophila has been widely used to investigate FMR1 functions at genetic, cellular, and molecular levels since dFMR1 mutants have many phenotypes in common with the wide spectrum of FMR1 functions that underlay the disease. In this review, we present very recent Drosophila studies investigating FMRP functions at genetic, cellular, molecular, and electrophysiological levels in addition to research on pharmacological treatments in the fly model. These studies have the potential to aid the discovery of pharmacological therapies for FXS.

Modeling Fragile X syndrome in neurogenesis: An unexpected phenotype and a novel tool for future therapies.

FMRP is an RNA-binding protein involved in synaptic translation. Its absence causes a form of intellectual disability, the Fragile X syndrome (FXS). Small neuroanatomical abnormalities, present both in human and mouse FMRP-deficient brains, suggest a subtle critical role of this protein in neurogenesis. Stable depletion of FMRP has been obtained in a mouse embryonic stem cell line Fmr1 (shFmr1 ES) that does not display morphological alterations, but an abnormal expression of a subset of genes mainly involved in neuronal differentiation and maturation. Inducing the differentiation of shFmr1 ES cells into the neuronal lineage results in an accelerated generation of neural progenitors and neurons during the first steps of neurogenesis. This transient phenotype is due to an elevated level of the Amyloid Precursor Protein (APP), whose mRNA is a target of FMRP. APP is processed by the BACE-1 enzyme, producing the ß-amyloid (Aß) peptide accelerating neurogenesis by activating the expression of Ascll. Inhibition of the BACE-1 enzyme rescues the phenotype of shFmr1 ES cells. Here we discuss the importance of the shFmr1 ES line not only to understand the physiopathology of FXS but also as a tool to screen biomolecules for new FXS therapies.

A new paramutation-like example at the Delta gene of Drosophila.

The hereditary transmission of a phenotype independent from DNA sequence implies epigenetic effects. Paramutation is a heritable epigenetic phenomenon observed in plants and animals. To investigate paramutation in Drosophila, we used the P{ry+t7.2 = PZ}Dl05151 P-element insertion in the Drosophila melanogaster genome that causes a dominant visible phenotype: the presence of characteristic extra-veins in the fly wings. This extra-vein phenotype presents variable expressivity and incomplete penetrance. The insert is a PZ element located 680 bp upstream from the ATG of the Delta (Dl) gene, encoding the Notch ligand involved in wing vein development, and acts as a null allele. In the G2 offspring from a cross between the heterozygous transgenic stock and wild-type flies, we observed the transmission of the extra-vein phenotype to wild-type flies without the transgene, independently of gender and across many generations. This is a "paramutation-like" example in the fly: the heritable transmission of a phenotypic change not linked to a classical genetic mutation. A "paramutagenic" allele in heterozygotes transmits the phenotype of the heterozygotes to the wild-type allele ("paramutant") in a stable manner through generations. Distinct from paramutation events so far described in Drosophila, here we deal with a dominant effect on a single gene involving variable hereditary signals.

Trans-nonachlor decreases miR-141-3p levels in human melanocytes in vitro promoting melanoma cell characteristics and shows a multigenerational impact on miR-8 levels in Drosophila.

Epidemiological association studies have revealed a role for pesticides in cancer occurrence, while a growing number of reports have highlighted the deleterious epigenetic modifications that can be produced by environmental factors. However, epidemiological data currently lack molecular support to unravel the epigenetic impact of pesticides on carcinogenesis. Based on epidemiological studies of melanoma, our data show for the first time that trans-nonachlor (TNC), a component of the pesticide chlordane, modulates the microRNA miR-141-3p in human melanocytic cells in vitro, with effects on melanomagenesis parameters. TNC downregulates the level of miR-141-3p in normal melanocytes to levels found endogenously in several melanoma cell lines. Ectopic expression of either a synthetic miRNA mimic or inhibitor in human melanocytic cells revealed that TNC counteracts the inhibitory effects of miR-141-3p on melanoma cell anchorage-independent growth ability, their invasive potential, and expression of a multipotent, embryonic-like, aggressive cancer phenotype (termed vasculogenic mimicry), involving VE-Cadherin. In addition, the data suggest that miR-141-3p regulates vasculogenic mimicry through extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol-3-kinase (PI3K). Notably, in the Drosophila animal model, TNC also decreased the level of miR-8, the sole miR-141-3p fly ortholog. Importantly, the downregulation of miR-8 levels induced by TNC in ancestors was transmitted through multigenerations, with a progressive reversion over time. Such a decrease in miR-8 levels translated to a loss-of-weight phenotype in offspring. Providing support to epidemiological data, these results altogether suggest that TNC may favor melanomagenesis by lowering the levels of miR-141-3p, thereby activating melanoma cell processes. Although any such conclusions in humans are yet to be determined, these experiments in Drosophila demonstrate that TNC can promote an epigenetic multigenerational inheritance of the miR-141-3p/miR-8 defect. This study therefore justifies the development of molecular investigations to decipher the toxic epigenetic heritable impact of pesticides on cancer occurrence.

Functional Gustatory Role of Chemoreceptors in Drosophila Wings.

Neuroanatomical evidence argues for the presence of taste sensilla in Drosophila wings; however, the taste physiology of insect wings remains hypothetical, and a comprehensive link to mechanical functions, such as flight, wing flapping, and grooming, is lacking. Our data show that the sensilla of the Drosophila anterior wing margin respond to both sweet and bitter molecules through an increase in cytosolic Ca(2+) levels. Conversely, genetically modified flies presenting a wing-specific reduction in chemosensory cells show severe defects in both wing taste signaling and the exploratory guidance associated with chemodetection. In Drosophila, the chemodetection machinery includes mechanical grooming, which facilitates the contact between tastants and wing chemoreceptors, and the vibrations of flapping wings that nebulize volatile molecules as carboxylic acids. Together, these data demonstrate that the Drosophila wing chemosensory sensilla are a functional taste organ and that they may have a role in the exploration of ecological niches.

Nano-architecture of gustatory chemosensory bristles and trachea in Drosophila wings.

In the Drosophila wing anterior margin, the dendrites of gustatory neurons occupy the interior of thin and long bristles that present tiny pores at their extremities. Many attempts to measure ligand-evoked currents in insect wing gustatory neurons have been unsuccessful for technical reasons. The functions of this gustatory activity therefore remain elusive and controversial. To advance our knowledge on this understudied tissue, we investigated the architecture of the wing chemosensory bristles and wing trachea using Raman spectroscopy and fluorescence microscopy. We hypothesized that the wing gustatory hair, an open-ended capillary tube, and the wing trachea constitute biological systems similar to nano-porous materials. We present evidence that argues in favour of the existence of a layer or a bubble of air beneath the pore inside the gustatory hair. We demonstrate that these hollow hairs and wing tracheal tubes fulfil conditions for which the physics of fluids applied to open-ended capillaries and porous materials are relevant. We also document that the wing gustatory hair and tracheal architectures are capable of trapping volatile molecules from the environment, which might increase the efficiency of their spatial detection by way of wing vibrations or during flight.

Tissue and stage-specific expression of the Tolls in Drosophila embryos.

The Drosophila transmembrane receptor Toll plays a key role in specifying the dorsoventral axis of the embryo. At later stages of development, it controls the immune response of the fly to fungal and Gram-positive bacterial infections. The Drosophila genome has a total of nine Toll-like genes, including the previously characterized Toll (Toll-1) and 18-wheeler (Toll-2). Here we describe the embryonic expression patterns of the seven Toll-like genes Toll-3 through Toll-9. We find that these genes have distinct expression domains and that their expression is dynamically changing throughout embryonic development. This complex and tissue-specific regulation of Toll-like gene expression strongly suggests a role in embryonic development for most Drosophila Tolls. The evolving picture on the Toll family members in Drosophila contrasts with that of mammalian Toll-like receptors, which are predominantly expressed in immune responsive cells where their activation occurs via microbial structural determinants.

graal: a Drosophila gene coding for several mosaic serine proteases.

Serine proteases play vital roles in several biological processes such as development and immunity. We have characterized Graal, a large multi-domain serine protease from Drosophila. Graal is spliced in at least three transcripts that are present throughout development. The domains found in Graal proteins are: chitin-binding domains (CBD), scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein receptor cysteine-rich (LDLR-CR) domains, histidine and proline-rich domains, a NGGYQPP-repeat domain and a serine protease domain. The last 2370 nucleotides of these RNAs are identical and encode a His-rich domain, two SRCR domains, two LDLR-CR domains and a protease domain. The transcription of graal is upregulated after fungal or bacterial infection. Analysis of the Iso1 (y;cn,sp,bw) strain shows that graal transcription is impaired in this fly line due to the insertion of a retrotransposon in the sixth exon. However, no phenotype could be observed consecutive to the absence of graal full length transcripts, particularly in the context of an immune challenge.

Ndae1 expression and regulation in Drosophila embryos.

The construction and prediction of cell fate maps at the whole embryo level require the establishment of an accurate atlas of gene expression patterns throughout development and the identification of the corresponding cis-regulatory sequences. However, while the expression and regulation of genes encoding upstream developmental regulators such as transcription factors or signaling pathway components have been analyzed in detail, up to date the number of cis-regulatory sequences identified for downstream effector genes, like ion channels, pumps and exchangers, is very low. The control and regulation of ion homeostasis in each cell, including at blastoderm stages, are essential for normal embryonic development. In this study, we analyzed in detail the embryonic expression pattern and cis-regulatory modules of the Drosophila Na+-driven anion exchanger 1 (Ndae1) gene, involved in the regulation of pH homeostasis. We show that Ndae1 is expressed in a tight and complex spatial-temporal pattern. In particular, we report that this downstream effector gene is under the control of the canonical dorsal-ventral patterning cascade through dorsal, Toll, twist and snail at early embryogenesis. Moreover, we identify several cis-regulatory modules, some of which control discrete and non-overlapping aspects of endogenous gene expression throughout development.

Environmentally selected aphid variants in clonality context display differential patterns of methylation in the genome.

Heritability of acquired phenotypic traits is an adaptive evolutionary process that appears more complex than the basic allele selection guided by environmental pressure. In insects, the trans-generational transmission of epigenetic marks in clonal and/or sexual species is poorly documented. Aphids were used as a model to explore this feature because their asexual phase generates a stochastic and/or environment-oriented repertoire of variants. The a priori unchanged genome in clonal individuals prompts us to hypothesize whether covalent methyl DNA marks might be associated to the phenotypic variability and fitness selection. The full differential transcriptome between two environmentally selected clonal variants that originated from the same founder mother was mapped on the entire genomic scaffolds, in parallel with the methyl cytosine distribution. Data suggest that the assortments of heavily methylated DNA sites are distinct in these two clonal phenotypes. This might constitute an epigenetic mechanism that confers the robust adaptation of insect species to various environments involving clonal reproduction.

Light- induced electron transfer and ATP synthesis in a carotene synthesizing insect.

A singular adaptive phenotype of a parthenogenetic insect species (Acyrthosiphon pisum) was selected in cold conditions and is characterized by a remarkable apparition of a greenish colour. The aphid pigments involve carotenoid genes well defined in chloroplasts and cyanobacteria and amazingly present in the aphid genome, likely by lateral transfer during evolution. The abundant carotenoid synthesis in aphids suggests strongly that a major and unknown physiological role is related to these compounds beyond their canonical anti-oxidant properties. We report here that the capture of light energy in living aphids results in the photo induced electron transfer from excited chromophores to acceptor molecules. The redox potentials of molecules involved in this process would be compatible with the reduction of the NAD+ coenzyme. This appears as an archaic photosynthetic system consisting of photo-emitted electrons that are in fine funnelled into the mitochondrial reducing power in order to synthesize ATP molecules.