Time filter

Source Type

Garcia-Alcover I.,Valentia Biopharma | Garcia-Alcover I.,University of Valencia | Colonques-Bellmunt J.,Valentia Biopharma | Garijo R.,Valentia Biopharma | And 9 more authors.
DMM Disease Models and Mechanisms | Year: 2014

Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1-MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery. © 2014. Published by The Company of Biologists Ltd

Iyer J.,The Pennsylvania State University | Wang Q.,The Pennsylvania State University | Le T.,The Pennsylvania State University | Pizzo L.,The Pennsylvania State University | And 15 more authors.
G3: Genes, Genomes, Genetics | Year: 2016

About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions. © 2016 Iyer et al.

Bargiela A.,University of Valencia | Bargiela A.,INCLIVA Health Research Institute | Cerro-Herreros E.,University of Valencia | Cerro-Herreros E.,INCLIVA Health Research Institute | And 8 more authors.
DMM Disease Models and Mechanisms | Year: 2015

Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1) disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor) or muscleblind, or by RNA interference (RNAi)-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis. © 2015. Published by The Company of Biologists Ltd.

Llamusi B.,University of Valencia | Llamusi B.,INCLIVA Health Research Institute | Munoz-Soriano V.,University of Valencia | Paricio N.,University of Valencia | And 2 more authors.
Biochemistry and Molecular Biology Education | Year: 2014

In situ hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the experiments, students analyzed the repressive effect of Snail over rhomboid expression using reporter lines containing different constructs of the rhomboid neuroectodermal enhancer fused to the lacZ gene. In the second experiment, the epistatic relationship between the cabut and decapentaplegic genes was analyzed. These simple experiments allowed students to (1) understand the role of transcription factors and cis-regulatory elements over gene expression regulation and (2) practice a widespread laboratory technique, in situ hybridization with nonradioactive labeled probes, to detect gene expression patterns. These experiments required 12 hr and were organized into four daily sessions that included the discussion of the results with students. Examples of the results obtained and their relevance are shown and discussed herein. The methods described in these laboratory exercises can be easily adapted to model organisms other than Drosophila. © 2014 The International Union of Biochemistry and Molecular Biology.

Bargiela A.,University of Valencia | Bargiela A.,INCLIVA Health Research Institute | Llamusi B.,University of Valencia | Llamusi B.,INCLIVA Health Research Institute | And 4 more authors.
PLoS ONE | Year: 2014

The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene. © 2014 Bargiela et al.

Discover hidden collaborations