Drosophila as a model system for genetic and genomic research

The sequencing of the Drosophila genome allowed the identification of most coding sequences, highlighting the necessity for a functional assignation of the identified genes. The information extracted from the sequence directly classified a considerable fraction of genes into known molecular categories, although there is still a large proportion of them that, due to poor sequence conservation, are not included into any informative class. Furthermore, in many instances the molecular nature of a protein is not particularly revealing about its functional requirements and network of interactions. In this manner, complementary genomic approaches to gene identification by sequence conservation are fundamental both in Drosophila and other organisms to assign particular functions to annotated genes. The approach more successful in the Drosophila field is the undertaking of genetic screenings to identify sets of interacting genes and genes controlling particular cellular processes. Classic genetic screens comprise all those based on a “phenotypic” paradigm, where the generation of large collections of mutant chromosomes is followed by their mapping. This approach has been recently expanded to include “genomic” tools, such as the use of microarrays and interference RNA, as well as reverse-genetics techniques, seeding the way to a “functional” annotation of the Drosophila genome.Peer reviewe

A Drosophila model of myeloproliferative neoplasm reveals a feed-forward loop in the JAK pathway mediated by p38 MAPK signalling

Myeloproliferative neoplasms (MPNs) of the Philadelphia-negative class comprise polycythaemia vera, essential thrombocythaemia and primary myelofibrosis (PMF). They are associated with aberrant numbers of myeloid lineage cells in the blood, and in the case of overt PMF, with development of myelofibrosis in the bone marrow and failure to produce normal blood cells. These diseases are usually caused by gain-of-function mutations in the kinase JAK2. Here, we use Drosophila to investigate the consequences of activation of the JAK2 orthologue in haematopoiesis. We have identified maturing haemocytes in the lymph gland, the major haematopoietic organ in the fly, as the cell population susceptible to induce hypertrophy upon targeted overexpression of JAK. We show that JAK activates a feed-forward loop, including the cytokine-like ligand Upd3 and its receptor, Domeless, which are required to induce lymph gland hypertrophy. Moreover, we present evidence that p38 MAPK signalling plays a key role in this process by inducing expression of the ligand Upd3. Interestingly, we also show that forced activation of the p38 MAPK pathway in maturing haemocytes suffices to generate hypertrophic organs and the appearance of melanotic tumours. Our results illustrate a novel pro-tumourigenic crosstalk between the p38 MAPK pathway and JAK signalling in a Drosophila model of MPNs. Based on the shared molecular mechanisms underlying MPNs in flies and humans, the interplay between Drosophila JAK and p38 signalling pathways unravelled in this work might have translational relevance for human MPNs

Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling

De novo mutations in CHD8 are strongly associated with autism spectrum disorder, but the basic biology of CHD8 remains poorly understood. Here we report that Chd8 knockdown during cortical development results in defective neural progenitor proliferation and differentiation that ultimately manifests in abnormal neuronal morphology and behaviors in adult mice. Transcriptome analysis revealed that while Chd8 stimulates the transcription of cell cycle genes, it also precludes the induction of neural-specific genes by regulating the expression of PRC2 complex components. Furthermore, knockdown of Chd8 disrupts the expression of key transducers of Wnt signaling, and enhancing Wnt signaling rescues the transcriptional and behavioral deficits caused by Chd8 knockdown. We propose that these roles of Chd8 and the dynamics of Chd8 expression during development help negotiate the fine balance between neural progenitor proliferation and differentiation. Together, these observations provide new insights into the neurodevelopmental role of Chd8.National Institutes of Health (U.S.) (Grant UH1-MH106018-03

Búsqueda y análisis de genes candidatos a regular la expresión génica en respuesta a la ruta de señalización EGRF/RAS en el ala de Drosophila Melanogaster

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-06-2008The development of the Drosophila melanogaster wing is a model system to analyse the genetic control of organ size, shape and pattern. Many known genes participate in a variety of processes, including cell survival, growth, proliferation and differentiation, which govern the generation of the adult wing. In this work we pursuit two main objectives, to identify new genes participating in wing development and to analyse the contribution of chromatin remodelling in the regulation of gene expression in response to EGFR signalling. In the first part we discuss the generation, mapping and functional analysis of new mutations induced in the 2L chromosomic arm which in homozygosis result in a wing phenotype affecting its size, margin and the pattern of veins. We have obtained 12 complementation groups corresponding to genes with a proven role in wing development such as smoothened, thick veins, Mothers against dpp, expanded and fat, and 73 new complementation groups represented by at least one allele. We studied in more detail a novel complementation group corresponding to the gene med15, a Mediator Complex member, and aimed to elucidate the role of Med15 in the network of gene expression regulation controlling the development of the veins. In the second part we analyse the functional requirements of Osa, a component of the SWI/SNF chromatin-remodelling complex, during Drosophila wing development. We found that osa is needed for imaginal cells growth and survival, and for the correct patterning of sensory organs and veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one key aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling result in similar wing vein phenotypes, and Osa is required for the expression of several nuclear targets of EGFR signalling, such as Delta, rhomboid and argos. We suggest that Osa facilitates the transcriptional responses to EGFR signalling in the wing, likely after EGFR-specific transcription factors are attached to their promoters, making the regulatory regions of EGFR target genes stably activated. The function of Osa is also shared by other members of the BAP complex, such as Snr1, Bap55, Mor and Brm, indicating that chromatin remodelling is a key component of transcriptional regulation in response to EGFR signalling

Análisis funcional del gen nab en el desarrollo de drosophila melanogaster

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-12-2006Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA binding domains and act by associating with other transcription factors, but their precise roles in development are not known. In this project we analyze the role of Nab in Drosophila development. By employing genetic and molecular approaches we found that nab is required for proximodistal patterning of the wing imaginal discs and also for determining specific neuronal fates in the embryonic Central Nervous System. We identified two targets of Nab: the products of the zinc finger- encoding genes rotund and squeeze. Both gene products belong to the Kruppel family, as the vertebrate partners of Nab: the EGR transcription factors. Nab is co-expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. Our results indicate that Nab acts as a co- activator of Squeeze, so is required to limit the number of neurons that express the LIM-homeodomain gene apterous, and to specify the Tv neuronal fate through the activation of the FMRFa neuropeptide. Conversely, Nab act as a co-repressor of Rotund in wing development, and is required to limit the expression of the gene wingless/WNT in the wing hinge, where Wg plays a mitogenic role. We demonstrate the requirement of vg in the activation of nab in the wing disk, and the limitation of its expression by zfh2. We show by pull-down assays that Nab binds directly to Rotund and Squeeze via their conserved C-terminal domain. Going further in the molecular mechanism underling the repression of the targets of Rn by Nab, we show preliminary results that involve the general corepressor dCtBP as a possible partner of NabPeer reviewe

The role of tarsal-less in Drosophila wing imaginal disc development

Trabajo presentado en el 1st PhD and Postdoc Symposium (From Cells to Organs), celebrado en Barcelona el 16 y 17 de abril de 2012.N

A Drosophila \textit{Drosophila } model of myeloproliferative neoplasm reveals a feed-forward loop in the JAK pathway mediated by p38 MAPK signalling.

Myeloproliferative neoplasms (MPNs) of the Philadelphia-negative class comprise polycythaemia vera, essential thrombocythaemia and primary myelofibrosis (PMF). They are associated with aberrant numbers of myeloid lineage cells in the blood, and in the case of overt PMF, with development of myelofibrosis in the bone marrow and failure to produce normal blood cells. These diseases are usually caused by gain-of-function mutations in the kinase JAK2. Here, we use Drosophila to investigate the consequences of activation of the JAK2 orthologue in haematopoiesis. We have identified maturing haemocytes in the lymph gland, the major haematopoietic organ in the fly, as the cell population susceptible to induce hypertrophy upon targeted overexpression of JAK. We show that JAK activates a feed-forward loop, including the cytokine-like ligand Upd3 and its receptor, Domeless, which are required to induce lymph gland hypertrophy. Moreover, we present evidence that p38 MAPK signalling plays a key role in this process by inducing expression of the ligand Upd3. Interestingly, we also show that forced activation of the p38 MAPK pathway in maturing haemocytes suffices to generate hypertrophic organs and the appearance of melanotic tumours. Our results illustrate a novel pro-tumourigenic crosstalk between the p38 MAPK pathway and JAK signalling in a Drosophila model of MPNs. Based on the shared molecular mechanisms underlying MPNs in flies and humans, the interplay between Drosophila JAK and p38 signalling pathways unravelled in this work might have translational relevance for human MPNs.This work was supported by grants from the European Commission [ERC 294665] and Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) [2014 SRG-535] to A.R.N.; Ministerio de Economía y Competitividad (MINECO) [Government of Spain, SIGNAGROWTH-BFU2013-44485 and INTERGROWTH-BFU2016-77587-P] and FEDER ‘Una manera de hacer Europa’ to M.M.; the European Union Seventh Framework Programme [FP7/Marie Curie-Skłodowska Actions/COFUND/IRBPostPro 2.0 2013] and the European Molecular Biology Organization (EMBO) [ASTF 369-2016] to A.T.-F

Identification of Genes Affecting Wing Patterning Through a Loss-of-Function Mutagenesis Screen and Characterization of med15 Function During Wing Development

The development of the Drosophila melanogaster wing depends on the correct regulation of cell survival, growth, proliferation, differentiation, and pattern formation. These processes, and the genes controlling then, are common to the development of epithelia in many different organisms. To identify additional genes contributing to wing development we have carried out a genetic screen in mosaic wings carrying clones of homozygous mutant cells. We obtained 12 complementation groups corresponding to genes with a proven role in wing formation such as smoothened, thick veins, mothers against dpp, expanded, and fat and 71 new complementation groups affecting the pattern of veins and the size of wing. We mapped one of these groups to the mediator15 gene (med15), a component of the Mediator complex. We show that Med15 and other members of the Mediator complex are required, among other processes, for the transcription of decapentaplegic target genes

A conserved function of the chromatin ATPase Kismet in the regulation of hedgehog expression

11 páginas, 7 figuras.The development of the Drosophila melanogaster wing depends on its subdivision into anterior and posterior compartments, which constitute two independent cell lineages since their origin in the embryonic ectoderm. The anterior–posterior compartment boundary is the place where signaling by the Hedgehog pathway takes place, and this requires pathway activation in anterior cells by ligand expressed exclusively in posterior cells. Several mechanisms ensure the confinement of hedgehog expression to posterior cells, including repression by Cubitus interruptus, the co-repressor Groucho and Master of thick veins. In this work we identified Kismet, a chromodomain-containing protein of the SNF2-like family of ATPases, as a novel component of the hedgehog transcriptional repression mechanism in anterior compartment cells. In kismet mutants, hedgehog is ectopically expressed in a domain of anterior cells close to the anterior–posterior compartment boundary, causing inappropriate activation of the pathway and changes in the development of the central region of the wing. The contribution of Kismet to the silencing of hedgehog expression is limited to anterior cells with low levels of the repressor form of Cubitus interruptus. We also show that knockdown of CHD8, the kismet homolog in Xenopus tropicalis, is also associated with ectopic sonic hedgehog expression and up-regulation of one of its target genes in the eye, Pax2, indicating the evolutionary conservation of Kismet/CHD8 function in negatively controlling hedgehog expression.We acknowledge grants BFU2009-09403 to J.F.dC; BFU2007-60042/BMC, Petri PET2007_0158, Proyecto de Excelencia CVI-3488 to J-L.G-S; CSD2007-00008 to J.F.dC. and JL.G-S; and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa”.Peer reviewe