A Transient Expression of Prospero Promotes Cell Cycle Exit of Drosophila Postembryonic Neurons through the Regulation of Dacapo

Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs) divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC) which divides only once to generate two postmitotic cells called ganglion cells (GCs) that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor PROSPERO is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that PROSPERO is not expressed in postmitotic neuronal progeny. Thus, PROSPERO is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. Here we focus on postembryonic neurogenesis and show that the expression of PROSPERO is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, we provide evidence that this expression of PROSPERO in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI) DACAPO. These findings imply that PROSPERO, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development

(CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCUG)16, in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG)106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. Our data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening

Segregation of postembryonic neuronal and glial lineages inferred from a mosaic analysis of the Drosophila larval brain

Due to its intermediate complexity and its sophisticated genetic tools, the larval brain of Drosophila is a useful experimental system to study the mechanisms that control the generation of cell diversity in the CNS. In order to gain insight into the neuronal and glial lineage specificity of neural progenitor cells during postembryonic brain development, we have carried an extensive mosaic analysis throughout larval brain development. In contrast to embryonic CNS development, we have found that most postembryonic neurons and glial cells of the optic lobe and central brain originate from segregated progenitors. Our analysis also provides relevant information about the origin and proliferation patterns of several postembryonic lineages such as the superficial glia and the medial-anterior Medulla neuropile glia. Additionally, we have studied the spatio-temporal relationship between gcm expression and gliogenesis. We found that gcm expression is restricted to the post-mitotic cells of a few neuronal and glial lineages and it is mostly absent from postembryonic progenitors. Thus, in contrast to its major gliogenic role in the embryo, the function of gcm during postembryonic brain development seems to have evolved to the specification and differentiation of certain neuronal and glial lineages.This work was supported by grants from the Spanish Ministry of Science and Technology, the Spanish Ministry of Education and Science, and the Generalitat Valenciana to F.J.T. J. Colonques and J. Ceron were recipients of predoctoral fellowships from the Spanish Ministry of Science and Technology.Peer reviewe

Molecular basis of Down syndrome’s neuropathologies: implication of the Minibraingene

[ES] El síndrome de Down (SD) genera un amplio número de anomalías, de las cuales las más graves son las neuropatologías que hacen del SD la causa más frecuente de retraso mental. El SD se debe a la triplicación del cromosoma 21. En base a estudios genéticos y a la secuenciación de este cromosoma se han podido identificar los genes posiblemente más relevantes para la generación del SD, entre los cuales destaca Minibrain (Mnb). El objetivo de este trabajo ha sido generar modelos experimentales in vivopara estudiar si las bases moleculares de las neuropatologías asociadas con el SD podían resistir en la alteración del gen Mnb.Con este fin, y para poder utilizar embriones de pollo como modelo experimental, se clonó el ortólogo de Mnben pollo. Estudios de expresión de Mnbjunto con experimentos de sobreexpresión llevados a cabo en transgénicos, sugieren que este gen está implicado en diversas funciones como proliferación y diferenciación a lo largo del desarrollo del cerebro.[EN] Down’s syndrome (DS) generates a large number of abnormalities. Neuropathologies are among the most severe abnormalities and make DS the most frequent cause of mental retardation. DS is originated by a triplication of chromosome 21. Based on genetic studies and the sequencing of chromosome 21, genes, which localize at the DS critical region have been identified. Among these genes, Minibrain (Mnb) appears as the most likely candidate to explain some DS neuropathologies. The goal of this work has been to generate in vivoexperimental models to study the molecular basis of the possible involvement of Mnbon DS neuropathologies. In order to use chick embryos as an experimental model, the chick orthologue of Mnbhas been cloned. Studies of Mnbexpression together with overexpression experiments carried out on transgenics, suggest that this gene is involved in different functions like proliferation and differentation along brain development.Trabajo financiado con una Ayuda a la Investigación de la Fundación MAPFRE Medicina.Peer reviewe

A new hypothesis for the neuronal deficit and the alterations in neuronal differentiation associated to Down syndrome: implication of the Minibrain gene

[ES] La disminución de neuronas, diversos defectos en la diferenciación neuronal y la aparición de síntomas neurodegenerativos están entre las alteraciones neuropatológicas que hacen del síndrome de Down (SD) la causa más frecuente de retraso mental. El SD se debe a la triplicación del cromosoma 21. En base a estudios genéticos y a la secuenciación de este cromosoma se han podido identificar los genes posiblemente más relevantes para la generación del SD, entre los cuales destaca Minibrain (Mnb). Dos han sido los objetivos de este trabajo: estudiar si Mnb podría estar implicado en la diferenciación neuronal y ver si la sobreexpresión de Mnb tiene efectos sobre muerte neuronal. Paralelamente se han intentado ver las relaciones de estas funciones del gen Mnb con las neuropatologías asociadas al SD. Experimentos llevados a cabo en modelos experimentales transgénicos demuestran que la sobreexpresión de Mnb genera muerte neuronal. Asimismo, los estudios de expresión de Mnb durante el desarrollo tardío del cerebro sugieren un papel de las Mnb-quinasas como elemento de señalización celular en el proceso de diferenciación neuronal. Todo ello contribuye a confeccionar una nueva hipótesis sobre las bases moleculares del déficit neuronal y las alteraciones de la diferenciación neuronal que se producen en el SD.[EN] The decrease of neuronal number, diverse defects in neuronal differentiation, and neurodegeneration are among the neuropathologic alterations which make DS the most frequent cause of mental retardation. DS is originated by triplication of chromosome 21. Based on genetic studies and the sequencing of chromosome 21, the possible most relevant genes for DS generation have been identified. Among them Minibrain (Mnb) appears the most likely candidate to explain some DS neuropathologies. Our work has approached two objectives: to study if Mnb could be involved in neuronal differentiation and find out if the overexpression of Mnb has an effect on cell death. In parallel, we have tried to establish the correlation of these functions of Mnb with the DS associated neuropathologies. By using transgenic experimental models, we have found that overexpression of Mnb induces neuronal death. Also, the expression of Mnb during late brain development suggests a role of Mnb-kinases as an important signaling element within the pro- cess of neuronal differentiation. All together, these results contribute to build a new hypothesis for the molecular basis of the neuronal deficit and alterations of neuronal differentiation associated to DS.Trabajo financiado con una Ayuda a la Investigación de la Fundación MAPFRE Medicina.Peer reviewe

Minibrain drives the Dacapo-dependent cell cycle exit of neurons in the Drosophila brain by promoting asense and prospero expression

A key aim of neurodevelopmental research is to understand how precursor cells decide to stop dividing and commence their terminal differentiation at the correct time and place. Here, we show that minibrain (mnb), the Drosophila ortholog of the Down syndrome candidate gene DYRK1A, is transiently expressed in newborn neuronal precursors known as ganglion cells (GCs). Mnb promotes the cell cycle exit of GCs through a dual mechanism that regulates the expression of the cyclin-dependent kinase inhibitor Dacapo, the homolog of vertebrate p27(Kip1) (Cdkn1b). Mnb upregulates the expression of the proneural transcription factor (TF) Asense, which promotes Dacapo expression. Mnb also induces the expression of Prospero, a homeodomain TF that in turn inhibits the expression of Deadpan, a pan-neural TF that represses dacapo In addition to its effects on Asense and Prospero, Mnb also promotes the expression of the neuronal-specific RNA regulator Elav, strongly suggesting that Mnb facilitates neuronal differentiation. These actions of Mnb ensure the precise timing of neuronal birth, coupling the mechanisms that regulate neurogenesis, cell cycle control and terminal differentiation of neurons.This work was supported by grants from the Dirección General de Investigación Científica y Técnica [BFU2009-08831, BFU2012-38892 to F.J.T.]; the Generalitat Valenciana [ACOMP/2012/047 to F.J.T.]; and the Fundación Inocente Inocente [050666100002 to F.J.T.]. J. Ceron and J. Colonques were recipients of PhD fellowships from the Ministerio de Educación y Ciencia. M.N.S. was recipient of a Santiago Grisolia Fellowship from the Generalitat Valenciana.Peer reviewe

Natural Compound Boldine Lessens Myotonic Dystrophy Type 1 Phenotypes in DM1 Drosophila Models, Patient-Derived Cell Lines, and HSA^LR Mice

Myotonic dystrophy type 1 (DM1) is a complex rare disorder characterized by progressive muscle dysfunction, involving weakness, myotonia, and wasting, but also exhibiting additional clinical signs in multiple organs and systems. Central dysregulation, caused by an expansion of a CTG trinucleotide repeat in the DMPK gene’s 3’ UTR, has led to exploring various therapeutic approaches in recent years, a few of which are currently under clinical trial. However, no effective disease-modifying treatments are available yet. In this study, we demonstrate that treatments with boldine, a natural alkaloid identified in a large-scale Drosophila-based pharmacological screening, was able to modify disease phenotypes in several DM1 models. The most significant effects include consistent reduction in nuclear RNA foci, a dynamic molecular hallmark of the disease, and noteworthy anti-myotonic activity. These results position boldine as an attractive new candidate for therapy development in DM1

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1

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

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1

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