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Cardiomyopathy, disease of the heart muscle, is a significant contributor to heart failure. The pathogenesis of cardiomyopathy is multifactorial and involves genetic, environmental, and lifestyle factors. Identifying and characterizing novel genes that contribute to cardiac pathophysiology are crucial for understanding cardiomyopathy and effective therapies. In this study, we investigated the role of a novel gene, Obg-like ATPase 1 ( Ola1 ), in cardiac pathophysiology using a cardiac-specific knockout mouse model as well as a Drosophila model. Our previous work demonstrated that OLA1 modulates the hypertrophic response of cardiomyocytes through the GSK-beta/beta-catenin signaling pathway. Furthermore, recent studies have suggested that OLA1 plays a critical role in organismal growth and development. For example, Ola1 null mice exhibit increased heart size and growth retardation. It is not known, however, if loss of function for Ola1 leads to dilated cardiomyopathy. We generated cardiac-specific Ola1 knockout mice (OLA1-cKO) to evaluate the role of OLA1 in cardiac pathophysiology. We found that Ola1 -cKO in mice leads to dilated cardiomyopathy (DCM) and left ventricular (LV) dysfunction. These mice developed severe LV dilatation, thinning of the LV wall, reduced LV function, and, in some cases, ventricular wall rupture and death. In Drosophila, RNAi-mediated knock-down specifically in developing heart cells led to the change in the structure of pericardial cells from round to elongated, and abnormal heart function. This also caused significant growth reduction and pupal lethality. Thus, our findings suggest that OLA1 is critical for cardiac homeostasis and that its deficiency leads to dilated cardiomyopathy and dysfunction. Furthermore, our study highlights the potential of the Ola1 gene as a therapeutic target for dilated cardiomyopathy and heart failure.
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The T-box (Tbx) proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. They are highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report posttranscriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid-mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla luciferase reporter significantly reduced the activity of the Luciferase, whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity, but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid-mRNA between the 2 transgenes, with and without the 3'UTR, indicating the absence of posttranscriptional regulation of mid in MP2. Moreover, while elevated mid-RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Overexpression of the 2 transgenes resulted in MP2-lineage defects at about the same frequency. These results indicate a translational/posttranslational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid-RNA and the protein had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell- and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the 2 transgenes.
Assuntos
Regiões 3' não Traduzidas , Proteínas de Drosophila , Proteínas com Domínio T , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Estabilidade de RNA/genética , Drosophila melanogaster/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Processamento Pós-Transcricional do RNA , Especificidade de Órgãos , Asas de Animais/metabolismo , Asas de Animais/crescimento & desenvolvimento , Linhagem Celular , Drosophila/genética , Drosophila/metabolismoRESUMO
Replication fork arrest-induced DNA double strand breaks (DSBs) caused by lesions are effectively suppressed in cells due to the presence of a specialized mechanism, commonly referred to as DNA damage tolerance (DDT). In eukaryotic cells, DDT is facilitated through translesion DNA synthesis (TLS) carried out by a set of DNA polymerases known as TLS polymerases. Another parallel mechanism, referred to as homology-directed DDT, is error-free and involves either template switching or fork reversal. The significance of the DDT pathway is well established. Several diseases have been attributed to defects in the TLS pathway, caused either by mutations in the TLS polymerase genes or dysregulation. In the event of a replication fork encountering a DNA lesion, cells switch from high-fidelity replicative polymerases to low-fidelity TLS polymerases, which are associated with genomic instability linked with several human diseases including, cancer. The role of TLS polymerases in chemoresistance has been recognized in recent years. In addition to their roles in the DDT pathway, understanding noncanonical functions of TLS polymerases is also a key to unraveling their importance in maintaining genomic stability. Here we summarize the current understanding of TLS pathway in DDT and its implication for human health.
Assuntos
DDT , Reparo do DNA , Humanos , Replicação do DNA , DNA/genética , Dano ao DNA , Instabilidade GenômicaRESUMO
In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. Here we show that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization.
Assuntos
Proteínas de Drosophila , Células-Tronco Neurais , Animais , Ciclina E/genética , Ciclina E/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/genéticaRESUMO
Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. Here, we show that the T-box transcription factor, Midline (Mid), couples these events. We find that in mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. Our results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.
Assuntos
Divisão Celular/genética , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Ciclo Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Hormônios Juvenis/genética , Hormônios Juvenis/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas com Domínio T/genética , Fatores de Transcrição/genéticaRESUMO
Axon-guidance by Slit-Roundabout (Robo) signaling at the midline initially guides growth cones to synaptic targets and positions longitudinal axon tracts in discrete bundles on either side of the midline. Following the formation of commissural tracts, Slit is found also in tracts of the commissures and longitudinal connectives, the purpose of which is not clear. The Slit protein is processed into a larger N-terminal peptide and a smaller C-terminal peptide. Here, I show that Slit and Slit-N in tracts interact with Robo to maintain the fasciculation, the inter-tract spacing between tracts and their position relative to the midline. Thus, in the absence of Slit in post-guidance tracts, tracts de-fasciculate, merge with one another and shift their position towards the midline. The Slit protein is proposed to function as a gradient. However, I show that Slit and Slit-N are not freely present in the extracellular milieu but associated with the extracellular matrix (ECM) and both interact with Robo1. Slit-C is tightly associated with the ECM requiring collagenase treatment to release it, and it does not interact with Robo1. These results define a role for Slit and Slit-N in tracts for the maintenance and fasciculation of tracts, thus the maintenance of the hardwiring of the CNS.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Axônios/metabolismo , Drosophila/embriologia , Drosophila/genética , Matriz Extracelular/metabolismo , Fasciculação/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Cones de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo , Proteínas RoundaboutRESUMO
Slit proteins act as repulsive axon guidance cues by activating receptors of the Roundabout (Robo) family. During early neurogenesis in Drosophila melanogaster, Slit prevents the growth cones of longitudinal tract neurons from inappropriately crossing the midline, thus restricting these cells to trajectories parallel to the midline. Slit is expressed in midline glial cells, and Robo is present in longitudinal axon tracts and growth cones. We showed that the enzyme Mummy (Mmy) controlled Slit-Robo signaling through mechanisms that affected both the ligand and the receptor. Mmy was required for the glycosylation of Slit, which was essential for Slit secretion. Mmy was also required for maintaining the abundance and spatial distribution of Robo through an indirect mechanism that was independent of Slit secretion. Moreover, secretion of Slit was required to maintain the fasciculation and position of longitudinal axon tracts, thus maintaining the hardwiring of the nervous system. Thus, Mmy is required for Slit secretion and for maintaining Robo abundance and distribution in the developing nervous system in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Cruzamentos Genéticos , Drosophila melanogaster , Glicosilação , Cones de Crescimento/metabolismo , Ligantes , Neuroglia/metabolismo , Neurônios/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Proteínas RoundaboutRESUMO
Asymmetric cell divisions in the central nervous system generate neurons of diverse fates. In Drosophila melanogaster, the protein Numb localizes asymmetrically to dividing neural precursor cells such that only one daughter cell inherits Numb. Numb inhibits Notch signaling in this daughter cell, resulting in a different cell fate from the Notch-induced fate in the other-Numb-negative-daughter cell. Precursor cells undergo asymmetric cytokinesis generating daughter cells of different sizes. I found that inactivation of Notch in fly embryonic neural precursor cells disrupted the asymmetric positioning of the cleavage furrow and produced daughter cells of the same size and fate. Moreover, inactivation of Notch at different times altered the degree of asymmetric Numb localization, such that earlier inactivation of Notch caused symmetric distribution of Numb and later inactivation produced incomplete asymmetric localization of Numb. The extent of asymmetrically localized Numb positively correlated with the degree of asymmetric cytokinesis and the size disparity in daughter cells. Loss of Numb or expression of constitutively active Notch led to premature specification of the precursor cells into the fate of one of the daughter cells. Thus, in addition to its role in the specification of daughter cell fate after division, Notch controls Numb localization in the precursor cells to determine the size and fate of daughter cells. Numb also inhibits Notch signaling in precursor cells to prevent Notch-induced differentiation of the precursor cell, forming an autoregulatory loop.
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Divisão Celular/fisiologia , Proteínas de Drosophila/metabolismo , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Tamanho Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Hormônios Juvenis/genética , Hormônios Juvenis/metabolismo , Células-Tronco Neurais/citologia , Receptores Notch/genéticaRESUMO
Parkinson's disease (PD) is one of the most common neurodegenerative disease characterized by the clinical triad: tremor, akinesia and rigidity. Several studies have suggested that PD patients show disturbances in olfaction at the earliest onset of the disease. The fruit fly Drosophila melanogaster is becoming a powerful model organism to study neurodegenerative diseases. We sought to use this system to explore olfactory dysfunction, if any, in PINK1 mutants, which is a model for PD. PINK1 mutants display many important diagnostic symptoms of the disease such as akinetic motor behavior. In the present study, we describe for the first time, to the best of our knowledge, neurophysiological and neuroanatomical results concerning the olfactory function in PINK1 mutant flies. Electroantennograms were recorded in response to synthetic and natural volatiles (essential oils) from groups of PINK1 mutant adults at three different time points in their life cycle: one from 3-5 day-old flies, from 15-20 and from 27-30 days. The results obtained were compared with the same age-groups of wild type flies. We found that mutant adults showed a decrease in the olfactory response to 1-hexanol, α-pinene and essential oil volatiles. This olfactory response in mutant adults decreased even more as the flies aged. Immunohistological analysis of the antennal lobes in these mutants revealed structural abnormalities, especially in the expression of Bruchpilot protein, a marker for synaptic active zones. The combination of electrophysiological and morphological results suggests that the altered synaptic organization may be due to a neurodegenerative process. Our results indicate that this model can be used as a tool for understanding PD pathogensis and pathophysiology. These results help to explore the potential of using olfaction as a means of monitoring PD progression and developing new treatments.
Assuntos
Percepção Olfatória , Doença de Parkinson/fisiopatologia , Olfato , Animais , Antenas de Artrópodes/metabolismo , Antenas de Artrópodes/patologia , Antenas de Artrópodes/fisiopatologia , Comportamento Animal , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Potenciais Evocados , Regulação da Expressão Gênica , Longevidade/genética , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mutação , Bulbo Olfatório/fisiopatologia , Doença de Parkinson/genética , Terminações Pré-Sinápticas/ultraestrutura , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.
Assuntos
Axônios/fisiologia , Sistema Nervoso Central/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Neurogênese/genética , Proteínas com Domínio T/genética , Animais , Axônios/metabolismo , Padronização Corporal/genética , Linhagem da Célula/genética , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas com Domínio T/metabolismoRESUMO
BACKGROUND: The objective of the study was to determine if community surveys, conducted 3 times over a period of 20 years in a small district of Sardinia (Italy), confirm the increase in depressive disorders reported in the recent literature. METHODS: Three community surveys were carried out on randomized samples of the same Sardinian mining area in 1988, 1998 and 2008. The surveys were conducted using the interview "Present State Examination" in 1988 (depression diagnosed with ICD-IX) and the CIDI-S in 1998 and 2008 (major depression diagnosed with ICD-X). The three surveys produced estimates of one-month prevalence and of lifetime prevalence in 1998 and 2008. RESULTS: Our work found a substantial decrease in depressive disorders from the survey conducted in 1998 to the survey in 2008 using a similar methodology, except in the youngest age group, which showed an increase in the rate. A decrease in the frequency of depressive disorders compared to what was found 20 years ago was also observed. However, in this case the comparison is more problematic because of use of different diagnostic systems. DISCUSSION: The research seems to show a decrease in depressive disorders over the past two decades. While the small population examined makes it difficult to generalize the overall findings, this study suggests that the hypothesis of an increase in the incidence of depressive disorders since the 1980s in western countries, should have exceptions. A complex interaction between socio-economic (mining closure and large migration) and biological factors (possible selective migration) is likely to influence changes in the prevalence of mood disorders. However, due to certain limitations of this study, this hypothesis may be considered from a heuristic perspective.
Assuntos
Depressão/epidemiologia , Transtorno Depressivo Maior/epidemiologia , Migração Humana , Mineração , Adolescente , Adulto , Distribuição por Idade , Idoso , Feminino , Inquéritos Epidemiológicos , Humanos , Incidência , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Relatório de Pesquisa , Distribuição por Sexo , Adulto JovemRESUMO
The Hem/Kette/Nap1 protein is involved in many biological processes. We have recently reported that Hem is required for the normal migration of neurons in the Drosophila embryo. In this paper, we report that Hem regulates the asymmetric division of neural precursor cells. We find that a well-studied Hem/Kette mutant allele produces at least two main, but possibly more, phenotypic classes of mutant embryos, and these phenotypes correlate with variable levels of maternal wild type Hem protein in the developing embryo. While the weaker class exhibits weak axon guidance defect and the mis-migration of neurons, the stronger class causes severe axon guidance defects, mis-migration of neurons and symmetric division of ganglion mother cells (GMC) of the RP2/sib lineage. We also show that the basis for the loss of asymmetric division is due to non-localization of Inscuteable and Numb in GMC-1. A non-asymmetric Numb segregates to both daughter cells of GMC-1, which then prevents Notch signaling from specifying a sib fate. This causes both cells to adopt an RP2 fate. Furthermore, loss of function for Abelson tyrosine kinase also causes loss of asymmetric localization of Inscuteable and Numb and symmetric division of GMC-1, the loss of function for WAVE has a very weakly penetrant loss of asymmetry defect. These results define another role for Hem/Kette/Nap1 in a neural precursor cell during neurogenesis.
Assuntos
Divisão Celular/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Gânglios/citologia , Hormônios Juvenis/metabolismo , Células-Tronco Neurais/citologia , Proteína 1 de Modelagem do Nucleossomo/genética , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Animais , Linhagem da Célula , Drosophila/embriologia , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Gânglios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Receptor Notch1/metabolismoRESUMO
Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm.
Assuntos
Divisão Celular , Sistema Nervoso Central/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Comportamento Animal/fisiologia , Linhagem Celular , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Loci Gênicos/genética , Larva/genética , Larva/fisiologia , Masculino , Mutação , Ovário/citologia , Ovário/embriologia , Ovário/metabolismo , Óvulo/citologia , Óvulo/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Espermatozoides/citologia , Espermatozoides/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. We have previously shown that RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. Here, we show that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at al and fail to send out its axon projection. We report that Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, our data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. We also find that Abelson tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. Our results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl.
Assuntos
Movimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas dos Microfilamentos/metabolismo , Neurônios/citologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Animais , Regulação para Baixo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microfilamentos/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Neurônios/enzimologia , Fenótipo , Transporte ProteicoRESUMO
In the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1âRP2/sib lineage, we found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. We further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. Our results tie Numb and Notch-signaling through a single player, Neur, thus giving us a more complete picture of the events surrounding asymmetric division of precursor cells. We also show that Neur and Numb are interdependent for their asymmetric-localizations.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Hormônios Juvenis/fisiologia , Células-Tronco Neurais/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Divisão Celular , Gânglios/citologiaRESUMO
The tumor suppressor morphogen, Patched (Ptc), has an extensive homology to the Niemann-Pick-C 1 (NPC1) protein. The NPC disease is a paediatric, progressive and fatal neurodegenerative disorder thought to be due to an abnormal accumulation of cholesterol in neurons. Here, we report that patched mutant adults develop a progressive neurodegenerative disease and their brain contains membranous and lamellar inclusions. There is also a significant reduction in the number of synaptic terminals in the brain of the mutant adults. Interestingly, feeding cholesterol to wild type flies generates inclusions in the brain, but does not cause the disease. However, feeding cholesterol to mutant flies increases synaptic connections and suppresses the disease. Our results suggest that sequestration of cholesterol in the mutant brain in the form of membranous material and inclusions affects available pool of cholesterol for cellular functions. This, in turn, negatively affects the synaptic number and contributes to the disease-state. Consistent with this, in ptc mutants there is a reduction in the pool of cholesterol esters, and cholesterol-mediated suppression of the disease accompanies an increase in cholesterol esters. We further show that Ptc does not function directly in this process since gain of function for Hedgehog also induces the same disease with a reduction in the level of cholesterol esters. We believe that loss of function for ptc causes neurodegeneration via two distinct ways: de-repression of genes that interfere with lipid trafficking, and de-repression of genes outside of the lipid trafficking; the functions of both classes of genes ultimately converge on synaptic connections.
Assuntos
Proteínas de Drosophila/genética , Drosophila/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Terminações Pré-Sinápticas/metabolismo , Receptores de Superfície Celular/genética , Animais , Colesterol/metabolismo , Proteínas de Drosophila/metabolismo , Microscopia Eletrônica , Doenças Neurodegenerativas/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
In the Drosophila CNS, combinatorial, interdependent, sequential genetic programs in neuroectodermal (NE) cells, prior to the formation of neuroblasts (NBs), determine the initial identity of NBs. Temporal factors are then sequentially expressed to change the temporal identity. It is unclear at what levels this positional and temporal information integrates to determine progeny cell identity. One idea is that this is a top-down linear process: the identity of a NB determines the identity of its daughter, the ganglion mother cell (GMC), the asymmetric division of the GMC and the fate specification of daughter cells of the GMC. Our results with midline (mid), which encodes a T-box protein, in a typical lineage, NB4-2-->GMC-1-->RP2/sib, suggest that at least part of the process operates in GMCs. That is, a GMC or a neuronal identity need not be determined at the NB or NE level. This is demonstrated by showing that Mid is expressed in a row 5 GMC (M-GMC), but not in its parent NB or NE cell. In mid mutants, M-GMC changes into GMC-1 and generates an RP2 and a sib without affecting the expression of key genes at the NE/NB levels. Expression of Mid in the M-GMC in mid mutants rescues the fate change, indicating that Mid specifies neurons at the GMC level. Moreover, we found a significant plasticity in the temporal window in which a neuronal lineage can develop. Although the extra GMC-1 in mid mutants is born approximately 2 hours later than the bona fide GMC-1, it follows the same developmental pattern as the bona fide GMC-1. Thus, a GMC identity can be independent of parental identity and GMC formation and elaboration need not be strictly time-bound.
Assuntos
Sistema Nervoso Central/embriologia , Drosophila/embriologia , Neurogênese , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , DNA/genética , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epistasia Genética , Genes de Insetos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Mutação , Neurogênese/genética , Neurônios/citologia , Proteínas Nucleares/genética , Fenótipo , Regiões Promotoras Genéticas , Proteínas com Domínio T/genética , Fatores de Tempo , Transativadores/genética , Fatores de Transcrição/metabolismo , Proteína Wnt1/genéticaRESUMO
Neurons and their precursor cells are formed in different regions within the developing CNS, but they migrate and occupy very specific sites in the mature CNS. The ultimate position of neurons is crucial for establishing proper synaptic connectivity in the brain. In Drosophila, despite its extensive use as a model system to study neurogenesis, we know almost nothing about neuronal migration or its regulation. In this paper, I show that one of the most studied neuronal pairs in the Drosophila nerve cord, RP2/sib, has a complicated migratory route. Based on my studies on Wingless (Wg) signaling, I report that the neuronal migratory pattern is determined at the precursor cell stage level. The results show that Wg activity in the precursor neuroectodermal and neuroblast levels specify neuronal migratory pattern two divisions later, thus, well ahead of the actual migratory event. Moreover, at least two downstream genes, Cut and Zfh1, are involved in this process but their role is at the downstream neuronal level. The functional importance of normal neuronal migration and the requirement of Wg signaling for the process are indicated by the finding that mislocated RP2 neurons in embryos mutant for Wg-signaling fail to properly send out their axon projection.
Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linhagem da Célula , Sistema Nervoso Central/anatomia & histologia , Sistema Nervoso Central/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Neurônios/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Wnt1RESUMO
Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient of Slit from the midline interacts with three different Robo receptors to specify the axon tract positioning. However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has no effect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of the midline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independent manner, is initially taken up by the commissural axon tracts when they cross the midline and is transported along the commissural tracts into the longitudinal connectives. These results are not consistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos that are genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryos with normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutant embryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3 homozygous individuals fail to develop but have axon tracts ranging from normal to various defects: robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role for these genes than what has been proposed. Finally, our previous results indicated that netrin phenotype is epistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistatic relationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our results argue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of the mutants involved is complete.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Animais , Transporte Axonal , Axônios/metabolismo , Axônios/ultraestrutura , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Primers do DNA/genética , Drosophila/genética , Proteínas de Drosophila/genética , Epistasia Genética , Feminino , Genes de Insetos , Masculino , Mutação , Proteínas do Tecido Nervoso/genética , Receptores de Netrina , Fenótipo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Proteínas RoundaboutRESUMO
At some point during the history of organismal evolution, unicellular, unipotent and mitotically active cells acquired an ability to undergo a special type of cell division called asymmetric division. By this special type of cell division, these cells could divide to generate two different progeny or to self-renew and at the same time generate a progeny that is committed to become a cell different from the mother cell. This type of cell division, which forms the basis for the functioning of totipotent or multipotent stem cells, underlies the fundamental basis for the developmental evolution of organisms. It is not clear if the asymmetric division without self-renewal preceded the asymmetric division with self-renewal. It is reasonable to assume that the asymmetric division without self-renewal preceded the asymmetric division with self-renewal. In this review we explore the genetic regulation of these two types of asymmetric divisions using the Drosophila central nervous system (CNS) as a model system. The results from recent studies argue that for cells to undergo a self-renewing asymmetric division, certain "stem cell" proteins must be maintained or up-regulated, while genes encoding proteins responsible for differentiation must be repressed or down-regulated. As long as a balance between these two classes of proteins is maintained via asymmetric segregation and activation/repression, the progeny that receives stem cell proteins/maintains stem cell competence will have the potential to undergo self-renewing asymmetric division. The other progeny will commit to differentiate. In non-self-renewing asymmetric division, down-regulation of stem cell proteins/competence combined with asymmetric segregation of cell identity specifying factors (either cell-autonomous or a combination of cell autonomous and non-cell autonomous signals) cause the two progeny to assume different differentiated identities. Identification of mutations that confer a stem cell type of division to nonstem cell precursors, or mutations that eliminate asymmetric division, has led the way in elucidating the molecular basis for these divisions. Given that there is a considerable degree of conservation of genes and their function, these studies should provide clear insight into how the self-renewing asymmetric division of stem cells in neural and other lineages is regulated not only in Drosophila but also in vertebrates including humans.