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1.
J Neurogenet ; 33(3): 157-163, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-30955404

RESUMEN

Axonal extension and synaptic targeting are usually completed during early development, but the axonal length and synaptic integrity need to be actively maintained during later developmental stages and the adult life. Failure in the axonal length maintenance and the subsequent axonal degeneration have been associated with neurological disorders, but currently little is known about the genetic factors controlling this process. Here, we show that regulated intracellular levels of cAMP-dependent protein kinase A (PKA) are critical for the axon maintenance during the transition from the early to the later larval stages in the Drosophila class IV dendritic arborization (da) sensory neurons. Our data indicate that when the intracellular levels of PKA are increased via genetic manipulations, these peripheral neurons initially form synapses with wild-type appearance, at their predicted ventral nerve cord (VNC) target sites (in the first and second instar larval stages), but that their synapses disintegrate, and the axons retract and become fragmented in the subsequent larval stages (third larval stage). The affected axonal endings at the disintegrated synaptic sites still express the characteristic presynaptic and cytoskeletal markers such as Bruchpilot and Fascin, indicating that the synapse had been initially properly formed, but that it later lost its integrity. Finally, the phenotype is significantly more prominent in the axons of the neurons whose cell bodies are located in the posterior body segments. We propose that the reason for this is the fact that during the larval development the posterior neurons face a much greater challenge while trying to keep up with the fast-paced growth of the larval body, and that PKA is critical for this process. Our data reveal PKA as a novel factor in the axonal length and synapse integrity maintenance in sensory neurons. These results could be of help in understanding neurological disorders characterized by destabilized synapses.


Asunto(s)
Axones/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neurogénesis/fisiología , Células Receptoras Sensoriales/enzimología , Sinapsis/enzimología , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Larva/enzimología , Larva/crecimiento & desarrollo
2.
J Biol Chem ; 291(12): 6200-17, 2016 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-26792857

RESUMEN

SCCRO (squamous cell carcinoma-related oncogene; also known as DCUN1D1) is a highly conserved gene that functions as an E3 in neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO(-/-) mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led us to assess whether compensation by SCCRO paralogues rescues lethality in SCCRO(-/-) mice. Using murine and Drosophila models, we assessed the in vivo activities of SCCRO and its paralogues in cullin neddylation. We found that SCCRO family members have overlapping and antagonistic activity that regulates neddylation and cell proliferation activities in vivo. In flies, both dSCCRO and dSCCRO3 promote neddylation and cell proliferation, whereas dSCCRO4 negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones with a net decrease in neddylation activity. We found that dSCCRO and, to a lesser extent, dSCCRO3 rescue the neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promote, and human SCCRO3 inhibits, neddylation and proliferation when expressed in flies. Our findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate neddylation and cell proliferation.


Asunto(s)
Proteínas Cullin/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/fisiología , Animales , Proliferación Celular , Proteínas de Drosophila/fisiología , Drosophila melanogaster , Femenino , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones Noqueados , Especificidad de Órganos
3.
J Neurogenet ; 29(2-3): 135-43, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26100104

RESUMEN

Autism spectrum disorder (ASD) is a neurodevelopmental disorder in humans characterized by complex behavioral deficits, including intellectual disability, impaired social interactions, and hyperactivity. ASD exhibits a strong genetic component with underlying multigene interactions. Candidate gene studies have shown that the neurobeachin (NBEA) gene is disrupted in human patients with idiopathic autism ( Castermans et al., 2003 ). The NBEA gene spans the common fragile site FRA 13A and encodes a signal scaffold protein ( Savelyeva et al., 2006 ). In mice, NBEA has been shown to be involved in the trafficking and function of a specific subset of synaptic vesicles. ( Medrihan et al., 2009 ; Savelyeva et al., 2006 ). Rugose (rg) is the Drosophila homolog of the mammalian and human NBEA. Our previous genetic and molecular analyses have shown that rg encodes an A kinase anchor protein (DAKAP 550), which interacts with components of the epidermal growth factor receptor or EGFR and Notch-mediated signaling pathways, facilitating cross talk between these and other pathways ( Shamloula et al., 2002 ). We now present functional data from studies on the larval neuromuscular junction that reveal abnormal synaptic architecture and physiology. In addition, adult rg loss-of-function mutants exhibit defective social interactions, impaired habituation, aberrant locomotion, and hyperactivity. These results demonstrate that Drosophila NBEA (rg) mutants exhibit phenotypic characteristics reminiscent of human ASD and thus could serve as a genetic model for studying ASDs.


Asunto(s)
Proteínas de Anclaje a la Quinasa A/genética , Conducta Animal/fisiología , Proteínas de Drosophila/genética , Locomoción/genética , Actividad Motora/genética , Conducta Social , Sinapsis/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Animales Modificados Genéticamente , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Mutación , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Transducción de Señal/genética , Sinapsis/metabolismo
4.
Micromachines (Basel) ; 11(4)2020 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-32244321

RESUMEN

Contemporary regenerative therapies have introduced stem-like cells to replace damaged neurons in the visual system by recapitulating critical processes of eye development. The collective migration of neural stem cells is fundamental to retinogenesis and has been exceptionally well-studied using the fruit fly model of Drosophila Melanogaster. However, the migratory behavior of its retinal neuroblasts (RNBs) has been surprisingly understudied, despite being critical to retinal development in this invertebrate model. The current project developed a new microfluidic system to examine the collective migration of RNBs extracted from the developing visual system of Drosophila as a model for the collective motile processes of replacement neural stem cells. The system scales with the microstructure of the Drosophila optic stalk, which is a pre-cursor to the optic nerve, to produce signaling fields spatially comparable to in vivo RNB stimuli. Experiments used the micro-optic stalk system, or µOS, to demonstrate the preferred sizing and directional migration of collective, motile RNB groups in response to changes in exogenous concentrations of fibroblast growth factor (FGF), which is a key factor in development. Our data highlight the importance of cell-to-cell contacts in enabling cell cohesion during collective RNB migration and point to the unexplored synergy of invertebrate cell study and microfluidic platforms to advance regenerative strategies.

5.
Genetics ; 178(4): 2003-16, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18430931

RESUMEN

Glia mediate a vast array of cellular processes and are critical for nervous system development and function. Despite their immense importance in neurobiology, glia remain understudied and the molecular mechanisms that direct their differentiation are poorly understood. Rap/Fzr is the Drosophila homolog of the mammalian Cdh1, a regulatory subunit of the anaphase-promoting complex/cyclosome (APC/C). APC/C is an E3 ubiquitin ligase complex well characterized for its role in cell cycle progression. In this study, we have uncovered a novel cellular role for Rap/Fzr. Loss of rap/fzr function leads to a marked increase in the number of glia in the nervous system of third instar larvae. Conversely, ectopic expression of UAS-rap/fzr, driven by repo-GAL4, results in the drastic reduction of glia. Data from clonal analyses using the MARCM technique show that Rap/Fzr regulates the differentiation of surface glia in the developing larval nervous system. Our genetic and biochemical data further indicate that Rap/Fzr regulates glial differentiation through its interaction with Loco, a regulator of G-protein signaling (RGS) protein and a known effector of glia specification. We propose that Rap/Fzr targets Loco for ubiquitination, thereby regulating glial differentiation in the developing nervous system.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Proteínas RGS/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Apoptosis , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Proteínas Cdh1 , Recuento de Células , Diferenciación Celular , Linaje de la Célula , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Modelos Biológicos , Neuronas/citología , Unión Proteica , Transporte de Proteínas
6.
Cells ; 8(10)2019 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-31652654

RESUMEN

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by the misdirected and/or insufficient migration of transplanted cells. RPCs must achieve appropriate spatial and functional positioning in host retina, collectively, to restore vision, whereas movement of clustered cells differs substantially from the single cell migration studied in classical chemotaxis models. Defining how RPCs interact with each other, neighboring cell types and surrounding extracellular matrixes are critical to our understanding of retinogenesis and the development of effective, cell-based approaches to retinal replacement. The current article describes a new bio-engineering approach to investigate the migratory responses of innate collections of RPCs upon extracellular substrates by combining microfluidics with the well-established invertebrate model of Drosophila melanogaster. Experiments utilized microfluidics to investigate how the composition, size, and adhesion of RPC clusters on defined extracellular substrates affected migration to exogenous chemotactic signaling. Results demonstrated that retinal cluster size and composition influenced RPC clustering upon extracellular substrates of concanavalin (Con-A), Laminin (LM), and poly-L-lysine (PLL), and that RPC cluster size greatly altered collective migratory responses to signaling from Fibroblast Growth Factor (FGF), a primary chemotactic agent in Drosophila. These results highlight the significance of examining collective cell-biomaterial interactions on bio-substrates of emerging biomaterials to aid directional migration of transplanted cells. Our approach further introduces the benefits of pairing genetically controlled models with experimentally controlled microenvironments to advance cell replacement therapies.


Asunto(s)
Técnicas Analíticas Microfluídicas , Modelos Biológicos , Regeneración , Retina/fisiología , Células Madre/metabolismo , Animales , Drosophila melanogaster , Retina/citología , Células Madre/citología
7.
PLoS One ; 14(12): e0226250, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31835272

RESUMEN

Collective behaviors of retinal progenitor cells (RPCs) are critical to the development of neural networks needed for vision. Signaling cues and pathways governing retinal cell fate, migration, and functional organization are remarkably conserved across species, and have been well-studied using Drosophila melanogaster. However, the collective migration of heterogeneous groups of RPCs in response to dynamic signaling fields of development remains incompletely understood. This is in large part because the genetic advances of seminal invertebrate models have been poorly complemented by in vitro cell study of its visual development. Tunable microfluidic assays able to replicate the miniature cellular microenvironments of the developing visual system provide newfound opportunities to probe and expand our knowledge of collective chemotactic responses essential to visual development. Our project used a controlled, microfluidic assay to produce dynamic signaling fields of Fibroblast Growth Factor (FGF) that stimulated the chemotactic migration of primary RPCs extracted from Drosophila. Results illustrated collective RPC chemotaxis dependent on average size of clustered cells, in contrast to the non-directional movement of individually-motile RPCs. Quantitative study of these diverse collective responses will advance our understanding of retina developmental processes, and aid study/treatment of inherited eye disease. Lastly, our unique coupling of defined invertebrate models with tunable microfluidic assays provides advantages for future quantitative and mechanistic study of varied RPC migratory responses.


Asunto(s)
Movimiento Celular , Microambiente Celular , Drosophila melanogaster/crecimiento & desarrollo , Neuronas/citología , Retina/citología , Células Madre/citología , Animales , Diferenciación Celular , Células Cultivadas , Quimiotaxis , Drosophila melanogaster/fisiología , Microfluídica , Neuronas/fisiología , Retina/fisiología , Transducción de Señal , Células Madre/fisiología
8.
Curr Biol ; 12(16): 1435-41, 2002 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-12194827

RESUMEN

Proteolysis of mitotic regulators like securins and cyclins requires Fizzy(FZY)/Cdc20 and Fizzy-related(FZR)/Hct1/Cdh1 proteins. Budding yeast Cdh1 acts not only during G1, but is also required for B-type cyclin degradation during exit from mitosis when Cdh1 is a target of the mitotic exit network controlling progression through late mitosis and cytokinesis. In contrast, observations in frog and Drosophila embryos have suggested that the orthologous FZR is not involved during exit from mitosis. However, the potential involvement of minor amounts of maternally derived FZR was not excluded in these studies. Similarly, the reported absence of severe mitotic defects in chicken Cdh1(-/-) cells might be explained by the recent identification of multiple Cdh1 genes [10]. Here, we have carefully analyzed the FZR requirement during exit from mitosis in Drosophila, which, apart from fzr, has only one additional homolog. We find that this fzr2 gene, although expressed in the male germline, is not expressed during mitotic divisions. Moreover, by characterizing fzr alleles, we demonstrate that completion of mitosis including Cyclin B degradation does not require FZR. However, fzr is an essential gene corresponding to the rap locus, and FZR, which accumulates predominantly in the cytoplasm, is clearly required during G1.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Cdh1 , Proteínas de Ciclo Celular/genética , Ciclina B/metabolismo , Proteínas de Drosophila/clasificación , Proteínas de Drosophila/genética , Embrión no Mamífero/fisiología , Femenino , Hibridación in Situ , Masculino , Meiosis/fisiología , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tubulina (Proteína)/metabolismo
9.
J Neurosci Methods ; 262: 32-40, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26738658

RESUMEN

BACKGROUND: The developing visual system in Drosophila melanogaster provides an excellent model with which to examine the effects of changing microenvironments on neural cell migration via microfluidics, because the combined experimental system enables direct genetic manipulation, in vivo observation, and in vitro imaging of cells, post-embryo. Exogenous signaling from ligands such as fibroblast growth factor (FGF) is well-known to control glia differentiation, cell migration, and axonal wrapping central to vision. NEW METHOD: The current study employs a microfluidic device to examine how controlled concentration gradient fields of FGF are able to regulate the migration of vision-critical glia cells with and without cellular contact with neuronal progenitors. RESULTS: Our findings quantitatively illustrate a concentration-gradient dependent chemotaxis toward FGF, and further demonstrate that glia require collective and coordinated neuronal locomotion to achieve directionality, sustain motility, and propagate long cell distances in the visual system. COMPARISON WITH EXISTING METHOD(S): Conventional assays are unable to examine concentration- and gradient-dependent migration. Our data illustrate quantitative correlations between ligand concentration/gradient and glial cell distance traveled, independent or in contact with neurons. CONCLUSIONS: Microfluidic systems in combination with a genetically-amenable experimental system empowers researchers to dissect the signaling pathways that underlie cellular migration during nervous system development. Our findings illustrate the need for coordinated neuron-glia migration in the Drosophila visual system, as only glia within heterogeneous populations exhibited increasing motility along distances that increased with increasing FGF concentration. Such coordinated migration and chemotactic dependence can be manipulated for potential therapeutic avenues for NS repair and/or disease treatment.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Microfluídica/métodos , Células-Madre Neurales/efectos de los fármacos , Vías Visuales/citología , Animales , Animales Modificados Genéticamente , Células Cultivadas , Simulación por Computador , Relación Dosis-Respuesta a Droga , Drosophila , Proteínas de la Matriz Extracelular/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Modelos Biológicos
10.
Genetics ; 161(2): 693-710, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12072466

RESUMEN

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/fisiología , Proteínas de Drosophila/fisiología , Drosophila/embriología , Quinasas MAP Reguladas por Señal Extracelular , Células Fotorreceptoras de Invertebrados/embriología , Transducción de Señal/fisiología , Proteínas ras , Proteínas de Anclaje a la Quinasa A , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/genética , Proteínas de Unión al GTP/genética , Genes erbB-1 , Proteínas de la Membrana/genética , Mutación , Fenotipo , Células Fotorreceptoras de Invertebrados/metabolismo , Retina/embriología
11.
Int J Dev Neurosci ; 31(7): 624-33, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23933137

RESUMEN

The assembly of functional synapses requires the orchestration of the synthesis and degradation of a multitude of proteins. Protein degradation and modification by the conserved ubiquitination pathway has emerged as a key cellular regulatory mechanism during nervous system development and function (Kwabe and Brose, 2011). The anaphase promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin ligase complex primarily characterized for its role in the regulation of mitosis (Peters, 2002). In recent years, a role for APC/C in nervous system development and function has been rapidly emerging (Stegmuller and Bonni, 2005; Li et al., 2008). In the mammalian central nervous system the activator subunit, APC/C-Cdh1, has been shown to be a regulator of axon growth and dendrite morphogenesis (Konishi et al., 2004). In the Drosophila peripheral nervous system (PNS), APC2, a ligase subunit of the APC/C complex has been shown to regulate synaptic bouton size and activity (van Roessel et al., 2004). To investigate the role of APC/C-Cdh1 at the synapse we examined loss-of-function mutants of Rap/Fzr (Retina aberrant in pattern/Fizzy related), a Drosophila homolog of the mammalian Cdh1 during the development of the larval neuromuscular junction in Drosophila. Our cell biological, ultrastructural, electrophysiological, and behavioral data showed that rap/fzr loss-of-function mutations lead to changes in synaptic structure and function as well as locomotion defects. Data presented here show changes in size and morphology of synaptic boutons, and, muscle tissue organization. Electrophysiological experiments show that loss-of-function mutants exhibit increased frequency of spontaneous miniature synaptic potentials, indicating a higher rate of spontaneous synaptic vesicle fusion events. In addition, larval locomotion and peristaltic movement were also impaired. These findings suggest a role for Drosophila APC/C-Cdh1 mediated ubiquitination in regulating synaptic morphology, function and integrity of muscle structure in the peripheral nervous system.


Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdh1/metabolismo , Proteínas de Drosophila/metabolismo , Locomoción/fisiología , Unión Neuromuscular/metabolismo , Terminales Presinápticos/fisiología , Potenciales Sinápticos/genética , Ciclosoma-Complejo Promotor de la Anafase/genética , Animales , Animales Modificados Genéticamente , Proteínas Cdh1/genética , Drosophila/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Larva , Locomoción/genética , Microscopía Electrónica de Transmisión , Contracción Muscular/genética , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mutación/genética , Unión Neuromuscular/genética , Unión Neuromuscular/ultraestructura , Técnicas de Placa-Clamp , Terminales Presinápticos/ultraestructura , Receptores AMPA/metabolismo
12.
J Neurogenet ; 21(3): 105-51, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17849284

RESUMEN

In the developing Drosophila eye, Rap/Fzr plays a critical role in neural patterning by regulating the timely exit of precursor cells. Rap/Fzr (Retina aberrant in pattern/Fizzy related) is an activator of the E3 Ubiquitin ligase, the APC (Anaphase Promoting Complex-cyclosome) that facilitates the stage specific proteolytic destruction of mitotic regulators, such as cyclins and cyclin-dependent kinases. To identify novel functional roles of Rap/Fzr, we conducted an F(1) genetic modifier screen to identify genes which interact with the partial-loss-function mutations in rap/fzr. We screened 2741 single P-element, lethal insertion lines and piggyBac lines on the second and third chromosome for dominant enhancers and suppressors of the rough eye phenotype of rap/fzr. From this screen, we have identified 40 genes that exhibit dosage-sensitive interactions with rap/fzr; of these, 31 have previously characterized cellular functions. Seven of the modifiers identified in this study are regulators of cell cycle progression with previously known interactions with rap/fzr. Among the remaining modifiers, 27 encode proteins involved in other cellular functions not directly related to cell-cycle progression. The newly identified variants fall into at least three groups based on their previously known cellular functions: transcriptional regulation, regulated proteolysis, and signal transduction. These results suggest that, in addition to cell cycle regulation, rap/fzr regulates ubiquitin-ligase-mediated protein degradation in the developing nervous system as well as in other tissues.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Ojo/embriología , Perfilación de la Expresión Génica , Genes de Insecto , Animales , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Ojo/ultraestructura , Femenino , Regulación del Desarrollo de la Expresión Génica , Larva , Masculino , Microscopía Electrónica de Rastreo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ubiquitina-Proteína Ligasas/genética
13.
Clin Dev Immunol ; 13(2-4): 299-319, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17162372

RESUMEN

Wnt signaling has been reported to regulate thymocyte proliferation and selection at several stages during T cell ontogeny, as well as the expression of FoxN1 in thymic epithelial cells (TECs). Kremen1 (Krm1) is a negative regulator of the canonical Wnt signaling pathway, and functions together with the secreted Wnt inhibitor Dickkopf (Dkk) by competing for the lipoprotein receptor-related protein (LRP)-6 co-receptor for Wnts. Here krm1 knockout mice were used to examine krm1 expression in the thymus and its function in thymocyte and TEC development. Krm1 expression was detected in both cortical and medullary TEC subsets, as well as in immature thymocyte subsets, beginning at the CD25+CD44+ (DN2) stage and continuing until the CD4+CD8+(DP) stage. Neonatal mice show elevated expression of krm1 in all TEC subsets. krm1(-/-) mice exhibit a severe defect in thymic cortical architecture, including large epithelial free regions. Much of the epithelial component remains at an immature Keratin 5+ (K5) Keratin 8(+)(K8) stage, with a loss of defined cortical and medullary regions. A TOPFlash assay revealed a 2-fold increase in canonical Wnt signaling in TEC lines derived from krm1(-/-) mice, when compared with krm1(+/+) derived TEC lines. Fluorescence activated cell sorting (FACS) analysis of dissociated thymus revealed a reduced frequency of both cortical (BP1(+)EpCAM(+)) and medullary (UEA-1(+) EpCAM(hi)) epithelial subsets, within the krm1(-/-) thymus. Surprisingly, no change in thymus size, total thymocyte number or the frequency of thymocyte subsets was detected in krm1(-/-) mice. However, our data suggest that a loss of Krm1 leads to a severe defect in thymic architecture. Taken together, this study revealed a new role for Krm1 in proper development of thymic epithelium.


Asunto(s)
Proteínas de la Membrana/farmacología , Transducción de Señal/efectos de los fármacos , Timo/citología , Proteínas Wnt/antagonistas & inhibidores , Proteínas de Xenopus/farmacología , Animales , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Timo/inmunología , Timo/metabolismo , Proteínas Wnt/fisiología
14.
Dev Biol ; 285(2): 436-46, 2005 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-16098963

RESUMEN

The rap (retina aberrant in pattern) gene encodes the Fizzy-related protein (Fzr), which as an activator of the ubiquitin ligase complex; APC/C (anaphase promoting complex/cyclosome) facilitates the cell cycle stage-specific degradation of cyclins. Loss-of-function mutations in rap cause unscheduled accumulation of cyclin B in the developing eye imaginal disc, resulting in additional mitotic cycles and defective patterning of the developing Drosophila eye. Targeted mis-expression of rap/fzr in the eye primordial cells causes precocious cell cycle exit, and smaller primordial eye fields, which either eliminate or drastically reduce the size of the adult eye. Although mitosis is inhibited in the mis-expression animals, cells with abnormally large nuclei form tumor-like structures from continued endoreplication, cell growth and retinal differentiation. Interestingly, overexpression of Rap/Fzr in the eye primordia also increases the size of the antennal primordium resulting in the induction of ectopic antennae. These results suggest that Rap/Fzr plays an essential role in the timely exit of precursor cells from mitotic cycles and indicate that mechanisms that regulate cell cycle exit are critical during pattern formation and morphogenesis.


Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Ojo/embriología , Genes prv/genética , Mitosis/fisiología , Animales , Proteínas Cdh1 , Ciclina B/metabolismo , Drosophila/genética , Ojo/metabolismo , Ojo/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Microscopía Electrónica de Rastreo , Reacción en Cadena de la Polimerasa
15.
Proc Natl Acad Sci U S A ; 102(37): 13188-93, 2005 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-16129827

RESUMEN

A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximately 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.


Asunto(s)
Marcación de Gen/métodos , Vectores Genéticos , Mutagénesis Insercional/métodos , Animales , Embrión de Mamíferos/citología , Perfilación de la Expresión Génica , Genómica/métodos , Métodos , Ratones , Recombinación Genética , Células Madre/metabolismo
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