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1.
Cell ; 163(7): 1756-69, 2015 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-26687360

RESUMEN

Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity.


Asunto(s)
Proteínas de Drosophila/metabolismo , Inmunoglobulinas/metabolismo , Neuronas/metabolismo , Receptores Inmunológicos/metabolismo , Sinapsis , Animales , Drosophila , Citometría de Flujo , Análisis de Secuencia de ARN , Visión Ocular
2.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-33766917

RESUMEN

The layered compartmentalization of synaptic connections, a common feature of nervous systems, underlies proper connectivity between neurons and enables parallel processing of neural information. However, the stepwise development of layered neuronal connections is not well understood. The medulla neuropil of the Drosophila visual system, which comprises 10 discrete layers (M1 to M10), where neural computations underlying distinct visual features are processed, serves as a model system for understanding layered synaptic connectivity. The first step in establishing layer-specific connectivity in the outer medulla (M1 to M6) is the innervation by lamina (L) neurons of one of two broad, primordial domains that will subsequently expand and transform into discrete layers. We previously found that the transcription factor dFezf cell-autonomously directs L3 lamina neurons to their proper primordial broad domain before they form synapses within the developing M3 layer. Here, we show that dFezf controls L3 broad domain selection through temporally precise transcriptional repression of the transcription factor slp1 (sloppy paired 1). In wild-type L3 neurons, slp1 is transiently expressed at a low level during broad domain selection. When dFezf is deleted, slp1 expression is up-regulated, and ablation of slp1 fully rescues the defect of broad domain selection in dFezf-null L3 neurons. Although the early, transient expression of slp1 is expendable for broad domain selection, it is surprisingly necessary for the subsequent L3 innervation of the M3 layer. DFezf thus functions as a transcriptional repressor to coordinate the temporal dynamics of a transcriptional cascade that orchestrates sequential steps of layer-specific synapse formation.


Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Neuronas/fisiología , Proteínas Represoras/metabolismo , Sinapsis/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vías Visuales/crecimiento & desarrollo , Animales , Drosophila melanogaster/genética , Neuronas/metabolismo , Mutación Puntual , Proteínas Represoras/genética , Vías Visuales/citología
3.
Mol Biol Cell ; 17(12): 5372-80, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17050735

RESUMEN

Under artificial conditions Golgi enzymes have the capacity to rapidly accumulate in the endoplasmic reticulum (ER). These observations prompted the idea that Golgi enzymes constitutively recycle through the ER. We have tested this hypothesis under physiological conditions through use of a procedure that captures Golgi enzymes in the ER. In the presence of rapamycin, which induces a tight association between FKBP (FK506-binding protein) and FRAP (FKBP-rapamycin-associated protein), an FKBP-tagged Golgi enzyme can be trapped when it visits the ER by an ER-retained protein fused to FRAP. We find that although FKBP-ERGIC-53 of the ER-Golgi intermediate compartment (ERGIC) rapidly cycles through the ER (30 min), FKBP-Golgi enzyme chimeras remain stably associated with Golgi membranes. We also demonstrate that Golgi dispersion upon nocodazole treatment mainly occurs through a mechanism that does not involve the recycling of Golgi membranes through the ER. Our findings suggest that the Golgi apparatus, as defined by its collection of resident enzymes, exists independent of the ER.


Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Animales , Brefeldino A/farmacología , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/enzimología , Células HeLa , Humanos , Membranas Intracelulares/efectos de los fármacos , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Nocodazol/farmacología , Proteínas Quinasas/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Proteínas de Unión a Tacrolimus/metabolismo , Transfección
4.
Neuron ; 103(5): 865-877.e7, 2019 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-31300277

RESUMEN

The ability of neurons to identify correct synaptic partners is fundamental to the proper assembly and function of neural circuits. Relative to other steps in circuit formation such as axon guidance, our knowledge of how synaptic partner selection is regulated is severely limited. Drosophila Dpr and DIP immunoglobulin superfamily (IgSF) cell-surface proteins bind heterophilically and are expressed in a complementary manner between synaptic partners in the visual system. Here, we show that in the lamina, DIP mis-expression is sufficient to promote synapse formation with Dpr-expressing neurons and that disrupting DIP function results in ectopic synapse formation. These findings indicate that DIP proteins promote synapses to form between specific cell types and that in their absence, neurons synapse with alternative partners. We propose that neurons have the capacity to synapse with a broad range of cell types and that synaptic specificity is achieved by establishing a preference for specific partners.


Asunto(s)
Proteínas de Drosophila/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Sinapsis/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster , Inmunoglobulinas/genética , Proteínas de la Membrana/genética , Neuronas/citología , Lóbulo Óptico de Animales no Mamíferos/citología , Mapas de Interacción de Proteínas
5.
Neural Dev ; 13(1): 11, 2018 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-29875010

RESUMEN

A striking feature of neural circuit structure is the arrangement of neurons into regularly spaced ensembles (i.e. columns) and neural connections into parallel layers. These patterns of organization are thought to underlie precise synaptic connectivity and provide a basis for the parallel processing of information. In this article we discuss in detail specific findings that contribute to a framework for understanding how columns and layers are assembled in the Drosophila visual system, and discuss their broader implications.


Asunto(s)
Drosophila/anatomía & histología , Red Nerviosa/fisiología , Neuronas/fisiología , Vías Visuales/anatomía & histología , Animales , Sinapsis/fisiología
6.
J Vis Exp ; (139)2018 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-30320761

RESUMEN

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.


Asunto(s)
Drosophila/citología , Técnicas Genéticas , Animales , Técnicas de Cultivo de Célula , Linaje de la Célula/genética , Drosophila/genética , Proteínas de Drosophila/genética , Marcadores Genéticos/genética , Mosaicismo , Técnicas de Amplificación de Ácido Nucleico
7.
Elife ; 72018 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-29513217

RESUMEN

Laminar arrangement of neural connections is a fundamental feature of neural circuit organization. Identifying mechanisms that coordinate neural connections within correct layers is thus vital for understanding how neural circuits are assembled. In the medulla of the Drosophila visual system neurons form connections within ten parallel layers. The M3 layer receives input from two neuron types that sequentially innervate M3 during development. Here we show that M3-specific innervation by both neurons is coordinated by Drosophila Fezf (dFezf), a conserved transcription factor that is selectively expressed by the earlier targeting input neuron. In this cell, dFezf instructs layer specificity and activates the expression of a secreted molecule (Netrin) that regulates the layer specificity of the other input neuron. We propose that employment of transcriptional modules that cell-intrinsically target neurons to specific layers, and cell-extrinsically recruit other neurons is a general mechanism for building layered networks of neural connections.


Asunto(s)
Proteínas de Drosophila/genética , Netrinas/genética , Neurogénesis/genética , Neuronas/metabolismo , Factores de Transcripción/genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Bulbo Raquídeo/crecimiento & desarrollo , Bulbo Raquídeo/metabolismo , Red Nerviosa/crecimiento & desarrollo , Células Fotorreceptoras de Invertebrados/metabolismo , Sinapsis/genética , Vías Visuales/crecimiento & desarrollo
9.
Neuron ; 82(2): 320-33, 2014 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-24742459

RESUMEN

Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1-R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. Here we demonstrate that additional axon-derived signals selectively couple target survival with layer specificity. We show that Jelly belly (Jeb) produced by R1-R6 axons interacts with its receptor, anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. We propose that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system.


Asunto(s)
Axones/fisiología , Red Nerviosa/metabolismo , Neuronas/fisiología , Transducción de Señal/fisiología , Vías Visuales/citología , Vías Visuales/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Animales Modificados Genéticamente , Muerte Celular/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Conos de Crecimiento/metabolismo , Laminas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Neuronas/clasificación , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo
10.
Neuron ; 81(2): 280-93, 2014 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-24462095

RESUMEN

The study of synaptic specificity and plasticity in the CNS is limited by the inability to efficiently visualize synapses in identified neurons using light microscopy. Here, we describe synaptic tagging with recombination (STaR), a method for labeling endogenous presynaptic and postsynaptic proteins in a cell-type-specific fashion. We modified genomic loci encoding synaptic proteins within bacterial artificial chromosomes such that these proteins, expressed at endogenous levels and with normal spatiotemporal patterns, were labeled in an inducible fashion in specific neurons through targeted expression of site-specific recombinases. Within the Drosophila visual system, the number and distribution of synapses correlate with electron microscopy studies. Using two different recombination systems, presynaptic and postsynaptic specializations of synaptic pairs can be colabeled. STaR also allows synapses within the CNS to be studied in live animals noninvasively. In principle, STaR can be adapted to the mammalian nervous system.


Asunto(s)
Bulbo Raquídeo/citología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica de Transmisión , Proteínas del Tejido Nervioso/genética , Neuronas/ultraestructura , Receptores de Neurotransmisores/genética , Receptores de Neurotransmisores/metabolismo , Recombinasas/genética , Recombinasas/metabolismo , Recombinación Genética/genética , Sinapsis/genética , Sinapsis/ultraestructura , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vías Visuales/citología , Vías Visuales/metabolismo
11.
Neuron ; 77(2): 299-310, 2013 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-23352166

RESUMEN

How neurons form synapses within specific layers remains poorly understood. In the Drosophila medulla, neurons target to discrete layers in a precise fashion. Here we demonstrate that the targeting of L3 neurons to a specific layer occurs in two steps. Initially, L3 growth cones project to a common domain in the outer medulla, overlapping with the growth cones of other neurons destined for a different layer through the redundant functions of N-Cadherin (CadN) and Semaphorin-1a (Sema-1a). CadN mediates adhesion within the domain and Sema-1a mediates repulsion through Plexin A (PlexA) expressed in an adjacent region. Subsequently, L3 growth cones segregate from the domain into their target layer in part through Sema-1a/PlexA-dependent remodeling. Together, our results and recent studies argue that the early medulla is organized into common domains, comprising processes bound for different layers, and that discrete layers later emerge through successive interactions between processes within domains and developing layers.


Asunto(s)
Bulbo Raquídeo/metabolismo , Sinapsis/metabolismo , Vías Visuales/metabolismo , Animales , Animales Modificados Genéticamente , Comunicación Celular/fisiología , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/fisiología , Bulbo Raquídeo/citología , Bulbo Raquídeo/fisiología , Mapeo de Interacción de Proteínas , Sinapsis/genética , Sinapsis/fisiología , Vías Visuales/citología , Vías Visuales/fisiología
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