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 OcularRESUMEN
Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system development is poorly understood. We explored the function of the Drosophila ClC-a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. We found that ClC-a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss-of-function ClC-a mutants revealed that these animals had smaller brains and widespread wiring defects. We showed that ClC-a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and identified defects in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. We propose that glia-mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits.
Asunto(s)
Canales de Cloruro/metabolismo , Red Nerviosa/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Neuroglía/metabolismo , Nicho de Células Madre/fisiología , Animales , Animales Modificados Genéticamente , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Canales de Cloruro/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mutación con Pérdida de Función/fisiología , Red Nerviosa/citologíaRESUMEN
The assembly of neural circuits relies on the accurate establishment of connections between synaptic partners. Precise wiring results from responses that neurons elicit to environmental cues and cell-cell contact events during development. A common design principle in both invertebrate and vertebrate adult nervous systems is the orderly array of columnar and layered synaptic units of certain neuropils. This similarity is particularly striking in the visual system, both at the structural and cell-type levels. Given the powerful genetic approaches and tools available in Drosophila, the fly visual system has been extensively used to probe how specific wiring patterns are achieved during development. In this review, we cover the developmental principles and molecular strategies that govern the assembly of columnar units (lamina cartridges and medulla columns), the formation of layers, afferent specific layer selection, and synaptogenesis in Drosophila. The mechanisms include: sequential developmental steps that ensure coordinated assembly of synaptic partners; anterograde and autocrine signaling; interactions between cell-surface molecules, or secreted molecules and their receptors that take place among neurons; and glia signaling to neurons.
Asunto(s)
Drosophila/crecimiento & desarrollo , Red Nerviosa/crecimiento & desarrollo , Neuronas/fisiología , Células Fotorreceptoras de Invertebrados/fisiología , Vías Visuales/crecimiento & desarrollo , Animales , Drosophila/metabolismo , Red Nerviosa/metabolismo , Transducción de Señal/fisiología , Sinapsis/metabolismo , Vías Visuales/metabolismoRESUMEN
How neurons make specific synaptic connections is a central question in neurobiology. The targeting of the Drosophila R7 and R8 photoreceptor axons to different synaptic layers in the brain provides a model with which to explore the genetic programs regulating target specificity. In principle this can be accomplished by cell-type-specific molecules mediating the recognition between synaptic partners. Alternatively, specificity could also be achieved through cell-type-specific repression of particular targeting molecules. Here we show that a key step in the targeting of the R7 neuron is the active repression of the R8 targeting program. Repression is dependent on NF-YC, a subunit of the NF-Y (nuclear factor Y) transcription factor. In the absence of NF-YC, R7 axons terminate in the same layer as R8 axons. Genetic experiments indicate that this is due solely to the derepression of the R8-specific transcription factor Senseless (Sens) late in R7 differentiation. Sens is sufficient to control R8 targeting specificity and we demonstrate that Sens directly binds to an evolutionarily conserved DNA sequence upstream of the start of transcription of an R8-specific cell-surface protein, Capricious (Caps) that regulates R8 target specificity. We show that R7 targeting requires the R7-specific transcription factor Prospero (Pros) in parallel to repression of the R8 targeting pathway by NF-YC. Previous studies demonstrated that Sens and Pros directly regulate the expression of specific rhodopsins in R8 and R7. We propose that the use of the same transcription factors to promote the cell-type-specific expression of sensory receptors and cell-surface proteins regulating synaptic target specificity provides a simple and general mechanism for ensuring that transmission of sensory information is processed by the appropriate specialized neural circuits.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Rodopsina/metabolismo , Sinapsis/metabolismo , Animales , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Especificidad por Sustrato , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The formation of neuronal connections requires the precise guidance of developing axons toward their targets. In the Drosophila visual system, photoreceptor neurons (R cells) project from the eye into the brain. These cells are grouped into some 750 clusters comprised of eight photoreceptors or R cells each. R cells fall into three classes: R1 to R6, R7, and R8. Posterior R8 cells are the first to project axons into the brain. How these axons select a specific pathway is not known. Here, we used a microarray-based approach to identify genes expressed in R8 neurons as they extend into the brain. We found that Roundabout-3 (Robo3), an axon-guidance receptor, is expressed specifically and transiently in R8 growth cones. In wild-type animals, posterior-most R8 axons extend along a border of glial cells demarcated by the expression of Slit, the secreted ligand of Robo3. In contrast, robo3 mutant R8 axons extend across this border and fasciculate inappropriately with other axon tracts. We demonstrate that either Robo1 or Robo2 rescues the robo3 mutant phenotype when each is knocked into the endogenous robo3 locus separately, indicating that R8 does not require a function unique to the Robo3 paralog. However, persistent expression of Robo3 in R8 disrupts the layer-specific targeting of R8 growth cones. Thus, the transient cell-specific expression of Robo3 plays a crucial role in establishing neural circuits in the Drosophila visual system by selectively regulating pathway choice for posterior-most R8 growth cones.
Asunto(s)
Axones/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Conos de Crecimiento/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Receptores Inmunológicos/metabolismo , Vías Visuales/embriología , Animales , Axones/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Análisis por Micromatrices , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismoRESUMEN
During circuit assembly it is essential that neurons connect with their specific synaptic partners. To facilitate this process, a common strategy in many organisms is the organization of brain regions, including the fly visual system, in layers and columns. The atypical-cadherin Flamingo (Fmi) and the receptor Golden Goal (Gogo) were proposed to regulate both the temporary and final layer selection of the R8 photoreceptor, through the cytoplasmic domain of Gogo. Our data suggests that Fmi intracellular signaling is also relevant for R8 final layer selection. The LIM-domain cytoplasmic molecule Espinas (Esn) binds Fmi, and they cooperatively control dendritic self-avoidance in sensory neurons. We observed defects in R8 layer selection in esn mutants with axons overshooting the final target layer, and we demonstrated that the LIM domain is necessary for layer selection. fmi knockdown in photoreceptors results in most R8 axons stalling at the temporary layer, however, we also detected R8 axons projecting past the final-target layer, and showed that fmi and esn genetically interact. Based on the previously described physical and genetic interactions between Fmi/Esn and the findings presented here, we propose that Esn signals downstream of Fmi to stabilize R8 axons in their final target layer.
RESUMEN
Screens for genes that orchestrate neural circuit formation in mammals have been hindered by practical constraints of germline mutagenesis. To overcome these limitations, we combined RNA-seq with somatic CRISPR mutagenesis to study synapse development in the mouse retina. Here synapses occur between cellular layers, forming two multilayered neuropils. The outer neuropil, the outer plexiform layer (OPL), contains synapses made by rod and cone photoreceptor axons on rod and cone bipolar dendrites, respectively. We used RNA-seq to identify selectively expressed genes encoding cell surface and secreted proteins and CRISPR-Cas9 electroporation with cell-specific promoters to assess their roles in OPL development. Among the genes identified in this way are Wnt5a and Wnt5b. They are produced by rod bipolars and activate a non-canonical signaling pathway in rods to regulate early OPL patterning. The approach we use here can be applied to other parts of the brain.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Mutagénesis/fisiología , Neurópilo/metabolismo , Retina/metabolismo , Análisis de Secuencia de ARN/métodos , Vía de Señalización Wnt/fisiología , Animales , Animales Recién Nacidos , Femenino , Masculino , Ratones , Ratones Transgénicos , Neurópilo/química , Conejos , Retina/química , Retina/crecimiento & desarrolloRESUMEN
Neurons form precise patterns of connections. The cellular recognition mechanisms regulating the selection of synaptic partners are poorly understood. As final mediators of cell-cell interactions, cell surface and secreted molecules (CSMs) are expected to play important roles in this process. To gain insight into how neurons discriminate synaptic partners, we profiled the transcriptomes of 7 closely related neurons forming distinct synaptic connections in discrete layers in the medulla neuropil of the fly visual system. Our sequencing data revealed that each one of these neurons expresses a unique combination of hundreds of CSMs at the onset of synapse formation. We show that 21 paralogs of the defective proboscis extension response (Dpr) family are expressed in a unique cell-type-specific fashion, consistent with the distinct connectivity pattern of each neuron profiled. Expression analysis of their cognate binding partners, the 9 members of the Dpr interacting protein (DIP) family, revealed complementary layer-specific expression in the medulla, suggestive of interactions between neurons expressing Dpr and those expressing DIP in the same layer. Through coexpression analysis and correlation to connectome data, we identify neurons expressing DIP as a subset of the synaptic partners of the neurons expressing Dpr. We propose that Dpr-DIP interactions regulate patterns of connectivity between the neurons expressing them.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismo , Sinapsis/fisiología , Animales , Drosophila/crecimiento & desarrollo , Neuronas/citologíaRESUMEN
Several lines of evidence indicate that selenoproteins mainly act as cellular antioxidants. Here, we test this idea comparing the sensitivity to oxidative stress (paraquat and hydrogen peroxide) between wild type and heterozygous flies for the selenophosphate synthetase selD(ptuf) mutation. Whereas under normal laboratory conditions no difference in life span is observed, a significant decrease is seen in heterozygous flies treated with oxidant agents. In contrast, overexpression of the selD gene in motoneurons did not extend longevity. Our results strongly suggest that selD haploinsufficiency makes heterozygous flies more sensitive to oxidative stress and add further evidence to the role of selenoproteins as cellular antioxidants.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Dosificación de Gen , Estrés Oxidativo , Fosfotransferasas/genética , Animales , Drosophila melanogaster/efectos de los fármacos , Regulación de la Expresión Génica , Heterocigoto , Peróxido de Hidrógeno/toxicidad , Longevidad/genética , Masculino , Neuronas Motoras/fisiología , Paraquat/toxicidadRESUMEN
The cellular antioxidant defense systems neutralize the cytotoxic by-products referred to as reactive oxygen species (ROS). Among them, selenoproteins have important antioxidant and detoxification functions. The interference in selenoprotein biosynthesis results in accumulation of ROS and consequently in a toxic intracellular environment. The resulting ROS imbalance can trigger apoptosis to eliminate the deleterious cells. In Drosophila, a null mutation in the selD gene (homologous to the human selenophosphate synthetase type 1) causes an impairment of selenoprotein biosynthesis, a ROS burst and lethality. We propose this mutation (known as selDptuf) as a tool to understand the link between ROS accumulation and cell death. To this aim we have analyzed the mechanism by which selDptuf mutant cells become apoptotic in Drosophila imaginal discs. The apoptotic effect of selDptuf does not require the activity of the Ras/MAPK-dependent proapoptotic gene hid, but results in stabilization of the tumor suppressor protein Dmp53 and transcription of the Drosophila pro-apoptotic gene reaper (rpr). We also provide genetic evidence that the initiator caspase DRONC is activated and that the effector caspase DRICE is processed to commit selDptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selDptuf mutant cells. These observations indicate that selDptuf ROS-induced apoptosis in Drosophila is mainly driven by the caspase-dependent Dmp53/Rpr pathway.