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Dendrite enlightenment
No full textNeuronal dendrites acquire complex morphologies during development. These are not just the product of cell-intrinsic developmental programs; rather they are defined in close interaction with the cellular environment. Thus, to understand the molecular cascades that yield appropriate morphologies, it is essential to investigate them in vivo, in the actual complex tissue environment encountered by the differentiating neuron in the developing animal. Particularly, genetic approaches have pointed to factors controlling dendrite differentiation in vivo. These suggest that localized and transient molecular cascades might underlie the formation and stabilization of dendrite branches with neuron type-specific characteristics. Here, I highlight the need for studies of neuronal dendrite differentiation in the animal, the challenges provided by such an approach, and the promising pathways that have recently opened
Modulators of hormonal response regulate temporal fate specification in the Drosophila brain
No full textNeuronal diversity is at the core of the complex processing operated by the nervous system supporting fundamental functions such as sensory perception, motor control or memory formation. A small number of progenitors guarantee the production of this neuronal diversity, with each progenitor giving origin to different neuronal types over time. How a progenitor sequentially produces neurons of different fates and the impact of extrinsic signals conveying information about developmental progress or environmental conditions on this process represent key, but elusive questions. Each of the four progenitors of the Drosophila mushroom body (MB) sequentially gives rise to the MB neuron subtypes. The temporal fate determination pattern of MB neurons can be influenced by extrinsic cues, conveyed by the steroid hormone ecdysone. Here, we show that the activation of Transforming Growth Factor-β (TGF-β) signalling via glial-derived Myoglianin regulates the fate transition between the early-born α’β’ and the pioneer αβ MB neurons by promoting the expression of the ecdysone receptor B1 isoform (EcR-B1). While TGF-β signalling is required in MB neuronal progenitors to promote the expression of EcR-B1, ecdysone signalling acts postmitotically to consolidate theα’β’ MB fate. Indeed, we propose that if these signalling cascades are impaired α’β’ neurons lose their fate and convert to pioneer αβ. Conversely, an intrinsic signal conducted by the zinc finger transcription factor Krüppel-homolog 1 (Kr-h1) antagonises TGF-β signalling and acts as negative regulator of the response mediated by ecdysone in promoting α’β’ MB neuron fate consolidation. Taken together, the consolidation of α’β’ MB neuron fate requires the response of progenitors to local signalling to enable postmitotic neurons to sense a systemic signal.</div
Circuit reorganization in the Drosophila mushroom body calyx accompanies memory consolidation
No full textThe anterior paired lateral neuron normalizes odour-evoked activity at the mushroom body calyx
No full textThe anterior paired lateral neuron normalizes odour-evoked activity in the Drosophila mushroom body calyx
No full textTo identify and memorize discrete but similar environmental inputs, the brain needs to distinguish between subtle differences of activity patterns in defined neuronal populations. The Kenyon cells (KCs) of the Drosophila adult mushroom body (MB) respond sparsely to complex olfactory input, a property that is thought to support stimuli discrimination in the MB. To understand how this property emerges, we investigated the role of the inhibitory anterior paired lateral (APL) neuron in the input circuit of the MB, the calyx. Within the calyx, presynaptic boutons of projection neurons (PNs) form large synaptic microglomeruli (MGs) with dendrites of postsynaptic KCs. Combining electron microscopy (EM) data analysis and in vivo calcium imaging, we show that APL, via inhibitory and reciprocal synapses targeting both PN boutons and KC dendrites, normalizes odour-evoked representations in MGs of the calyx. APL response scales with the PN input strength and is regionalized around PN input distribution. Our data indicate that the formation of a sparse code by the KCs requires APL-driven normalization of their MG postsynaptic responses. This work provides experimental insights on how inhibition shapes sensory information representation in a higher brain centre, thereby supporting stimuli discrimination and allowing for efficient associative memory formation
Ceramide Synthase in Transcriptional Regulation and Lipid Sensing
Full text linkCeramide synthases (CerS) are highly conserved transmembrane proteins necessary for sphingolipid biosynthesis. CerSs carry a TLC (Tram-Lag1-CLN8) domain, containing a conserved Lag1p motif required for ceramide synthesis. Many of the CerS enzymes also contain a homeodomain. Although homeodomain proteins are known to regulate transcription via DNA binding, the homeodomain function of CerSs is frequently not investigated and previous studies doubted that it could act as a transcription factor. Recently, it has been shown that the Drosophila homologous of CerS called "Schlank", is not only involved in sphingolipid biosynthesis but also in the regulation of body fat. Moreover, the regulation of body fat is independent of CerS activity. Besides localizing at the endoplasmic reticulum, as each mammalian CerSs, Schlank also localizes at the inner nuclear membrane (INM). Two nuclear localization sites (NLS1, NLS2) are necessary for the nuclear localization of the protein. In order to distinguish the homeodomain function from the catalytic one, in vivo homeodomain animal models were generated and analyzed. The mutation in the NLS2 site severely affected lipolysis and development, demonstrating an essential function of the Schlank homeodomain in the regulation of body fat metabolism and growth. This work shows that Schlank binds the promoter regions of lipases (e.g., lip3) via the homeodomain and directly regulates transcription. Mutations of the NLS2 site led to loss of DNA binding and deregulated gene expression. This mechanism seems to be conserved in mammalian CerS2. Furthermore, Schlank adjusts transcriptional regulation according to the energy status. Upon starvation, nuclear localization and consequently DNA binding diminish. On the other hand, Schlank responds to changing fatty acid levels (essential molecules in sphingolipid biosynthesis), increasing nuclear localization and regulating gene expression. This study demonstrates a double function of CerS Schlank as an enzyme and a transcriptional regulator, sensing lipid levels and transducing the information to the level of gene expression. This function is required to adjust and maintain lipid homeostasis.Ceramid-Synthasen (CerS) sind hochkonservierte Transmembranproteine, die für die Sphingolipid-Biosynthese notwendig sind. CerSs tragen eine TLC (Tram-Lag1-CLN8)-Domäne, die ein konserviertes Lag1p-Motiv enthält, das für die Ceramidsynthese erforderlich ist. Viele der CerS-Enzyme enthalten auch eine Homöodomäne. Obwohl Homöodomain-Proteine bekanntermaßen die Transkription über die DNA-Bindung regulieren, ist die Homöodomain-Funktion von CerSs nicht genau untersucht und frühere Studien bezweifelten sogar, dass sie als Transkriptionsfaktor fungieren könnte. In jüngster Zeit konnte gezeigt werden, dass das Drosophila-Homolog namens "Schlank" nicht nur in der Sphingolipidbiosynthese, sondern auch in der Regulation von Körperfett involviert ist. Die Regulierung des Körperfettanteils ist zudem unabhängig von der CerS-Aktivität. Neben der Lokalisierung am Endoplasmatischen Retikulum, wie auch die Säuger CerS, ist Schlank auch an der inneren Kernmembran (INM) lokalisiert. Zwei nukleare Lokalisierungsstandorte (NLS1, NLS2) sind für die nukleare Lokalisierung des Proteins notwendig. Um die Homöodomänenfunktion von der katalytischen zu unterscheiden, wurden in vivo Homöodomänen-Fliegenmodelle generiert und analysiert. Die Mutation am NLS2-Standort wirkte sich stark auf die Lipolyse und Entwicklung aus und demonstrierte eine wesentliche Funktion der Schlank-Heimatdomäne bei der Regulierung des Körperfettstoffwechsels und des Wachstums. Diese Arbeit zeigt, dass Schlank die Promotor-Regionen von Lipasen (z.B. Lip3) über die Homöodomäne bindet und die Transkription direkt reguliert. Mutationen des NLS2-Standortes führten zum Verlust der DNA-Bindung und deregulierten Genexpression. Dieser Mechanismus scheint im Säuger CerS2 konserviert zu sein. Darüber hinaus passt Schlank die Transkriptionsregulation an den Energiestatus an. Werden die Fliegen gehungert nimmt die nukleare Lokalisation und damit die Bindung an die DNA ab. Auf der anderen Seite reagiert Schlank auf sich ändernde Fettsäurespiegel (essentielle Moleküle in der Sphingolipidbiosynthese), erhöht die nukleare Lokalisation und reguliert die Genexpression. Diese Studie demonstriert eine Doppelfunktion von CerS Schlank als Enzym und Transkriptionsregulator, indem sie den Lipidspiegel misst und die Informationen auf die Ebene der Genexpression transformiert. Diese Funktion ist erforderlich, um die Lipid-Homöostase zu regulieren und aufrechtzuerhalten
Augmin complex components control branching of sensory neuron dendrites in <em>Drosophila</em> larvae
Full text linkMicrotubule is the major architectural element to support proper neuronal structure. It is tightly organized with intrinsic polarity and affects not only neuronal morphology but also the transport property within the cell. In many cell types, the centrosome component γ-tubulin is the principal microtubule nucleator. However, the mechanism underlying neuronal microtubule nucleation and organization remains unknown. During neuronal development, the centrosome is inactivated and microtubule nucleation becomes acentrosomal. Whether the microtubule centrosomal-independent nucleation contributes to the establishment of polarity in neurons remains unclear and essential to answer. The purpose of this work is to reveal whether Augmin mediated microtubule nucleation plays a role in building up proper dendritic morphology and organizing dendritic microtubule polarity. To this purpose, I analyzed the dendrite morphology of class IV ddaC da neurons in Drosophila larvae carrying mutations for γ-tubulin and Augmin. I found that dendritic morphology and dendrite branch dynamics were changed in γ-tubulin, dgt5, dgt6 (Augmin) and Dgp71WD (γ-TuRC) mutants. Interestingly, the phenotypes of these various mutants were similar, suggesting the possibility that they might act in concert. To test this possibility, I performed genetic interaction experiments between γtub23C, dgt5, dgt6 and Dgp71WD and found these molecules play coordinate roles in dendrite morphology. In Augmin complex mutant neurons, the localization of fluorescently tagged α-tubulin and the microtubule minusend marker Nod were both altered, suggesting a role of Augmin in microtubule organization in these neurons. Taken together, my work suggests a role of the Augmin complex in the proper organization of microtubules in neuronal dendrites, which is important for achieving dendritic complexity in Drosophila PNS class IV da neuron
Perception and memories in the fly brain
Full text linkThe brain is an extremely complex organ that controls thoughts, memories, motor skills and every process that is required to maintain our body alive and healthy. Although this is common knowledge, the mechanisms by which the brain performs these tasks are currently not fully understood.
In this thesis, I focused on how sensory information is represented in higher brain regions, and how these representations are used to create and consolidate memories related to them. Precisely, using Drosophila melanogaster as a model, I investigated the circuitry of the mushroom body calyx, the input region of a neuropil involved in stimuli discrimination and memory formation in the fly brain. In the calyx, olfactory projection neurons synapse onto mushroom body intrinsic cells, the Kenyon cells, via synaptic complexes known as microglomeruli. Each microglomerulus is a microcircuit of his own, constituted by a central projection neuron presynaptic bouton surrounded by several dendritic endings of different Kenyon cells. This structural organization is believed to facilitate stimuli discrimination by transforming highly overlapping representations at the level of the projection neurons into sparse, decorrelated responses at the Kenyon cells one. Moreover, structural changes at the microglomerular level following associative memory formation have been reported in insect brains over the years. Nevertheless, the exact processes underlying such phenomena have not been described yet.
Here, I show that memory consolidation induces structural plasticity in a stimulus-specific way in the calyx. Specifically, I found that the microglomeruli involved in the representation of the stimulus presented in the behavioural task increased in number after long-term memory formation. This increase in microglomeruli was protein synthesis dependent and strictly linked to the consolidation of the memory, as control flies and mutants unable to consolidate memories did not show structural changes within the same time frame.
Furthermore, in this thesis I analyse the role of inhibitory synapses in microglomeruli of the calyx. Inhibition at the mushroom bodies is provided by the APL neuron, whose presence is required to maintain Kenyon cells odour responses sparse, hence facilitating odours discrimination in the fly. Here, I show that via inhibitory and reciprocal synapses targeting both projection neurons boutons and Kenyon cells dendrites, APL normalizes odour-evoked representations in microglomeruli of the calyx. In particular, I observed that APL inhibition scaled with the inputs strength and localized to the regions where those inputs were located within the calyx, leading to more homogenous responses in Kenyon cells dendrites. I confirmed this hypothesis by inhibiting output from the APL, which led to more variable activities in Kenyon cells dendrites.
Altoghether, this thesis provides insights on how stimuli are processed, represented and used to create associative memories in the fly brain. As similar network organizations can be found in brains of other species including humans, I believe that the principles here described can be potentially applied to all brain regions sharing conformational features with the Drosophila mushroom body
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