Carlos Oliva

Role of the maguk protein family in synapse formation and function

Synaptic function is crucially dependent on the spatial organization of the presynaptic and postsynaptic apparatuses and the juxtaposition of both membrane compartments. This precise arrangement is achieved by a protein network at the submembrane region of each cell that is built around scaffold proteins. The membrane‐associated guanylate kinase (MAGUK) family of proteins is a widely expressed and well‐conserved group of proteins that plays an essential role in the formation and regulation of this scaffolding. Here, we review general features of this protein family, focusing on the discs large and calcium/calmodulin‐dependent serine protein kinase subfamilies of MAGUKs in the formation, function, and plasticity of synapses.

The Specification of Cortical Subcerebral Projection Neurons Depends on the Direct Repression of TBR1 by CTIP1/BCL11a

The acquisition of distinct neuronal fates is fundamental for the function of the cerebral cortex. We find that the development of subcerebral projections from layer 5 neurons in the mouse neocortex depends on the high levels of expression of the transcription factor CTIP1; CTIP1 is coexpressed with CTIP2 in neurons that project to subcerebral targets and with SATB2 in those that project to the contralateral cortex. CTIP1 directly represses Tbr1 in layer 5, which appears as a critical step for the acquisition of the subcerebral fate. In contrast, lower levels of CTIP1 in layer 6 are required for TBR1 expression, which directs the corticothalamic fate. CTIP1 does not appear to play a critical role in the acquisition of the callosal projection fate in layer 5. These findings unravel a key step in the acquisition of cell fate for closely related corticofugal neurons and indicate that differential dosages of transcriptions factors are critical to specify different neuronal identities.

Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001

Proper connectivity of Drosophila motion detector neurons requires Atonal function in progenitor cells

BackgroundVertebrates and invertebrates obtain visual motion information by channeling moving visual cues perceived by the retina through specific motion sensitive synaptic relays in the brain. In Drosophila, the series of synaptic relays forming the optic lobe are known as the lamina, medulla, lobula and lobula plate neuropiles. The fly’s motion detection output neurons, called the T4 and T5 cells, reside in the lobula plate. Adult optic lobe neurons are derived from larval neural progenitors in two proliferating compartments known as the outer and inner proliferation centers (OPC and IPC). Important insight has been gained into molecular mechanisms involved in the development of the lamina and medulla from the OPC, though less is known about the development of the lobula and lobula plate.ResultsHere we show that the proneural gene Atonal is expressed in a subset of IPC progenitors that give rise to the higher order motion detection neurons, T4 and T5, of the lobula plate. We also show that Atonal does not act as a proneural gene in this context. Rather, it is required specifically in IPC neural progenitors to regulate neurite outgrowth in the neuronal progeny.ConclusionsOur findings reveal that a proneural gene is expressed in progenitors but is required for neurite development of their progeny neurons. This suggests that transcriptional programs initiated specifically in progenitors are necessary for subsequent neuronal morphogenesis.

Regulation of axonal development by the nuclear protein hindsight (pebbled) in the Drosophila visual system.

The molecules and networks involved in the process of acquisition and maintenance of the form of a mature neuron are not completely known. Using a misexpression screen we identified the gene hindsight as a gene involved in the process of acquisition of the neuronal morphogenesis in the Drosophila adult nervous system. hindsight encodes a transcription factor known for its role in early developmental processes such as embryonic germ band retraction and dorsal closure, as well as in the establishment of cell morphology, planar cell polarity, and epithelial integrity during retinal development. We describe here a novel function for HNT by showing that both loss and gain of function of HNT affects the pathfinding of the photoreceptors axons. By manipulating the timing and level of HNT expression, together with the number of cells manipulated we show here that the function of HNT in axonal guidance is independent of the HNT functions previously reported in retinal cells. Based on genetic interaction experiments we show that part of HNT function in axonal development is exerted through the regulation of genes involved in the dynamics of the actin cytoskeleton.

Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex

Summary The axonal wiring molecule Slit and its Round-About (Robo) receptors are conserved regulators of nerve cord patterning. Robo receptors also contribute to wiring brain circuits. Whether molecular mechanisms regulating these signals are modified to fit more complex brain wiring processes is unclear. We investigated the role of Slit and Robo receptors in wiring Drosophila higher-order brain circuits and identified differences in the cellular and molecular mechanisms of Robo/Slit function. First, we find that signaling by Robo receptors in the brain is regulated by the Receptor Protein Tyrosine Phosphatase RPTP69d. RPTP69d increases membrane availability of Robo3 without affecting its phosphorylation state. Second, we detect no midline localization of Slit during brain development. Instead, Slit is enriched in the mushroom body, a neuronal structure covering large areas of the brain. Thus, a divergent molecular mechanism regulates neuronal circuit wiring in the Drosophila brain, partly in response to signals from the mushroom body.

Hindsight regulates photoreceptor axon targeting through transcriptional control of jitterbug/Filamin and multiple genes involved in axon guidance in Drosophila

During axon targeting, a stereotyped pattern of connectivity is achieved by the integration of intrinsic genetic programs and the response to extrinsic long and short‐range directional cues. How this coordination occurs is the subject of intense study. Transcription factors play a central role due to their ability to regulate the expression of multiple genes required to sense and respond to these cues during development. Here we show that the transcription factor HNT regulates layer‐specific photoreceptor axon targeting in Drosophila through transcriptional control of jbug/Filamin and multiple genes involved in axon guidance and cytoskeleton organization.Using a microarray analysis we identified 235 genes whose expression levels were changed by HNT overexpression in the eye primordia. We analyzed nine candidate genes involved in cytoskeleton regulation and axon guidance, six of which displayed significantly altered gene expression levels in hnt mutant retinas. Functional analysis confirmed the role of OTK/PTK7 in photoreceptor axon targeting and uncovered Tiggrin, an integrin ligand, and Jbug/Filamin, a conserved actin‐ binding protein, as new factors that participate of photoreceptor axon targeting. Moreover, we provided in silico and molecular evidence that supports jbug/Filamin as a direct transcriptional target of HNT and that HNT acts partially through Jbug/Filamin in vivo to regulate axon guidance. Our work broadens the understanding of how HNT regulates the coordinated expression of a group of genes to achieve the correct connectivity pattern in the Drosophila visual system.

Temporal and spatial expression of Drosophila DLGS97 during neural development

The products of the Drosophila discs-large (dlg) gene are members of the MAGUK family of proteins, a group of proteins involved in localization, transport and recycling of receptors and channels in cell junctions, including the synapse. In vertebrates, four genes with multiple splice variants homologous to dlg are described. dlg originates two main proteins, DLGA, similar to the vertebrate neuronal protein PSD95, and DLGS97, similar to the vertebrate neuronal and epithelial protein SAP97. DLGA is expressed in epithelia, neural tissue and muscle. DLGS97 is expressed in neural tissue and muscle but not in epithelia. The distinctive difference between them is the presence in DLGS97 of an L27 domain. The differential expression between these variants makes the study of DLGS97 of key relevance to understand the in vivo role of synaptic MAGUKs in neurons. Here we present the temporal and spatial expression pattern of DLGS97 during embryonic and larval nervous system development, during eye development and in adult brain. Our results show that DLGS97 is expressed zygotically, in neurons in the embryo, larvae and adult, and is absent at all stages in glial cells. During eye development DLGS97 starts to be expressed in photoreceptor cells at early stages of differentiation and localizes basal to the basolateral junctions. In the brain, DLGS97 is expressed in the mushroom bodies and optic lobes at larval and adult stages; and in the antennal lobe in the adult stage. In addition we show that both, dlgS97 and dlgA transcripts, express during development multiple splice variants with differences in the use of exons in two sites.

Chapter Twelve – Receptor Tyrosine Kinases and Phosphatases in Neuronal Wiring: Insights From Drosophila

Tyrosine phosphorylation is at the crossroads of many signaling pathways. Brain wiring is not an exception, and several receptor tyrosine kinases (RTKs) and tyrosine receptor phosphates (RPTPs) have been involved in this process. Considerable work has been done on RTKs, and for many of them, detailed molecular mechanisms and functions in several systems have been characterized. In contrast, RPTPs have been studied considerably less and little is known about their ligands and substrates. In both families, we find redundancy between different members to accomplish particular wiring patterns. Strikingly, some RTKs and RPTPs have lost their catalytic activity during evolution, but not their importance in biological processes. In this regard, we have to keep in mind that these proteins have multiple domains and some of their functions are independent of tyrosine phosphorylation/dephosphorylation. Since RTKs and RPTPs are enzymes involved not only in early stages of axon and dendrite pathfinding but also in synapse formation and physiology, they have a potential as drug targets. Drosophila has been a key model organism in the search of a better understanding of brain wiring, and its sophisticated toolbox is very suitable for studying the function of genes with pleiotropic functions such as RTKs and RPTPs, from wiring to synaptic formation and function. In these review, we mainly cover findings from this model organism and complement them with discoveries in vertebrate systems.

Receptor Tyrosine Kinases and Phosphatases in Neuronal Wiring

Tyrosine phosphorylation is at the crossroads of many signaling pathways. Brain wiring is not an exception, and several receptor tyrosine kinases (RTKs) and tyrosine receptor phosphates (RPTPs) have been involved in this process. Considerable work has been done on RTKs, and for many of them, detailed molecular mechanisms and functions in several systems have been characterized. In contrast, RPTPs have been studied considerably less and little is known about their ligands and substrates. In both families, we find redundancy between different members to accomplish particular wiring patterns. Strikingly, some RTKs and RPTPs have lost their catalytic activity during evolution, but not their importance in biological processes. In this regard, we have to keep in mind that these proteins have multiple domains and some of their functions are independent of tyrosine phosphorylation/dephosphorylation. Since RTKs and RPTPs are enzymes involved not only in early stages of axon and dendrite pathfinding but also in synapse formation and physiology, they have a potential as drug targets. Drosophila has been a key model organism in the search of a better understanding of brain wiring, and its sophisticated toolbox is very suitable for studying the function of genes with pleiotropic functions such as RTKs and RPTPs, from wiring to synaptic formation and function. In these review, we mainly cover findings from this model organism and complement them with discoveries in vertebrate systems.