The adhesion molecule TAG-1 is required for proper migration of the superficial migratory stream in the medulla but not of cortical interneurons

AbstractThe neural cell adhesion molecule TAG-1 has been implicated in the tangential migration of neurons of the caudal medulla and of cortical interneurons. In the former case, protein is expressed by the neurons as they migrate, and blocking its function results in altered and reduced migration in vitro. In the latter case, protein is expressed, in part, by the pathway the interneurons use to reach the cortex, and in vitro experiments propose a role for TAG-1 in this system, as well. However, the in vivo requirement of TAG-1 in these migrations has not been investigated. In this report, we analyze the developmental phenotype of TAG-1-deficient animals in these two migratory systems. We show that mutant mice have smaller lateral reticular nuclei as a result of increased cell death in the superficial migratory stream of the caudal medulla. On the other hand, the absence of TAG-1 does not affect the number, morphology, timing and routes of GABAergic interneurons that migrate from the ganglionic eminences to the cortex. Therefore, TAG-1 function is required for the survival of the neurons of some precerebellar nuclei, while it is not required for cortical interneuron migration in vivo

Subdomain-mediated axon-axon signaling and chemoattraction cooperate to regulate afferent innervation of the lateral habenula

A dominant feature of neural circuitry is the organization of neuronal projections and synapses into specific brain nuclei or laminae. Lamina-specific connectivity is controlled by the selective expression of extracellular guidance and adhesion molecules in the target field. However, how (sub)nucleus-specific connections are established and whether axon-derived cues contribute to subdomain targeting are largely unknown. Here, we demonstrate that the lateral subnucleus of the habenula (lHb) determines its own afferent innervation by sending out efferent projections that express the cell adhesion molecule LAMP to reciprocally collect and guide dopaminergic afferents to the lHb-a phenomenon we term subdomain-mediated axon-axon signaling. This process of reciprocal axon-axon interactions cooperates with lHb-specific chemoattraction mediated by Netrin-1, which controls axon target entry, to ensure specific innervation of the lHb. We propose that cooperation between pretarget reciprocal axon-axon signaling and subdomain-restricted instructive cues provides a highly precise and general mechanism to establish subdomain-specific neural circuitry

Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons

During corticogenesis, pyramidal neurons (~80% of cortical neurons) arise from the ventricular zone, pass through a multipolar stage to become bipolar and attach to radial glia[superscript 1, 2], and then migrate to their proper position within the cortex[superscript 1, 3]. As pyramidal neurons migrate radially, they remain attached to their glial substrate as they pass through the subventricular and intermediate zones, regions rich in tangentially migrating interneurons and axon fibre tracts. We examined the role of lamellipodin (Lpd), a homologue of a key regulator of neuronal migration and polarization in Caenorhabditis elegans, in corticogenesis. Lpd depletion caused bipolar pyramidal neurons to adopt a tangential, rather than radial-glial, migration mode without affecting cell fate. Mechanistically, Lpd depletion reduced the activity of SRF, a transcription factor regulated by changes in the ratio of polymerized to unpolymerized actin. Therefore, Lpd depletion exposes a role for SRF in directing pyramidal neurons to select a radial migration pathway along glia rather than a tangential migration mode.Ruth L. Kirschstein National Research Service Award (grant F32- GM074507)National Institutes of Health (U.S.) (grant # GM068678

A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion

Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon guidance defects in vivo. Thus, Slit locally stimulates directional filopodial extension, a process that is required for subsequent axonal repulsion downstream of the Robo receptor.National Institutes of Health (U.S.) (Grant F32-CA165700)National Institutes of Health (U.S.) (Grant R01-GM068678)National Institutes of Health (U.S.) (Grant P30-CA014051

Πρότυπο έκφρασης του γονιδίου Gt12 στο κεντρικό νευρικό σύστημα του ποντικού σε εμβρυικά στάδια ανάπτυξης και στο ενήλικο άτομο

Recent work has uncovered a large number of imprinted genes, many of which are thought to play a role in neurodevelopment and behavior. In order to begin to understand the role of specific genes in these processes, their expression patterns will be the key to their functional elucidation. In this study we used in situ hybridization to study the developmental expression of Gtl2 in the mouse central nervous system (CNS) from E10.5 to adulthood, since preliminary data from a microarray study indicated differential expression between the ventral and dorsal telencephalon of the mouse at a critical time point in the generation and migration of cortical neuronal populations. Strong expression was observed in the diencephalon, ventral telencephalon, post mitotic cell layers of the neocortex and pyramidal cell layer of the hippocampus.Πρόσφατες μελέτες, έχουν αποκαλύψει ένα μεγάλο αριθμό γενετικά αποτυπωμένων γονιδίων (genetically imprinted), πολλά από τα οποία θεωρείται ότι εμπλέκονται στην ανάπτυξη του νευρικού συστήματος και συνεπώς στη συμπεριφορά. Βασική προϋπόθεση για την κατανόηση του ρόλου τέτοιων γονιδίων, είναι η διερεύνηση του προτύπου έκφρασής τους. Μicroarray αναλύσεις έδειξαν ότι το γονίδιο Gtl2, το οποίο παρουσιάζει μητρική αποτύπωση (maternally imprinted) εμφανίζει διαφορετική έκφραση στον κοιλιακό και ραχιαίο τελεγκέφαλο σε αναπτυξιακά στάδια κρίσιμα για την γέννηση και μετανάστευση των νευρωνικών πληθυσμών του φλοιού του εγκεφάλου. Στη συγκεκριμένη μελέτη χρησιμοποιήθηκαν τεχνικές όπως η υβριδοποίηση in situ και η ανοσοϊστοχημεία, προκειμένου να μελετηθεί η έκφραση του συγκεκριμένου γονιδίου στο κεντρικό νευρικό σύστημα του ποντικού, από την εμβρυϊκή ημέρα 10,5, μέχρι τα ενήλικα στάδια. Έντονη έκφραση του Gtl2 παρατηρείται στο διεγκέφαλο, στην κοιλιακή περιοχή του τελεγκεφάλου, στις μεταμιτωτικές στιβάδες του νεοφλοιού και στην στιβάδα των πυραμιδικών κυττάρων του ιπποκάμπου

Intracellular and extracellular modultors of cortical interneuron development

Cortical GABAergic interneurons provide inhibitory input to the pyramidal cells and are characterized by extraordinary neurochemical and functional diversity. Their dysfunction and abnormal development have been associated with severe disorders, like autism, epilepsy and schizophrenia. Although recent studies have uncovered some of the molecular components underlying interneuron development, including the cellular and molecular mechanisms guiding their migration to the cortex, the intracellular components involved are still unknown. Rac1, a member of the Rac subfamily of Rho GTPases, has been implicated in various aspects of cortical development such as cell cycle dynamics, axonogenesis and neuronal migration. In this study we have addressed the specific role of Rac1 in interneuron progenitors originating in the medial ganglionic eminence, the subpallial source of GABAergic interneurons, via Cre/loxP technology. Our findings show that in the absence of Rac1, only half of GABAergic interneurons populate the cortex. Their progenitors are delayed in exiting the cell cycle and as a consequence migrate towards the pallium later than control cells. The absence of Rac1 from postmitotic interneurons does not result in obvious defects. These data indicate that a cell autonomous and stage-specific requirement of Rac1 activity within proliferating progenitors of cortical interneurons is necessary for their development. We have also assessed the extracellular signals that facilitate interneuron migration to the cortex during development. TAG1, a cell adhesion molecule of the IgSF, is expressed by the corticofugal fibers that are thought to serve as a scaffold for interneurons to migrate on, during embryogenesis. Using the promoter of the gene encoding for TAG1 as a genetic tool, we have generated two transgenic mouse lines, to help us address questions regarding the role of corticofugal axons in interneuronal migration.Οι ενδονευρώνες του φλοιού των εγκεφαλικών ημισφαιρίων διαδραματίζουν βασικό ρόλο στα νευρικά κυκλώματα του φλοιού, δημιουργώντας ανασταλτικές συνάψεις με τους πυραμιδικούς νευρώνες. Χαρακτηρίζονται από αξιοθαύμαστη ποικιλομορφία, τόσο ως προς τα μοριακά, όσο και ως προς τα ηλεκτροφυσιολογικά και λειτουργικά χαρακτηριστικά τους και η δυσλειτουργία τους έχει εμπλακεί σε ποικίλες παθολογικές καταστάσεις, όπως ο αυτισμός, η επιληψία και η σχιζοφρένεια. Ωστόσο, τα ενδοκυττάρια και εξωκυττάρια σήματα που μετέχουν στην ανάπτυξη των κυττάρων αυτών, παραμένουν εν πολλοίς άγνωστα. Η πρωτεΐνη Rac1, μέλος της οικογένειας των Rho-GTPασών, εμπλέκεται σε πολλαπλές διεργασίες κατά την ανάπτυξη του νευρικού συστήματος, όπως στη δημιουργία νευραξόνων, στην κυτταρική μετανάστευση και στη ρύθμιση του κυτταρικού κύκλου. Στην παρούσα διατριβή, μελετήθηκε ο ρόλος της Rac1 στην ανάπτυξη των ενδονευρώνων του φλοιού. Η πρωτεΐνη απενεργοποιήθηκε in vivo σε μύες, μέσω της τεχνολογίας του Cre/loxP ανασυνδυασμού, μόνο στο συγκεκριμένο κυτταρικό πληθυσμό. Από την ανάλυση των μεταλλαγμένων ζώων, προκύπτει ότι τα κύτταρα καθυστερούν να εξέλθουν του κυτταρικού κύκλου. Κατά συνέπεια, υπάρχει μειωμένη μετανάστευση ενδονευρώνων προς το φλοιό, ενώ τελικά στον ώριμο εγκέφαλο, εντοπίζονται μόνο οι μισοί ενδονευρώνες. Τα αποτελέσματα αυτά οδηγούν στο συμπέρασμα ότι η Rac1 είναι απαραίτητη για την ανάπτυξη των ενδονευρώνων, ρυθμίζοντας την ομαλή έκβαση του κυτταρικού κύκλου τους. Σε πειράματα στα οποία η Rac1 απενεργοποιήθηκε in vivo, ειδικά σε μεταμιτωτικούς ενδονευρώνες, η απώλεια της πρωτεΐνης δεν επηρεάζει την ανάπτυξη των κυττάρων, υπογραμμίζοντας την ανάγκη για λειτουργική Rac1 στα πρόδρομα μιτωτικά νευρικά κύτταρα. Στα πλαίσια της συγκεκριμένης εργασίας, μελετήθηκαν και εξωκυττάρια καθοδηγητικά σήματα κατά την ανάπτυξη των ενδονευρώνων του φλοιού. Η πρωτεΐνη TAG1, μόριο κυτταρικής συνάφειας της οικογένειας των ανοσοσφαιρινών, εκφράζεται στους φλοιοθαλαμικούς άξονες κατά την εμβρυογένεση. Οι άξονες αυτοί θεωρούνται υπόστρωμα πάνω στο οποίο μετακινούνται οι οριζόντια μεταναστεύοντες ενδονευρώνες, στην πορεία τους προς το φλοιό. Χρησιμοποιώντας τον υποκινητή του γονιδίου της TAG1 ως γενετικό εργαλείο, δημιουργήθηκαν δύο σειρές γενετικά τροποποιημένων μυών, προκειμένου να μελετηθεί ο ρόλος των φλοιοθαλαμικών αξόνων στην καθοδήγηση των ενδονευρώνων

The absence of Rac1 and Rac3 significantly affects actin-microtubule dynamics in developing cortical interneurons.

The proper function of the CNS requires the correct integration of glutamatergic neurons and GABAergic interneurons. Cortical GABAergic interneurons are characterized by extraordinary neurochemical and functional diversity. Although recent studies have uncovered some of the molecular components underlying interneuron development, including the cellular and molecular mechanisms guiding their migration to the cortex, the intracellular components involved in these processes are still unknown. Rac-proteins are RhoGTPases that integrate multiple extracellular signals required for essential processes in diverse cell types as cytoskeleton organization, vesicle trafficking, transcription, cell cycle progression, and apoptosis. We examined the role of the ubiquitous Rac1 and neural-specific Rac3 in interneurons derived from the medial ganglionic eminence (MGE). Previously we used Cre/loxP technology to uncover a cell autonomous and stage-specific requirement for Rac1 activity within proliferating interneurons. Most Rac1 conditional mutant mice die after 3-6 weeks due to epileptic seizures as a result of the presence of 50% of GABAergic interneurons postnatally. In addition, Rac1 mutant MGE cells in vitro show cytoskeletal alterations in growth cone formation and a significant reduction of the leading process length (Vidaki et al., 2012). However, the simultaneous absence of Rac1 and Rac3 results not only in additive but also distinctive defects. Double mutant mice die earlier and display a dramatic (80%) loss of cortical interneurons, evident from embryonic stages. In addition to the Rac1 specific defects, Rac1/Rac3-deficient interneurons show gross cytoskeletal defects in vitro, with a prominent polarity-related defect (Tivodar et al., 2014). Stabilization of mictrotubules improves neuronal growth and polarity. We propose that in the absence of Rac1/Rac3 cortical interneurons fail to tangentially migrate towards the pallium due to defects in actin-microtubule cytoskeletal dynamics that we are currently analyzing

A Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons

During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local translation. Keywords: Mena; ENAH; Ena/VASP; developing axon; growth cone; axon guidance; local translation; ribonucleoprotein; Dyrk1aNational Institutes of Health (U.S.) (Grant U01-CA184897)National Cancer Institute (U.S.) (Grant P30-CA14051