Odorant receptors in axons of olfactory sensory neurons : in vitro studies in explant cultures

Olfaktorische Sinnesneurone (OSN), die mit einem bestimmten Odorantrezeptor (OR) ausgestattet sind, liegen weit verstreut im olfaktorischen Epithel (OE) der Nase und senden ihr Axon in gemeinsame Glomeruli im olfaktorischen Bulb (OB). In einem räumlich hochkonservierten Muster werden dort rezeptorspezifisch synaptische Kontakte mit nachgeschalteten Projektions- und Interneuronen hergestellt. Über die molekularen Mechanismen, die dieser höchstpräzisen Verdrahtung zugrunde liegen ist trotz intensiver Forschung noch immer wenig bekannt. Im Hinblick auf eine Aufklärung der komplexen Prozesse ist ein geeignetes experimentelles System erforderlich, das eine gezielte Manipulation der auswachsenden Axone ermöglicht. In der hier vorliegenden Arbeit wurde daher zunächst ein in vitro System mit Gewebeexplantaten des olfaktorischen Systems etabliert. Die Verwendung transgener Mauslinien, in denen spezifische Sinneszellpopulationen inklusive aller zellulären Ausläufer durch intrinsische Fluoreszenz visualisierbar sind, erlaubte eine kontinuierliche Beobachtung distinkter Axonpopulationen unter definierten und manipulierbaren Bedingungen. Zellen in Explantaten des OE, die zum Zeitpunkt des Embryonalstadiums 14 (E14) in Kultur gebracht wurden, entwickelten nach wenigen Tagen zahlreiche axonale Ausläufer, die radiär und unfaszikuliert aus den Explantaten auswuchsen. Die Explantate enthielten nach kurzer Kultivierung hauptsächlich Vorläuferzellen von olfaktorischen Sinneszellen; nach einigen Tagen konnten bereits zahlreiche reife Sinneszellen mit robustem Axonwachstum nachgewiesen werden. Durch den Einsatz einer rezeptorspezifischen transgenen Mauslinie konnte weiterhin die Expression distinkter OR Gene in Subpopulationen von Sinneszellen sichtbar gemacht werden. Die Kulturbedingungen erlaubten also die Differenzierung von Vorläuferzellen zu typischen olfaktorischen Sinnesneuronen mit charakteristischer Genexpression. Bezüglich der zentralen Frage zu Interaktionen zwischen olfaktorischen Axonen und ihrem Zielgewebe zeigten Kokultur-Experimente, dass ein Kontakt der Axone mit dem OB Gewebe zunächst eine repulsive Wirkung hatte. Erst eine Vorkultivierung des OB Gewebes resultierte in einer attraktiven Wirkung, wodurch die Axone sogar aus großen Entfernungen vom OB angezogen wurden. Die Anwesenheit des Bulbusgewebes hatte zudem einen positiven Einfluss auf die Wachstumsgeschwindigkeit der Axone. Dabei kam es zunächst zu einer starken Bündelung von Axonen, die in unmittelbarer Nähe des OB wieder in einzelne Fasern defaszikulierten. Diese Ergebnisse zeigten, dass mit dem in dieser Arbeit etablierten Explantatsystem charakteristische Parameter der Generierung olfaktorischer Sinneszellen, des Auswachsens von Axonen und der Interaktion der Axone mit ihrem Zielgewebe in vitro rekapituliert werden konnten. Somit wurden ideale Voraussetzungen dafür geschaffen, um zentrale Fragestellungen zu den molekularen Mechanismen zu bearbeiten, die der Etablierung der einzigartigen Verschaltung in diesem System zugrunde liegen. Die Explantatkulturen wurden im Folgenden dazu genutzt, die Rolle, die das olfaktorische Rezeptorprotein bei dem Prozess der olfaktorischen Wegfindung spielt, näher zu untersuchen. Durch Expression von genetisch modifizierten Varianten des Odorantrezeptors mOR256-17 in den Explataten konnten neue Einblicke in dessen subzelluläre Lokalisation gewonnen werden. Anhand eines mOR256-17-EGFP Fusionsproteins wurde ein vesikulärer Transport in die Dendriten der Sinneszellen sichtbar, welcher in einer Akkumulation des OR Proteins in den Cilien resultierte. Erstmals wurde mit dieser Technik das OR Protein in vesikulären Strukturen des Axons sichtbar, die anterograd und retrograd transportiert wurden. Der Rezeptor konnte in den Wachstumskegeln wachsender Axone und dort speziell in den Filopodien visualisiert werden. Mittels einer neuartigen Nachweismethode, mit der selektiv Proteine markiert werden können, die in der Plasmamembran lokalisiert sind, konnte ein retrograder Transport von internalisierten mOR256-17 Proteinen beobachtet werden. Die Generierung einer OR-Variante, bei der die Interaktionsdomäne des Rezeptors mit G-Proteinen mutiert wurde, resultierte in einer gestörten Verteilung des OR Proteins in den Sinneszellen. Insgesamt wurde mit dem in dieser Arbeit etablierten in vitro System ein entscheidendes Werkzeug geschaffen, mit dem bereits neue Einblicke in die Funktion distinkter molekularer Komponenten gewonnen wurde und dessen zukünftiger Einsatz zum weiteren Verständnis der komplexen Prozesse bei der Projektion olfaktorischer Sinneszellen beitragen kann.Olfactory sensory neurons (OSN) expressing a particular odorant receptor (OR) are widely scattered throughout the olfactory epithelium (OE) of the nose and send their axon into a small number of common glomeruli in the olfactory bulb (OB). In a spatially well conserved pattern these axons establish synaptic contacts to second order neurons. The molecular mechanisms underlying the precise wiring are still not well understood. To generate a system which may facilitate the investigation of distinct aspects of this complex process, an in vitro culture with tissue explants from the olfactory system was established in the present work. The use of tissues from transgenic mice which enabled the visualisation of OSN and their processes by intrinsic fluorescence allowed a continuous observation of distinct axonal populations under defined and manipulable conditions. Cells within an explant from the OE harvested at the embryonic stage 14 (E14) extended numerous axonal processes within a few days which grew out radially and without fasciculation. During the initial culture period the explants contained mainly progenitor cells; after several days in culture cells differentiated to OMP-positive, thus mature OSN. Using receptor specific transgenic mouse lines the expression of distinct OR genes in a subpopulation of OSN could be detected. Altogether, the culture conditions thus allowed the differentiation of progenitor cells into OSN with characteristic gene expression. Concerning the key question of how axons of OSN interact with their target tissue, co-culture experiments with OB tissue were performed; they showed that axons were initially repelled by their target. A precultivation of OB tissue, however, resulted in an attraction of axons even from larger distances. Moreover, the bulb tissue exerted a positive effect on the growth rate of OSN axons. During their growth these axons formed bundles which defasciculated in the vicinity of the OB explants. These results showed that characteristic parameters in the generation of OSN, their axonal growth and interactions with the target tissue were recapitulated by the in vitro culture system, thus, providing optimal conditions for the examination of key questions regarding the molecular mechanisms involved in establishing the unique projection pattern. Subsequently, the explant culture system was used to investigate the role of the odorant receptor protein in the process of path finding. Expression of genetically modified receptor variants in the explants revealed novel insights into the subcellular localisation of the odorant receptor mOR256-17. An mOR256-17-EGFP fusion protein could be detected in vesicles transported into the dendrite of OSN, resulting in an accumulation of the OR in the cilia. Using this technique it was possible to observe for the first time OR proteins in vesicles which were transported anterogradely and retrogradely along the entire axon. The OR could be visualised within the growth cones and the attached filopodia. Taking advantage of a novel detection method in which proteins integrated into the plasma membrane were selectively marked, retrogadely transported vesicles containing internalised mOR256-17 protein could be observed. The generation of an OR variant, in which the G-protein binding domain was mutated resulted in a disturbed localisation of the OR protein within OSN. Hence, by developing an improved in vitro explant system, an important tool was generated that allowed novel insights into the function of distinct molecular components and should be valuable for future studies aimed at understanding the complex processes that lead to the precise connection of OSN with their target

Reggies/flotillins regulate cytoskeletal remodeling during neuronal differentiation via CAP/ponsin and Rho GTPases

The reggies/flotillins were discovered as proteins upregulated during axon regeneration. Here, we show that expression of a trans-negative reggie-1/flotillin-2 deletion mutant, R1EA, which interferes with oligomerization of the reggies/flotillins, inhibited insulin-like growth factor (IGF)-induced neurite outgrowth in N2a neuroblastoma cells and impaired in vitro differentiation of primary rat hippocampal neurons. Cells expressing R1EA formed only short and broad membrane protrusions often with abnormally large growth cones. R1EA expression strongly perturbed the balanced activation of the Rho-family GTPases Rac1 and cdc42. Furthermore, focal adhesion kinase (FAK) activity was also enhanced by R1EA expression, while other signaling pathways like ERK1/2, PKC or PKB signaling were unaffected. These severe signaling defects were caused by an impaired recruitment of the reggie/flotillin-associated adaptor molecule CAP/ponsin to focal contacts at the plasma membrane. Thus, the reggies/flotillins are crucial for coordinated assembly of signaling complexes regulating cytoskeletal remodeling

Preformed reggie/flotillin caps : stable priming platforms for macrodomain assembly in T cells

T cell activation after contact with an antigen-presenting cell depends on the regulated assembly of the T cell receptor signaling complex, which involves the polarized assembly of a stable, raftlike macrodomain surrounding engaged T cell receptors. Here we show that the preformed reggie/flotillin caps present in resting T cells act as priming platforms for macrodomain assembly. Preformed reggie-1/flotillin-2 caps are exceptionally stable, as shown by fluorescence recovery after photobleaching (FRAP). Upon T cell stimulation, signaling molecules are recruited to the stable reggie/flotillin caps. Importantly, a trans-negative reggie-1/flotillin-2 deletion mutant, which interferes with assembly of the preformed reggie/flotillin cap, impairs raft polarization and macrodomain formation after T cell activation. Accordingly, expression of the trans-negative reggie-1 mutant leads to the incorrect positioning of the guanine nucleotide exchange factor Vav, resulting in defects in cytoskeletal reorganization. Thus, the preformed reggie/flotillin caps are stable priming platforms for the assembly of multiprotein complexes controlling actin reorganization during T cell activation

Trafficking of the microdomain scaffolding protein reggie-1/flotillin-2

The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrPc). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking

Engrailed 1 mediates correct formation of limb innervation through two distinct mechanisms.

Engrailed-1 (En1) is expressed in the ventral ectoderm of the developing limb where it plays an instructive role in the dorsal-ventral patterning of the forelimb. Besides its well-described role as a transcription factor in regulating gene expression through its DNA-binding domain, En1 may also be secreted to form an extracellular gradient, and directly impact on the formation of the retinotectal map. We show here that absence of En1 causes mispatterning of the forelimb and thus defects in the dorsal-ventral pathfinding choice of motor axons in vivo. In addition, En1 but not En2 also has a direct and specific repulsive effect on motor axons of the lateral aspect of the lateral motor column (LMC) but not on medial LMC projections. Moreover, an ectopic dorsal source of En1 pushes lateral LMC axons to the ventral limb in vivo. Thus, En1 controls the establishment of limb innervation through two distinct molecular mechanisms

Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification

During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Here, using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, we demonstrate that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. We provide evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, we observed major alterations in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, we identify Sip1 (Zeb2) as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision.status: publishe

Engrailed controls the dorsal-ventral choice of lateral LMC axons <i>in vivo</i>.

<p><b>(a)</b> An ectopic source of either En1, En2, or En1-SR was established by injecting the proteins into the dorsal mesenchyme of the developing chick wing. <b>(b)</b> Immunohistochemical detection of neurofilament (red) and EphA4 (green) visualizes the dorsal-ventral branching point and EphA4-positive LMCl axons. After injection of En1 into the dorsal wing bud more EphA4-positive axons are present in the ventral branch compared to mock (PBS) injection (arrows). <b>(c)</b> Quantification of axonal EphA4 signal results in a higher average EphA4 intensity in the ventral branch after En1 injection when compared to embryos where PBS was injected EphA4 signal was not altered significantly if En2 (28.75% ± 0.48 SEM, n = 4) or En1-SR (29.75% ± 1.25 SEM) were injected into the dorsal wing mesenchyme. <b>(d)</b> The expression patterns of the dorsal marker <i>Lmx1b</i> were not altered by injection of ectopic En1 into the dorsal wing. Scale bar in d equals 100μm for b and 250μm for d.</p

Lateral LMC axons are misguided in absence of En1.

<p><b>(a)</b> Retrograde tracing from dorsal forelimb mesenchyme labels motor neurons in the LMCl (Lim1, blue) and showed no significant increase of aberrantly projecting LMCm neurons (Isl1/2-positive, green) in <i>En1</i>^<i>-/-</i> mutant embryos when compared to wildtype littermates. <b>(b)</b> Retrograde tracing from ventral forelimb mesenchyme labels motor neurons in the LMCm (Isl1/2, blue) and revealed a significantly increased number of Lim1-positive LMCl motor neurons that aberrantly project to the ventral limb in <i>En1</i>^<i>-/-</i> mutant embryos (Lim1-positive, green and Rhodamine-positive, red). <b>(c)</b> Staining for neurofilament in E12.5 murine embryos shows bifurcation of limb innervating axons into a dorsal (yellow dashed line) and a ventral (cyan dashed line) branch. In <i>En1</i>^<i>-/-</i> mutant embryos, a significantly higher proportion of EphA4-positive axons is observed in the ventral branch, while in wildtypes EphA4 is largely restricted to the dorsally projecting branch. Scale bar in c equals 50μm for all panels.</p