Rostral growth of commissural axons requires the cell adhesion molecule MDGA2

Background: Long-distance axonal growth relies on the precise interplay of guidance cues and cell adhesion molecules. While guidance cues provide positional and directional information for the advancing growth cone, cell adhesion molecules are essential in enabling axonal advancement. Such a dependence on adhesion as well as guidance molecules can be well observed in dorsal commissural interneurons, which follow a highly stereotypical growth and guidance pattern. The mechanisms and molecules involved in the attraction and outgrowth towards the ventral midline, the axon crossing towards the contralateral side, the rostral turning after midline crossing as well as the guidance along the longitudinal axis have been intensely studied. However, little is known about molecules that provide the basis for commissural axon growth along the anterior-posterior axis. Results: MDGA2, a recently discovered cell adhesion molecule of the IgCAM superfamily, is highly expressed in dorsolaterally located (dI1) spinal interneurons. Functional studies inactivating MDGA2 by RNA interference (RNAi) or function-blocking antibodies demonstrate that either treatment results in a lack of commissural axon growth along the longitudinal axis. Moreover, results from RNAi experiments targeting the contralateral side together with binding studies suggest that homophilic MDGA2 interactions between ipsilaterally projecting axons and post-crossing commissural axons may be the basis of axonal growth along the longitudinal axis. Conclusions: Directed axonal growth of dorsal commissural interneurons requires an elaborate mixture of instructive (guidance) and permissive (outgrowth supporting) molecules. While Wnt and Sonic hedgehog (Shh) signalling pathways have been shown to specify the growth direction of post-crossing commissural axons, our study now provides evidence that homophilic MDGA2 interactions are essential for axonal extension along the longitudinal axis. Interestingly, so far each part of the complex axonal trajectory of commissural axons uses its own set of guidance and growth-promoting molecules, possibly explaining why such a high number of molecules influencing the growth pattern of commissural interneurons has been identified

Wnt signaling in commissural axon guidance

The population of dorsal commissural interneurons is a favoured model system to study the molecular mechanisms of axon guidance. The initial dorsal-to-ventral trajectory of commissural axons is well understood. However, the navigation of commissural axons after crossing the midline, when they abruptly turn into the longitudinal axis and extend towards the brain, is still poorly understood. Two distinct model organisms revealed two different molecular cues that guide these axons in the longitudinal axis. Wnt family members attract mouse commissural axon rostrally, while Sonic hedgehog (Shh) pushes chicken commissural axons towards the brain. We could show that the role of Wnt proteins is conserved in the chick. The molecular mechanism of Wnt action, however, shows peculiarities not seen in the mouse. Expression analysis of Wnt genes led to three candidates, Wnt4, Wnt5a, and Wnt7a, which were further characterized. Loss of function and gain of function experiments revealed that Wnt5a and Wnt7a, but not Wnt4, are commissural axon guidance cues. This is in contrast to mouse, where Wnt4 is the major guidance force. But more importantly, Wnt transcripts were distributed evenly in the chick, while displaying gradients in the mouse. Secreted frizzled- related proteins are known Wnt antagonists. We could show that Sfrp1 is expressed in a gradient and is able to modulate Wnt activity. Downregulation and upregulation of Sfrp1 phenocopies guidance errors seen after modulation of Wnt expression. Therefore, a Wnt activity gradient, shaped by Sfrp, guides postcrossing commissural axons in the chicken spinal cord. Die Zellpopulation der dorsalen Kommissuralneurone ist ein beliebtes Modellsystem, um die molekularen Mechanismen der axonalen Wegleitung zu studieren. Das initiale, dorso-ventral gerichtete Wachstum der Kommissuralaxone ist relative gut untersucht. Nachdem die Axone die embryonale Mittellinie überquert haben, wachsen sie, nach einem abrupten Richtungswechsel, in Richtung Gehirn. Über diesen Prozess ist noch wenig bekannt. Zwei verschiedene Modellorganismen haben zwei unterschiedliche molekulare Mechanismen für die Wegleitung in der Längsachse zu Tage gefördert. In der Maus werden Kommissuralaxone von Proteinen der Wnt Familie kopfwärts gezogen, während im Huhn dieselben Axone von Sonic hedgehog (Shh) Richtung Kopf gestossen werden. Wir konnten zeigen, dass die Rolle der Wnt Proteine im Huhn und in der Maus vergleichbar sind. Die molekularen Mechanismen sind jedoch unterschiedlich. Die Analyse der Expressionsmuster verschiedener Wnt Gene führte zu drei Kandidaten, Wnt4, Wnt5a und Wnt7a, welche weiter charakterisiert wurden. Funktionelle Experimente demonstrierten, dass Wnt5a und Wnt7a, aber nicht Wnt4, eine Rolle in der axonalen Wegleitung haben. Dies steht im Gegensatz zur Maus, in welcher Wnt4 diese Funktion übernimmt. Noch schwerwiegender ist jedoch, dass Wnt Transkripte im Huhn gleichmässig verteilt waren, während in der Maus Transkriptionsgradienten gefunden wurden. Wir konnten zeigen, dass der Wnt Antagonist Sfrp1 in einem Gradienten exprimiert ist und die Wnt Aktivität regulieren kann. Modulation der Sfrp1 Expression führte zu ähnlichen Phänotypen wie wir sie bei den Wnt Proteinen gesehen haben. Wir schliessen daraus, dass im Rückenmark des Huhnes ein Wnt Aktivitätsgradient, geformt durch die Wirkung von Sfrp, die Wegfindung der Kommissuralaxone steuert

Wiring the Vascular Network with Neural Cues: A CNS Perspective

The vascular and the nervous system are responsible for oxygen, nutrient, and information transfer and thereby constitute highly important communication systems in higher organisms. These functional similarities are reflected at the anatomical, cellular, and molecular levels, where common developmental principles and mutual crosstalks have evolved to coordinate their action. This resemblance of the two systems at different levels of complexity has been termed the "neurovascular link." Most of the evidence demonstrating neurovascular interactions derives from studies outside the CNS and from the CNS tissue of the retina. However, little is known about the specific properties of the neurovascular link in the brain. Here, we focus on regulatory effects of molecules involved in the neurovascular link on angiogenesis in the periphery and in the brain and distinguish between general and CNS-specific cues for angiogenesis. Moreover, we discuss the emerging molecular interactions of these angiogenic cues with the VEGF-VEGFR-Delta-like ligand 4 (Dll4)-Jagged-Notch pathway

Formin-mediated actin polymerization at endothelial junctions is required for vessel lumen formation and stabilization

During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis