Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system

<p>Abstract</p> <p>Background</p> <p>During spinal cord development, expression of chicken SEMAPHORIN6A (SEMA6A) is almost exclusively found in the boundary caps at the ventral motor axon exit point and at the dorsal root entry site. The boundary cap cells are derived from a population of late migrating neural crest cells. They form a transient structure at the transition zone between the peripheral nervous system (PNS) and the central nervous system (CNS). Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots. Based on its very restricted expression in boundary cap cells, we tested for a role of Sema6A as a gate keeper between the CNS and the PNS.</p> <p>Results</p> <p>Downregulation of Sema6A in boundary cap cells by <it>in ovo </it>RNA interference resulted in motoneurons streaming out of the spinal cord along the ventral roots, and in the failure of dorsal roots to form and segregate properly. PlexinAs interact with class 6 semaphorins and are expressed by both motoneurons and sensory neurons. Knockdown of PlexinA1 reproduced the phenotype seen after loss of Sema6A function both at the ventral motor exit point and at the dorsal root entry site of the lumbosacral spinal cord. Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point.</p> <p>Conclusion</p> <p>Sema6A acts as a gate keeper between the PNS and the CNS both ventrally and dorsally. It is required for the clustering of boundary cap cells at the PNS/CNS interface and, thus, prevents motoneurons from streaming out of the ventral spinal cord. At the dorsal root entry site it organizes the segregation of dorsal roots.</p

Expression patterns of plexins and neuropilins are consistent with cooperative and separate functions during neural development

BACKGROUND: Plexins are a family of transmembrane proteins that were shown to act as receptors for Semaphorins either alone or in a complex together with Neuropilins. Based on structural criteria Plexins were subdivided into 4 classes, A through D. PlexinAs are mainly thought to act as mediators of repulsive signals in cell migration and axon guidance. Their functional role in vertebrates has been studied almost exclusively in the context of Semaphorin signaling, i.e. as co-receptors for class 3 Semaphorins. Much less is known about Plexins of the other three classes. Despite the fact that Plexins are involved in the formation of neuronal circuits, the temporal changes of their expression patterns during development of the nervous system have not been analyzed in detail. RESULTS: Only seven plexins are found in the chicken genome in contrast to mammals, where nine plexins have been identified. Here, we describe the dynamic expression patterns of all known plexin family members in comparison to the neuropilins in the developing chicken spinal cord. CONCLUSION: Our in situ hybridization study revealed that the expression patterns of plexins and neuropilins are only partially overlapping, especially during early and intermediate stages of spinal cord development, supporting both cooperative and separate functions of plexins and neuropilins in neural circuit formation

Characterization of semaphorin 6A during development of the embryonic chicken spinal cord

Für die Bildung des informationsverarbeitenden Netzwerks, das unser Nervensystem darstellt, müssen Nervenzellen während der Embryonalentwicklung mit grosser Präzision verknüpft werden. Während der letzten 20 Jahre wurden die Prinzipien der axonalen Wegfindung erarbeitet. Insbesondere wurde eine Vielzahl von Wegweisermolekülen entdeckt, die für die axonale Wegfindung von entscheidender Bedeutung sind. Eine Familie von Wegweisermolekülen sind die Semaphorine. Sie wurden ursprünglich als repulsive Moleküle identifiziert, die axonale Wegfindung beeinflussen. Semaphorine kommen in löslicher und membrangebundener Form vor. In letzter Zeit wurden auch weitere Funktionen von Semaphorinen beschrieben, wie z. B. axonale Faszikulation, Auswahl der Zielzellen, Zellwanderung und Wegfindung von Dendriten. Ausserdem wurden Semaphorine auch mit Plastizität und Regeneration im adulten Nervensystem in Verbindung gebracht. Als Rezeptoren von Semaphorinen wurden Plexine und Neuropiline identifiziert. In meiner Doktorarbeit habe ich die Funktion des transmembranalen Semaphorins 6A während der Entwicklung des Nervensystems untersucht. Semaphorin6A ist in den sog. Boundary Cap Zellen exprimiert. Das Blockieren der Funktion von Semaphorin6A mittels in ovo RNAi (in ovo RNA Interferenz) führte zu einer fehlerhaften Anordnung der Axonbündel, die von den Spinalganglien ins Rückenmark wachsen. Motoneuronen, die normalerweise im ventralen Teil des Rückenmarks liegen, wurden ausserhalb des Rückenmarks entlang der Vorderwurzeln entdeckt. Diese Beobachtungen bestätigen somit die Hypothese, dass Semaphorin6A in den Boundary Cap Zellen als "Gate Keeper" Eintritt und Austritt aus dem Rückenmark kontrollieren. Neuronal connections are made during embryonic development with astonishing precision to ultimately form the information processing network of the nervous system. Over the past few decades, much has been learned about the general principles of axon guidance. Many molecular cues have been discovered which help establishing these connections accurately. One family of axon guidance cues, the semaphorins, was first identified as repellents for navigating axons during brain wiring. Semaphorins are secreted, membrane-attached or transmembrane in nature. Recent studies have implicated these molecules in many other processes of neuronal development, including axonal fasciculation, target selection, neuronal migration, and dendritic guidance, as well as in the remodeling and repair of the adult nervous system. The functions of Semaphorins are mediated by receptor complexes consisting of Plexins and Neuropilins. In my thesis, I characterized the function of a transmembrane semaphorin, SEMA6A, in the developing chicken spinal cord. Chicken SEMA6A is expressed in boundary cap cells. Silencing SEMA6A by in ovo RNAi (in ovo RNA interference) led to aberrant arrangement of the dorsal roots and emigration of motor neurons from the spinal cord. Thus, these data suggest a role for SEMA6A as a gatekeeper at the CNS/PNS interface

Ectopic expression of the Sema6A ectodomain or full-length Sema6A in motoneurons competes with BCC-derived Sema6A binding to motoneurons

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> The AP-tagged ectodomain of Sema6A binds to axons expressing PlexinAs. Both commissural axons (open arrowhead) and motor axons (arrowhead) express PlexinAs [20] and bind the Sema6A ectodomain. No binding of the AP-tag alone was detectable. Ectopic expression of both the ectodomain of Sema6A (not shown) and the full-length myc-tagged form resulted in motoneurons streaming out of the spinal cord along the ventral roots (arrows). Staining of the myc tag demonstrates expression of Sema6A in motor axons (arrowhead), consistent with a competitive role of motor axon-derived Sema6A with BCC-derived Sema6A in the periphery. As seen after downregulation of either Sema6A in BCCs (compare to Figure 7f) or PlexinA1 in motoneurons (compare to Figure 7g), ectopic expression of Sema6A resulted in the aberrant formation of BCC clusters. Bar: 100 μm

SEMA6A is expressed in neural crest cells that give rise to boundary cap cells

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> Neural crest cells that give rise to boundary cap cells express SEMA6A while they are still migrating ventrally (HH19; arrow). Boundary cap cells start to cluster first at the VMEP. Only those cells that have formed clusters at the VMEP express the BCC marker KROX20 (arrowhead). The neural crest markers SOX10 and the 1E8 epitope are expressed by many neural crest-derived cell populations and are not restricted to BCCs at HH19. Note that neither SOX10 (open arrow in (c)), nor 1E8 (open arrow in (d)) are expressed in BCCs while they are still migrating. First clusters of BCCs next to the DREZ marked by SEMA6A expression (open arrowhead) or KROX20 (open arrowhead) are detectable at lumbosacral levels by HH21. At this stage, many SEMA6A-expressing cells are still migrating along the neural tube to reach the ventral BCC cluster (arrow in (e)). A similar situation is found at HH24. SEMA6A expression is clearly detectable in dorsal BCCs (open arrowhead in (i); compare with (j)). After HH30, SEMA6A expression in dorsal (open arrowhead in (n)) and ventral (arrowhead in (n)) BCCs decreased but was still visible by HH36 (o). hybridizations on adjacent transverse sections of the lumbosacral spinal cord are shown for SEMA6A (a, e, i, m-p), KROX20 (b, f, j), and SOX10 (c, g, k) at HH19 (a-d), HH21 (e-h), HH24 (i-l) as indicated. Sections shown in (d, h, l) were stained for 1E8 (red) and neurofilament (green). Bars are 100 μm in (a-n), 200 μm in (o), and 500 μm in (p)

Downregulation of Sema6A in BCCs results in translocation of motoneurons out of the spinal cord

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> In the absence of Sema6A from BCCs, motoneurons stream out of the ventral spinal cord and migrate along the ventral roots (arrows). The open arrow points to a motoneuron that is located in the ventral funiculus. In control-treated embryos motoneurons along ventral roots or in the ventral funiculus were rarely seen. Motoneurons were identified by Isl-1 (red). An EGFP plasmid was co-injected with the dsRNA derived from SEMA6A. Axons were stained with an antibody against neurofilament (blue). Note that sensory neurons in the DRG (asterisk in (a, b)) are also stained by Isl-1. Perturbation of Sema6B or Sema6D did not enhance the number of motoneurons in the periphery compared to control-treated embryos injected only with the plasmid encoding EGFP. Three asterisks indicate < 0.0001 for the comparison between dsS6A and all other treatment groups. Values are given as mean ± standard error of mean. Bar: 50 μm

The expression of PLEXINA1 (PA1) and PLEXINA2 (PA2) differs between the thoracic and the lumbosacral levels of the spinal cord

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> Based on their expression pattern, none of the PlexinAs can be excluded as a binding partner for Sema6A [20]. In addition to the dynamic changes over time, the expression of PLEXINA1 and PLEXINA2 differs strongly between thoracic and lumbosacral levels of the spinal cord. PLEXINA1 is strongly expressed in the ventral spinal cord at HH20, but remains to be expressed strongly only at the lumbosacral but not the thoracic level at HH24 and HH25. Even more pronounced are the changes of PLEXINA2 expression. At HH20, expression is detectable in lateral motoneurons only at the thoracic but not at the lumbosacral level of the spinal cord. This difference is even more pronounced at older stages. AS, antisense probe; TH, thoracic level; LS lumbosacral level. Arrowheads indicate expression of either PA1 or PA2; open arrowheads indicate no or very weak expression. Asterisks label the hind limb to indicate that sections were taken from the lumbosacral level of the spinal cord. Bar: 200 μm

Lack of Sema6A and Sema6D in dorsal BCCs results in aberrant segregation of dorsal roots

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> In control embryos axon bundles from each dorsal root ganglion extend to the DREZ in a well organized manner. Roots from adjacent DRGs are segregated and they are all of the same length (dashed bars). In contrast, in embryos lacking Sema6A, roots from adjacent DRGs are no longer segregated (arrowheads). The arrangement of roots arising from individual DRGs is strongly disorganized and roots are often formed by fibers from two adjacent DRGs (arrowheads in (b)). Similarly, roots are disorganized in embryos lacking Sema6D (arrowheads). In addition the length of the roots varied more in the absence of Sema6D (compare dashed bars in (c)). Strong phenotypes were seen in 71% of the embryos lacking Sema6A and in 68% of the embryos lacking Sema6D. Only 13% of the embryos injected with an EGFP plasmid had a comparable phenotype. Downregulation of Sema6B resulted in aberrant DRG shapes and root arrangement in 30% of the embryos. The shapes of DRGs were classified as arc-like when the distance between the most anterior and the most posterior fiber emanating from the DRG was the same as the anteroposterior diameter of the DRG; as bell-shaped when the fibers spread an anteroposterior length that was bigger than the diameter of the DRG; and as mushroom-like when the fibers entered the dorsal spinal cord in a segment that was shorter than the diameter of the DRG. Note that the diameter of the mushroom-like DRGs was smaller than the diameter of arc-like or bell-shaped DRGs. Bar: 200 μm