Semaphorin 3B role in spinal cord neurogenesis

L'orientation des divisions cellulaires est un processus majeur impliqué dans la morphogenèse des tissus, le renouvellement et le contrôle du destin cellulaire. Au cours du développement du système nerveux chez les vertébrés, la croissance du tube neural et la génération des cellules neuronales et gliales résultent de la prolifération de progéniteurs neuraux organisés le long d'un neuroépithelium fermé autour d'un canal central. L'orientation du fuseau des progéniteurs en mitose par rapport au plan apical est cruciale pour la conservation de l'intégrité du neuroepithelium. Elle peut aussi influencer le destin des cellules filles. Jusqu’à présent, les études se sont principalement concentrées sur les mécanismes intracellulaires contrôlant l'orientation du fuseau mitotique, en revanche, l'existence de signaux extracellulaires y contribuant est mal définie à l’heure actuelle. Durant le développement de la moelle épinière, le canal du tube neural est une source de signaux extracellulaires majeurs comme les morphogènes. Pour la plupart des progéniteurs neuraux, la mitose a lieu au niveau apical à proximité du canal central. Nous avons donc émis l’hypothèse que le canal pourrait aussi délivrer des signaux extracellulaires régulant l'orientation des divisions des progéniteurs neuraux. Mes travaux de thèse révèlent que de tels signaux existent. Plus particulièrement je montre que la Sémaphorine 3B, un facteur initialement connu pour son rôle chimiotropique, joue un rôle majeur dans l'orientation des divisions des progéniteurs spinaux. Chez des embryons de souris E10.5 maintenus en incubation à court terme après ouverture de leur tube neural et dilution du liquide céphalorachidien, nous observons une forte augmentation du pourcentage de divisions obliques comparées aux embryons non ouverts. L’analyse d’une lignée de souris dans laquelle le canal central est scindé en deux sous-canaux indépendants, créant ainsi une obstruction du flux entre les parties dorsales et ventrales du canal révèle aussi une altération de l'orientation des divisions des progéniteurs neuraux. Des signaux provenant du canal sont donc nécessaires à l'orientation planaire de la division d'une population de progéniteurs spinaux. Par hybridation in situ et immuno-marquage, nous avons mis en évidence l'expression d'ARN et de protéines Sema3B dans des cellules de la plaque du plancher aux stades E10.5 et E11.5. Ce résultat suggèrait que cette Sema3 pouvait être sécrétée dans le canal de l’épendyme. L'invalidation du gène Sema3B a conduit à une diminution du pourcentage des divisions planaires à E10.5 sans changement de leur nombre ou de leur polarité. De plus, une exposition à court terme des tubes neuraux ouverts à de la Sema3B exogène, a rétabli des divisions planaires dans une large proportion de progéniteurs neuraux. Les défauts d’orientation des mutants Sema3B sont corrélés à une altération secondaire de la prolifération, de la croissance de la moelle et de la neurogenèse. Ces résultats révèlent ainsi qu'au-delà de son rôle de sécréteur de morphogène, la plaque du plancher fournit aussi un signal extracellulaire qui contrôle l'orientation de division de progéniteurs neuraux. Ce travail suggère aussi que la signalisation Sémaphorine, connue comme instructive dans le guidage des cellules et axones migrants, puisse être interprétée par des cellules neuroépitheliales comme des repères spatiaux extrinsèques permettant l'orientation de leur fuseau mitotiqueIn pluricellular organisms, the orientation of cell division has a major impact on tissue morphogenesis architecture and renewal, as well as on cell fate choices. During the development of the central nervous system in vertebrates, the growth of the neural tube and the generation of neuronal cells and glial cells result from the proliferation of neural progenitors organized in a neuroepithelium closed around a central canal. The orientation of progenitor mitotic spindle with respect to the apical plan is important for the conservation of the integrity of the neuroepithelium and influences the fate of daughter cells. Previous studies mainly focused on intracellular mechanisms controlling the mitotic spindle orientation, but whether extracellular signaling contributes to this process remains unknown. In the developing spinal cord, the lumen is a source of major extracellular signals like morphogens. For most neural progenitors, the mitosis takes place at the apical pole in tight vicinity of the central lumen. We hypothesized that canal-derived extracellular signals could regulate the orientation of neural progenitor divisions. My PhD work aimed at testing this hypothesis and identifying such factors. We show that dorsally open neural tubes from E10.5 mice, maintained in short term culture display a strong increase in the percentage of oblique divisions compared to un-open ones. The genetic disruption of the lumen fluid diffusion between the ventral and dorsal parts of the lumen leads to similar defects. Lumen-derived signals are thus required for neural progenitors to achieve planar divisions in the mouse spinal neuroepithelium at the onset of neurogenesis. By in situ hybridization, immunostaining and a knock-in mouse line, we detected Sema3B mRNA and proteins in floor plate cells at E10.5 and E11.5, which suggests that it could be secreted in the lumen of the spinal cord. The invalidation of Sema3B results in a decrease in the percentage of planar divisions in E10.5 spinal progenitors without alteration of progenitor number or polarity. Furthermore, a short term exposure of open neural tubes to exogenous Sema3B restores planar divisions in a large population of spinal progenitors. We observed that Sema3B knock out subsequently altered proliferation and neurogenesis steps. These results thus reveal that beyond its role as morphogen-releasing organizer, the floor plate also provides an extracellular signal which controls the orientation of neural progenitor division. This work also suggests that Sema signaling known as an instructive chemotropic cue in the guidance of migrating cells and axons also serves for neuroepithelial cells as an extrinsic cue to control the orientation of their divisio

Rôle de la Sémaphorine 3B dans la neurogenèse de la moelle épinière

In pluricellular organisms, the orientation of cell division has a major impact on tissue morphogenesis architecture and renewal, as well as on cell fate choices. During the development of the central nervous system in vertebrates, the growth of the neural tube and the generation of neuronal cells and glial cells result from the proliferation of neural progenitors organized in a neuroepithelium closed around a central canal. The orientation of progenitor mitotic spindle with respect to the apical plan is important for the conservation of the integrity of the neuroepithelium and influences the fate of daughter cells. Previous studies mainly focused on intracellular mechanisms controlling the mitotic spindle orientation, but whether extracellular signaling contributes to this process remains unknown. In the developing spinal cord, the lumen is a source of major extracellular signals like morphogens. For most neural progenitors, the mitosis takes place at the apical pole in tight vicinity of the central lumen. We hypothesized that canal-derived extracellular signals could regulate the orientation of neural progenitor divisions. My PhD work aimed at testing this hypothesis and identifying such factors. We show that dorsally open neural tubes from E10.5 mice, maintained in short term culture display a strong increase in the percentage of oblique divisions compared to un-open ones. The genetic disruption of the lumen fluid diffusion between the ventral and dorsal parts of the lumen leads to similar defects. Lumen-derived signals are thus required for neural progenitors to achieve planar divisions in the mouse spinal neuroepithelium at the onset of neurogenesis. By in situ hybridization, immunostaining and a knock-in mouse line, we detected Sema3B mRNA and proteins in floor plate cells at E10.5 and E11.5, which suggests that it could be secreted in the lumen of the spinal cord. The invalidation of Sema3B results in a decrease in the percentage of planar divisions in E10.5 spinal progenitors without alteration of progenitor number or polarity. Furthermore, a short term exposure of open neural tubes to exogenous Sema3B restores planar divisions in a large population of spinal progenitors. We observed that Sema3B knock out subsequently altered proliferation and neurogenesis steps. These results thus reveal that beyond its role as morphogen-releasing organizer, the floor plate also provides an extracellular signal which controls the orientation of neural progenitor division. This work also suggests that Sema signaling known as an instructive chemotropic cue in the guidance of migrating cells and axons also serves for neuroepithelial cells as an extrinsic cue to control the orientation of their divisionL'orientation des divisions cellulaires est un processus majeur impliqué dans la morphogenèse des tissus, le renouvellement et le contrôle du destin cellulaire. Au cours du développement du système nerveux chez les vertébrés, la croissance du tube neural et la génération des cellules neuronales et gliales résultent de la prolifération de progéniteurs neuraux organisés le long d'un neuroépithelium fermé autour d'un canal central. L'orientation du fuseau des progéniteurs en mitose par rapport au plan apical est cruciale pour la conservation de l'intégrité du neuroepithelium. Elle peut aussi influencer le destin des cellules filles. Jusqu’à présent, les études se sont principalement concentrées sur les mécanismes intracellulaires contrôlant l'orientation du fuseau mitotique, en revanche, l'existence de signaux extracellulaires y contribuant est mal définie à l’heure actuelle. Durant le développement de la moelle épinière, le canal du tube neural est une source de signaux extracellulaires majeurs comme les morphogènes. Pour la plupart des progéniteurs neuraux, la mitose a lieu au niveau apical à proximité du canal central. Nous avons donc émis l’hypothèse que le canal pourrait aussi délivrer des signaux extracellulaires régulant l'orientation des divisions des progéniteurs neuraux. Mes travaux de thèse révèlent que de tels signaux existent. Plus particulièrement je montre que la Sémaphorine 3B, un facteur initialement connu pour son rôle chimiotropique, joue un rôle majeur dans l'orientation des divisions des progéniteurs spinaux. Chez des embryons de souris E10.5 maintenus en incubation à court terme après ouverture de leur tube neural et dilution du liquide céphalorachidien, nous observons une forte augmentation du pourcentage de divisions obliques comparées aux embryons non ouverts. L’analyse d’une lignée de souris dans laquelle le canal central est scindé en deux sous-canaux indépendants, créant ainsi une obstruction du flux entre les parties dorsales et ventrales du canal révèle aussi une altération de l'orientation des divisions des progéniteurs neuraux. Des signaux provenant du canal sont donc nécessaires à l'orientation planaire de la division d'une population de progéniteurs spinaux. Par hybridation in situ et immuno-marquage, nous avons mis en évidence l'expression d'ARN et de protéines Sema3B dans des cellules de la plaque du plancher aux stades E10.5 et E11.5. Ce résultat suggèrait que cette Sema3 pouvait être sécrétée dans le canal de l’épendyme. L'invalidation du gène Sema3B a conduit à une diminution du pourcentage des divisions planaires à E10.5 sans changement de leur nombre ou de leur polarité. De plus, une exposition à court terme des tubes neuraux ouverts à de la Sema3B exogène, a rétabli des divisions planaires dans une large proportion de progéniteurs neuraux. Les défauts d’orientation des mutants Sema3B sont corrélés à une altération secondaire de la prolifération, de la croissance de la moelle et de la neurogenèse. Ces résultats révèlent ainsi qu'au-delà de son rôle de sécréteur de morphogène, la plaque du plancher fournit aussi un signal extracellulaire qui contrôle l'orientation de division de progéniteurs neuraux. Ce travail suggère aussi que la signalisation Sémaphorine, connue comme instructive dans le guidage des cellules et axones migrants, puisse être interprétée par des cellules neuroépitheliales comme des repères spatiaux extrinsèques permettant l'orientation de leur fuseau mitotiqu

Brain Tumor promotes axon growth across the midline through interactions with the microtubule stabilizing protein Apc2

<div><p>Commissural axons must cross the midline to establish reciprocal connections between the two sides of the body. This process is highly conserved between invertebrates and vertebrates and depends on guidance cues and their receptors to instruct axon trajectories. The DCC family receptor Frazzled (Fra) signals chemoattraction and promotes midline crossing in response to its ligand Netrin. However, in Netrin or <i>fra</i> mutants, the loss of crossing is incomplete, suggesting the existence of additional pathways. Here, we identify Brain Tumor (Brat), a tripartite motif protein, as a new regulator of midline crossing in the <i>Drosophila</i> CNS. Genetic analysis indicates that Brat acts independently of the Netrin/Fra pathway. In addition, we show that through its B-Box domains, Brat acts cell autonomously to regulate the expression and localization of Adenomatous polyposis coli-2 (Apc2), a key component of the Wnt canonical signaling pathway, to promote axon growth across the midline. Genetic evidence indicates that the role of Brat and Apc2 to promote axon growth across the midline is independent of Wnt and Beta-catenin-mediated transcriptional regulation. Instead, we propose that Brat promotes midline crossing through directing the localization or stability of Apc2 at the plus ends of microtubules in navigating commissural axons. These findings define a new mechanism in the coordination of axon growth and guidance at the midline.</p></div

Model for how brain tumor interacts with Apc2 to promote axon growth across the midline.

<p>We propose that Brat maintains Apc2 at the plus-ends of microtubules at the periphery of the growth cone resulting in axon extension across the midline.</p

Brat acts in parallel to the <i>Netrin-Fra</i> pathway.

<p>(A-D) Stage 15–16 embryos of the indicated genotypes carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-GFP (green) (A-D) or anti-HRP (magenta) (A’-D’) antibodies. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW), Anti-HRP reveals all of the CNS axons. Scale bar represents 10μm (A). Arrowheads indicate segments with non-crossing EW axons (A-D) or thin commissures (A’-D’). (A) EW neurons cross in the posterior commissure in 100% of segments in wild-type embryos. (A’) In every segment thick anterior and posterior commissures are formed as axons cross the midline. (B) In <i>fra</i> mutants EW neurons fail to cross in 36% of segments. (B’) <i>fra</i> mutants show thinner commissures. (C) and (C’) <i>brat</i> homozygous mutants show no obvious signs of commissural guidance defects: EW neurons fail to cross in only 4% of segments. (D) In <i>fra</i>, <i>brat</i> double mutants EW axons fail to cross the midline in 56% of segments. (D’) <i>fra</i>, <i>brat</i> double mutants also show thinner commissures. (E) Quantification of EW midline crossing defects in the genotypes shown in (B-D). Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗∗p < 0.0001). (F) Schematic diagrams of the EW axon trajectories observed in each genotype; the EW axons can cross the midline (Cross), grow ipsilaterally (Ipsi) or stall (Stall). (G, I, K) In <i>fra</i> mutants, 55% of the EW axons cross the midline (G), 29% grow ipsilaterally (I) and 16% remain stalled (K). (H, J, L) In <i>fra</i>, <i>brat</i> double mutants, 38% of the EW axons cross the midline (H), 28% grow ipsilaterally (J) and 34% stall (L). (M) Quantification of the distribution of the EW axon trajectories in the genotypes shown in (G-L). Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed using Chi-squared test (****p < 0.0001).</p

Brain Tumor is a positive regulator of midline crossing.

<p>(A-F) Stage 15–16 embryos of the indicated genotypes carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-GFP antibody. Anti-GFP reveals cell bodies and axons of the eagle neurons (EG and EW). Anterior is up in all images. Scale bar represents 10μm (A). EG neurons project through the anterior commissure of each segment, while EW neurons project through the posterior commissure. Arrowheads indicate segments with non-crossing EW axons. (A) In wild-type embryos EW axons cross in the posterior commissure in 100% of segments. (B) In <i>fra</i> mutants EW axons fail to cross in 36% of segments (arrowheads). (C) EW axons fail to cross in 28% of segments when UAS-FraΔC is selectively expressed in eagle neurons. (D) In a FraΔC background the heterozygosity for <i>brat</i> enhances the EW crossing defects to 53%. (E) Complete loss of <i>brat</i> enhances the EW crossing defect to 69% of segments in FraΔC background. (F) EW crossing defects in <i>brat/brat;</i> FraΔC embryos are rescued (69% versus 41%) when UAS-Brat is expressed in eagle neurons. (G) Quantification of EW midline crossing defects in the genotypes shown in (B-F). Df (2L) Exel8040 is a chromosomal deficiency containing <i>brat</i>. Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗p < 0.001). (H) Schematic diagrams of the EW axon trajectories observed in each genotype; the EW axons can cross the midline (Cross), grow ipsilaterally (Ipsi) or stall (Stall). (I, K, M) When UAS-FraΔC is selectively expressed in eagle neurons, 72% of the EW axons cross the midline (I), 22% grow ipsilaterally (K) and 6% stall (M). (J,L,N) Heterozygosity for <i>brat</i> in a FraΔC background enhances the EW crossing defects, 47% of the EW cross the midline (J), 23% grow ipsilaterally (L) and 30% stall (N). (O) Quantification of the distribution of the EW axon trajectories in the genotypes shown in (I-N). The enhanced EW crossing defects in <i>brat/+;</i> FraΔC embryos are rescued when UAS-Brat is expressed in eagle neurons. Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed using Chi-squared test (****p < 0.0001).</p

The B-box domains of Brat are required for its midline crossing function.

<p>(A-F) Stage 15–16 embryos of the indicated genotype carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-GFP antibody. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10μm (A). Arrowheads indicate segments with non-crossing EW axons. (A-C) EW crossing defects in the heterozygous <i>brat</i> mutant expressing FraΔC are rescued when (A) UAS-Brat (50% versus 24%), (B) UAS-Brat^ΔNHL (50% versus 32%) or (C) UAS-Brat^ΔCC (50% versus 29%) are expressed in eagle neurons. (D-F) In the heterozygous <i>brat</i> mutant expressing FraΔC, expression of (D) UAS-Brat^ΔBB, (E) UAS-Brat^ΔBB1 or (F) UAS-Brat^ΔBB2 fail to rescue the EW midline crossing defects (respectively for (D) (E) and (F): 50% versus 54%, 50% versus 40% and 50% versus 37%). (G) Schematic representation of Brat full-length protein and Brat deletion domain mutants used to identify the domain required for midline crossing. (H) Quantification of EW midline crossing defects in the genotypes shown in (A-F). Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗∗p < 0.0001).</p

Brat acts independently of the Nanos/Pumilio complex and of d4EHP.

<p>(A-D) Stage 15–16 embryos of the indicated genotype carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-GFP antibody. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10μm (A). Arrowheads indicate segments with non-crossing EW axons. (A) Heterozygosity for <i>brat</i> enhances the EW crossing defects to 50% in a FraΔC background. (B-D) EW crossing defects in heterozygous <i>brat</i> mutants expressing FraΔC are rescued when (B) UAS-Brat (50% versus 34%), (C) UAS-Brat^GD (50% versus 36%) or (D) UAS-Brat^RD (50% versus 39%) are expressed in eagle neurons. (E) Schematic representation of Brat protein and its different domains. The G774D and R837D point mutations are indicated with arrows. (F) Quantification of EW midline crossing defects in the genotypes shown in (A-D). Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗p < 0.001).</p

Apc2 expression co-localizes with EB1 in growing axon and cell bodies of Eagle neurons and is reduced in <i>brat</i> mutant embryos.

<p>(A-F’) Stage 13 and 16 embryos carrying eg-GAL4, UAS-Apc2GFP and UAS-EB1RFP transgenes, stained with anti-GFP (green) and anti-RFP (red) antibodies. Anti-GFP and anti-RFP label cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10μm (A) or 2μm (C’ and F’). (A-C’) At stage 13, Apc2 and EB1 expression co-localize in the growing axon and the cell body of the Eagle neurons. (D-F’) At stage 16, Apc2 and EB1 expression co-localize in the elongated axon of the Eagle neurons. (G-H’) Stage 15–16 embryos of the indicated genotype carrying eg-GAL4 and UAS-Apc2GFP transgenes, stained with anti-GFP antibodies. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10μm (G) or 5 μm (G’). (G) and (G’) In control embryos the average of the GFP signal intensity reflecting the Apc2 transgene expression, corresponds to 89% in cell bodies and 79% in axons. (H) and (H’) <i>brat</i> homozygous mutant embryos, show a decrease of the GFP signal intensity to 50% in cell bodies and 25% in axons, reflecting a reduction of the Apc2 transgene expression. (I) Quantification of the GFP staining signal intensity shown in (G-H’). Data are presented as mean ± SEM. 10 embryos were scored for each genotype. Significance was assessed using the Student’s t-test (^∗∗∗∗p < 0.0001).</p

Midline crossing is sensitive to reduced Apc2 and Arm function and does not require Arm transcriptional activity.

<p>(A-D) Stage 15–16 embryos of the indicated genotype carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-GFP (grey or green) (A-C) or anti-HRP (magenta) (D) antibodies. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW), Anti-HRP reveals all of the CNS axons. Scale bar represents 10μm (A). Arrowheads indicate segments with non-crossing EW axons (A-C) or thin commissures (D). (A) In a FraΔC background the heterozygosity for <i>Apc2</i> enhances the EW crossing defects to 59%. (B) In the embryos double heterozygous for <i>Apc2</i> and <i>brat</i> expressing UAS-FraΔC selectively in eagle neurons, EW axons fail to cross in the posterior commissure in 72% of segments. (C) In <i>Apc2</i> and <i>brat</i> double mutant embryos, EW axons fail to cross in the posterior commissure in 20% of segments and show thinner commissures in some segments (D). (E) Quantification of EW midline crossing defects in the genotypes shown in (A-D). Df (2L) Exel6168 is a chromosomal deficiency containing <i>Apc2</i>. Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗∗p< 0.0001). (F) Quantification of EW midline crossing defects in the indicated genotypes. Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗p < 0.001).</p