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Vav3-deficient mice exhibit a transient delay in cerebellar development.

Quevedo C, Sauzeau V, Menacho-Márquez M, Castro-Castro A, Bustelo XR - Mol. Biol. Cell (2010)

Bottom Line: We report here that Vav3 is expressed at high levels in Purkinje and granule cells, suggesting additional roles for this protein in the cerebellum.Using primary neuronal cultures, we show that Vav3 is important for dendrite branching, but not for primary dendritogenesis, in Purkinje and granule cells.These results indicate that Vav3 function contributes to the timely developmental progression of the cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Campus Unamuno, E-37007 Salamanca, Spain.

ABSTRACT
Vav3 is a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases that has been involved in functions related to the hematopoietic system, bone formation, cardiovascular regulation, angiogenesis, and axon guidance. We report here that Vav3 is expressed at high levels in Purkinje and granule cells, suggesting additional roles for this protein in the cerebellum. Consistent with this hypothesis, we demonstrate using Vav3-deficient mice that this protein contributes to Purkinje cell dendritogenesis, the survival of granule cells of the internal granular layer, the timely migration of granule cells of the external granular layer, and to the formation of the cerebellar intercrural fissure. With the exception of the latter defect, the dysfunctions found in Vav3(-/-) mice only occur at well-defined postnatal developmental stages and disappear, or become ameliorated, in older animals. Vav2-deficient mice do not show any of those defects. Using primary neuronal cultures, we show that Vav3 is important for dendrite branching, but not for primary dendritogenesis, in Purkinje and granule cells. Vav3 function in the cerebellum is functionally relevant, because Vav3(-/-) mice show marked motor coordination and gaiting deficiencies in the postnatal period. These results indicate that Vav3 function contributes to the timely developmental progression of the cerebellum.

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Deficient dendritic arborization of Vav3−/− Purkinje cells. (A) Sagittal cerebellar slices from P6 wild-type (left) and Vav3−/− (right) mice were stained with an anti-calbindin antibody to visualize Purkinje cells. Representative fluorescence images for the lateral part of lobule V and VI are shown in low- (top; bar, 60 μm) and high (bottom; bar, 30 μm)-power views. Signals derived from calbindin are seen in green color in the images. Asterisks label two Purkinje cells with two dendrite main trunks branching out independently from the soma. A clear defect in the arborization of the dendritic tree of Purkinje cells is observed in Vav3-deficient mice. Note also that the thickness of the molecular layer is reduced in those animals (compare top panels). (B and C) Detection by immunofluorescence techniques of the expression of synaptophysin (B, top; for a magnification of those images, see C), GAD67 (B; second row of panels from top), and GAT1 (B; third row of panels from top) in sagittal cerebellar sections obtained from P6 wild-type mice (B and C; left) and Vav3−/− mice (B and C; right). Signals from synaptophysin (B and C) and GAD67 (B) are shown in green. Those from GAT1 are shown in red (B). Pictures show representative images obtained in cerebellar lobule IV. Similar results were obtained in the rest of cerebellar lobules analyzed (data not shown). Bar, 60 μm. (D) Detection by immunofluorescence techniques of the expression of the presynaptic Bassoon marker in cerebellar sections obtained from P6 wild-type mice (left) and Vav3-deficient mice (right). Signals from Bassoon protein are shown in red. Those from the 4,6-diamidino-2-phenylindole counterstaining are shown in blue. Bar, 100 μm. The cerebellar regions corresponding to the external granular cell layer (egl), the molecular cell layer (mcl), and the internal granular cell layer (igl) are indicated. (E) Quantification of Bassoon immunoreactivity in the molecular layer of the cerebella obtained from mice of the indicated genotypes (n = 4 animals). **p < 0.01 compared with wild-type controls. a.u., arbitrary units. It is observed that Vav3-deficient mice show lower levels of Bassoon immunoreactivity in their cerebellar molecular layers. (F) Examples of Golgi-stained Purkinje cells present in sections of adult mice cerebella obtained from wild-type mice (left) and Vav3-deficient mice (middle and right). Bar, 50 μm. With the exception of a Purkinje cell shown in the middle bottom panel, the structure of wild-type and Vav3-deficient Purkinje cells is similar (compare rest of panels).
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Figure 3: Deficient dendritic arborization of Vav3−/− Purkinje cells. (A) Sagittal cerebellar slices from P6 wild-type (left) and Vav3−/− (right) mice were stained with an anti-calbindin antibody to visualize Purkinje cells. Representative fluorescence images for the lateral part of lobule V and VI are shown in low- (top; bar, 60 μm) and high (bottom; bar, 30 μm)-power views. Signals derived from calbindin are seen in green color in the images. Asterisks label two Purkinje cells with two dendrite main trunks branching out independently from the soma. A clear defect in the arborization of the dendritic tree of Purkinje cells is observed in Vav3-deficient mice. Note also that the thickness of the molecular layer is reduced in those animals (compare top panels). (B and C) Detection by immunofluorescence techniques of the expression of synaptophysin (B, top; for a magnification of those images, see C), GAD67 (B; second row of panels from top), and GAT1 (B; third row of panels from top) in sagittal cerebellar sections obtained from P6 wild-type mice (B and C; left) and Vav3−/− mice (B and C; right). Signals from synaptophysin (B and C) and GAD67 (B) are shown in green. Those from GAT1 are shown in red (B). Pictures show representative images obtained in cerebellar lobule IV. Similar results were obtained in the rest of cerebellar lobules analyzed (data not shown). Bar, 60 μm. (D) Detection by immunofluorescence techniques of the expression of the presynaptic Bassoon marker in cerebellar sections obtained from P6 wild-type mice (left) and Vav3-deficient mice (right). Signals from Bassoon protein are shown in red. Those from the 4,6-diamidino-2-phenylindole counterstaining are shown in blue. Bar, 100 μm. The cerebellar regions corresponding to the external granular cell layer (egl), the molecular cell layer (mcl), and the internal granular cell layer (igl) are indicated. (E) Quantification of Bassoon immunoreactivity in the molecular layer of the cerebella obtained from mice of the indicated genotypes (n = 4 animals). **p < 0.01 compared with wild-type controls. a.u., arbitrary units. It is observed that Vav3-deficient mice show lower levels of Bassoon immunoreactivity in their cerebellar molecular layers. (F) Examples of Golgi-stained Purkinje cells present in sections of adult mice cerebella obtained from wild-type mice (left) and Vav3-deficient mice (middle and right). Bar, 50 μm. With the exception of a Purkinje cell shown in the middle bottom panel, the structure of wild-type and Vav3-deficient Purkinje cells is similar (compare rest of panels).

Mentions: Despite the conservation of the overall histological structure of the cerebellum in Vav3-deficient mice, a closer analysis of this tissue indicated significant, time-dependent defects in Purkinje cells. When we visualized those cells using antibodies to calbindin (Christakos et al., 1987), we observed that they displayed aberrant cell morphologies in P6 Vav3−/− mice. Thus, although the Purkinje cells were properly aligned in the expected cerebellar layer (Figure 3A, right), they had smaller and poorly branched dendritic trees (Figure 3A, right). Furthermore, we found Purkinje cells containing two or more thin dendritic stems sprouting from the somas that were clearly different from the single primary stem present in wild-type cells at this developmental stage (Figure 3A, bottom on the right). As a consequence of this deficient arborization, the thickness of the molecular layer was significantly reduced in Vav3-deficient cerebella compared with their wild-type counterparts (Figure 3A). Despite these defects, we found that Purkinje cells had normal numbers (data not shown) and were arranged in a wild-type-like laminar distribution in Vav3−/− mice independently of the cerebellar lobule analyzed (Figure 3, A and B). Likewise, we could also observe that each Purkinje cell projected axons to the underlying white matter tracks (Supplemental Figure S1), indicating that axogenesis was not impaired in the absence of Vav3 expression. Consistent with this, we found normal levels of calbindin staining in the fastigial deep nucleus (data not shown, but see below; and Supplemental Figure S3), the main target of the GABAergic axons of Purkinje cells located in the anterior lobules of the cerebellum.


Vav3-deficient mice exhibit a transient delay in cerebellar development.

Quevedo C, Sauzeau V, Menacho-Márquez M, Castro-Castro A, Bustelo XR - Mol. Biol. Cell (2010)

Deficient dendritic arborization of Vav3−/− Purkinje cells. (A) Sagittal cerebellar slices from P6 wild-type (left) and Vav3−/− (right) mice were stained with an anti-calbindin antibody to visualize Purkinje cells. Representative fluorescence images for the lateral part of lobule V and VI are shown in low- (top; bar, 60 μm) and high (bottom; bar, 30 μm)-power views. Signals derived from calbindin are seen in green color in the images. Asterisks label two Purkinje cells with two dendrite main trunks branching out independently from the soma. A clear defect in the arborization of the dendritic tree of Purkinje cells is observed in Vav3-deficient mice. Note also that the thickness of the molecular layer is reduced in those animals (compare top panels). (B and C) Detection by immunofluorescence techniques of the expression of synaptophysin (B, top; for a magnification of those images, see C), GAD67 (B; second row of panels from top), and GAT1 (B; third row of panels from top) in sagittal cerebellar sections obtained from P6 wild-type mice (B and C; left) and Vav3−/− mice (B and C; right). Signals from synaptophysin (B and C) and GAD67 (B) are shown in green. Those from GAT1 are shown in red (B). Pictures show representative images obtained in cerebellar lobule IV. Similar results were obtained in the rest of cerebellar lobules analyzed (data not shown). Bar, 60 μm. (D) Detection by immunofluorescence techniques of the expression of the presynaptic Bassoon marker in cerebellar sections obtained from P6 wild-type mice (left) and Vav3-deficient mice (right). Signals from Bassoon protein are shown in red. Those from the 4,6-diamidino-2-phenylindole counterstaining are shown in blue. Bar, 100 μm. The cerebellar regions corresponding to the external granular cell layer (egl), the molecular cell layer (mcl), and the internal granular cell layer (igl) are indicated. (E) Quantification of Bassoon immunoreactivity in the molecular layer of the cerebella obtained from mice of the indicated genotypes (n = 4 animals). **p < 0.01 compared with wild-type controls. a.u., arbitrary units. It is observed that Vav3-deficient mice show lower levels of Bassoon immunoreactivity in their cerebellar molecular layers. (F) Examples of Golgi-stained Purkinje cells present in sections of adult mice cerebella obtained from wild-type mice (left) and Vav3-deficient mice (middle and right). Bar, 50 μm. With the exception of a Purkinje cell shown in the middle bottom panel, the structure of wild-type and Vav3-deficient Purkinje cells is similar (compare rest of panels).
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Figure 3: Deficient dendritic arborization of Vav3−/− Purkinje cells. (A) Sagittal cerebellar slices from P6 wild-type (left) and Vav3−/− (right) mice were stained with an anti-calbindin antibody to visualize Purkinje cells. Representative fluorescence images for the lateral part of lobule V and VI are shown in low- (top; bar, 60 μm) and high (bottom; bar, 30 μm)-power views. Signals derived from calbindin are seen in green color in the images. Asterisks label two Purkinje cells with two dendrite main trunks branching out independently from the soma. A clear defect in the arborization of the dendritic tree of Purkinje cells is observed in Vav3-deficient mice. Note also that the thickness of the molecular layer is reduced in those animals (compare top panels). (B and C) Detection by immunofluorescence techniques of the expression of synaptophysin (B, top; for a magnification of those images, see C), GAD67 (B; second row of panels from top), and GAT1 (B; third row of panels from top) in sagittal cerebellar sections obtained from P6 wild-type mice (B and C; left) and Vav3−/− mice (B and C; right). Signals from synaptophysin (B and C) and GAD67 (B) are shown in green. Those from GAT1 are shown in red (B). Pictures show representative images obtained in cerebellar lobule IV. Similar results were obtained in the rest of cerebellar lobules analyzed (data not shown). Bar, 60 μm. (D) Detection by immunofluorescence techniques of the expression of the presynaptic Bassoon marker in cerebellar sections obtained from P6 wild-type mice (left) and Vav3-deficient mice (right). Signals from Bassoon protein are shown in red. Those from the 4,6-diamidino-2-phenylindole counterstaining are shown in blue. Bar, 100 μm. The cerebellar regions corresponding to the external granular cell layer (egl), the molecular cell layer (mcl), and the internal granular cell layer (igl) are indicated. (E) Quantification of Bassoon immunoreactivity in the molecular layer of the cerebella obtained from mice of the indicated genotypes (n = 4 animals). **p < 0.01 compared with wild-type controls. a.u., arbitrary units. It is observed that Vav3-deficient mice show lower levels of Bassoon immunoreactivity in their cerebellar molecular layers. (F) Examples of Golgi-stained Purkinje cells present in sections of adult mice cerebella obtained from wild-type mice (left) and Vav3-deficient mice (middle and right). Bar, 50 μm. With the exception of a Purkinje cell shown in the middle bottom panel, the structure of wild-type and Vav3-deficient Purkinje cells is similar (compare rest of panels).
Mentions: Despite the conservation of the overall histological structure of the cerebellum in Vav3-deficient mice, a closer analysis of this tissue indicated significant, time-dependent defects in Purkinje cells. When we visualized those cells using antibodies to calbindin (Christakos et al., 1987), we observed that they displayed aberrant cell morphologies in P6 Vav3−/− mice. Thus, although the Purkinje cells were properly aligned in the expected cerebellar layer (Figure 3A, right), they had smaller and poorly branched dendritic trees (Figure 3A, right). Furthermore, we found Purkinje cells containing two or more thin dendritic stems sprouting from the somas that were clearly different from the single primary stem present in wild-type cells at this developmental stage (Figure 3A, bottom on the right). As a consequence of this deficient arborization, the thickness of the molecular layer was significantly reduced in Vav3-deficient cerebella compared with their wild-type counterparts (Figure 3A). Despite these defects, we found that Purkinje cells had normal numbers (data not shown) and were arranged in a wild-type-like laminar distribution in Vav3−/− mice independently of the cerebellar lobule analyzed (Figure 3, A and B). Likewise, we could also observe that each Purkinje cell projected axons to the underlying white matter tracks (Supplemental Figure S1), indicating that axogenesis was not impaired in the absence of Vav3 expression. Consistent with this, we found normal levels of calbindin staining in the fastigial deep nucleus (data not shown, but see below; and Supplemental Figure S3), the main target of the GABAergic axons of Purkinje cells located in the anterior lobules of the cerebellum.

Bottom Line: We report here that Vav3 is expressed at high levels in Purkinje and granule cells, suggesting additional roles for this protein in the cerebellum.Using primary neuronal cultures, we show that Vav3 is important for dendrite branching, but not for primary dendritogenesis, in Purkinje and granule cells.These results indicate that Vav3 function contributes to the timely developmental progression of the cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Campus Unamuno, E-37007 Salamanca, Spain.

ABSTRACT
Vav3 is a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases that has been involved in functions related to the hematopoietic system, bone formation, cardiovascular regulation, angiogenesis, and axon guidance. We report here that Vav3 is expressed at high levels in Purkinje and granule cells, suggesting additional roles for this protein in the cerebellum. Consistent with this hypothesis, we demonstrate using Vav3-deficient mice that this protein contributes to Purkinje cell dendritogenesis, the survival of granule cells of the internal granular layer, the timely migration of granule cells of the external granular layer, and to the formation of the cerebellar intercrural fissure. With the exception of the latter defect, the dysfunctions found in Vav3(-/-) mice only occur at well-defined postnatal developmental stages and disappear, or become ameliorated, in older animals. Vav2-deficient mice do not show any of those defects. Using primary neuronal cultures, we show that Vav3 is important for dendrite branching, but not for primary dendritogenesis, in Purkinje and granule cells. Vav3 function in the cerebellum is functionally relevant, because Vav3(-/-) mice show marked motor coordination and gaiting deficiencies in the postnatal period. These results indicate that Vav3 function contributes to the timely developmental progression of the cerebellum.

Show MeSH
Related in: MedlinePlus