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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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Vav3 regulates dendritic outgrowth in Purkinje and granular cells. (A) Examples of the morphology of Purkinje cells obtained in mixed cultures derived from wild-type mice (left) and Vav3-deficient mice (right). To obtain these images, cell cultures were fixed, stained with anti-calbindin antibodies, and subjected to microscopy analysis. Less dendritic arborization is apparent in cultures generated from Vav3−/− mice. (B) Quantification of the Purkinje cell morphology in cultures derived from cerebella of the indicated genotypes. Quantification included the total dendritic tree area (left), the length of the longest dendrite (middle), and the number of branching points (right) in Purkinje cells (n = 4 independent experiments). **p < 0.01 compared with wild-type controls. A reduced number of tips is observed in Vav3-deficient Purkinje cells (right). No statistically significant differences were observed in the other two parameters evaluated in these experiments. (C) Expression levels of MAP2 in cultures of granule cell cultures of the indicated genotypes (top). At the indicated divisions in vitro (DIV, top), cellular lysates were obtained, electrophoretically fractionated in 6% SDS-PAGE gels, transferred onto nitrocellulose, and MAP2 levels were determined by anti-MAP2 Western blot (WB) analysis (top). Equal loading was demonstrated by the detection of a nonspecific band recognized by the anti-MAP2 antibody (middle) and by reblotting with an anti-vinculin antibody (bottom). (D) Quantification of MAP2 protein expression in granular cell cultures. Bands from the anti-MAP2 immunoblots obtained in three independent experiments were quantified by densitometry analysis and statistically analyzed. Data are presented as mean and SEM and were normalized taking into consideration the protein levels of vinculin detected in each sample. *p < 0.05 compared with wild-type controls. Reduced numbers of MAP2 are seen in cultures from Vav3-deficient mice. (E) Example of wild-type granule cells expressing EGFP alone (left l) or in combination with wild-type Vav3 (right). Cells were processed and transfected as indicated in Materials and Methods, fixed at division 4, and photographed using an inverted fluorescence microscope. Bar, 30 μm. Increased dendrite ramifications are seen in the granular cell expressing the Vav3 protein. (F) Quantitation of the total dendritic length (left) and the number of tips (right) of granular cells that were transfected with plasmids encoding the indicated proteins (n = 50 independent cells in each transfection). ***p < 0.001 compared with wild-type controls. Vav3 overexpression promotes an increase in the total length and number of tips in the transfected cells.
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Figure 6: Vav3 regulates dendritic outgrowth in Purkinje and granular cells. (A) Examples of the morphology of Purkinje cells obtained in mixed cultures derived from wild-type mice (left) and Vav3-deficient mice (right). To obtain these images, cell cultures were fixed, stained with anti-calbindin antibodies, and subjected to microscopy analysis. Less dendritic arborization is apparent in cultures generated from Vav3−/− mice. (B) Quantification of the Purkinje cell morphology in cultures derived from cerebella of the indicated genotypes. Quantification included the total dendritic tree area (left), the length of the longest dendrite (middle), and the number of branching points (right) in Purkinje cells (n = 4 independent experiments). **p < 0.01 compared with wild-type controls. A reduced number of tips is observed in Vav3-deficient Purkinje cells (right). No statistically significant differences were observed in the other two parameters evaluated in these experiments. (C) Expression levels of MAP2 in cultures of granule cell cultures of the indicated genotypes (top). At the indicated divisions in vitro (DIV, top), cellular lysates were obtained, electrophoretically fractionated in 6% SDS-PAGE gels, transferred onto nitrocellulose, and MAP2 levels were determined by anti-MAP2 Western blot (WB) analysis (top). Equal loading was demonstrated by the detection of a nonspecific band recognized by the anti-MAP2 antibody (middle) and by reblotting with an anti-vinculin antibody (bottom). (D) Quantification of MAP2 protein expression in granular cell cultures. Bands from the anti-MAP2 immunoblots obtained in three independent experiments were quantified by densitometry analysis and statistically analyzed. Data are presented as mean and SEM and were normalized taking into consideration the protein levels of vinculin detected in each sample. *p < 0.05 compared with wild-type controls. Reduced numbers of MAP2 are seen in cultures from Vav3-deficient mice. (E) Example of wild-type granule cells expressing EGFP alone (left l) or in combination with wild-type Vav3 (right). Cells were processed and transfected as indicated in Materials and Methods, fixed at division 4, and photographed using an inverted fluorescence microscope. Bar, 30 μm. Increased dendrite ramifications are seen in the granular cell expressing the Vav3 protein. (F) Quantitation of the total dendritic length (left) and the number of tips (right) of granular cells that were transfected with plasmids encoding the indicated proteins (n = 50 independent cells in each transfection). ***p < 0.001 compared with wild-type controls. Vav3 overexpression promotes an increase in the total length and number of tips in the transfected cells.

Mentions: Given the defective dendrite arborization found in the developing Vav3−/− cerebellum, we decided to investigate this process in more detail using in vitro cultures of both Purkinje and granule cells. We observed that cultured Purkinje cells from Vav3-deficient mice could develop the primary dendrite trunks in vitro and expand them to generate dendritic tree areas similar to those induced in wild-type cells (Figure 6, A and B). However, the Vav3 gene deficiency induced severe defects in the branching of those dendrite trunks to generate secondary and tertiary dendrites (Figure 6A). As a consequence, the number of branching points per Purkinje cell decreased ∼7-fold compared with those formed by wild-type cells (Figure 6B, right). When we analyzed primary granule cell cultures, we observed no gross alterations in the dendrite formation using immunofluorescence techniques (data not shown). However, the analysis of protein extracts from these cells at different divisions in culture (DIV) times indicated that Vav3−/− granule cells had a consistent reduction in the levels of MAP2) (Figure 6, C and D). These results were indicative of dendrite dysfunctions in Vav3-deficient granule cells, because MAP2 is an important regulatory protein involved in dendrite morphogenesis (Friedrich and Aszodi, 1991). To verify this possibility, we carried out transient transfections in primary cultures of granule cells using mammalian expression vectors expressing the enhanced green fluorescent protein (EGFP) or coexpressing the EGFP plus a c-Myc–tagged version of wild-type Vav3. After transfection, cells were fixed and examined by immunofluorescence microscopy to identify EGFP-positive cells and quantify their dendritic processes (Figure 6E). These experiments indicated that the overexpression of wild-type Vav3 increased the total dendrite length per cell (596 ± 176 vs. 406 ± 178.8 μm of cells expressing EGFP alone), as well as the total number of dendritic branching tips (12 ± 0.6 vs. 8 ± 0.5 of cells expressing EGFP alone; Figure 6F). These results indicate that Vav3 affects dendrite branching in both Purkinje and granule cells.


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)

Vav3 regulates dendritic outgrowth in Purkinje and granular cells. (A) Examples of the morphology of Purkinje cells obtained in mixed cultures derived from wild-type mice (left) and Vav3-deficient mice (right). To obtain these images, cell cultures were fixed, stained with anti-calbindin antibodies, and subjected to microscopy analysis. Less dendritic arborization is apparent in cultures generated from Vav3−/− mice. (B) Quantification of the Purkinje cell morphology in cultures derived from cerebella of the indicated genotypes. Quantification included the total dendritic tree area (left), the length of the longest dendrite (middle), and the number of branching points (right) in Purkinje cells (n = 4 independent experiments). **p < 0.01 compared with wild-type controls. A reduced number of tips is observed in Vav3-deficient Purkinje cells (right). No statistically significant differences were observed in the other two parameters evaluated in these experiments. (C) Expression levels of MAP2 in cultures of granule cell cultures of the indicated genotypes (top). At the indicated divisions in vitro (DIV, top), cellular lysates were obtained, electrophoretically fractionated in 6% SDS-PAGE gels, transferred onto nitrocellulose, and MAP2 levels were determined by anti-MAP2 Western blot (WB) analysis (top). Equal loading was demonstrated by the detection of a nonspecific band recognized by the anti-MAP2 antibody (middle) and by reblotting with an anti-vinculin antibody (bottom). (D) Quantification of MAP2 protein expression in granular cell cultures. Bands from the anti-MAP2 immunoblots obtained in three independent experiments were quantified by densitometry analysis and statistically analyzed. Data are presented as mean and SEM and were normalized taking into consideration the protein levels of vinculin detected in each sample. *p < 0.05 compared with wild-type controls. Reduced numbers of MAP2 are seen in cultures from Vav3-deficient mice. (E) Example of wild-type granule cells expressing EGFP alone (left l) or in combination with wild-type Vav3 (right). Cells were processed and transfected as indicated in Materials and Methods, fixed at division 4, and photographed using an inverted fluorescence microscope. Bar, 30 μm. Increased dendrite ramifications are seen in the granular cell expressing the Vav3 protein. (F) Quantitation of the total dendritic length (left) and the number of tips (right) of granular cells that were transfected with plasmids encoding the indicated proteins (n = 50 independent cells in each transfection). ***p < 0.001 compared with wild-type controls. Vav3 overexpression promotes an increase in the total length and number of tips in the transfected cells.
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Figure 6: Vav3 regulates dendritic outgrowth in Purkinje and granular cells. (A) Examples of the morphology of Purkinje cells obtained in mixed cultures derived from wild-type mice (left) and Vav3-deficient mice (right). To obtain these images, cell cultures were fixed, stained with anti-calbindin antibodies, and subjected to microscopy analysis. Less dendritic arborization is apparent in cultures generated from Vav3−/− mice. (B) Quantification of the Purkinje cell morphology in cultures derived from cerebella of the indicated genotypes. Quantification included the total dendritic tree area (left), the length of the longest dendrite (middle), and the number of branching points (right) in Purkinje cells (n = 4 independent experiments). **p < 0.01 compared with wild-type controls. A reduced number of tips is observed in Vav3-deficient Purkinje cells (right). No statistically significant differences were observed in the other two parameters evaluated in these experiments. (C) Expression levels of MAP2 in cultures of granule cell cultures of the indicated genotypes (top). At the indicated divisions in vitro (DIV, top), cellular lysates were obtained, electrophoretically fractionated in 6% SDS-PAGE gels, transferred onto nitrocellulose, and MAP2 levels were determined by anti-MAP2 Western blot (WB) analysis (top). Equal loading was demonstrated by the detection of a nonspecific band recognized by the anti-MAP2 antibody (middle) and by reblotting with an anti-vinculin antibody (bottom). (D) Quantification of MAP2 protein expression in granular cell cultures. Bands from the anti-MAP2 immunoblots obtained in three independent experiments were quantified by densitometry analysis and statistically analyzed. Data are presented as mean and SEM and were normalized taking into consideration the protein levels of vinculin detected in each sample. *p < 0.05 compared with wild-type controls. Reduced numbers of MAP2 are seen in cultures from Vav3-deficient mice. (E) Example of wild-type granule cells expressing EGFP alone (left l) or in combination with wild-type Vav3 (right). Cells were processed and transfected as indicated in Materials and Methods, fixed at division 4, and photographed using an inverted fluorescence microscope. Bar, 30 μm. Increased dendrite ramifications are seen in the granular cell expressing the Vav3 protein. (F) Quantitation of the total dendritic length (left) and the number of tips (right) of granular cells that were transfected with plasmids encoding the indicated proteins (n = 50 independent cells in each transfection). ***p < 0.001 compared with wild-type controls. Vav3 overexpression promotes an increase in the total length and number of tips in the transfected cells.
Mentions: Given the defective dendrite arborization found in the developing Vav3−/− cerebellum, we decided to investigate this process in more detail using in vitro cultures of both Purkinje and granule cells. We observed that cultured Purkinje cells from Vav3-deficient mice could develop the primary dendrite trunks in vitro and expand them to generate dendritic tree areas similar to those induced in wild-type cells (Figure 6, A and B). However, the Vav3 gene deficiency induced severe defects in the branching of those dendrite trunks to generate secondary and tertiary dendrites (Figure 6A). As a consequence, the number of branching points per Purkinje cell decreased ∼7-fold compared with those formed by wild-type cells (Figure 6B, right). When we analyzed primary granule cell cultures, we observed no gross alterations in the dendrite formation using immunofluorescence techniques (data not shown). However, the analysis of protein extracts from these cells at different divisions in culture (DIV) times indicated that Vav3−/− granule cells had a consistent reduction in the levels of MAP2) (Figure 6, C and D). These results were indicative of dendrite dysfunctions in Vav3-deficient granule cells, because MAP2 is an important regulatory protein involved in dendrite morphogenesis (Friedrich and Aszodi, 1991). To verify this possibility, we carried out transient transfections in primary cultures of granule cells using mammalian expression vectors expressing the enhanced green fluorescent protein (EGFP) or coexpressing the EGFP plus a c-Myc–tagged version of wild-type Vav3. After transfection, cells were fixed and examined by immunofluorescence microscopy to identify EGFP-positive cells and quantify their dendritic processes (Figure 6E). These experiments indicated that the overexpression of wild-type Vav3 increased the total dendrite length per cell (596 ± 176 vs. 406 ± 178.8 μm of cells expressing EGFP alone), as well as the total number of dendritic branching tips (12 ± 0.6 vs. 8 ± 0.5 of cells expressing EGFP alone; Figure 6F). These results indicate that Vav3 affects dendrite branching in both Purkinje and granule cells.

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