Limits...
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.

Show MeSH

Related in: MedlinePlus

Vav3 is expressed in Purkinje and granular cells of the mouse cerebellum. (A) Anti-Vav3 immunohistochemical analysis of sagittal cerebellar sections obtained from wild-type (left) and Vav3−/− (right) mice. mcl, molecular cell layer; gcl, granular cell layer. Bar, 50 μm. Detection of Vav3 immunoreactivity is observed in sections from wild-type animals in both Purkinje cells and in scattered areas of the granular cell layer. (B) Total RNA samples obtained from wild type and Vav3−/− granular cell cultures were subjected to RT-PCR analysis using oligonucleotide primers specific for the mouse Vav3 cDNA (top). As control, aliquots of the same RNAs were amplified using oligonucleotide primers specific for the mouse P36b4 cDNA (bottom). Final PCR products were separated electrophoretically in agarose gels and photographed. (C) Total RNAs obtained from the cerebella of wild-type mice at the indicated postnatal (P) stages were subjected to RT-PCR analysis using oligonucleotide primers for the mouse Vav3 (top) and P36b4 (bottom) cDNAs and processed as indicated above. These results of this figure show that Vav3 is expressed at different levels in the cerebellum in a developmental-dependent manner (C). Furthermore, Vav3 is expressed in this tissue at least in Purkinje (A) and granule cells (B).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2836963&req=5

Figure 1: Vav3 is expressed in Purkinje and granular cells of the mouse cerebellum. (A) Anti-Vav3 immunohistochemical analysis of sagittal cerebellar sections obtained from wild-type (left) and Vav3−/− (right) mice. mcl, molecular cell layer; gcl, granular cell layer. Bar, 50 μm. Detection of Vav3 immunoreactivity is observed in sections from wild-type animals in both Purkinje cells and in scattered areas of the granular cell layer. (B) Total RNA samples obtained from wild type and Vav3−/− granular cell cultures were subjected to RT-PCR analysis using oligonucleotide primers specific for the mouse Vav3 cDNA (top). As control, aliquots of the same RNAs were amplified using oligonucleotide primers specific for the mouse P36b4 cDNA (bottom). Final PCR products were separated electrophoretically in agarose gels and photographed. (C) Total RNAs obtained from the cerebella of wild-type mice at the indicated postnatal (P) stages were subjected to RT-PCR analysis using oligonucleotide primers for the mouse Vav3 (top) and P36b4 (bottom) cDNAs and processed as indicated above. These results of this figure show that Vav3 is expressed at different levels in the cerebellum in a developmental-dependent manner (C). Furthermore, Vav3 is expressed in this tissue at least in Purkinje (A) and granule cells (B).

Mentions: Previous Northern blot analysis indicated that the human VAV3 gene is expressed in the cerebellum (Movilla and Bustelo, 1999). Furthermore, the Allen Brain Atlas (http://mouse.brain-map.org) also records high levels of expression of the mouse Vav3 gene, but not of Vav1 or Vav2 genes, in the granular cell layer of the cerebellum. To confirm and expand the expression pattern of that gene in this tissue, we performed immunohistochemical experiments using a home-made, Vav3-specific rabbit polyclonal antibody (Movilla and Bustelo, 1999). These analyses indicated high levels of Vav3 in Purkinje cells and in cells located in the granular cell layer of the cerebellum (Figure 1A, left). In Purkinje cells, Vav3 protein was detected both in the cytoplasm of the somas and in dendrites (Figure 1A, left). No expression of Vav3 was detected instead in molecular cell layer cells (Figure 1A, left). This immunoreactivity was specific for Vav3, because the antibody did not detect those signals in cerebellar sections obtained from Vav3-deficient mice (Figure 1A, right). To verify whether the detection of Vav3 signals in the granular cell layer was due to its expression in Purkinje cell axons or in granular cells, we carried out RT-PCRs using primary granular cells cultured in vitro. As shown in Figure 1B (top), the band specific for the amplified Vav3 cDNA was detected in the cultures obtained from wild-type cells but not from Vav3−/− granular cells. We performed further RT-PCR experiments to analyze the expression kinetics of the Vav3 mRNA during cerebellar development. We observed that the expression of the Vav3 transcript peaked at the time of birth (P0) and at the end of cerebellar development (P21 and adulthood). Moreover, we found that the Vav3 mRNA underwent a sharp decrease during the developmental interval between P7 and P14 (Figure 1C, top). As control, the P36b4 mRNA showed a constitutive expression pattern in all the developmental stages surveyed in this study (Figure 1C, bottom). These results indicate that the Vav3 gene is transcribed both in Purkinje and granule cells and that its expression pattern fluctuates during cerebellar development.


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 is expressed in Purkinje and granular cells of the mouse cerebellum. (A) Anti-Vav3 immunohistochemical analysis of sagittal cerebellar sections obtained from wild-type (left) and Vav3−/− (right) mice. mcl, molecular cell layer; gcl, granular cell layer. Bar, 50 μm. Detection of Vav3 immunoreactivity is observed in sections from wild-type animals in both Purkinje cells and in scattered areas of the granular cell layer. (B) Total RNA samples obtained from wild type and Vav3−/− granular cell cultures were subjected to RT-PCR analysis using oligonucleotide primers specific for the mouse Vav3 cDNA (top). As control, aliquots of the same RNAs were amplified using oligonucleotide primers specific for the mouse P36b4 cDNA (bottom). Final PCR products were separated electrophoretically in agarose gels and photographed. (C) Total RNAs obtained from the cerebella of wild-type mice at the indicated postnatal (P) stages were subjected to RT-PCR analysis using oligonucleotide primers for the mouse Vav3 (top) and P36b4 (bottom) cDNAs and processed as indicated above. These results of this figure show that Vav3 is expressed at different levels in the cerebellum in a developmental-dependent manner (C). Furthermore, Vav3 is expressed in this tissue at least in Purkinje (A) and granule cells (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836963&req=5

Figure 1: Vav3 is expressed in Purkinje and granular cells of the mouse cerebellum. (A) Anti-Vav3 immunohistochemical analysis of sagittal cerebellar sections obtained from wild-type (left) and Vav3−/− (right) mice. mcl, molecular cell layer; gcl, granular cell layer. Bar, 50 μm. Detection of Vav3 immunoreactivity is observed in sections from wild-type animals in both Purkinje cells and in scattered areas of the granular cell layer. (B) Total RNA samples obtained from wild type and Vav3−/− granular cell cultures were subjected to RT-PCR analysis using oligonucleotide primers specific for the mouse Vav3 cDNA (top). As control, aliquots of the same RNAs were amplified using oligonucleotide primers specific for the mouse P36b4 cDNA (bottom). Final PCR products were separated electrophoretically in agarose gels and photographed. (C) Total RNAs obtained from the cerebella of wild-type mice at the indicated postnatal (P) stages were subjected to RT-PCR analysis using oligonucleotide primers for the mouse Vav3 (top) and P36b4 (bottom) cDNAs and processed as indicated above. These results of this figure show that Vav3 is expressed at different levels in the cerebellum in a developmental-dependent manner (C). Furthermore, Vav3 is expressed in this tissue at least in Purkinje (A) and granule cells (B).
Mentions: Previous Northern blot analysis indicated that the human VAV3 gene is expressed in the cerebellum (Movilla and Bustelo, 1999). Furthermore, the Allen Brain Atlas (http://mouse.brain-map.org) also records high levels of expression of the mouse Vav3 gene, but not of Vav1 or Vav2 genes, in the granular cell layer of the cerebellum. To confirm and expand the expression pattern of that gene in this tissue, we performed immunohistochemical experiments using a home-made, Vav3-specific rabbit polyclonal antibody (Movilla and Bustelo, 1999). These analyses indicated high levels of Vav3 in Purkinje cells and in cells located in the granular cell layer of the cerebellum (Figure 1A, left). In Purkinje cells, Vav3 protein was detected both in the cytoplasm of the somas and in dendrites (Figure 1A, left). No expression of Vav3 was detected instead in molecular cell layer cells (Figure 1A, left). This immunoreactivity was specific for Vav3, because the antibody did not detect those signals in cerebellar sections obtained from Vav3-deficient mice (Figure 1A, right). To verify whether the detection of Vav3 signals in the granular cell layer was due to its expression in Purkinje cell axons or in granular cells, we carried out RT-PCRs using primary granular cells cultured in vitro. As shown in Figure 1B (top), the band specific for the amplified Vav3 cDNA was detected in the cultures obtained from wild-type cells but not from Vav3−/− granular cells. We performed further RT-PCR experiments to analyze the expression kinetics of the Vav3 mRNA during cerebellar development. We observed that the expression of the Vav3 transcript peaked at the time of birth (P0) and at the end of cerebellar development (P21 and adulthood). Moreover, we found that the Vav3 mRNA underwent a sharp decrease during the developmental interval between P7 and P14 (Figure 1C, top). As control, the P36b4 mRNA showed a constitutive expression pattern in all the developmental stages surveyed in this study (Figure 1C, bottom). These results indicate that the Vav3 gene is transcribed both in Purkinje and granule cells and that its expression pattern fluctuates during cerebellar development.

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