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The RhoGEF DOCK10 is essential for dendritic spine morphogenesis.

Jaudon F, Raynaud F, Wehrlé R, Bellanger JM, Doulazmi M, Vodjdani G, Gasman S, Fagni L, Dusart I, Debant A, Schmidt S - Mol. Biol. Cell (2015)

Bottom Line: We found a strong increase in the expression of the Cdc42-specific GEF DOCK10.Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs.We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Biochimie Macromoléculaire, CNRS-UMR 5237, Université de Montpellier, 34293 Montpellier, France.

No MeSH data available.


Related in: MedlinePlus

Gene expression profiling of all DOCK RhoGEFs in purified postnatal Purkinje neurons. (A, B) Sorting of GFP-expressing postnatal Purkinje neurons by FACS. Dissociated cells from cerebella of Pcp2-GFP mice at P3, P7, P15, and P20 were plated before (A) and after (B) sorting and fixed. Nuclei were stained with Hoechst stain to visualize the total cell number in the sample, and Purkinje neurons were revealed with a calbindin antibody (red) and by direct fluorescence of the GFP (green). Micrographs show representative sorting results of cerebellar cells from mice at P7. (C) Histogram representing the enrichment of Purkinje neurons within the cell population upon sorting, expressed as relative percentages. Data are expressed as mean enrichment value of all age groups combined ± SD of at least three independent experiments. **p < 0.01 (Student's t test). (D) RT-qPCR performed before and after FACS on the mRNA of specific marker genes of the main cerebellar cell types: calbindin (Purkinje cells), NeuroD1 (cerebellar granule neurons), GFAP (astrocytes), and Tcfap2a (interneurons). Data are expressed as mean ± SD of at least three experiments performed on P7 Pcp2-GFP mice in the example shown. **p < 0.01 and ***p < 0.001 (Student's t test). (E) RT-qPCR performed on purified PC mRNAs of all 11 mammalian DOCK-family RhoGEFs. Data are expressed as mean ± SD of at least three experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 (Student's t test).
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Figure 1: Gene expression profiling of all DOCK RhoGEFs in purified postnatal Purkinje neurons. (A, B) Sorting of GFP-expressing postnatal Purkinje neurons by FACS. Dissociated cells from cerebella of Pcp2-GFP mice at P3, P7, P15, and P20 were plated before (A) and after (B) sorting and fixed. Nuclei were stained with Hoechst stain to visualize the total cell number in the sample, and Purkinje neurons were revealed with a calbindin antibody (red) and by direct fluorescence of the GFP (green). Micrographs show representative sorting results of cerebellar cells from mice at P7. (C) Histogram representing the enrichment of Purkinje neurons within the cell population upon sorting, expressed as relative percentages. Data are expressed as mean enrichment value of all age groups combined ± SD of at least three independent experiments. **p < 0.01 (Student's t test). (D) RT-qPCR performed before and after FACS on the mRNA of specific marker genes of the main cerebellar cell types: calbindin (Purkinje cells), NeuroD1 (cerebellar granule neurons), GFAP (astrocytes), and Tcfap2a (interneurons). Data are expressed as mean ± SD of at least three experiments performed on P7 Pcp2-GFP mice in the example shown. **p < 0.01 and ***p < 0.001 (Student's t test). (E) RT-qPCR performed on purified PC mRNAs of all 11 mammalian DOCK-family RhoGEFs. Data are expressed as mean ± SD of at least three experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 (Student's t test).

Mentions: To identify novel RhoGEFs involved in dendritic tree morphogenesis, we undertook gene expression profiling of all DOCK-family RhoGEFs in Purkinje cells. Because these neurons represent only 2–3% of the whole cerebellum, we took advantage of a mouse strain carrying the green fluorescent protein (GFP) under the Purkinje-specific promoter Pcp2 to purify the PCs by FACS (Tomomura et al., 2001). Neurons were isolated from cerebella of postnatal day 3 (P3), P7, P15, and P20 Pcp2-GFP mice, corresponding to the time frame of development of the Purkinje cell dendritic tree. Purity of the Purkinje cell samples was assessed both by visual counting of the calbindin-positive cells (calbindin is a Purkinje cell–specific marker) before and after sorting (Figure 1, A-C) and by real-time quantitative PCR (RT-qPCR) of marker genes specific for the Purkinje cells (calbindin) and for the other main cerebellar cell types, namely granule cells (NeuroD1), astrocytes (glial fibrillary acidic protein [GFAP]), and interneurons (Tcfap2a; Figure 1D). After sorting, Purkinje cells represented ∼78% of the cell population.


The RhoGEF DOCK10 is essential for dendritic spine morphogenesis.

Jaudon F, Raynaud F, Wehrlé R, Bellanger JM, Doulazmi M, Vodjdani G, Gasman S, Fagni L, Dusart I, Debant A, Schmidt S - Mol. Biol. Cell (2015)

Gene expression profiling of all DOCK RhoGEFs in purified postnatal Purkinje neurons. (A, B) Sorting of GFP-expressing postnatal Purkinje neurons by FACS. Dissociated cells from cerebella of Pcp2-GFP mice at P3, P7, P15, and P20 were plated before (A) and after (B) sorting and fixed. Nuclei were stained with Hoechst stain to visualize the total cell number in the sample, and Purkinje neurons were revealed with a calbindin antibody (red) and by direct fluorescence of the GFP (green). Micrographs show representative sorting results of cerebellar cells from mice at P7. (C) Histogram representing the enrichment of Purkinje neurons within the cell population upon sorting, expressed as relative percentages. Data are expressed as mean enrichment value of all age groups combined ± SD of at least three independent experiments. **p < 0.01 (Student's t test). (D) RT-qPCR performed before and after FACS on the mRNA of specific marker genes of the main cerebellar cell types: calbindin (Purkinje cells), NeuroD1 (cerebellar granule neurons), GFAP (astrocytes), and Tcfap2a (interneurons). Data are expressed as mean ± SD of at least three experiments performed on P7 Pcp2-GFP mice in the example shown. **p < 0.01 and ***p < 0.001 (Student's t test). (E) RT-qPCR performed on purified PC mRNAs of all 11 mammalian DOCK-family RhoGEFs. Data are expressed as mean ± SD of at least three experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 (Student's t test).
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Figure 1: Gene expression profiling of all DOCK RhoGEFs in purified postnatal Purkinje neurons. (A, B) Sorting of GFP-expressing postnatal Purkinje neurons by FACS. Dissociated cells from cerebella of Pcp2-GFP mice at P3, P7, P15, and P20 were plated before (A) and after (B) sorting and fixed. Nuclei were stained with Hoechst stain to visualize the total cell number in the sample, and Purkinje neurons were revealed with a calbindin antibody (red) and by direct fluorescence of the GFP (green). Micrographs show representative sorting results of cerebellar cells from mice at P7. (C) Histogram representing the enrichment of Purkinje neurons within the cell population upon sorting, expressed as relative percentages. Data are expressed as mean enrichment value of all age groups combined ± SD of at least three independent experiments. **p < 0.01 (Student's t test). (D) RT-qPCR performed before and after FACS on the mRNA of specific marker genes of the main cerebellar cell types: calbindin (Purkinje cells), NeuroD1 (cerebellar granule neurons), GFAP (astrocytes), and Tcfap2a (interneurons). Data are expressed as mean ± SD of at least three experiments performed on P7 Pcp2-GFP mice in the example shown. **p < 0.01 and ***p < 0.001 (Student's t test). (E) RT-qPCR performed on purified PC mRNAs of all 11 mammalian DOCK-family RhoGEFs. Data are expressed as mean ± SD of at least three experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 (Student's t test).
Mentions: To identify novel RhoGEFs involved in dendritic tree morphogenesis, we undertook gene expression profiling of all DOCK-family RhoGEFs in Purkinje cells. Because these neurons represent only 2–3% of the whole cerebellum, we took advantage of a mouse strain carrying the green fluorescent protein (GFP) under the Purkinje-specific promoter Pcp2 to purify the PCs by FACS (Tomomura et al., 2001). Neurons were isolated from cerebella of postnatal day 3 (P3), P7, P15, and P20 Pcp2-GFP mice, corresponding to the time frame of development of the Purkinje cell dendritic tree. Purity of the Purkinje cell samples was assessed both by visual counting of the calbindin-positive cells (calbindin is a Purkinje cell–specific marker) before and after sorting (Figure 1, A-C) and by real-time quantitative PCR (RT-qPCR) of marker genes specific for the Purkinje cells (calbindin) and for the other main cerebellar cell types, namely granule cells (NeuroD1), astrocytes (glial fibrillary acidic protein [GFAP]), and interneurons (Tcfap2a; Figure 1D). After sorting, Purkinje cells represented ∼78% of the cell population.

Bottom Line: We found a strong increase in the expression of the Cdc42-specific GEF DOCK10.Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs.We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Biochimie Macromoléculaire, CNRS-UMR 5237, Université de Montpellier, 34293 Montpellier, France.

No MeSH data available.


Related in: MedlinePlus