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First published online 18 January 2006
doi: 10.1242/dev.02230

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Regulation of somitogenesis by Ena/VASP proteins and FAK during Xenopus development

Department of Genetics, Cell Biology and Development and Developmental Biology Center, University of Minnesota, Minneapolis, MN 55455, USA.

View larger version (132K): [in a new window]   Fig. 1. Xena localization during somitogenesis. (A) At stage 17, Xena is enriched at the PSM/notochord (no) boundary (arrow) and at the cell periphery in the PSM and somitic mesoderm during somite formation (asterisks). (B) Longitudinal view of a stage 22 embryo showing accumulation of Xena at intersomitic boundaries. (C-E) In anterior regions (stage 18 embryo is shown), Xena and FN accumulate at presumptive somite boundaries prior to rotation. Arrow indicates accumulation at a newly formed boundary and arrowheads indicate accumulation at the next forming boundary. (F) In posterior regions (stage 22 embryo is shown), Xena accumulation at somite boundaries coincides with rotation. Arrow indicates accumulation at forming boundary; arrowhead shows lack of Xena enrichment in the PSM where the next boundary will form. (G) Cross-section of a stage 22 embryo showing Xena localization to cell-cell contacts within somites. (H) Xena is enriched at intermyotomal boundaries in stage 45 embryos. Anterior is towards the left in all images except G. Xena localization was visualized with antibodies raised against Mena (A,B) or Xena (C,E-H). Scale bars: 100 µm.

View larger version (113K): [in a new window]   Fig. 2. Xena colocalizes with components of integrin adhesion complexes at intersomitic and intermyotomal junctions. Confocal images of stage 22 (A-C,G-I,M-O) and stage 35 (D-F,J-L) embryos stained for Xena (A,D,G,J,M; red in C,F,I,L,O), ß1-integrin (B,E, green in C,F), vinculin (H,K, green in I,L) and FAK (N, green in O). Xena colocalizes with ß1-integrin, vinculin and FAK at intersomitic boundaries and with ß1-integrin and vinculin at intermyotomal junctions (co-localization appears yellow). Xena staining was performed with anti-Xena antibodies. Anterior is to the left in all images. Scale bars: 100 µm.

View larger version (103K): [in a new window]   Fig. 3. FP4-mito causes mis-localization of Xena in the PSM and somites. (A-C) Expression of FP4-mito results in the mis-localization of Xena to the surface of mitochondria within the cell where it co-localizes with GFP. (D-F) Xena localization is unaffected in AP4-mito-expressing cells. Embryos were double-stained for GFP to detect FP4-mito (A, green in C) and AP4-mito (D, green in F) and Xena (B,E, red in C,F). Anterior is towards the left. Scale bar: 100 µm.

View larger version (69K): [in a new window]   Fig. 4. Inhibition of Ena/VASP function does not perturb patterning of the PSM. (A,B) Embryos stained for ß-tubulin, which outlines the periphery of cells, shows that expression of AP4-mito (A) or FP4-mito (B) does not alter the distribution or morphology of cells in the PSM following gastrulation. (C-H) Inhibition of Ena/VASP function does not affect the intensity or distribution of paraxial protocadherin (PAPC; C-E) or MyoD (F-H). Injected side is marked with an asterisk in each panel. Anterior is at the top. PAPC: AP4-mito, n=10; FP4-mito, n=17. MyoD: AP4-mito, n=7; FP4-mito, n=17. Scale bar: 100 µm.

View larger version (59K): [in a new window]   Fig. 5. Inhibition of Ena/VASP function disrupts somite rotation and leads to expanded somite area. (A-D) Dorsal explants of stage 22 embryos were co-stained for 12/101 (A,C; red in B,D) to visualize somite morphology and GFP (green in B,D) to visualize FP4-mito and AP4-mito expression. Expression of FP4-mito (A,B) resulted in disruption of somite organization and cohesion, whereas somite morphology appeared normal in AP4-mito (C,D) expressing embryos. Scale bar: 100 µm. (E) Percentage of stage 22 embryos showing normal somite morphology and mild or major disruption of somites, as assessed by 12/101 immunostaining. Results are pooled from three independent experiments; n=24-33 embryos/group. Anterior is at the top in A-D. (F-I) Somite area is increased and cells are misoriented in FP4-mito (F,G), but not AP4-mito (H,I) injected embryos. Scale bar: 100 µm. (J) Quantitative analysis of cross-sectional area of somites from uninjected, AP4-mito or FP4-mito injected embryos reveals that inhibition of Ena/VASP results in a significant increase in somite area (see Materials and methods). *P<0.001 (Student's t-test). Error bars indicate s.e.m. Results shown in graph are from four independent experiments, n=10-14 embryos per treatment group.

View larger version (113K): [in a new window]   Fig. 6 . Inhibition of Ena/VASP results in defects in somite rotation and boundary formation. Dorsal explants of stage 25 embryos expressing FP4-mito (A-C) or AP4-mito (D-F) were coimmunostained for Xena (A,D; green in C,F) and ß1-integrin (B,E; red in C,F). (A-C) FP4-mito expression leads to mis-localization of Xena that is accompanied by the presence of misoriented cells within somites and disruption of ß1-integrin localization at intersomitic boundaries. (D-F)AP4-mito expression does not alter Xena or ß1-integrin localization, and is accompanied by normal somite formation. Scale bar: 100 µm.

View larger version (157K): [in a new window]   Fig. 7. Inhibition of Ena/VASP leads to disruption of the FN matrix. (A-D) Stacked z-series of FN staining reveals a fragmentation of the matrix (arrowheads), separation of somites from overlying ectoderm (arrows), gaps in the intersomitic FN matrix (asterisks) and loss of defined boundaries between the somite and neural tube or notochord in FP4-mito (bracket) (A), but not AP4-mito (C) injected embryos. Injected side is on the right. High magnification images of the boundary between the dorsal somite and neural tube reveals a disruption in the integrity of the FN matrix in FP4-mito (B) but not AP4-mito (D) injected embryos. (E-G) Stacked z-series show a dense FN matrix (white) on the interior BCR surface of an uninjected embryo (green, E) and an embryo injected with AP4-mito (green, F). By contrast, expression of FP4-mito (G) blocks FN matrix assembly as revealed by a thin or absent FN matrix in areas of GFP signal. s, somite; nt, neural tube. Scale bars: 100 µm.

View larger version (16K): [in a new window]   Fig. 8. Inhibition of Ena/VASP function impairs spreading of somitic cells on FN. Percentage of cells with a rounded phenotype from uninjected embryos and embryos injected with AP4-mito or FP4-mito with (+) or without (-) detectable GFP signal show a significant increase in the occurrence of a rounded phenotype in cells expressing FP4-mito. *P<0.05 (Student's t-test). Error bars indicate s.e.m. Results are from four independent experiments, n=864-888 cells per treatment group.

View larger version (86K): [in a new window]   Fig. 9. Inhibition of Ena/VASP function leads to a decrease in FAK-Y397 phosphorylation. Protein lysates prepared from stage 20 dorsal explants were probed with anti-FAK-pY397, anti-FAK, or anti--fodrin antibodies. Expression of FP4-mito caused a significant decrease in the levels of FAK-pY397.

View larger version (90K): [in a new window]   Fig. 10. FAK is required for somitogenesis and FN matrix deposition. (A) Immunoblot showing levels of FAK and FRNK in control (left lane) and FRNK-injected (right lane) embryos. (B) FRNK expression inhibits autophosphorylation at Y397; -fodrin serves as a loading control. (C) Inhibition of FAK results in somite defects including misorientation of cells (arrow) and impaired somite boundary formation (arrowheads). FRNK-injected side is at the bottom and anterior is to the left. (D,E) Immunostaining of FN matrix in control (D) and FRNK-injected (E) BCRs show that FAK is required for FN matrix deposition. Scale bars: 100 µm.

View larger version (72K): [in a new window]   Fig. 11. Inhibition of FAK alters Xena localization. (A) Xena is enriched at the cortex in control animal caps. (B) Inhibition of FAK results in decrease in the levels of Xena at the cortex. (C) Plot of pixel intensity in control animal caps showing enrichment of Xena at the cortex. (D) Plot of pixel intensity in FRNK-injected animal caps reveals that Xena is de-localized from the cortex and displays a more uniform distribution. (E) Quantitative analysis of Xena staining in control (GFP) and FRNK injected BCRs. For 10 randomly selected cells, pixel intensity was measured along a line extending across the region of cell-cell contact. Average pixel intensity at the membrane (two peak intensities 0.3 µm apart) was compared with the average pixel intensity of the juxtamembrane region (3.3 µm adjacent to membrane). The ratio of the average membrane intensity to average juxtamembrane intensity was significantly lower for FRNK-expressing embryos. P<0.001 (Student's t-test). Error bars indicate s.d. n=50 cells per treatment.