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1 Figure S1 Treatment with both Sema6D and Plexin-A1 sirnas induces the phenotype essentially identical to that induced by treatment with Sema6D sirna alone or Plexin-A1 sirna alone. (a,b) The cardiac tube of an HH stage 10 chick embryo was electroporated in ovo with sirna (40 pmol/µl)and then allowed to develop to HH stage 32. Embryos were treated with the following conditions: (a) csema6d and control sirnas; (b) csema6d and cplexin- A1 sirnas. csema6d sirna suppresses cardiac expansion and trabecular formation (a). Embryos treated with both csema6d and cplexin-a1 sirnas display the suppression of trabeculation, which is compatible with those treated with either csema6d or cplexin-a1 sirnas (b). 1

2 Figure S2 Plexin-A2 and Plexin-A4 fail to compensate a function of Plexin-A1 in the trabecular formation. As shown in Fig. 1, over-expression of Plexin-A1-EC could rescue the suppressed trabeculation in embryo treated with cplexin-a1 sirna but not in embryo treated with csema6d sirna, indicating the possibility that Plexin-A1 may function as a ligand in trabeculation. However, there is also a possibility that Plexin-A2 and Plexin-A4, which are expressed in developing hearts, are similarly involved in trabeculation. To address this possibility, the effect of the extracellular domain of Plexin-A2 and Plexin-A4 (Plexin-A2-EC and Plexin-A4-EC) on trabeculation was analyzed. (a-f) The cardiac tube of an HH stage 10 chick embryo was electroporated in ovo with sirna (40 pmol/µl). Transfectants (10 5 cells) were then injected into the amniotic cavity of HH stage 12 embryos, which were then allowed to develop to HH stage 32. Embryos were treated with the following conditions: (a) control cells; (b) Plexin-A2- EC cells; (c) Plexin-A4-EC cells; (d) cplexin-a1 sirna and control cells; (e) cplexin-a1 sirna and Plexin-A2-EC cells; (f) cplexin-a1 sirna and Plexin-A4-EC cells. Neither of Plexin-A2-EC nor Plexin-A4-EC affect the trabecular formation of cardiac ventricle in not only control embryos (a, b, c) but also embryos treated with cplexin-a1 sirna (d, e, f). C and T indicate the compact and trabecular layers, respectively. Scale bar, 100 µm. (g) Ratio of the thickness of trabecular layer to that of the compact layer in cardiac ventricles treated with transfectants were measured in the left ventricular free wall near the apex. Data are the means ± SEM. *p<0.05, vs. embryo treated with control cells. Thus, the role of Plexin-A1 in the trabecular formation of cardiac ventricle is specific. 2

3 Figure S3 Effect of Sema6D or Plexin-A1 RNAi on the proliferation and apoptosis of myocardial cells in developing cardiac ventricle. The embryos treated with csema6d or cplexin-a1 sirnas displayed the defects of expansion of cavity and trabeculation in cardiac ventricle. Although our study indicates the changes in migration of myocardial cells by Sema6D and Plexin-A1 interaction, the alternative possibility that this interaction may regulate the proliferation and apoptosis of myocardial cells remaines. To address this possibility, we analyzed growth and apoptosis of myocardial cells of embryos treated with Sema6D or Plexin-A1 sirna. (a) The cardiac tube of an HH stage 10 chick embryo was electroporated in ovo with csema6d or Plexin-A1 sirnas (40 pmol/µl), and then allowed to develop to HH stage 32. TUNEL staining was performed in sections of cardiac tube using enhanced fluorescent detection by nick-end labeling of cleaved double stranded DNA with terminal deoxynucleotidyl transferase-mediated digoxigenin-dntp (1 hour at 37 C), followed by detection using fluoresceinlabeled anti-digoxigenin antibody. Sections were also stained for nuclei (DiI). Representative fluorescent photomicrographs depicting TUNEL (green) and DiI (red) staining of cardiac ventricle. Scale bars, 10 µm. (Lower panel) The quantitative data showing the number of TUNEL-positive nuclei expressed as N per 1000 total nuclei. Data are mean±sem, *P<0.05 versus Control. TUNEL-positive nuclei are not more frequent than in embryos treated with csema6d and cplexin-a1 sirnas. (b) The cardiac tube of an HH stage 10 chick embryo was electroporated in ovo with csema6d or Plexin-A1 sirnas (40 pmol/µl). 5-Bromo-2 -deoxyuridine (BrdU, Sigma) was injected into the amniotic cavity of HH stage 32 embryos. After 4 hour, embryos were removed, fixed, and sectined. For detection of BrdU in cardiac ventricle, sections were immunstained with mouse anti-brdu antibody and counterstained with DiI for total nuclei. Scale bars, 10 µm. (Lower panel) The quantitative data showing the number of BrdU-positive nuclei expressed as N per 100 total nuclei. Data are mean±sem, *P<0.05 versus Control. BrdUpositive nuclei are not more frequent than in embryos treated with csema6d and cplexin-a1 sirnas. Thus, Sema6D-Plexin-A1 signaling pathway does not regulate the proliferation either or apoptosis of myocardial cells, but regulate the migration properties of myocardial cells in the cardiac ventricle. 3

4 Figure S4 (a-d) There is no evidence for direct interaction between Sema6D and Plexin-A1 in Cis. (e) The localization of full-length and cleaved Sema6D in transfected cells. Since myocardial cells in the compact layer express both Sema6D and Plexin-A1, there is a possibility that they may interact in Cis and this interaction may have an influence on downstream signals of Sema6D. To address this possibility, we examined the effect of coexpression of Sema6D and Plexin-A1 on downstream signals of Sema6D (a) and the possible interaction between Sema6D and Plexin-A1 through their cytoplasmic regions (b-d). (a) HEK293 cells expressing Sema6D and HEK293 cells co-expressing Sema6D and Plexin-A1 were lysed and immunoprecipitated with anti-v5, anti-abl kinase, or anti-mena antibodies, and immunoprecipitates were immunoblotted with the indicated antibodies. Cells co-expressing Sema6D and Plexin-A1 show enhanced binding of endogenous Abl kinase with its tyrosine-phosphorylation and reduced binding of endogenous Mena with its tyrosine-phosphorylation to Sema6D, compared with cells expressing only Sema6D. The pattern of Abl kinase and Mena association with Sema6D in cells expressing both Sema6D and Plexin- A1 is essentially identical to that seen when cells expressing only Sema6D were cultured with cells expressing Plexin-A1 (Fig. 2a). The result suggests that co-expression of Sema6D and Plexin-A1 does not affect downstream signals of Sema6D, such as Abl kinase activation. (b) Lysates from cardiac tubes of HH stage 32 chick embryos were immunoprecipitated with anti- Sema6D antibody and immunoprecipitates and lysates were blotted with the indicated antibodies. However, anti-plexin-a1 failed to co-imunoprecipitate Sema6D, suggesting that endogenous Sema6D may not interact with endogenous Plexin-A1 in Cis. (c, d) To further study whether the physical interaction occurs between Sema6D and Plexin-A1 through their cytoplasmic region, cells expressing the cytoplasmic regions of each molecule were used for immunoprecipitate-immunoblot assay. (c) The constructs used for in vitro binding assays diagrammed here are FLAG-tagged Sema6D-CD [Sema6D lacking the extracellular region (Arg2 to Val1080)] and myc-tagged Plexin- A1-CD [Plexin-A1 lacking the extracellular region (Pro2 to Ser1241)]. (d) HEK293 cells were transfected with the indicated constructs. Proteins were immunoprecipitated from transfected cell lysates and immunoblotted with the indicated antibodies. Sema6D-CD dose not bind to Plexin-A1-CD. Thus, the cytoplasmic interaction between Sema6D and Plexin-A1 does not occur, suggesting that Sema6D-Plexin-A1 interaction in Cis is more probable than that in Trans. (e) To analyze the distribution of Sema6D at different molecular weights to membrane and cytosolic fraction, cells expressing Sema6D were homogenized in the absence of detergent. The cytosolic fraction and Triton X-100-soluble membrane fraction were obtained by serial centrifugation and extraction with 1% Triton X-100, and then immunoblotted with anti-sema6d antibody. The full-length and truncated Sema6D are almost exclusively detected in the membrane fraction. Thus, upon binding to Plexin-A1, truncated Sema6D remains at the plasma membrane, suggesting that after proteolysis the truncated Sema6D is still functional. 4

5 Figure S5 Abl kinase regulates the trabecular formation in developing cardiac ventricle. As shown in Fig. 4h, constitutively active Abl kinase stimulated migration of myocardial cells toward the trabeculae in the cardiac ventricle, indicating that Abl kinase, which is activated by Sema6D-Plexin-A1 interaction, is important in trabecular formation. To study the functional role of endogenous Abl kinase in the developing heart, endogenous Abl kinase activity was inhibited with STI-571. (a, b) An HH stage 10 chick embryo was incubated in ovo with STI-571 (50 µm), and then embryos were allowed to develop at HH stage 32. Incubation with STI-571 suppresses the trabecular formation in cardiac ventricle. Scale bar, 100 µm. (c) Ratio of the thickness of trabecular layer to that of the compact layer in cardiac ventricles treated with STI-571 were measured in the left ventricular free wall near the apex. Data are the means ± SEM. *p<0.05, vs. embryo treated with control vehicle. Thus, Abl kinase functions as a downstream signal molecule of Sema6D and its function is important in the physiological condition. 5

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