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Phosphorylation and de-phosphorylation of Marcksb is required for the activation of BMP signaling.(A) Overexpression of marcksb at high dosage (1000 pg per embryo) caused ventralization defects. “asterisk” shows the notochord; “arrow” indicates the disappearance of notochord; “arrow head” indicates the enlarged blood island. (B) The percentage of embryos with normal-like, moderate ventralization or severe ventralization. “n” represents the number of embryos we observed. (C) Phosphorylation and de-phosphorylation mutation types of Marcksb altered the sub-cellular location of Marcksb. (C-a) a diagram showing mutated regions of S4D-Marcksb and S4N-Marcksb; (C-b) A schematic showing the procedure of mosaic overexpression assay; (C- c, d and e) confocal microscopy analysis at shield stage showed that wildtype Marcksb and S4N-Marcksb co-localized with memGFP while S4D-Marcksb did not. (D) Representative images showing the expression of szl in wildtype embryos (D-a), and the embryos injected with marcksb mRNA (D-b), S4D-marcksb mRNA (D-c) and S4N-marcksb mRNA (D-d) at shield stage. Embryos are animal-pole view with dorsal to the right. (E) The percentage of embryos with normal-like, decreased or mildly increased expression of szl in different experimental groups indicated in the (D). “n” represents the number of embryos we observed. (F, G) Genetic interaction was examined by co-injection of sub-dose marcksb mRNA (200 pg/embryo) and chd_MO. (F) representative embryos showing for phenotypes of normal-like, moderate ventralization (V1-V2, ventralization type 1 to type 2) and severe ventralization (V3-V4, ventralization type 3 to type 4); (G) The percentage of embryos with normal-like, moderate ventralization or severe ventralization. “n” represents the number of embryos we observed. (H) Representative images showing the expression of szl in the shield-stage embryos injected with chd_MO (H-a), chd_MO plus marcksb mRNA (H-b), chd_MO plus S4D-marcksb mRNA (H-c), and chd_MO plus S4N-marcksb (H-d). Embryos are animal-pole view with dorsal to the right. (I) The percentage of embryos with rescued, increased or dramatically increased expression of szl in different experimental groups indicated in the (H). “n” represents the number of embryos we observed.
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