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Figure S1. ATP4a Expression, MO Targeting and Morphant Phenotypes, Related to Figure 1(A�D) Expression of ATP4a mRNA (A) and protein (B-D). (A) RT-PCR analysis. Actin served as loading control. Note that mRNA levels decreased but expression persisted through stage 17. (B-D) Immunohistochemistry with an antibody previously used for ATP4a localization in Xenopus (Aw et al., 2008). (B, C) Z-stack projections; (D) individual frames. (B) Gastrula embryo at stage 10.5. ATP4a (B′) was ubiquitously expressed, including the superficial mesoderm on top of the dorsal lip of the blastopore (dotted line). (C) Dorsal explant at stage 17. Ventral view of the GRP at the boundary between the GRP and lateral endodermal crest (LEC) cells (dotted line). Double staining with an antibody against acetylated tubulin (green) in (C′) revealed cilia at the GRP and midbodies in the area of the LECs (shown at higher magnification in insets). (D) Optical sections of region outlined in (C) at levels indicated in schematic drawing demonstrated localized staining at membranes as well as in vesicle-like cytoplasmic structures.(E) Injection of lineage tracer rhodamine-B dextran into the marginal region (prospective C-tier of 32-cell embryo) of 4-cell embryos revealed specific targeting of GRP tissue only when dorsal blastomeres were injected close to the dorsal pole (dorsal marginal zone, DMZ, top). More lateral injections of dorsal blastomeres (C2 lineage) or injection of ventral blastomeres (C3 and C4) targeted the intermediate mesoderm (C2/C3), lateral plate mesoderm (C2/C3), and ventral mesoderm (VMZ), respectively.(F and G) Pitx2c expression patterns (F) and organ situs (G) encountered in ATP4a morphants. The outflow tract of the heart and the position of the gall bladder are indicated by green and red arrowheads, respectively, and the direction of gut looping is marked by yellow arrows. Note that morphants occasionally (and dose-dependently) developed cysts, and therefore organ situs could not be determined. |
