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Highlights:
â–º ATP4 is required during gastrulation/neurulation for cilia-driven leftward flow to occur
â–º ATP4 acts on canonical and noncanonical Wnt signaling
► Wnt/β-catenin is necessary and sufficient for gastrula Foxj1 expression
â–º Leftward flow is the conserved mode of symmetry breakage in Xenopus
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Figure 1. ATP4a Is Symmetrically Expressed and Required Post-MBT for LR Development(A�E) Whole-mount in situ hybridization of staged embryos at the (A) two-cell, (B) four-cell, and (C) 32-cell stages. Note that ATP4a mRNA was enriched in the animal cytoplasm, as shown in the bisected four-cell embryo in (B), viewed from the inner surface in (B′) and (B�), but symmetrically expressed with respect to the LR axis. (D) Zygotic ATP4a expression in the stomach of a stage (st.) 45 tadpole; see plane of section in (D′) indicated by dashed line in (D). Arrowhead indicates gastric epithelium in blow-up. (E) Four-cell embryo stained with sense control RNA.(F and G) (F) Pitx2c expression and (G) organ situs in wild-type embryos at tadpole stages (26�32) and in specimens injected or treated as specified. The image in (D′) was reconstructed from several images taken from the same histological section. ∗∗Highly significant. ∗∗∗Very highly significant. ns, not significant; n, number of embryos analyzed; wt, wild-type.See also Figure S1.
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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.
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Figure 2. Impaired Ciliation and Leftward Flow upon ATP4 Loss of Function(A�F) Flow analysis. (A) Directionality and (B) velocity of fluorescent beads added to GRP explants at stage 17 were drastically reduced in ATP4a morphants or SCH28080-treated specimens, as compared to wild-type, CoMO-injected, or DMSO-treated embryos. n represents number of explants analyzed. (C�F) Frequency distribution of trajectory angles in representative explants injected with (C) CoMO, (D) ATP4aMO, (E) DMSO, and (F) SCH28080. Dashed circles indicate maximum frequency in histogram specified in percent. a, anterior; l, left; n, number of particles above threshold; p, posterior; r, right; v, average velocity of particles; Ï, quality of flow.(G�K) SEM analysis of GRP ciliation and morphology. (G and H) Representative dorsal explants reveal shorter cilia, fewer ciliated cells, and polarization defects in (H) ATP4a morphant (H) as compared to (G) CoMO-injected specimen. Cell boundaries are indicated by dashed orange lines in higher magnification of SEM pictures in (G′) and (H′). Blowups in (G′) and (H′) illustrate a long and posteriorly polarized cilium in (G′), indicated by a green arrowhead, and two short cilia in (H′), of which one emerges in a central position, indicated by a yellow arrowhead. (G� and H�) Evaluation of cilia polarization. Green, posterior; yellow, other; red, no cilium. (I�K) Quantification of (I) ciliation rate and (J) cilia length of GRP cilia in defined areas, as indicated by white squares in (G) and (H). (K) Cilia polarization was assessed in areas of defined size, as indicated by dashed boxes in (G) and (H). ∗∗Highly significant. ∗∗∗Very highly significant. ns, not significant. Numbers indicate number of dorsal explants (in parentheses) or cilia (in brackets) analyzed.See also Figure S2 and Movie S1.
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Figure S2. Frequency Distribution of Trajectory Angles in Dorsal Explants, Related to Figure 2(A) Uninjected controls.(B) CoMo-injected controls.(C) ATP4aMO-injected specimens.(D) DMSO- and (E) SCH28080-treated embryos. Dashed circles mark maximum frequency in histogram specified in percent. a, anterior; l, left; n, number of particles above threshold; p, posterior; r, right; v, average velocity of particles; rho, quality of flow.
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Figure 3. Turbulent Flow in ATP4a Morphants Causes Bilateral Nodal Cascade Induction(A�C) Analysis of bead trajectories in time-lapse movies of dorsal explants from representative (A) CoMO-injected or (B) ATP4aMO-injected embryos and (C) specimen treated with SCH28080. Flow is displayed as GTTs of 25 s length; cf. color bar in (A). Note that trajectories in (B) and (C) project to the left side (indicated with blue arrows) and right side (indicated with pink arrows) of the GRP, whereas GTTs in (A) point uniformly to the left. White arrows represent trajectories running anteriorly or posteriorly.(D) Whole-mount in situ hybridization of stage 20 dorsal explants with probes specific for (top) Coco and (bottom) Xnr1. Left-sided Coco repression was lost in (middle) ATP4a morphants and inverted upon (right) parallel left-sided knockdown of flow. Xnr1 expression was unaffected.(E) Quantification of Coco expression patterns.(F) Quantification of Pitx2c expression patterns in stage 26�32 tadpoles following MO injections into the C1 lineage (dorsal midline-GRP) of four-cell embryos as indicated. Note that the bilateral induction (cf. Figure 1F) in ATP4a morphants was dependent on both the presence of GRP-Xnr1 and ciliary motility.See also Movie S1.
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Figure 4. ATP4 Is Required for Canonical and Noncanonical Wnt Signaling(A) Phenotypes of ATP4a morphant tadpoles, which were less pigmented, displayed shortened AP axes, small heads, and reduced eyes.(B and C) Canonical Wnt signaling. (B) Reduced En2 expression at the mid-hindbrain boundary of (bottom) morphant stage 26 tadpole. (C) Xwnt8- and Dvl2-mediated, but not β-cat-induced, twinning requires ATP4a.(D) Noncanonical Wnt signaling. Convergent-extension movements of activin-induced animal cap explants: Reduced elongation in ATP4aMO-injected explants was rescued by coinjection of constitutively active (CA) RhoA. ∗∗∗Very highly significant. ns, not significant.See also Figure S3.
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Figure S3. ATP4a Acts on Noncanonical Wnt Signaling, Related to Figure 4(A and B) Convergent extension. (A) Neural tube closure: widening of the neural tube in ATP4a morphants. Embryos were injected unilaterally into the animal right blastomeres at the 4 cell stage, fixed and processed for Sox3 expression to visualize the neural plate by WMISH at stage 18. Staging was according to the progress of neural tube closure on the uninjected (left) side. Note that the widening of the neural plate was partially rescued upon ATP4a co-injection. (B) Shortening of the anterior-posterior (AP) axis. Embryos were bilaterally injected into the DMZ at the 4 cell stage and the AP extension was determined at stage 32. Note that MO-induced shortening was partially rescued by ATP4a co-injection. The width of the neural plate (A) and the length of embryos (B) were measured (as indicated by dashed lines). Results are depicted as box plots.(C) Neural crest cell specification and migration: right-sided ATP4aMO-injection resulted in reduced and altered Twist1 mRNA expression at stage 26 (n1-n3/4; neural crest migratory streams).(D) Right-sided pronephric tubule (pnt) defects upon unilaterally right-sided ATP4aMO-injection, as demonstrated by WMISH using the pronephros marker gene ATP1b1. d, dorsal; l, left; pnd, pronephric duct; pnt, pronephric tubule; r, right; v, ventral. ∗∗∗, very highly significant. Numbers in brackets represent number of analyzed specimens.
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Figure 5. ATP4 Is Required for Wnt/β-cat-Mediated Induction of Foxj1 During Gastrulation(A�C). Reduced mRNA expression of (A) Foxj1 in the SM of ATP4a-morphant or SCH28080-treated embryos was rescued upon coinjection of (B) a β-cat DNA expression construct. (C) Ectopic expression of Foxj1 on the ventral lip following injection of a β-cat DNA expression construct into ventral blastomeres at the four-cell stage (VMZ lineage).(D) Quantification of results.(E) Partial rescue of Pitx2c expression in ATP4a morphants upon coinjection of a Foxj1 DNA expression construct.See also Figure S4.
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Figure S4. Rescue of Ciliation Rate but Not Cilia Polarization by Foxj1 in ATP4a Morphants, Related to Figure 5Embryos were injected at the 4-cell stage into the DMZ and dorsal explants were prepared at stage 17. Specimens were processed for IHC to assess cilia polarization (A-D), or for SEM analysis to determine the GRP ciliation rate (E).(A�C) IHC using antibodies against acetylated tubulin to visualize cilia (red) and actin (green) to outline cell boundaries. (A) Control uninjected specimen. (B) ATP4a morphant. (C) Coinjection of ATP4aMo and Foxj1 mRNA. (A′-C′) Evaluation of results.(D) Quantification of cilia polarization. Note that co-injection of Foxj1 aggravated polarization defects.(E) Ciliation rate. Note that ciliation rate was partially rescued by Foxj1 co-injection.
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atp4a ( ATPase, H+/K+ exchanging, alpha polypeptide ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 2, animal view.
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atp4a ( ATPase, H+/K+ exchanging, alpha polypeptide ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal view.
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atp4a ( ATPase, H+/K+ exchanging, alpha polypeptide ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 2-, animal view.
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atp4a ( ATPase, H+/K+ exchanging, alpha polypeptide ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, animal view.
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atp4a (ATPase, H+/K+ exchanging, alpha polypeptide ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 45, ventral view, anterior up.
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