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Fig. 1 Alterations in Rab11 function randomize laterality. (A) 1-cell embryos were injected with mRNAs encoding WTor
DN Rab constucts and scored at stage 45 based on the situs of three organs: the heart, the gut coil and the gallbladder. (A)
Wildtype organ situs (situs solitus) in an untreated embryo. The gut coils to the tadpole left (yellow arrow), the gallbladder is
on the right (green arrow), and the heart curves to the left (red arrow). (B) An example of heterotaxic organ situs in an embryo
injected at 1-cell with DNRab11, with inverted positioning of the heart and gallbladder. The gut is positioned properly. (C)
Embryo with situs inversus, a form of heterotaxia where the position of all three organs is reversed, after 1-cell injection of
DNRab11. (D) Embryos injected with various doses of either DNRab11 or WT Rab11 at 1-cell displayed significantly increased
rates of heterotaxia. For all graphs, numbers on bars indicate sample size. **p < 0.001 relative to uninjected controls (X2 test),
#p < 0.001 comparing treated groups (X2 test). (E) Embryos injected at 1-cell with DNRab11 were scored for Nodal (Xnr-1) mRNA
localization at stage 21. Left-sided Nodal expression was significantly decreased in DNRab11 injected embryos compared to
controls. *p < 0.01 relative to uninjected controls (X2 test).
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Fig. 2 DNRab11 alters LR patterning, regardless of where it is expressed, if injected at 1-cell. (A) 1-cell embryos were coinjected
in a purposefully biased manner with DNRab11 and a b-gal lineage tracer, or injected with b-gal alone. In this
schematic, the dot represents the north pole (animal-most point) of the embryo, and the needle shows an example of a
biased injection location. These embryos were used to compare left/right (B, C) and dorsal/ventral (D, E) patterns of b-gal
expression. (B) Embryos were raised to stage 45 and scored for situs of organs. Then, localization of b-gal expression
(indicated by red arrows) was utilized to indicate whether the injections were targeted to the left, right, or both sides of the
embryo. (C) Similar rates of heterotaxia were observed regardless of whether DNRab11 was targeted to the left, right, or both
sides of the embryo. *p < 0.001 compared to controls injected with b-gal alone (X2 test). (D) Similar to what was done for LR
localization, embryoswere examined to determine whether the localization of b-gal expression (indicated by red arrows) was
limited to dorsal, ventral, or both structures. (E) DNRab11 was effective at randomizing the LR axis when targeted to dorsal,
ventral or both structures, although statistical significance was only achieved when DNRab11 was targeted to ventral
structures. *p<0.001 compared to controls injected with b-gal alone (X2 test).
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Fig. 4 Rab11 mRNA and protein expression during development. (A) Whole mount in situ hybridization was performed for Rab11 on albino embryos at 1-cell (i), 4-cell (ii), blastula (iii), gastrula (iv) and neurula stages (v). At all stages, a diffuse signal (purple) was visible throughout the embryo with the strongest signal localized in the animal pole. Sense probes show little signal at all stages examined (vi, data not shown). Red arrows indicate signal, blue arrow on blastula stage embryo indicates
areas with lower signal due to contraction of the hollowembryo during fixation; this is an artifact. bp = blastopore, nf = neural folds. (B) Immunohistochemical analysis was performed for Rab11 protein on 100 lm sections collected from embryos at 1 cell (i and ii), 4-cell (iii and iv), blastula (v), gastrula (vi) and neurula (vii). At 1-cell, Rab11 protein is localized to the animal
hemisphere, with the strongest expression near the cleavage furrow (i) and no other visible biases (ii). At 4-cell Rab11
remains localized to the animal hemisphere (iii) and is relatively symmetrical along the LR and dorsalentral axes (iv). A
small portion of embryos showed slight asymmetries along the LR axis at 4-cell, but these were not consistently biased (data
not shown). Rab11 is highly localized within cells of blastula (v) and gastrula stage embryos (vi). This strong expression likely
corresponds to the perinuclear region of the cell (see v', vi'). In neurula stages, Rab11 is visible in the neural folds and
notochord, and a diffuse signal is also visible throughout the endoderm (vii). Green arrows indicate positive signal.
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Fig. 5 Rab11 and ductin have a close intracellular localization. Embryoswere injected at the 1-cell stage with Rab11-Tom and
Ductin-YFP. At stage 45, cells fromthe tail expressing both constructswere viewed at 100magnification. Whenever possible,
single cells or a small cluster of cells with tomato and YFP signal were examined. (A) DuctinFP (shown in green), Rab11
Tom (shown in red) and a merged image (with overlapping regions indicated in yellow) were examined. Z stacks were
analyzed for intensity with the plot profile feature in ImageJ along the six lines indicated (representative of different regions
within the cells). (B) Quantitative plots of Rab11om (red) and DuctinFP (green) expression corresponding to each region of
the cells (indicated by lines in panel A). Regions of high intensities of DuctinFP and Rab11om expression were found to
overlap. Furthermore, strong Rab11-Tom expression was often found surrounding strong ductin expression, as indicated by
the arrows in plot 2.
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Fig. 6 DNRab11 alters asymmetric ion transporter localization at the 4-cell stage. (A) 4-cell embryos were oriented,
sectioned, and immunostained for ductin protein. Biases in expression were quantified by identifying the strongest
expressing blastomere(s). The control embryo shown (i) has the strongest expression in the ventral right cell, verified with
ImageJ thresholding tools (ii). The embryo injected with DNRab11 shown (iii) has the strongest expression in the dorsal left
cell, verified with ImageJ thresholding tools (iv). (B) A total of 110 control and 45 DNRab11-injected embryos were
quantitatively analyzed. In controls, the most common blastomere scored as having the highest expression of ductin was the
ventral right; ventral right localization was decreased in DNRab11 injected embryos, although this decrease did not reach
statistical significance. Dorsal left expression was rare in control embryos, and the incidence of dorsal left expression was
significantly higher in DNRab11-injected embryos (*p < 0.05). (C) The same protocol was used for identifying biases in the
expression of KCNQ1. The control embryo shown (i) has the strongest expression of KCNQ1 in the ventral right cell, verified
with ImageJ thresholding tools (ii). The embryo injected with DNRab11 shown (iii) has the strongest expression in the dorsal
left cell, verified with ImageJ thresholding tools (iv). (D) A total of 85 control and 40 DNRab11-injected embryos were
quantitatively analyzed. In controls, the most common blastomere scored as having the highest expression of KCNQ1 was
the ventral right; ventral right localization was decreased in DNRab11 injected embryos, although this decrease did not reach
statistical significance. Dorsal left expression was relatively rare in control embryos, and the incidence of dorsal left
expression was significantly higher in DNRab11-injected embryos (*p < 0.05).
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Fig. 7 Experimental results support an assembly-line model of Rab11-mediated vesicular traffic in the ion flux model of LR
asymmetry. Schematic representation of the assembly line model of Rab11-mediated transport of vesicles in the early
embryo. (A) Rab11 associates with ion transporter-containing vesicles throughout the embryo. (B) Rab11 molecules attached
to vesicles move toward the + end of the cytoskeletal structure for a short distance, and then dissociate from the vesicles.
Other Rab11 molecules then attach to the vesicles in the exchanges shown. (C) Vesicles containing ion transporters
accumulate in the right ventral cell, but Rab11 remains distributed evenly, as indicated by the relatively symmetric
expression of endogenous Rab11 mRNA and protein (Fig. 4). (D) The biased localization of ion transporters establishes the
asymmetric bioelectrical properties that have been reported previously in early Xenopus embryos. The pumping of positive
ions out of the right ventral cell causes it to be relatively more negative than other blastomeres. (E) Membrane voltage and pH
gradients drive the asymmetric localization of serotonin, a positively charged molecule, by the 32-cell stage (not shown).
Serotonin localized to the right side of the embryo actively suppresses Xnr-1 expression on the that side at stage 22.
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Fig. 3 Rab11 acts cooperatively with planar cell polarity
(PCP) in LR patterning. Previous studies indicate that
altering expression of the PCP protein Vangl2 via
morpholinos (Vangl2MO) disrupts LR patterning
(Vandenberg and Levin, 2012). Epistasis experiments were
conducted to determine whether Rab11 acts on the same LR
pathway as PCP. DNRab11 mRNA, Vangl2MO and a mixture
of the two treatments were tested for their effects on organ
situs; three replicates were examined and data were
normalized to the incidence of heterotaxia induced by
DNRab11 to allow for comparisons between treatments.
Both DNRab11 and Vangl2MO induced significant levels of
heterotaxia compared to untreated controls. A mixture
of the two reagents produced intermediate levels of
heterotaxia. ANOVA p < 0.001, different letters indicate
significant differences (p < 0.05) in Bonferroni posthoc
analysis.
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kcnq1 (potassium voltage-gated channel, KQT-like subfamily, member 1) gene expression in Xenopus laevis embryos, NF stage 3, as assayed by immunohistochemistry, ventral side up.
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atp6v0c (ATPase, H+ transporting, lysosomal 16kDa, V0 subunit c) gene expression in Xenopus laevis embryos, NF stage 3, as assayed by immunohistochemistry.
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rab11a (RAB11A, member RAS oncogene family) gene expression in Xenopus laevis embryo, assayed via immunohistochemistry,in a 100 um sections through NF stage 1 (1cell) embryo, animal pole up.
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rab11a (RAB11A, member RAS oncogene family) gene expression in Xenopus laevis embryo, assayed via immunohistochemistry, in a 100 um sections through NF stage 3 (4 cell) embryo, animal pole up.
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