Vandenberg LN et al. (2013), Rab GTPases are required for early orientation ...

XB-ART-46581

Mech Dev 2013 Jan 01;1304-5:254-71. doi: 10.1016/j.mod.2012.11.007.

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Rab GTPases are required for early orientation of the left-right axis in Xenopus.

Vandenberg LN , Morrie RD , Seebohm G , Lemire JM , Levin M .

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The earliest steps of left-right (LR) patterning in Xenopus embryos are driven by biased intracellular transport that ensures a consistently asymmetric localization of maternal ion channels and pumps in the first 2-4 blastomeres. The subsequent differential net efflux of ions by these transporters generates a bioelectrical asymmetry; this LR voltage gradient redistributes small signaling molecules along the LR axis that later regulate transcription of the normally left-sided Nodal. This system thus amplifies single cell chirality into a true left-right asymmetry across multi-cellular fields. Studies using molecular-genetic gain- and loss-of-function reagents have characterized many of the steps involved in this early pathway in Xenopus. Yet one key question remains: how is the chiral cytoskeletal architecture interpreted to localize ion transporters to the left or right side? Because Rab GTPases regulate nearly all aspects of membrane trafficking, we hypothesized that one or more Rab proteins were responsible for the directed, asymmetric shuttling of maternal ion channel or pump proteins. After performing a screen using dominant negative and wildtype (overexpressing) mRNAs for four different Rabs, we found that alterations in Rab11 expression randomize both asymmetric gene expression and organ situs. We also demonstrated that the asymmetric localization of two ion transporter subunits requires Rab11 function, and that Rab11 is closely associated with at least one of these subunits. Yet, importantly, we found that endogenous Rab11 mRNA and protein are expressed symmetrically in the early embryo. We conclude that Rab11-mediated transport is responsible for the movement of cargo within early blastomeres, and that Rab11 expression is required throughout the early embryo for proper LR patterning.

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Species referenced: Xenopus laevis
Genes referenced: atp6v0c gal.2 kcnq1 nodal nodal1 rab11a rab11b rab9a vangl2
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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).
	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).
	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.
	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.
	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).
	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.
	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.
	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.
	atp6v0c (ATPase, H+ transporting, lysosomal 16kDa, V0 subunit c) gene expression in Xenopus laevis embryos, NF stage 3, as assayed by immunohistochemistry.
	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.
	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.

References [+] :

Adams, Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates. 2006, Pubmed, Xenbase