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

Abstract

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.

1. Introduction

Externally, vertebrates display bilateral symmetry along their midline such that their left and right sides appear to be mirror images of each other. However, the internal arrangement and morphology of several organs are asymmetric, including the heart, liver, spleen, stomach, intestine, and brain (Dawson, 1977; Neville, 1976). The origin of this biased organ distribution has fascinated mankind for millenia (McManus, 2002), and there is significant medical relevance for uncovering the mechanisms behind its establishment, as approximately 1 in 8000–10,000 people are born with a randomization in organ situs (Francis et al., 2012; Palmer, 2004; Peeters and Devriendt, 2006). Normal organ patterning is referred to as situs solitus, while defects in laterality can take on many forms (Hackett, 2002). A full reversal in the placement of all organs is referred to as situs inversus while heterotaxia is the term given to situations in which the placement of each organ is randomized, which can lead to a vast array of medical complications.

There are three major steps to establishing bodily asymmetry: (1) the initial symmetry breaking event, which must be amplified to (2) induce asymmetric gene expression cascades on the L and R sides of the midline, which later (3) regulate morphogenesis to establish asymmetric positioning of individual organs (Brownet al.,1991). Despite significant success in understanding and characterizing components of the asymmetry pathway, many questions still remain with respect to the earliest symmetry breaking and amplification steps.

One model proposes that LR asymmetry is generated by the movement of monocilia localized to a small ‘node’ in a region of the posterior notochord (known as the gastrocoel roof plate, or GRP in Xenopus) (Basu and Brueckner, 2008; Blum et al., 2009). An asymmetric fluid flow generated by ciliary motion has been postulated to cause the asymmetric distribution of a morphogen and/or asymmetric activation of sensory cilia on the left side of the embryo which initiate left-sided Nodalexpression (McGrath and Brueckner, 2003; McGrath et al., 2003; Schweickert et al., 2012; Tabin and Vogan, 2003). Although experiments have implicated cilia in laterality determination in Xenopus (Schweickert et al., 2007; Vick et al., 2009), numerous molecular-genetic functional data indicate that cytoplasmic (intracellular) events set up the LR axis much earlier in development (Klar, 1987, 2008; Levin, 2005; Lobikin et al., 2012; Vandenberg and Levin, 2010). A number of maternal ion channels and pumps are consistently asymmetrically localized by the 4-cell stage, and the physiological gradients they create are required for normal LR asymmetry (Adams et al., 2006; Aw et al., 2008; Levin et al., 2002; Morokuma et al., 2008). Moreover, it has recently been shown that components of the early cytoskeleton are involved in ensuring the LR localization of such molecules in a wide range of species from plant to mammalian cells (Aw et al., 2008; Lobikin et al., 2012).

The implication of multiple motor proteins in LR patterning by genetic experiments (Armakolas and Klar, 2007; Hozumi et al., 2006; Marszalek et al., 1999; Nonaka et al., 1998; Qiu et al., 2005; Supp et al., 1997) and the known role of intracellular transport in guiding ion channels and pumps to different parts of polarized cells (Hamm-Alvarez and Sheetz, 1998; Zheng et al., 2008) suggest that the LR axis could be established by the directed intracellular transport of LR determinants at early stages. One such model proposes that ion channel/pump cargo within early blastomeres is translocated to the L or R sides by motor proteins whose directionality is governed by biased cytoskeletal tracks (Aw and Levin, 2009; Levin, 2005). The inherent chirality of the cytoskeleton (Aw et al., 2008; Danilchik et al., 2006; Lobikin et al., 2012), oriented with respect to the main body axes by an organizing center such as the MTOC (Vandenberg and Levin, 2009), can establish the asymmetric localization of ion transporters including the H⁺K⁺-ATPase, the K⁺ channel Kir4.1, the H⁺-V-ATPase, and the K⁺ channel component KCNQ1 (Adams et al., 2006; Aw et al., 2008; Morokuma et al., 2008). While the steps linking the resulting LR voltage gradient to asymmetric transcription of the left-sided gene Nodal have been worked out in some detail (Carneiro et al., 2011; Vandenberg and Levin, 2010), it still remains to work out exactly how the maternal protein cargo reaches its appropriate side during early cleavage stages.

The main coordinators of motor proteins and membranes in cells are a family of proteins known as Rab GTPases (Stenmark, 2009). Rab GTPases are a large subclass of the Ras GTPase family and thus are molecular switches that alter between active (GTP bound) and inactive (GDP bound) states. In the inactive GDP-bound state, Rabs dimerize at the switch region of the protein; upon activation, they release from each other (Pfeffer, 2005). Rabs serve to regulate membrane trafficking and intracellular signaling events through temporal and spatial cues via their indirect interactions with coat components, motors, and SNAREs (Schwartz et al., 2007; Stenmark, 2009). Because ion transporters are transmembrane proteins and thus are often moved to and from the plasma membrane via vesicles (Forte and Zhu, 2010), we hypothesized that Rab GTPases could be involved in the shuttling of ion transporters to localized areas of the early embryo. Indeed, many interactions between ion transporters, including those with roles in LR patterning, and Rab GTPases have been shown to occur (Gidon et al., 2012; Seebohm et al., 2007; van de Graaf et al., 2006). Moreover, LR asymmetry is a kind of planar polarity (Aw and Levin, 2009) and it is now known that a number of Rab proteins function in setting up planar cell polarization (Classen et al., 2005; Gault et al., 2012; Gordon et al., 2012; Purvanov et al., 2010). Thus, we tested one prediction of the ion flux model: that a Rab protein would be functionally involved in LR patterning during embryogenesis.

We screened several Rabs to determine whether they play a role in LR patterning and found that alterations in Rab11, Rab4, Rab7, and Rab9 signaling all influence laterality. We focused on Rab11, which regulates the recycling of endosomes to the plasma membrane, and is required in the secretory pathway from the trans golgi network to the plasma membrane (Forte and Zhu, 2010). Our results show that Rab11 is expressed in the early embryo and closely associates with at least one sub-unit of the H⁺-V-ATPase, that Rab11 loss-of-function reagents disrupt the asymmetric localization of LR-relevant ion transporters, and that Rab11 influences LR asymmetry in cells that do not contribute to ciliated node cells. Taken together, our results implicate Rab GTPases as the molecules that link the chiral cytoskeleton to the asymmetric trafficking of LR-relevant ion transporters in the early embryo.

2. Results

2.1. A molecular screen of Rab constructs identifies a role for Rab11 in LR patterning

To determine if Rab GTPases are involved in LR patterning, various mRNA constructs encoding dominant negative and wild-type versions of several Rab GTPases were injected into 1-cell embryos (Table 1). mRNAs were titered to levels that allowed the scoring of embryos with perfect overall development (except for LR patterning defects) and a dorsoanterior index of 5 (30–50 pg mRNA). After raising these embryos to stage 45, each group was scored for heterotaxia by examining the looping of the heart, the position of the gall bladder, and the direction of gut coiling (Fig. 1A–C). Expression of several loss-of-function Rab constructs altered LR patterning, and the most effective were those that were expected to prevent localization of proteins (including ion transporters) to the plasma membrane (Table 1). From these results we conclude that multiple components of endocytotic recycling are important for LR patterning. Because both WT and DN Rab11 mRNAs affected LR patterning, which suggests the importance of a proper balance in delivery of LR-relevant cargo to the plasma membrane (Kessler et al., 2012), we focused our subsequent analyses on Rab11.

Table 1 –

Screening Rab mRNA constructs for effects on LR patterning.

Normal location of Rab GTPase	Construct	Expectation of construct	% Heterotaxia
Rab 11 shuttles vesicles from the TGN and recycling endosomes to plasma membrane	DNRab 11	Prevent localization of ion transporters to plasma membrane	18*
	WTRab 11	Increase movement of ion transporters to plasma membrane	11*
Rab 4 shuttles vesicles from early endosomes to plasma membrane	DNRab 4	Prevent localization of ion transporters to plasma membrane	13*
	WTRab 4	Increase movement of ion transporters to plasma membrane	2
Rab 7 shuttles vesicles from late endosomes to lysosomes, also from early endosomes to late endosomes	DNRab 7	Prevent movement of ion transporters away from plasma membrane	2
	WTRab 7	May increase movement away from plasma membrane, may also lead to increased break-down of ion transporters in lysosomes	11*
Rab 9 shuttles vesicles from late endosomes back to the TGN	DNRab 9	Prevent movement of ion transporters away from plasma membrane	2
	WTRab 9	May increase movement of ion transporters away from plasma membrane	11*

TGN = trans golgi network.

^*p < 0.01.

Fig. 1 –

Alterations in Rab11 function randomize laterality. (A–C) 1-cell embryos were injected with mRNAs encoding WT or 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’s 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).

2.2. Dose-dependent effects of dominant negative (DN)Rab11 on LR patterning

In our initial screen of Rab constructs, we observed that 30–50 pg of DNRab11 mRNA induced randomized organ situs at stage 45 (18% versus 2% in uninjected controls, p < 0.01, Fig. 1D). Although our main analysis was limited to healthy embryos with a normal dorsoanterior index, we did note that if the concentration of DNRab11 mRNA was increased to a dose that was more toxic (approx. 50% of embryos died, 100–140 pg), significantly more randomization of organ situs occurred (41% heterotaxia, p ≪ 0.001, Fig. 1D). In contrast, higher doses of WTRab11 mRNA had no significant additional effect on randomized organ situs (13%, p < 0.01 relative to controls, Fig. 1D), suggesting that altering the delicate balance of protein transport in the early embryo via overexpression of Rab11 can disrupt LR patterning, but that loss of Rab11 function has the greatest effect on laterality. From these results we conclude that integrity of Rab11 function is necessary for the establishment of LR asymmetry.

To determine whether Rab11 exerts its effect on laterality prior to, and through regulation of, the expression of the frog homolog of Nodal (Xnr1), we analyzed Xnr-1 expression in control embryos and embryos injected at 1-cell with 50 pg DNRab11. Whereas uninjected controls displayed a strong left-sided bias in Xnr-1 expression at stage 22, expression was found significantly less often on the left side of DNRab11 injected embryos, with over 40% of injected embryos lacking any Xnr-1 expression (Fig. 1E). From these results, we conclude that Rab11 acts prior to the initiation of, and functions in the same pathway as, the Nodal signaling cascade in the establishment of LR asymmetry.

2.3. Effects of Rab11 signaling are early, and independent of ciliary flow at the GRP

Because many exogenous mRNA constructs are translated within an hour of injection (Lobikin et al., 2012), the effects of altered Rab11 signaling could be due to actions as early as the first cleavage stages. To further address the issue of timing, we injected 2-cell embryos with the high dose of DNRab11 mRNA (100–140 pg) and examined organ situs at stage 45. Regardless of whether injections were introduced into a single blastomere, or into both blastomeres, injections performed at the 2-cell stage had no effect on LR patterning (Table 2). While mRNA and its protein can persist for days, the fact that injections made at 2-cell are already too late to affect asymmetry suggests that the effect on asymmetry is taking place extremely early.

Table 2 –

Injection of DNRab11 mRNA after the 1-cell stage does not affect LR patterning.

Treatment	Sample size (n)	% Heterotaxia, X2 p-value
Uninjected controls	219	1
DNRab11, 1 of 2 cell	247	2, p = 0.62
Uninjected controls	159	1
DNRab11, 2 of 2 cell	437	4, p = 0.11
Uninjected controls	164	2
DNRab11, 1 of 4 or 1 of 8 cell at dorsal marginal zone (targeting GRP)	119	6, p = 0.24

Although later injections did not affect LR patterning, we also specifically targeted DNRab11 mRNA to the dorsal–marginal zone at the 4–8 cell stages; ciliary flow during neurulation has been suggested to regulate LR patterning in Xenopus (Blum et al., 2006, 2009), and the ciliated organ derives from these dorsal blastomeres. We injected DNRab11 mRNA on the left side, because only cilia on the left side of the GRP are required for LR-relevant flow (Vick et al., 2009). Even when the GRP was directly targeted, we found no effect of DNRab11 on LR patterning (Table 2). Taken together, the above data indicate that Rab11 acts very early in development, within the first few cell cleavages, in a GRP-independent manner.

While targeting specific blastomeres at later stages is the typical way to influence expression of a protein in a particular region of the embryo, off-center injections at the 1-cell stage allow for the earliest expression of constructs while still limiting the region of expression in the developing embryo (Aw et al., 2010). To determine whether the location of early Rab11 activity is important for LR patterning, we co-injected 1-cell embryos with DNRab11 and β-gal mRNAs, a lineage label, in a purposefully unilateral manner (Fig. 2A). Post-sorting the embryos based on the location of the β-gal signal (which revealed whether the early mRNA injections had been made on the left, right, or both sides of the 1 cell embryo), we observed no correlation between the mRNA localization and the ability to induce heterotaxia (Fig. 2B,C). Further, when separating injections based on their localization to dorsal, ventral, ordorsal + ventral sides of the embryo, we found that targeting the construct to either dorsal or ventral regions induced heterotaxia, although statistical significance was achieved only when DNRab11 was targeted to the ventral region (p < 0.001, Fig. 2D and E). From these results, we conclude that disruption of Rab11 activity in any region of cleavage stage embryos is sufficient to alter LR patterning, and that Rab11 function is important along the length of the entire early embryo.

Fig. 2 –

DNRab11 alters LR patterning, regardless of where it is expressed, if injected at 1-cell. (A) 1-cell embryos were co-injected in a purposefully biased manner with DNRab11 and a β-gal lineage tracer, or injected with β-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 β-gal expression. (B) Embryos were raised to stage 45 and scored for situs of organs. Then, localization of β-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 β-gal alone (X² test). (D) Similar to what was done for LR localization, embryos were examined to determine whether the localization of β-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 β-gal alone (X² test).

2.4. Epistasis experiments indicate Rab11 acts cooperatively with planar cell polarity to orient the LR axis

Previous studies have implicated a role for planar cell polarity (PCP) proteins in LR patterning (Antic et al., 2010; Borovina et al., 2010; Ferrante et al., 2009; Maisonneuve et al., 2009; May-Simera et al., 2010; Oteiza et al., 2010; Song et al., 2010), including studies that indicate a role for PCP independent of ciliary flow (Vandenberg and Levin, 2012; Zhang and Levin, 2009). PCP proteins are necessary to maintain cytoskeletal integrity (Etienne-Manneville and Hall, 2003), and may control cell behavior through coordinated vesicle trafficking (Gray et al., 2009). Importantly, previous studies indicate that Rab11 interacts with the PCP pathway in the drosophila wing (Strutt and Vincent, 2010) and the Xenopus embryonic epidermis, where it is required for intercalation of ciliated cell precursors (Kim et al., 2012). To determine whether Rab11’s actions in LR orientation are due to interactions with the PCP pathway, we performed epistasis experiments with Vangl2 morpholinos (Antic et al., 2010; Vandenberg and Levin, 2012) and our DNRab11 construct. Each one of these treatments alone induced heterotaxia, with the DNRab11 construct somewhat more effective than the Vangl2 morpholinos (Fig. 3). If these two pathways (Rab and PCP) had distinct roles in LR patterning, we would expect to see additive effects by combining DNRab11 and the Vangl2 morpholino. Instead, a combination of these two treatments, at the same doses as tested individually, produced intermediate levels of heterotaxia. From these results, we conclude that Rab11 acts in a complimentary fashion to the PCP pathway, and may have overlapping roles with Vangl2.

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.

2.5. Rab11 is expressed during early development

Because our results indicated a role for Rab11 in LR patterning before the onset of the Nodal signaling cascade, we next asked when and where Rab11 was expressed in the early embryo. To determine the timing and localization of Rab11 mRNA expression, we performed in situ hybridization on whole embryos using a probe against the Xenopus Rab11 mRNA. During early cleavage stages, we observed Rab11 mRNA expressed throughout the animal pole of the embryo, without any bias in the LR or dorsal–ventral axes (Fig. 4Ai and ii). At blastula and gastrula stages, Rab11 mRNA remained expressed in the animal pole, with somewhat less expression in the cells that contribute to the blastopore (Fig. 4Aiii and iv). Finally, Rab11 mRNA was expressed throughout the neurula stage embryo including in the neural folds and notochord (Fig. 4Av).

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 hollow embryo 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 dorsal–ventral 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.

To determine whether Rab11 protein was present in these same stages, a western blot was run on whole embryo cell lysates of various stages with a commercial antibody against Rab11. A band corresponding to 25 kDa, equivalent to the known size of Rab11, was found for all stages analyzed (1-cell, 2-cell, 4-cell, 8-cell, st. 6.5, st. 10.5, st. 18 and st. 25; data not shown). To determine the specific localization of Rab11, especially during cleavage stages, embryos of various stages were sectioned, and a hydrogen-peroxidase immunohistochemistry reporting system (as described in Levin (2004)) was used to visualize Rab11 protein (Fig. 4B). In agreement with our western blot results, we observed Rab11 expression at all stages analyzed. In 1-cell embryos, Rab11 was localized to the animal-most region, with intense expression around the developing cleavage furrow (Fig. 4Bi, arrow), consistent with the hypothesis that Rab11 is necessary to deliver new membranes to this region (Horgan and McCaffrey, 2009, 2012). Dorsal/ventral cross sections revealed strong Rab11 expression throughout the 1-cell embryo (Fig. 4Bii). In every 4-cell stage embryo examined, all four blastomeres expressed Rab11 in the animal region, and in most embryos no obvious LR or dorsal–ventral biases were revealed (Fig. 4Biii and iv). Some embryos showed slight biases in Rab11 protein expression along the LR axis, but these were not consistent (data not shown). This mostly symmetric expression of Rab11 was not unexpected, as Rab11 is necessary for the operation of the recycling endocytotic pathway (Horgan and McCaffrey, 2009, 2011, 2012), and therefore is likely to be expressed in all cells to perform housekeeping functions. At stage 8/9 (blastula), Rab11 protein was localized in a punctate manner within individual cells (see Fig 4Bv and v′). This pronounced expression in the perinuclear region remained at stage 10 (gastrula), but there was also diffuse localization throughout the cell body (Fig. 4Bvi and vi′). In most cell lines, Rab11 localizes to the perinuclear region (Duman et al., 1999; Eisfeld et al., 2011), which is closely associated with the trans-Golgi, microtubule organizing center, and apical recycling compartments. Finally, we observed that during neurula stages, Rab11 protein was diffusely expressed in cells throughout the embryo, with the strongest expression in the developing endoderm, the notochord and the developing somites (Fig. 4Bvii). We conclude that Rab11 is present at very early developmental stages, in a spatio-temporal pattern consistent with the functional data presented above (Sections 2.1–2.3).

2.6. Rab11 co-localizes with ductin

In Xenopus, proton efflux via the H⁺-V-ATPase is necessary for proper laterality (Adams et al., 2006). The c subunit (ductin) of the H⁺-V-ATPase is essential for its function; four ductin subunits work together in the plasma membrane spanning domain to bind and translocate protons (Nishi and Forgac, 2002). We hypothesized that Rab11 functions to bring ductin to the cell surface, where it acts in the LR pathway. Therefore, we sought to determine if these two proteins interact directly. Embryos were co-injected with mRNA constructs encoding Rab11 fused to a Tomato fluorescent protein (Rab11-Tom) and an mRNA encoding ductin fused to a yellow fluorescent protein (ductin-YFP, Vandenberg et al., 2011a). The localization of the two constructs was then visualized with fluorescent microscopy. We found that the dark pigmentation of the early Xenopus embryo, light scattering, and autofluorescence of the yolk platelets made in vivo visualization impossible. In stage 45 tadpole tails, the tissue is transparent. Thus, we analyzed the localization of ductin and Rab11 at this stage. We selected cells that were expressing both Rab11-Tom and ductin-YFP (Fig. 5A) and quantified the localization of these proteins to determine whether they were expressed in the same regions of the cell. Using the plot profile feature of ImageJ, we found that the patterns of fluorescence corresponding to Rab11 and ductin change in parallel, indicating that Rab11 and ductin are often physically localized to the same areas within cells (Fig. 5A and B). Additionally, our quantification analyses indicate that the regions expressing Rab11-Tom were often larger than – but still overlapping with – their ductin–YFP counterparts (Fig. 5B, see arrows in panel 2). From these results we conclude that Rab11 and ductin are in close proximity, and that ductin may be enriched in Rab11-positive vesicles.

Fig. 5 –

Rab11 and ductin have a close intracellular localization. Embryos were injected at the 1-cell stage with Rab11-Tom and Ductin-YFP. At stage 45, cells from the tail expressing both constructs were viewed at 100× magnification. Whenever possible, single cells or a small cluster of cells with tomato and YFP signal were examined. (A) Ductin–YFP (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 Rab11–Tom (red) and Ductin–YFP (green) expression corresponding to each region of the cells (indicated by lines in panel A). Regions of high intensities of Ductin–YFP and Rab11–Tom 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.

2.7. Ion transporters are asymmetrically expressed in early cleavage stages

An asymmetric distribution of ion transporters has been shown to occur by the 4-cell stage of embryonic development in Xenopus (Adams et al., 2006; Aw et al., 2008; Morokuma et al., 2008). However, quantitative data on degree of bias among cohorts had not been published. Because 2-cell injections of DNRab11 did not induce heterotaxia, we hypothesized that Rab11’s effect on organ situs was due to a role in the localization of these ion transporters during the first few cleavage stages. Therefore, to determine whether DNRab11 affects LR patterning by altering their asymmetric distribution, we first sought to quantify the localization of several ion transporters previously described, focusing our efforts on two transporters that are required for laterality and have been shown to be expressed asymmetrically at the 4-cell stage (Adams et al., 2006; Morokuma et al., 2008). To determine their localization, immunohistochemistries for ductin and KCNQ1 were performed on cross sections of 4-cell embryos made perpendicular to the anterior–posterior axis (Fig. 6).

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).

Immunohistochemistry for ductin on oriented, sectioned 4-cell embryos showed that ductin expression was highly asymmetric, as only 4% of embryos analyzed had symmetric expression. In accordance with the ion flux model of LR asymmetry, of those embryos with strong ductin localization to two or fewer blastomeres (i.e., embryos with an asymmetric localization of ductin), 55% were consistently scored as having the highest level of ductin expression in regions encompassing the ventral right blastomere, while regions that included the dorsal left blastomere had the least (11%, X² = 45.6, p ≪ 0.001, Fig. 6A and B). From these data, we conclude that ductin is most highly concentrated in the ventral right blastomere.

We also quantified the localization of KCNQ1, a 6-trans-membrane protein, whose tetramer forms an ion channel that is implicated in cardiac arrhythmia, hearing loss, and gastric acid secretion (Seebohm et al., 2007), in addition to LR asymmetry in Xenopus (Morokuma et al., 2008). We observed that 27% of embryos had symmetric expression of KCNQ1. Of the embryos with an asymmetric localization of KCNQ1 to two or fewer blastomeres (i.e., embryos with asymmetric localization of KCNQ1), 34% were consistently scored as having the highest level of expression in regions encompassing the ventral right blastomere, while regions that included the dorsal left blastomere had the least (18%, X² = 5.2, p = 0.02, Fig. 6C and D). Thus, our quantification results support the conclusion that KCNQ1 is most highly concentrated in the ventral right blastomere of 4-cell embryos.

2.8. DNRab11 alters the biased localization of ductin and KCNQ1

Because the biases in ion transporter localization discussed in Section 2.7 above are necessary to establish the electrochemical gradient that directs laterality (Vandenberg and Levin, 2010), and Rab GTPases are involved in the directed transport of membranes along the cytoskeleton (Stenmark, 2009), we hypothesized that Rab11 could affect laterality by acting as the mediator of the asymmetric transport of KCNQ1 and ductin. To determine whether Rab11 is necessary for proper ion transporter localization, we injected embryos with DNRab11 at the 1-cell stage and then analyzed the localization of ion transporters at the 4-cell stage as described in Section 2.7. Of note, DNRab11-injected embryos were slower to undergo cytokinesis than their uninjected counterparts (data not shown), which is consistent with the aforementioned role for Rab11 at the cleavage furrow (Horgan and McCaffrey, 2009) and indicates that DNRab11 has effects at early cleavage stages. In uninjected controls, ductin is preferentially localized to the ventral right cell, but this biased localization is disrupted by injections of DNRab11, which reduced ventral right expression and significantly increased dorsal left expression (2.5-fold, X² = 4.9, p = 0.03, Fig. 6A and B). There was no change in the percent of embryos with symmetric ductin expression (4% versus 4%, data not shown). Our results indicate that Rab11 is necessary for the directional transport of ductin toward the ventral right cell, a crucial upstream event in generating the electrochemical gradient involved in LR patterning (Adams et al., 2006).

We next analyzed the effects of DNRab11 on KCNQ1 localization. Like ductin, the frequency of symmetric KCNQ1 expression was similar in DNRab11-injected embryos and uninjected controls (25% versus 27% respectively, data not shown). Similar to what was observed for ductin expression, the preferential localization of KCNQ1 to the ventral right cell in uninjected controls was disrupted by injections of DNRab11, which significantly increased dorsal left expression (2.1-fold, X² = 4.8, p = 0.03, Fig. 6C and D). From these results, we conclude that Rab11 is necessary for the localization of KCNQ1 to the ventral right blastomere, and loss of Rab11 function leads to increased expression of KCNQ1 on the opposite side of the embryo.

3. Discussion

Several recent reviews discuss the numerous molecular pathways that have been implicated in LR patterning in vertebrate and invertebrate phyla (Basu and Brueckner, 2008; Pohl, 2011; Speder et al., 2007; Wood, 2005). In a number of systems including Arabidopsis, Caenorhabditis elegans, snail, and Drosophila, intracellular components such as cytoskeletal and motor protein molecules are involved in very early stages of the establishment of laterality (Chang et al., 2011; Kuroda et al., 2009; Speder et al., 2006; Thitamadee et al., 2002). The involvement of tubulins in this process appears to be widely conserved as well among plant (Arabidopsis), nematode, human cells, and amphibian embryos (Lobikin et al., 2012). In Xenopus, the establishment of LR asymmetry is dependent upon an electrochemical gradient created by the asymmetric distribution of ion transporter proteins (Adams et al., 2006; Aw et al., 2008; Morokuma et al., 2008; Vandenberg and Levin, 2010).

Given the widely-conserved roles of pathways involved in regulating the spatial arrangement of intracellular machinery, we hypothesized that Rab GTPases, key regulators of membrane trafficking (Schwartz et al., 2007), could be involved in the directed movement of LR-relevant ion transporters in the early embryo. Our molecular screen using DN or WT Rab GTPase mRNA constructs indicated that many alterations in transport machinery serving the plasma membrane could affect LR asymmetry, and strongly implicated Rab11 in LR specification. Our data are consistent with the known roles of Rab11 in regulation of endosomal transport and interactions with the actin cytoskeleton (Hoekstra et al., 2004; Mottola et al., 2010; Schwartz et al., 2007), but uniquely implicate Rab machinery in large-scale pattern formation. It is interesting that, as with other pathways previously thought to be “house-keeping” machinery (gap junctions, resting potential regulators, microtubules, etc.) (Levin, 2005), it has proven possible to dissociate subtle patterning functions from basic cell upkeep roles.

3.1. Rab11 is necessary for the asymmetric shuttling of ion transporters

Rab11 influences the localization of ion transporters in many biological systems, including three transporters implicated in LR patterning (Adams et al., 2006; Aw et al., 2008; Morokuma et al., 2008). For example, Rab11 is a key regulator of the movement of vesicles containing the H⁺K⁺-ATPase to the apical membrane of parietal cells upon external acidification (Duman et al., 1999), is involved in the movement of KCNQ1 subunits in cardiac muscle (Seebohm et al., 2007), and directs H⁺-V-ATPase localization in human salivary duct cells through direct interactions with the epsilon subunit (Oehlke et al., 2011). We analyzed the effect of DNRab11 expression on the localization of KCNQ1 and the H⁺-V-ATPase subunit ductin, both of which are known to be involved in LR patterning (Adams et al., 2006; Morokuma et al., 2008). We first confirmed and quantified the localization biases of these ion transporter subunits (Fig. 6) and determined that the expression of DNRab11 altered these biases, reducing strong expression in the ventral right cell, and significantly increasing expression in the opposite, dorsal left blastomere (Fig. 6B and D). This reversal in localization is indicative of a directed, Rab11-mediated transport of KCNQ1 and ductin to the right ventral blastomere. Specifically, our results indicate that Rab11 is required to localize ion transporters along both the dorsal–ventral and LR axes (Fig. 6). These results are consistent with a role for the cytoskeleton in the asymmetric transport of molecules along all three axes in the early cleavage stage Xenopus embryo (Aw et al., 2008).

Because expression of DNRab11 did not produce symmetrical localization of ion transporters, there may be an as yet unidentified secondary mechanism that works in opposition to Rab11 to direct protein localization to the dorsal left side of the embryo. Other Rab GTPases are certainly candidate molecules for this opposing director, and a screen could be conducted to determine if this is the case. It should be noted that a similar, still puzzling result was obtained in the mouse mutant for Inversin protein; under this mutation (thought to be a null), asymmetry is not lost but largely reversed (Morgan et al., 1998). As with Inversin, it may be that Rab11 perturbation results in uncoupling the normally consistent direction of an existing asymmetry with the other two axes rather than affecting the generation of asymmetry.

In addition to establishing that Rab11 directs ion transporter localization, we also sought to determine if Rab11’s effect on ductin localization was a result of a direct or indirect interaction between these proteins. We observed a clear concordance between Rab11 and ductin expression, indicating that the two proteins are located in the same subcellular regions (Fig. 5). Their parallel expression indicates that Rab11’s effect on ductin transport may occur via a direct interaction, or an indirect mechanism mediated by effector proteins, as opposed to being a secondary result of another (primary) Rab11 function.

3.2. Rab11 is required in the early embryo for LR patterning, independent of the GRP

Although Rab11 is required for ciliogenesis in some systems (Knodler et al., 2010; Qin, 2012; Westlake et al., 2011), our results indicate that the effect of this Rab on LR patterning is not cilia-dependent. First, DNRab11 injections made after the 1-cell stage did not induce a significant amount of heterotaxia, indicating that Rab11 has a very early role in the context of LR patterning. If the effects of DNRab11 were due to altered ciliary flow, injections at the 2-cell stage would be equally effective in inducing heterotaxia. Furthermore, targeting DNRab11 specifically to the GRP progenitors (Blum et al., 2009; Schweickert et al., 2007) had no effect on organ situs, and biased 1-cell DNRab11 injections randomized the LR axis even when they were localized to the right side, which does not affect LR relevant ciliary flow (Vick et al., 2009). In this, Rab11 function resembles several other pathways (e.g., serotonin and tubulin) that have now been shown to function in asymmetry independent of ciliary flow (Lobikin et al., 2012; Vandenberg et al., 2012). Together, these results indicate that Rab11 is required in the early embryo, likely within the first 60–90 min post-fertilization, in a GRP-independent manner, for proper LR patterning in Xenopus. These results are also consistent with the observed expression patterns of Rab11 in the early embryo.

3.2. A role for Rab GTPases in the ion flux model of LR patterning

The ion flux model proposes that the inherent chirality of the early cytoskeleton directs the consistent asymmetric distribution of maternal ion channel and pump proteins (Levin, 2005). Their differential activity on the left and right sides generates a LR biased gradient of membrane voltage and pH; this gradient, exerted over cells coupled by open gap junctions, drives the redistribution of the positively charged molecule serotonin, which ultimately suppresses asymmetric gene expression on the right side (Vandenberg and Levin, 2010). Many molecular-genetic gain- and loss-of-function experiments support this model in Xenopus, as well as chick, sea urchin, C. elegans, snails, and zebrafish, among others (Chuang et al., 2007; Duboc et al., 2005; Hibino et al., 2006; Levin, 1998; Shibazaki et al., 2004; Shimeld and Levin, 2006). As in snail and C. elegans embryos (Kuroda et al., 2009; Pohl, 2011), this physiological system is a powerful way to convert cellular polarity into embryo-wide asymmetry.

In Xenopus, one of the main questions concerns how the early embryo translates the intrinsic chirality of the cytoskeleton (Aw et al., 2008; Danilchik et al., 2006; Lobikin et al., 2012) into an asymmetric distribution of ion transporters. Three fundamental possibilities exist: (1) asymmetric degradation of endogenous proteins and mRNA, (2) asymmetric anchoring of protein or mRNA in certain portions of the early embryo, or (3) asymmetrically directed transport of protein or mRNA to certain sides of the cell/embryo. While the first two have not been directly tested and cannot be ruled out (Klar, 1994), our data support the third hypothesis: that asymmetric localization of ion transporters is accomplished through directed transport of proteins, specifically to the right ventral side of the embryo. Rab GTPases associate with specific membranes and motor proteins (Stenmark, 2009) and thus can indirectly link ion transporters embedded in membranes with the cytoskeleton. It is also worth noting that overexpression of Rab11 can induce heterotaxia as well, albeit to a lesser extent than DNRab11 (Fig. 1D). These results may indicate that a delicate balance in endosomal transport is required for proper patterning, and any perturbation in this system can alter the electrochemical gradient required for LR orientation (Fig. 7).

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.

Because of the multitude of studies implicating intracellular functions of motor proteins in the establishment of LR asymmetry (Armakolas and Klar, 2007; Armakolas et al., 2010; Hozumi et al., 2006; Klar, 2008; Levin, 2005; Qiu et al., 2005; Speder et al., 2006), further characterization of which motor proteins associate with which Rab GTPases during early development, as well as in which direction those transporters move along cytoskeletal tracks (toward the + or – end), will prove essential. For example, Rab11 associates with MyosinVb, which moves cargo toward the + ends of actin (Schuh, 2011), both directly and through the Rab11–FIP2 effector protein (Hales et al., 2002). In addition to the inherent directionality that is known to exist in the microtubule network of the Xenopus embryo (Aw et al., 2008), a recent discovery of long-range actin transport (Schuh, 2011) suggests that actin may also be key to the biased distribution of ion transporters. Indeed, embryos exposed to latrunculin, an agent that prevents actin polymerization, were unable to asymmetrically localize ion transporters and other LR-relevant proteins (Aw et al., 2008; Qiu et al., 2005).

Because our results were consistent with Rab11 directing ion transporter localization, we sought to determine whether Rab11 complexes were moving ion transporters via a “highway” model, whereby one or more Rab11 complexes would attach to vesicular cargo containing ion transporters and shuttle them the length of the cell, or whether an “assembly line” model was more appropriate, in which each Rab11 complex would be responsible for the movement of cargo within a small intracellular space, passing the cargo onto the next Rab11 complex to continue directional movement (Fig. 7). Importantly, endogenous Rab11 protein and mRNA were not found to have LR biases (Fig. 4), in line with the latter model. Further support for an “assembly line” type transport of ductin and KCNQ1 comes from biased co-injections of DNRab11 with a β-gal lineage tracer. We found that injecting DNRab11 on either the left or right side of the 1-cell embryo induces heterotaxia (Fig. 2), and thus Rab11 function is necessary on both sides of the embryo; alterations at any point in the “assembly line” can prevent correct shuttling of ion transporters.

3.4. Does Rab11 fit in an evolutionarily conserved model of LR patterning?

Recent data implicating the same tubulin mutations in asymmetry of models as evolutionarily distant as Arabidopsis, C. elegans, Xenopus, and human cells (Lobikin et al., 2012) suggest a highly conserved origin for asymmetry, although different bodyplans necessarily amplify that asymmetry in somewhat different ways (Vandenberg and Levin, 2009). In fact, even single cells, from yeast to mammalian somatic cells, have an inherent ability to establish a LR axis (Chen et al., 2012; Klar, 1987, 2008; Wan et al., 2011; Xu et al., 2007), supporting the conclusion that asymmetry is an ancient, cytoplasmically-based phenomenon that does not require multicellular structures or ciliary flow. One study of individual cells in culture implicated apical–basal polarity machinery in LR asymmetry (Xu et al., 2007), and the related machinery for PCP is required for LR patterning in several species, including chick, which lacks a ciliated node (Antic et al., 2010; May-Simera et al., 2010; Oteiza et al., 2010; Song et al., 2010; Vandenberg and Levin, 2012; Zhang and Levin, 2009). PCP is an especially attractive mechanism for orienting the LR axis because it can be applied across a wide range of embryonic architectures (Aw and Levin, 2009; Vandenberg and Levin, 2012), and is a good candidate for a conserved amplification mechanism for LR patterning.

PCP proteins control cellular behavior through coordinated vesicle trafficking (Gray et al., 2009), a process that intimately involves Rab GTPases. In particular, the endocytotic recycling of the Frizzled-1 (Fz1) receptor involves Rab11, and has been proposed to be involved in PCP-dependent Drosophila wing morphogenesis (Strutt and Vincent, 2010). For this reason, we asked whether the effects we observed by altering Rab11 expression on LR patterning could be due to its interactions with the PCP pathway. While previous studies have shown that epistasis experiments can reveal that reagents act in the same or different pathways to affect LR patterning (Vandenberg et al., 2011b), here we observed no additive effects of altering Rab11 signaling and the PCP pathway simultaneously (Fig. 3). These results may indicate that Rab11 affects LR patterning by interacting with some components of the cell polarity complexes previously implicated in LR patterning, but future experiments analyzing epstatic effects with a larger array of PCP proteins are needed to provide insight into how PCP-dependent membrane trafficking is coordinated in the early embryo to create an asymmetric distribution of ion transporters.

3.5. Conclusions

Elucidating the process by which LR asymmetry is established in species with various embryonic architectures has significant implications for both evolutionary and developmental biology, and identifying the best model to understand LR patterning in humans is essential for the medical community. Here we demonstrate, through molecular loss-of-function and overexpression experiments, that Rab11 is necessary for proper LR patterning in Xenopus, independent of ciliary flow at the GRP. We have also quantitatively demonstrated that ion transporters implicated in the orientation of the LR axis are asymmetrically distributed in the early embryo, and that this biased distribution is dependent on Rab11 function.

The involvement of Rab GTPase-directed transport in the early establishment of laterality provides a mechanism by which the chiral nature of the cytoskeleton in the early embryo can be translated into the asymmetric localization of ion transporters, filling in a previous uncertainty in the ion flux model of LR asymmetry. It should be noted that Rab GTP-dependent transport of ion translocators and other physiological coordination mechanisms such as planar cell polarity are ideally suited to orient and amplify fundamental asymmetries such as differential chromatid segregation (Armakolas and Klar, 2007; Klar, 1994, 2008) or the chirality of microtubule organizing centers (Levin and Palmer, 2007; Vandenberg and Levin, 2010) and actin cytoskeletal structures (Ali et al., 2002; Danilchik et al., 2006; Nishizaka et al., 1993; Pyrpassopoulos et al., 2012; Shibazaki et al., 2004). Future experiments designed to elucidate whether Rab GTPases direct the transport of other proteins implicated in LR patterning in the early embryo, including those in the PCP pathway, will provide significant insight into this field, whose central question is far from solved.

4. Materials and methods

4.1. Animal husbandry

Xenopus embryos were collected and fertilized according to standard protocols (Sive et al., 2000) in 0.1× Modified Marc’s Ringers (MMR) pH 7.8 + 0.1% Gentamicin and staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Tufts University’s Institutional Animal Care and Use Committee (#M2008–08).

4.2. Xenopus microinjection

For microinjections, embryos were placed in 3% Ficoll in 1× MMR. Capped, synthetic mRNAs (Sive et al., 2000) were dissolved in water containing rhodamine labeled dextran and injected into embryos following standard protocols. For our initial screen, mRNAs encoding a WT human Rab11, DN human Rab11 with a serine to asparagine mutation at amino acid 25, WT human Rab4, DN human Rab4 with an asparagine to isoleucine mutation at amino acid 121, WT human Rab7, DN human Rab7 with a threonine to asparagine mutation at amino acid 22, WT human Rab 9, and DN human Rab9 with a serine to asparagine mutation at amino acid 21. The dominant negative nature of several of these constructs has been previously characterized (Choudhury et al., 2002; Seebohm et al., 2008). When necessary, β-galactosidase mRNA was used as a lineage label. For epistasis experiments, Vangl2 morpholinos (Antic et al., 2010; Mitchell et al., 2009; Vandenberg and Levin, 2012) were injected alone or co-injected with DNRab11.

Injections were performed at the 1-cell stage in the north pole (animal-most point) of the embryo unless otherwise noted. For 2-cell stage injections, one or both of the two blastomeres were injected, as indicated in specific experiments. For injections at the 4–8 cell stage, we injected a single blastomere on the left side, specifically at the dorsal–marginal zone, which has previously been shown to target cilia at the GRP that are implicated in LR-relevant flow (Vick et al., 2009). After injections, embryos were washed and maintained in 0.1× MMR at 14–22 °C until desired stages.

4.3. Laterality assay

At stage 45, Xenopus embryos were analyzed for position (situs) of the heart (looping to the left), stomach (coiling to the left) and gall bladder (positioned on the right) as shown in Fig. 1. Heterotaxia was defined as the reversal in position of one or more organs. Animals with abnormal dorsoanterior index (DAI) as described in (Kao and Elinson, 1988) are known to have increased incidence of LR patterning defects (Danos and Yost, 1995); therefore only embryos with a normal DAI (DAI = 5) were scored. Percent heterotaxia was calculated as the absolute number of heterotaxic embryos divided by the total number of scorable embryos. A χ² test with Pearson correction for increased stringency was used to compare absolute counts of heterotaxic embryos.

4.4. In situ Hybridization

A DIG labeling kit (Roche, Branford, CT) was used to generate in situ hybridization probes against Xnr-1 (the Xenopus Nodal homolog) mRNAs (Sampath et al., 1997), Rab11 mRNAs, and a Rab11 sense probe. Whole mount in situ hybridization was performed using standard protocols (Harland, 1991) on fixed embryos collected at 1-cell, 4-cell, NF stages 8, 10.5 and 14–17 (Rab11) and NF stages 21–22 (Xnr-1). Albino embryos were used for Rab11 and Rab11 sense probes to allow for clearer visualization of signal; embryos treated with sense and antisense probes were exposed to reagents for the same length of time, allowing for suitable comparisons in expression. A χ² test with Pearson correction for increased stringency was used to compare absolute counts of embryos with normal (left-sided) and abnormal (right, bilateral or absent) expression of Xnr-1.

4.5. Western blot

Western blot analysis was performed according to standard protocols using a commercial anti-Rab11 antibody (Cell Signaling) at 1:1000 dilution and a goat anti-rabbit secondary antibody with HRP conjugation (Jackson Immunologicals) at 1:5000. This analysis was performed on embryos collected at various stages from 1 cell to stage 25 and immunosignals were visualized with chemiluminescence.

4.6. Embedding, sectioning and Immunohistochemistry

Embryos were embedded in gelatin-albumin according to a previously established protocol (Levin, 2004), but substituting fish gelatin (Sigma) for bovine gelatin. Embryos were oriented so that they could be cut along the dorsal–ventral or anterior–posterior/animal–vegetal axes. 100 μm sections were cut from the blocks with a Leica vibratome.

Immunohistochemistry was carried out as described (Levin, 2004) using several primary antibodies and a goat anti-rabbit secondary antibody with HRP conjugation (Jackson Immunologicals) at 1:500. After several washes, detection was carried out with peroxidase substrate (Moss Biologicals, Inc.). After a dark blue/purple signal appeared, embryos were washed until background signal was no longer visible in sections treated without primary antibody. Primary antibodies used were monoclonal Rab11 antibodies (Cell Signaling) against the human peptide sequences around Arg30 at 1:100; an antibody against ductin (H⁺-V-ATPase c subunit) (Vandenberg et al., 2011a) used at 1:200; and an antibody against KCNQ1 (Morokuma et al., 2008) used at 1:500.

4.7. Cell lineage tracers

For some experiments, mRNAs encoding β-galactosidase (β-gal) were co-injected with other mRNAs or injected alone in a purposefully biased manner at the 1-cell stage (as described in Aw et al. (2010)). At stage 45, tadpoles were scored for heterotaxia, overdosed with tricaine, fixed for 45 min and stained with X-gal (Roche Applied Sciences, Indianapolis, IN).

4.8. Analysis of ion transporter localization

Because no quantitative scoring of ion transporter localization at the 4-cell stage had previously been carried out, we developed a novel protocol. Embryos with obvious segregation of pigmentation into the ventral portion (Klein, 1987) were selected for accurate orientation upon sectioning. After performing immunohistochemistry for an ion transporter, both sides of the section were analyzed to visually score the relative amount of ion transporter expression; we analyzed both the intensity and range of staining in each individual blastomere. When all 4 blastomeres had the same level of expression, the embryo was scored as “symmetric”. Otherwise, the blastomere with the greatest level of expression (as defined both by intensity and area of staining) was identified visually, and verified with ImageJ (http://rsb.info.nih.gov/ij/) thresholding for a number of examples to determine visual accuracy. In the event of relatively equal expression between blastomeres, both were identified as strongly expressing the protein of interest. A χ² test with Pearson correction for increased stringency was used to compare absolute counts of embryos with strong ventral right, strong dorsal left, or symmetric expression of KCNQ1 and ductin.

4.9. Analysis of subcellular localization of target proteins

To compare the subcellular localization of Rab11 and ductin, mRNA constructs encoding fluorescently tagged versions of each protein (Rab11-Tom and Ductin-YFP) were co-injected into 1-cell stage embryos. Embryos were raised to stage 45, anesthetized with 1% tricaine in MMR and their tails were amputated between the anus and the tip. These tail portions were mounted on slides with 50% glycerol and imaged. To analyze images, the plot profile feature of ImageJ was used to determine the intensity of each signal. The plots were then overlaid to compare patterning.

4.10. Microscopy and image collection

For immunohistochemistry and the visualization of tomato or β-gal lineage tracer signals, images were collected with a Nikon SMZ1500 dissection microscope with a Retiga 2000R camera and ImageQ software. For protein co-localization experiments, an Olympus BX-61 with a Hamamatsu ORCA AG CCD camera, controlled by MetaMorph software, was used for imaging. Adobe Photoshop^™ and Illustrator^™ were used to orient, scale, and brighten images.