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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Biol. Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
PMCID: PMC2801549
NIHMSID: NIHMS154593

The involvement of lethal giant larvae and Wnt signaling in bottle cell formation in Xenopus embryos

Abstract

Lethal giant larvae (Lgl) plays a critical role in establishment of cell polarity in epithelial cells. While Frizzled/Dsh signaling has been implicated in the regulation of the localization and activity of Lgl, it remains unclear whether specific Wnt ligands are involved. Here we show that Wnt5a triggers the release of Lgl from the cell cortex into the cytoplasm with the concomitant decrease in Lgl stability. The observed changes in Lgl localization were independent of atypical PKC (aPKC), which is known to influence Lgl distribution. In ectodermal cells, both Wnt5a and Lgl triggered morphological and molecular changes characteristic of apical constriction, whereas depletion of their functions prevented endogenous and ectopic bottle cell formation. Furthermore, Lgl RNA partially rescued bottle cell formation in embryos injected with a dominant negative Wnt5a construct. These results suggest a molecular link between Wnt5a and Lgl that is essential for apical constriction during vertebrate gastrulation.

Keywords: Lgl, Wnt5a, apical constriction, bottle cell, Xenopus

Introduction

Cell polarity is essential for a variety of biological processes including asymmetric cell division, directed cell migration and epithelial morphogenesis in development and normal physiology of multicellular organisms (Goldstein and Macara, 2007; Humbert et al., 2006). Lethal giant larvae (Lgl) is a central participant in the establishment and maintenance of cell polarity (Vasioukhin, 2006; Wirtz-Peitz and Knoblich, 2006). In epithelial cells, Lgl is localized to the basolateral cell cortex and was proposed to maintain the identity of the basolateral domain by regulating targeted vesicular trafficking (Musch et al., 2002; Peng et al., 2000; Wirtz-Peitz and Knoblich, 2006; Zhang et al., 2005). Loss of Lgl activity expands the apical membrane domain, whereas overexpression of Lgl leads to the expanded basolateral membrane domain (Chalmers et al., 2005; Dollar et al., 2005). Lgl also regulates asymmetric cell division of Drosophila neuroblasts and neural progenitor cells in mice (Klezovitch et al., 2004; Peng et al., 2000). In Lgl-deficient neuroblasts, cell fate determinants fail to be targeted unequally to the basal cell cortex and therefore are inherited by both daughter cells during cytokinesis (Lee et al., 2006a).

The mechanism restricting Lgl localization to the basolateral domain includes negative regulation by the apical PAR6/PAR3/aPKC complex (Betschinger et al., 2003; Plant et al., 2003; Yamanaka et al., 2003). aPKC phosphorylates Lgl, thereby dissociating it from the apical cell cortex (Betschinger et al., 2005). The Wnt pathway components Frizzled and Dishevelled (Dsh) were also implicated in the regulation of Lgl localization in Xenopus and Drosophila embryos (Dollar et al., 2005). Knockdown of Dsh reduces the stability and the cortical localization of Lgl, whereas overexpression of Frizzled 8 (Fz8) causes relocalization of Lgl from the basolateral cortex into the cytoplasm (Dollar et al., 2005). While these results implicate Wnt signaling in Lgl regulation, specific Wnt ligands that affect the localization and function of Lgl remain to be identified.

Due to their critical functions in cell polarity and cell shape regulation, several PAR (partitioning defective) proteins, including PAR-1, PAR-6 and aPKC, and planar cell polarity (PCP) proteins, were implicated in early morphogenetic processes of gastrulation and neurulation. This role has been clearly established for cell migration, epithelial-mesenchymal transformation and convergent extension movements (Kinoshita et al., 2008; Kusakabe and Nishida, 2004; Lee et al., 2008; Ozdamar et al., 2005; Sokol, 2000; Wallingford et al., 2002). Apical constriction is another morphogenetic process that accompanies blastopore formation at the beginning of gastrulation (Davidson et al., 2002; Hardin and Keller, 1988; Shook and Keller, 2003). Genetic studies identified the Folded gastrulation ligand acting through the Gα12/13-Concertina pathway to locally activate Rho-kinase and myosin II during apical constriction in Drosophila ventral furrow formation (Costa et al., 1994; Dawes-Hoang et al., 2005; Kolsch et al., 2007). The relevance of this pathway to vertebrate gastrulation is currently unknown, and the involvement of apical-basal polarity proteins, such as PAR proteins and Lgl, in apical constriction has not been investigated.

In gastrulating Xenopus embryos, apical constriction is evident during blastopore formation by the appearance of bottle cells (Davidson et al., 2002; Hardin and Keller, 1988; Shook and Keller, 2003). Bottle cells can be identified by the appearance of accumulated pigment granules at the involution boundary (blastopore) at the onset of gastrulation. During bottle cell formation, actin and myosin become concentrated at the apical surface and microtubule arrays are aligned along the apicobasal axis (Lee and Harland, 2007). Interestingly, Lgl was shown was shown to interact with myosin II (Barros et al., 2003; Strand et al., 1994) and cause induce pigment aggregation in ectodermal cells that is indicative of bottle cell formation (Dollar et al., 2005). Moreover, recent work implicated components of the Wnt/PCP pathway in archenteron invagination in the sea urchin embryo (Croce et al., 2006), the apical constriction of Xenopus neural plate cells (Kinoshita et al., 2008) and ingressing endodermal precursors in C. elegans (Lee et al., 2006b). In this study, we examined the involvement of Lgl and Wnt signaling in apical constriction and bottle cell formation. Our experiments identify Wnt5a as a specific ligand that regulates Lgl localization and stability and suggest that the observed regulation is relevant for bottle cell formation during vertebrate gastrulation.

Materials and methods

Embryo culture, microinjection and lineage tracing

In vitro fertilization, embryo microinjection and culture were carried out as described previously (Hikasa and Sokol, 2004). Developmental stages of embryo were determined according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1994). Microinjections were done in the animal pole or the dorso-vegetal region of four or eight-cell stage embryos. Animal pole explants were dissected at late blastula or early gastrula stages. RNAs for microinjection were in vitro synthesized using mMessage mMachine RNA transcription kit (Ambion). For lineage tracing, nuclear β-galactosidase (100 pg) or membrane-targeted mCherry (400 pg) RNA was injected along with morpholinos anti-sense oligonucleotides (MOs) or RNAs, and the β-galactosidase activity was visualized with the Red-Gal substrate (Research Organics).

DNA constructs and morpholinos

The following previously described constructs were used: pXT7-GFP-Lgl and pXT7-Myc-Lgl (Dollar et al., 2005), pCS2-GFP-CAAX, pCS2-aPKC-CAAX and pCS2-aPKC-N (Ossipova et al., 2007), XWnt11 (Tada and Smith, 2000), XWnt5a (Moon et al., 1993), activin βB and XWnt8 (Sokol et al., 1991). The GFP-Lgl6SA construct (Dollar and Sokol, unpublished) was generated by substituting alanines for serines at positions 647, 651, 655, 659, 666 and 669 in pXT7-GFP-Lgl using single primer-based Pfu-mediated mutagenesis (Itoh et al., 2005). DN-XWnt5a (comprising amino acids 1-309) was generated by removing the C-terminal region of the protein as described (Tada and Smith, 2000). The PCR product encoding DN-XWnt5a was cloned into the Eco RI and Spe I sites of pXT7. pXT7-DN-XWnt5a was linearized with Not I for RNA synthesis. Anti-sense MOs were obtained from Gene Tools. Lgl MO1 and MO2 target non-overlapping sequences in the Lgl mRNA 5′ untranslated region and open reading frame, respectively. The sequence and efficacy of Lgl MO1 were reported previously (Dollar et al., 2005). Lgl MO2 had the following sequence: 5′-GCCGCCTGAACCGAA ACTTCATCAT-3′. The efficacy and specificity of this MO are shown in Suppl. Fig. 6. Control MO (Co MO) has a similar base composition, but a different sequence: 5′-AGCTGACGCACCCGCTGCCAG TACA-3′.

RT-PCR analysis

Total RNA was extracted from whole embryos and animal cap explants using TRI-reagent (Molecular Research Center) and treated with RNase-free DNase I to remove genomic DNA. cDNAs were prepared using the Superscript first strand synthesis system (Invitrogen). PCR primers for Goosecoid, Chordin, Xbra, Xnr3 and FGFR were described by (Hikasa and Sokol, 2004) and by the De Robertis laboratory homepage (http://www.lifesci.ucla.edu/hhmi/derobertis/index.html). The number of PCR cycles for each primer pair was determined empirically to maintain amplification in the linear range.

Fluorescence microscopy

To examine subcellular localization of proteins in animal cap tissue, animal explants expressing GFP-tagged constructs were dissected at stages 9.5 or 10, fixed in 4% paraformaldehyde (PFA) in PBS for 1 hour, washed and mounted for observation with the Vectashield mounting medium (Vector). For cryosections, embryos injected with RNAs and/or MOs were manually devitellinized at stage 10.5 and fixed in Dent's solution, MEMFA (0.1M MOPS, 2mM EGTA, 1mM MgSO4, 3.7% formaldehyde) or 4% paraformaldehyde buffer for 2 hours (Lee and Harland, 2007). Fixation for F-actin or phospho-myosin light chain staining was described previously (Lee and Harland, 2007). To examine protein localization at or near the blastopore, whole embryos were hand-sectioned midsagittally using a razor blade after fixation. Indirect immunofluorescence on cryosections and bisected embryos was performed using the following antibodies: mouse anti-GFP (BD Biosciences, JL-8, 1:500), rabbit anti-aPKC (Santa Cruz, 1:200), rabbit anti-β-catenin (Santa Cruz, 1:200) and rabbit anti-phospho-S20-myosin light chain (Abcam, 1:300). For F-actin staining, Alexa 488-conjugated phalloidin (5 units/ml, Molecular Probes) was used. If applicable, cryosections were double-stained using a combination of polyclonal and monoclonal primary antibodies. Secondary antibodies were Alexa 488-conjugated anti-rabbit or anti-mouse (Molecular Probes, 1:200) and Cy3-conjugated anti-rabbit (Jackson ImmunoResearch, 1:200). Imaging was performed on a Zeiss Axiophot microscope with the Apotome attachment. Cell length and apical width were measured using Carl Zeiss AxioVision software, and apical staining intensity measurement was performed using Image J 1.40g software. Statistical significance was determined using T-test using Microsoft Excel software.

Western blot analysis

Western blotting was performed as previously described (Gloy et al., 2002). Briefly, whole embryos or animal cap explants were lysed in a buffer containing 1% Triton X-100, 50 mM sodium chloride, 50 mM TrisHCl at pH 7.6, 1 mM EDTA, 0.6 mM phenylmethylsulphonyl fluoride (PMSF), 10 mM sodium fluoride and 1 mM sodium orthovanadate, and the lysates were subjected to SDS-PAGE. The following primary antibodies were used: mouse monoclonal anti-Myc (9E10), anti-β-tubulin antibodies (Sigma), rabbit polyclonal anti-Dvl2 antibody (Itoh et al., 2005), rabbit polyclonal anti-XLgl1 (Dollar et al., 2005).

Results

Wnt5a triggers Lgl redistribution

Overexpressed Fz8 receptor was reported to alter the basolateral localization of Lgl (Dollar et al., 2005). To investigate whether this effect of Frizzled on Lgl distribution mimics the action of a specific Wnt ligand, embryos were injected with RNAs encoding Wnt3a, Wnt5a, Wnt7b, Wnt8 and Wnt11 together with GFP-Lgl and examined Lgl distribution in Xenopus ectodermal cells (Fig. 1). Without Wnt ligands, GFP-Lgl was restricted to the basolateral cell surfaces in animal cap explants and in embryo cross-sections (Figs. 1A, B, G). Interestingly, overexpression of Wnt5a, but not Wnt3a, Wnt7b, Wnt8 or Wnt11, displaced GFP-Lgl from the cell cortex into the cytoplasm (Figs. 1C, D, H, I and data not shown), similarly to the effect of Fz8 (Dollar et al., 2005). The effect was observed with high penetrance in the majority of injected embryos (over 90%, n > 100). This redistribution was specific to Lgl, because Wnt5a did not affect the localization of membrane-targeted GFP (Figs. 1E, F) or basolaterally localized β-catenin (Suppl. Fig. 1).

Fig. 1
Wnt5a affects localization and stability of Lgl

As aPKC has been shown to exclude Lgl from the apical region of epithelial cells (Betschinger et al., 2003; Plant et al., 2003), we wanted to test whether this kinase mediates the observed effect of Wnt5a on Lgl localization. We examined the effect of Wnt5a on the localization of GFP-Lgl6SA, a form of Lgl, in which the six conserved aPKC phosphorylation sites were replaced by alanine residues complex (Betschinger et al., 2003; Plant et al., 2003; Yamanaka et al., 2003). Consistent with the assumption that this mutant is resistant to aPKC-mediated phosphorylation, GFP-Lgl6SA was present at both apical and basolateral cortical domains and, unlike wild-type Lgl, was not dissociated from the cell cortex in response to constitutively active aPKC (aPKC-CAAX, Suppl. Fig. 2). Notably, coexpression of Wnt5a could still relocalize Lgl6SA into the cytoplasm (Figs. 1J, K), suggesting that aPKC is not involved in the redistribution of Lgl in response to Wnt5a. Consistent with this conclusion, a dominant interfering form of aPKC (aPKC-N) did not suppress the release of Lgl from the cell cortex in response to Wnt5a (Suppl. Fig. 3).

In addition to the change in Lgl intracellular localization in response to Wnt5a, we found that Lgl protein levels decreased in embryos overexpressing Wnt5a, but not in those injected with Wnt8 (Fig. 1L). Thus, the dissociation of Lgl from the cortex in cells overexpressing Wnt5a may lead to its destabilization, an effect reminiscent to the effect of Fz8 on Lgl (Dollar et al., 2005).

Wnt5a and Lgl induce morphological changes that are characteristic of bottle cells

Ectodermal cells revealed striking pigment accumulation after microinjection of Wnt5a RNA, as compared to Wnt11 RNA (Figs. 2A, B). This effect was similar to the phenotype caused by Lgl or Activin βB RNA (Figs. 2C, D; (Dollar et al., 2005). In cross-sections, many superficial cells were apically constricted and elongated in the apicobasal direction (Figs. 2E, F, and data not shown), resembling the morphological characteristics of ectopic bottle cells induced by activin (Kurth and Hausen, 2000). Thus, we next examined whether Wnt5a or Lgl trigger the accumulation of cytoskeletal proteins such as actin and activated phospho-myosin light chain (MLC) at the apical side of the cell, as shown for ectopic and endogenous bottle cells (Kurth and Hausen, 2000; Lee and Harland, 2007). Compared to uninjected control cells, ectodermal cells expressing exogenous activin βB, Wnt5a or Lgl revealed increased apical accumulation of F-actin and phospho-MLC (Figs. 3A, B). Together, these data suggest that, like activin, both Wnt5a and Lgl are sufficient on their own to induce cytoskeletal reorganization and cell shape changes, leading to ectopic bottle cell formation.

Fig. 2
Wnt5a and Lgl induce bottle cell morphology in Xenopus ectoderm
Fig. 3
Activin βB, Lgl and Wnt5a cause ectopic apical accumulation of F-actin and phospho-myosin in superficial ectoderm

Wnt5a and Lgl are essential for bottle cell formation

Next, we investigated whether Wnt5a and/or Lgl are required for ectopic bottle cell formation in response to activin. Indeed, Activin βB RNA caused marked pigmentation of injected ectodermal cells at high penetrance (over 90% of injected embryos revealed this phenotype, n > 50), indicative of ectopic bottle cell formation (Fig. 4A). This pigment redistribution was inhibited by two Lgl MO (MO1 and MO2) with unrelated sequences and a truncated form of Wnt5a (DN-Wnt5a), which dominantly blocked Wnt5a activity (Fig. 4A and Suppl. Fig. 4), suggesting that both Lgl and Wnt5a are essential for ectopic induction of bottle cells in embryonic ectoderm. In cross-sections, apical constriction and apicobasal elongation of activin-expressing cells was inhibited by coinjection of DN-Wnt5a or Lgl MOs (Figs. 4B-D).

Fig. 4
Wnt5a and Lgl are required for activin-dependent induction of ectopic bottle cells

We next tested whether the inhibitory effects of DN-Wnt5a and Lgl MO on bottle cell formation are due to impaired activin signaling activity. Using RT-PCR analysis, we observed that Activin βB RNA activated several target genes, such as Chordin, Goosecoid and Xnr3, in ectodermal cells (Fig. 4E). Coinjection of DN-Wnt5a, Lgl MO1 and Lgl MO2 had no effect on ectopic gene expression by activin (Fig. 4E). These results suggest that Wnt5a and Lgl functions are required for activing to trigger ectopic bottle cell formation, independently of gene activation.

In Xenopus embryos, bottle cells are present in the blastopore lip that originates dorsally at stage 10 and then spreads to the lateral and ventral regions of embryo (Hardin and Keller, 1988). To examine the role for Wnt5a and Lgl in endogenous bottle cell formation, we injected DN-Wnt5a RNA or two different Lgl MOs along with β-galactosidase as a lineage tracer and observed blastopore ring formation. DN-Wnt5a, Lgl MO1 and Lgl MO2 strongly inhibited blastopore formation in 81% (n=82), 65% (n=49) and 76% (n=47) of injected embryos, respectively (Figs. 5A, B).

Fig. 5
Wnt5a and Lgl are essential for endogenous bottle cell formation

We next examined whether the depletion of Wnt5a or Lgl function influences the localization of F-actin and phospho-MLC in bottle cells. F-actin accumulated at the apical surface of bottle cells in control MO-injected embryos, but not in DN-Wnt5a-expressing embryos (Fig. 6A). Notably, Lgl MO1 and MO2 did not affect F-actin accumulation, even though the blastopore groove in Lgl-depleted embryos appeared shallower, when compared to control embryos (Fig. 6A). Phospho-MLC also accumulated apically in bottle cells of uninjected or control MO-injected embryos (Fig. 6B and data not shown). This apical staining was decreased in DN-Wnt5a or Lgl MO-injected embryos (Fig. 6B). Importantly, the inhibitory effect of DN-Wnt5a on blastopore formation was partially reversed by Lgl RNA (Figs. 7A, B). Consistent with this, depletion of Lgl could inhibit apical constriction and apicobasal elongation of Wnt5a-expressing cells (Suppl. Fig. 5). In addition, Lgl RNA recovered apical F-actin accumulation in bottle cells expressing DN-Wnt5a (Fig. 7C). Together, these results suggest that both Wnt5a and Lgl are involved in apical constriction during Xenopus gastrulation and that Lgl is likely to function in this process downstream or in parallel to Wnt5a.

Fig. 6
Roles for Wnt5a and Lgl in apical F-actin and phospho-MLC accumulation in endogenous bottle cells
Fig. 7
Lgl acts downstream of Wnt5a in regulating bottle cell formation

Discussion

In bottle cell morphogenesis, apical constriction and apicobasal elongation are driven by actomyosin contractility (Lee and Harland, 2007). Our experiments show that overexpressed Lgl induces apical myosin accumulation and ectopic bottle cells, whereas its depletion inhibits this accumulation and blastopore formation, suggesting that Lgl regulates bottle cell shape by controlling myosin localization and activity. In bottle cells, Lgl may act to exclude myosin or specific myosin regulators such as Rho kinase (Barros et al., 2003; Kinoshita et al., 2008) from the basolateral cortex. In agreement with this possibility, Lgl has been reported to bind myosin II and negatively regulate its localization in Drosophila neuroblasts (Barros et al., 2003; Strand et al., 1994), and myosin II was mislocalized in Lgl1-/- mouse neural progenitor cells (Klezovitch et al., 2004). On the other hand, Lgl could also affect cell shape by additional means, as it has been hypothesized to function in protein vesicular trafficking (Vasioukhin, 2006; Wirtz-Peitz and Knoblich, 2006), suggesting that Lgl might direct polarized transport of some proteins that regulate apical localization of myosin or its activators. Further studies are necessary to discriminate these possibilities and determine the precise mechanism by which Lgl influences apical constriction and blastopore formation.

We identified a candidate Wnt ligand that may be responsible for the regulation of Lgl by Frizzled and Dishevelled in Xenopus ectoderm cells (Dollar et al., 2005). We found that Wnt5a, but not Wnt8 or Wnt11, causes the dissociation of Lgl from the basolateral cortex. As Wnt5a is expressed in the deep layer of dorsal marginal zone during gastrulation (Schambony and Wedlich, 2007), it is a likely candidate for an endogenous Wnt protein that regulates blastopore formation in a gastrulating embryo. This hypothesis is further supported by our observation that DN-Wnt5a inhibits endogenous bottle cells during gastrulation. Nevertheless, since DN-Wnt5a inhibits both Wnt5a and Wnt11 signaling as shown in Suppl. Fig. 4, we cannot exclude a possible role for a Wnt11-like protein in bottle cell formation without additional experiments. Consistent with our findings, Wnt5a has been shown to polarize actomyosin filaments, induce epithelial-mesenchymal transition and stimulate directed migration of melanoma cells in a pathway that involves Frizzled3, Dsh, PKC and RhoB (Dissanayake et al., 2007; Witze et al., 2008). Moreover, a Wnt pathway involving Wnt11, Dsh and Rho kinase was shown to be critical for neural tube closure in chick and frog embryos (Kinoshita et al., 2008).

Whereas the link between cell polarity and apical constriction remains poorly understood, we show that Lgl is required for Wnt5a-triggered apical constriction as well as blastopore formation in vivo, suggesting that the observed effect of Wnt5a on Lgl localization may be relevant to this process. As Lgl is likely to regulate polarized protein trafficking (Vasioukhin, 2006; Wirtz-Peitz and Knoblich, 2006), it may function to define apical versus basolateral cell surface properties. We hypothesize that after being relocalized by Wnt5a, cytoplasmic Lgl triggers apical accumulation of specific proteins, including regulators of F-actin and phospho-MLC that trigger apical constriction and apicobasal elongation, similarly to what we observe in Lgl-overexpressing cells (Dollar et al., 2005). Future experiments are warranted to identify additional molecular components that function to regulate apical constriction in response to Wnt5a and Lgl signaling and establish their connection to the known regulators of bottle cell formation such as the actin-binding protein Shroom3 (Haigo et al., 2003), as well as activin/nodal signaling (Kurth and Hausen, 2000).

Supplementary Material

01

Supplementary Fig. 1. Wnt5a has no effect on β-catenin localization:

Cryosections of embryos injected in the dorsal margin with GFP RNA (400 pg) alone or with Wnt5a RNA (1 ng) were double-stained with anti-GFP- and anti-β-catenin antibodies. Endogenous bottle cells in the dorsal embryonic region at the beginning of gastrulation are shown.

02

Supplementary Fig. 2. Lgl6SA mutant is not released from the cell cortex in response to a constitutively active aPKC:

Ectodermal tissues from embryos injected animally with RNAs encoding GFP-Lgl (500 pg) or GFP-Lgl6SA (500 pg) or along with CA-aPKC RNA (10 pg) were cryosectioned and double-stained with anti-GFP and anti-β-catenin antibodies.

03

Supplementary Fig. 3. aPKC does not function downstream of Wnt5a in redistributing Lgl:

Four cell stage embryos were injected animally with RNAs, encoding GFP-Lgl (500 pg), XWnt5a (1 ng), or DN-aPKC (2 ng) as indicated. GFP-Lgl fluorescence was examined in animal cap explants (A-D) at stage 10 using a Zeiss Axiophot microscope with the Apotome attachment.

04

Supplementary Fig. 4. DN-Wnt5a inhibits both Wnt5a and Wnt11 signaling:

Four cell stage embryos were injected animally with RNAs encoding XWnt5a (600 pg), XWnt11 (500 pg) and DN-Wnt5a (2 ng) as indicated, and animal cap explants were dissected at stage 9.5, cultured to stage 12 and then subjected to western blot analysis using anti-Dvl2 antibody (1:500 dilution). P-Dsh, phosphorylated form of Dishevelled.

05

Supplementary Fig. 5. Depletion of Lgl inhibits morphological changes caused by Wnt5a:

(A-E) Sectioned superficial ectoderm from embryos injected animally with mCherry RNA (400 pg) alone as a lineage tracer or along with XWnt5a RNA (1 ng), Co MO (40 ng), Lgl MO1 (40 ng) and Lgl MO2 (5 ng) as indicated. (F) Apical index in the cells shown in (A-E). Error bars indicate standard deviation.

06

Supplementary Fig. 6. Specific depletion of endogenous Lgl by Lgl MO2:

Animal cap explants dissected at stage 9.5 from embryos injected with Lgl MO2 (4 ng) or Co MO (4 ng) were cultured to stage 25 and harvested for western blotting analysis with anti-Lgl1 antibody. β-tubulin serves as a loading control.

Acknowledgments

We thank Gretchen Dollar for the construction and initial characterization of the GFP-Lgl6SA construct, Keiji Itoh and Andrew Sproul for critical comments on the manuscript and other members of the Sokol laboratory for helpful discussions. This study was supported by NIH grants to S. S.

Footnotes

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