Daam1 regulates the endocytosis of EphB during the convergent extension of the zebrafish notochord

Abstract

Convergent extension (CE) movement of cells is one of the fundamental processes that control the organized morphogenesis of tissues and organs. The molecular events connecting the noncanonical Wnt pathway and CE movement, however, are not well understood. We show that subcellular localization of Daam1, an essential component of noncanonical Wnt signaling, changes dynamically during notochord formation. In the early phases, Daam1 complexes with EphB receptors and Disheveled 2. This complex is incorporated into endocytic vesicles in a dynamin-dependent manner, thereby resulting in the removal of EphB from the cell surface with subsequent switching of cell adhesiveness. In the next step, Daam1 colocalizes with the actin cytoskeleton to induce morphological extension of cells. We elucidate the molecular mechanism underlying the CE movement of notochord cells with Daam1 as a dynamic coordinator of endocytosis and cytoskeletal remodeling.

The establishment of cell polarity is one of the fundamental processes critical for cell division, migration, and convergent extension (CE) cell movement during development (1, 2). Polarity within the cells in the epithelial plane, planar cell polarity (PCP), has been characterized extensively in Drosophila, highlighting two core PCP genes, frizzled (Dfz) and disheveled (dsh) (1, 3). Both genes are highly conserved in vertebrates, playing essential roles in the establishment of cell polarity and cell movement (4, 5). In invertebrates and vertebrates, the Fz and Dsh (Dvl) families control cell fate and proliferation as part of the canonical Wnt signaling pathway involving β-catenin (6). In contrast, Fz and Dvl are involved in noncanonical Wnt signaling in the PCP/CE pathway. Fz and Dv1 activate another core PCP molecule, RhoA, a member of the Rho family of GTPases (2, 7). Its activation requires a formin-homology (FH) protein, Daam1 (disheveled-associated activator of morphogenesis) (8). Daam1 binds to both RhoA and Dvl, making a pivotal complex that is essential for PCP/CE signaling during Xenopus gastrulation. Hence, Daam1 relays the noncanonical Wnt signal to the cytoskeleton, which causes it to initiate its remodeling to change cell behaviors.

During gastrulation, cells migrate away from their original position in a concerted fashion specifically through modulation of cell adhesion (2). Wnt11, which acts in the noncanonical Wnt cascade, is involved in this process, controlling the cohesion of mesoendodermal cells through the Rab5c-mediated endocytosis of E-cadherin (9).

In addition to the noncanonical Wnt cascade, multiple signaling pathways, such as BMP (10), Stat3 (11), and Eph/Ephrin (12–14), regulate key processes of gastrulation. Eph receptor tyrosine kinases and Ephrins play important roles in cell migration and adhesion during development. Activation of Eph leads to opposite effects, deadhesion/repulsion or adhesion/attraction of cells, depending on the cell type and signaling contexts (15). Consequently, this cascade regulates diverse developmental processes, including gastrulation, segmentation, angiogenesis, migration of neural crest cells, and axonal pathfinding (16). It is unclear which mechanisms enable switching between these distinct responses. EphB/EphrinB-mediated repulsion is known to be controlled by a mechanism involving endocytosis of Eph receptors (17, 18), yet upstream signaling cascades controlling this endocytic event remain to be solved.

In this study, we show that Daam1 regulates endocytosis of EphB molecules and cytoskeletal remodeling to coordinate cell behavior during notochord development.

Results

Subcellular Localization of Daam1.

Using an anti-Daam1 antibody to label HEK293 cells, we found Daam1 in vesicle-like dot structures where early endosomal autoantigen 1 (EEA1) colocalized (Fig. 1A). In addition, Daam1 and the fluorescent styryl dye (FM1–43FX) were both located in the vesicles, indicating incorporation of FM1–43FX into Daam1-positive endocytic vesicles (Fig. 1B). When stimulated by Wnt1-conditioned medium, cells changed their shape and developed cytoplasmic protrusions in which the Daam1-positive vesicles accumulated (yellow arrowheads, Fig. 1C). Colocalization with Dvl2 and incorporated transferrin was also confirmed [see supporting information (SI) Fig. 6].

Fig. 1.

Localization of Daam1 in the endocytic vesicles. (A) In HEK293 cells, endogenous Daam1 was observed in the vesicles, which were visualized by staining with anti-Daam1 and anti-EEA1 antibodies. (B) After exposure to fluorescent FM1–43FX, cells were fixed and stained with the anti-Daam1 antibody and DAPI. Three spots of incorporated FM1–43FX (arrows) colocalized with the Daam1-positive vesicles. A small area indicated by white dotted lines was magnified and shown (Inset). (C) When stimulated by Wnt1-conditioned medium, HEK293 cells changed cell shape to form protrusions. The Daam1-positive endocytic vesicles accumulated in the protrusion (arrows). (D) After GFP-tagged RhoB was expressed in HEK293 cells, cells were stained with the anti-Daam1 antibody. Colocalization of Daam1 and RhoB was evident in the vesicles (arrows). (E) Introduction of a dominant-negative RhoB (N19) tagged with GFP reduced the number of vesicles and resulted in the formation of a large vacuole, in which Daam1 colocalized (arrows). (F) Expression of a kinase-dead mutant of GFP-Dnm1 [Dnm1(K44A)] disrupted the vesicle-like distribution of Daam1. Instead, Daam1 localized at the cell membrane (arrows). In a Dnm1(K44A)-negative cell, Daam1 localized in the endocytic vesicles (arrowheads).

Endocytosis serves numerous functions in cells, including internalization of ligand-receptor molecules, signal transduction, and cell–cell communication. It has been shown that small Rho GTPases, such as RhoB, RhoD, and Rab5, play pivotal roles in cell migration (19), which is also regulated by endocytosis. When the EGFP-tagged RhoB protein was expressed in HEK293 cells, the Daam1 and RhoB proteins colocalized in the vesicles and at the cell membrane (Fig. 1D). When RhoB(N19), a dominant-negative mutant RhoB (20), was expressed, the number of endocytic vesicles was decreased. Instead, a large vacuole that was both Daam1- and RhoB-positive was formed (Fig. 1E). These results confirm that Daam1 and RhoB colocalize in the endocytic vesicles even when endocytosis is abnormally disrupted.

The repulsion of cells is controlled by dynamin, a large guanosine triphosphatase that functions during endocytic trafficking of membrane molecules (21). When a dominant-negative mutant of dynamin [Dnm1(K44A)], defective in GTP binding and hydrolysis (21), was expressed, Daam1 was not distributed in the vesicles, but rather localized at the cell membrane (yellow arrows, Fig. 1F). Daam1 was present in the vesicles of a cell not expressing Dnm1(K44A) (yellow arrowheads, Fig. 1F). These observations indicate that Daam1 colocalizes in the endocytic vesicles with dynamin. In addition, Daam1 interacts with RhoA (8), RhoB, and Dnm1 (data not shown). These data again support the involvement of Daam1 in the trafficking of the endocytic vesicle. Localization of overexpressed Myc-tagged Daam1 was also confirmed (SI Fig. 6).

EphB1 Interacts with Daam1.

Ephrins and Eph receptors play a pivotal role in the regulation of cell migration, with the endocytosis of the EphB/EphrinB complex being a key event for termination of cell–cell adhesion and subsequent repulsive movement (17, 18). It has been shown that EphB receptors interact with Dvl2 in the Xenopus eye (22). Thus, it is likely that EphB receptors are directly involved in endocytosis in which Daam1 and Dvl2 participate. To confirm this possibility, we performed an immunoprecipitation assay using HEK293 cells. In the absence of EphrinB1-FC stimulation, HA-tagged EphB2 and Myc-Daam1 proteins associated weakly, whereas most of EphB2 protein coprecipitated along with Daam1 from cells stimulated by EphrinB1-FC (Fig. 2A). This indicates that ligand-stimulated EphB2 preferentially associates with Daam1. The same result was obtained when EphB1 was used (data not shown). Finally, we confirmed that the N-terminal of Daam1 interacted with the C-terminal domain of EphB1 (SI Fig. 7).

Fig. 2.

Association of phosphorylated EphB2 with Daam1 and Wnt signaling. (A) HA-tagged EphB2 and Myc-tagged Daam1 were coexpressed in HEK293 cells. When EphrinB1-FC was added, Daam1 and EphB2 associated, resulting in coprecipitation with Daam1 by an anti-Myc tag antibody. (B) The EphB2 proteins associated with Daam1 were phosphorylated when coprecipitated with Daam1 and detected with an anti-phospho-tyrosine antibody (lane 1). In contrast, phosphorylation of EphB2 molecules associated with Dvl2 was low (lane 2). In the presence of Daam1 and Wnt1, phosphorylation of EphB2 associated with Daam1 was enhanced (lane 3) compared with that of Dvl2-associated EphB2 stimulated by Wnt3a (lane 4).

Noncanonical Wnt Signal Induces Association of Phosphorylated EphB and Daam1.

In HEK293 cells, the EphB2 molecules that coprecipitated with Daam1 were phosphorylated, as evidenced by anti-phospho-tyrosine antibody binding (Fig. 2B, lane 1). In contrast, the EphB2 proteins that coprecipitated with Dvl2 were phosphorylated at a lower level (Fig. 2B, lane 2). To address the putative effects of Wnt signaling on the phosphorylation of EphB2, we analyzed its phosphorylation level in cells stimulated by either Wnt1 or Wnt3a. Wnt1 activates both the canonical and noncanonical Wnt cascades (8). Nonetheless, canonical signaling, as measured with the TOP-flash reporter plasmid, was not stimulated in Wnt1-stimulated HEK293 cells expressing Daam1. In contrast, Wnt3a activated the TOP-flash reporter activity in Wnt3a-stimulated cells expressing Dvl2 (22) (SI Fig. 8).

EphB2 proteins were extensively phosphorylated in the presence of noncanonical Wnt signaling, compared with those stimulated by the canonical Wnt pathway (Fig. 2B, lanes 3 and 4). These data suggest that the noncanonical Wnt signal induces the preferential association of Daam1 and phosphorylated EphB. Consistent with this, the nonphosphorylated mutant of EphB1 (K631F/K660F) does not bind to Daam1 (SI Fig. 7).

Disruption of the Noncanonical Wnt or the EphB Signaling Cascades Resulted in a Phenotype Consistent with CE.

To explore the Wnt and EphB signaling cascades further, we performed loss-of-function experiments with zebrafish embryos. Injection of mRNA encoding zNDaam1a resulted in a short body axis, a kinked tail, and cyclopia (Fig. 3B). NDaam1, lacking its C-terminal half, acted as a dominant-negative form (8). The same phenotype was induced by injection of an mRNA encoding Xdd1, a mutant Dvl2 lacking the C-terminal part of its PDZ domain (data not shown). Injection of another mRNA encoding a soluble form of EphB1 comprising only its extracellular domain again resulted in a similar phenotype (Fig. 3C) (12, 14). The phenotypes of Daam1a- and EphB4-morphants were milder than those produced by the zNDaam1a and soluble EphB1 injection experiments (SI Fig. 9). These results may be explained by the redundancy of the Daam and EphB genes. Indeed, not only Daam1b, Daam2, EphB2, and EphB3 but also EphA4b and EphA3 are expressed in the notochord (ZFIN web). Injection of Dnm1(K44A) mRNA induced a similar phenotype (Fig. 3D).

Fig. 3.

Disruption of the noncanonical Wnt pathway or the EphB signaling cascade induces the same CE phenotype. (A–D) Morphology [72 hours postfertilization (hpf)] of the wild-type embryo (A), embryo injected with zebrafish NDaam1a mRNA (B), mRNA encoding a soluble form of mouse EphB1 (C), and mRNA encoding a K44A mutant of zebrafish Dnm1 (D). These embryos share a short and bent body axis (red arrowheads), as well as cyclopia (blue arrowheads). (E) EGFP protein was expressed in the notochord cells to visualize their shape. In the wild-type embryo, the notochord cells elongate to show the intercalation movement. When zNDaam1 was expressed, the notochord cells became round and did not develop a polarized shape. Dorsal views at 11 hpf are shown. Rostral to the left, caudal to the right. (F) At 14 hpf, myoD is expressed in the somites and the adaxial cells in the wild type. (G) Injection of zNDaam1 mRNA resulted in a short body axis with compressed somites. (H and I) Injection of mRNA encoding the soluble form of mouse EphB1 (H) or the dominant-negative dynamin1 [Dnm1(K44A)] (I) induced a similar phenotype. (J) Injection of Dnm1-MO resulted in similar but more severe defects. (K) Coinjection of EphB4-MO and EphB2-MO induced severe defects. The notochord was short, broader, and bent. Pictures shown in F–K are shown at the same magnification. (L) Injection of mRNA encoding zNDaam1 or the soluble form of EphB1 induced the abnormalities of CE movement in 19.2% or 11.6% of injected embryos, respectively, as judged by expression of myoD at 14 hpf. Coinjection of these mRNAs induced the same abnormality in 49.4% of embryos. (M) Injection of a soluble mEphB1-FC mRNA induced the CE defect in 29.8% of injected embryos. Coinjection with zCDaam1a mRNA rescued this defect, and the CE abnormality was observed in only 11.4% of embryos. Injection of zCDaam1 mRNA alone induced the defect in 9.0% of embryos.

To visualize notochord cell shape, EGFP protein was expressed with the promoter of the floating head (flh) gene (22) (Fig. 3E). In the wild-type embryo, notochord cells elongated to show the CE cell movement. In contrast, when the flh promoter–EGFP construct was coinjected with zNDaam1a mRNA, cells became round and failed to show either polarized cell shape or the typical CE movement. This indicates that inactivation of Daam1a results in an abnormal cell shape and a loss of polarized cell movement.

Next, we examined the expression patterns of myoD in embryos injected with the mutant mRNAs and morpholino oligonucleotides (MOs). This gene is normally expressed in adaxial cells and segmented somites (Fig. 3F). In the zNDaam1-injected embryo, expression of myoD in the somites became compressed (Fig. 3G). Expression of the soluble form of EphB1 and the Dnm1 mutant K44A resulted in the same phenotype (Fig. 3 H and I). Injection of MO against Dnm1 also produced a similar short and bent notochord (Fig. 3J). Coinjection of MOs against EphB2 and EphB4, both of which are expressed in the notochord, also produced similar defects with changes in gene expression. These results indicate that the abnormal CE movement results from the disruption of Daam1 and EphB functions (additional supporting data are shown in SI Figs. 9 and 10).

To determine whether a functional relationship existed between Daam1 and EphB1, we coexpressed NDaam1 and the soluble form of EphB1 in embryos. Coexpression of these two proteins produced defects in 49.4% of injected embryos (n = 97), whereas the defects were observed at lower rates when either NDaam1 (19.2%, n = 52) or the soluble EphB1 (11.6%, n = 42) were expressed individually (Fig. 3L). The relationship was also explored with a rescue experiment using a constitutively active form of Daam1 (zCDaam1) (Fig. 3M). Misexpression of the soluble form of EphB1 resulted in defects in 28.8% of embryos (n = 59). Coexpression with zCDaam1 rescued the abnormalities in a significant number (11.4%, n = 44). Injection of CDaam1 mRNA alone induced slight defects in only 9.0% of embryos (n = 67).

Dynamic Subcellular Localization of Daam1 in the Zebrafish Notochord.

To further confirm the roles of Daam1 in the CE movement, we observed the dynamic subcellular localization of EGFP-fused Daam1 proteins, which were expressed from an EGFP-Daam1 fusion gene driven by the Flh promoter (22) in the zebrafish notochord. EGFP-Daam1 proteins were detected in some vesicles in the cortex of cells at the early intercalation stages (Fig. 4A). In addition to this, a bright accumulation of signals was observed in the center of the cells. Although we failed to stain this organelle with an antibody, we suspected it was the Golgi apparatus, because colocalization of Daam1 with the Golgi apparatus was confirmed in HEK293 cells in vitro (data not shown). Ten hours after injection of this construct, GFP-positive vesicle-like dots actively internalized in the caudal half of the cells (Fig. 4B). As the CE movement proceeded, the cells became compressed, and GFP signals accumulated in fibers elongating laterally near the caudal surface (Fig. 4C). These findings indicate that the Daam1 protein changes its subcellular localization dynamically. We also found that the subcellular localization of Daam1 was asymmetrical, with the caudal half active in endocytosis (Fig. 4A and SI Movies 1 and 2).

Fig. 4.

Subcellular localization of Daam1 in notochord cells. (A) Serial pictures of a notochord cell taken at 20-min intervals. EGFP-Daam1-positive vesicles (red arrows) moved from the caudal cell surface to the center (green arrowhead). Daam1-positive fibers were formed on the caudal side of the cell (blue line). (B) Serial pictures of another notochord cell taken at 4-min intervals. EGFP-Daam1-positive vesicles (red arrows) moved from the caudal cell surface to the center (green arrowhead). (C) Serial pictures of the cell shown in (A). GFP-tagged Daam1 accumulated in the fibers near the caudal cell surface (blue bars). (D and E) Subcellular localization of GFP-tagged Daam1 is shown in green. At this stage, Daam1 colocalized with F-actin in the cell cortex and the fibers formed near the caudal surface (yellow arrowheads). Note that the colocalization is more evident at the lateral ends that make tight junctions with surrounding tissues (blue arrowheads in E). (F) When wnt11 MO was injected, the notochord cells did not elongate but, rather, formed small spiky protrusions where Daam1 colocalized. Note that Daam1 did not localize in the endocytic vesicles or the fibers in the cell cortex and, instead, stayed at the cell membrane.

Cytoskeleton and Daam1.

Next, we explored the nature of the Daam1-positive fibers formed in the later stages. As the CE cell movement proceeded, cells became compressed. In these elongating notochord cells, phalloidin-positive F-actin structures were evident at 12 hpf. In these stages, Daam1 colocalized with the cytoskeleton formed at the cell cortex (Fig. 4D) and the lateral ends (Fig. 4E, blue arrowheads). When the two images were merged, colocalization of these two distinct components was evident (Fig. 4D). This dynamic change of localization implies that Daam1-mediated Eph endocytosis releases the adhesive properties of the caudal cell surface of the notochord cells. In the next step, Daam1 is involved in the elongation of cell shape, associating with F-actin that aligns along the direction of cell extension and contributes to tight adhesive connections with surrounding tissues.

When MO against wnt11, which plays important roles during the CE cell movement (23), was injected, the notochord cells changed their cell shape, forming small protrusions where Daam1 colocalized (Fig. 4E). In these cells, Daam1 did not localize in the endocytic vesicles or the fibers in the cell cortex. This indicates that the subcellular localization depends on the noncanonical Wnt cascade.

Discussion

Based on our data, we propose the following model to describe the role of EphB and Dvl2 in noncanonical Wnt signaling (Fig. 5). EphB receptor tyrosine kinases are phosphorylated after clustering triggered by binding to their cognate ligands EphrinBs. In the absence of noncanonical Wnt signaling or in the presence of canonical Wnt signaling, the EphB receptors do not make a complex with Daam1. In the presence of noncanonical Wnt signaling, phosphorylated EphB molecules are recruited to make a ternary complex with Dvl2 and Daam1. This complex is transported to the endocytic vesicles, whose formation depends on dynamin. The subsequent endocytic removal of EphB molecules induces cell repulsion, followed by the initiation of CE cell movement (Fig. 5). In CE cell movement, dissociation and elongation of cells are mediated through the EphB/Dvl2/Daam1 ternary complex and the Dvl2/Daam1/ROCK cascade (8), respectively. The two cascades are coordinated to achieve the orchestrated morphogenetic movement of cells (Fig. 5).

Fig. 5.

Noncanonical Wnt signaling and Daam1-mediated endocytosis of EphB. On binding to EphrinBs, EphB receptor molecules are phosphorylated and recruited to make a ternary complex with Dvl2 and Daam1. This complex is incorporated into the endocytic vesicles in a dynamin-dependent manner. Active noncanonical Wnt signaling is necessary for the formation of this ternary complex. After the endocytic removal of EphB, cells dissociate to enter the next steps of CE cell movement, migration and elongation. In the absence of the noncanonical Wnt signal or in the presence of the canonical Wnt signal, EphB receptors associate with Dvl2 alone to form a nonfunctional complex that remains on the cell surface. This likely takes place after dephosphorylation by an unknown phophatase activity. Consequently, cells do not dissociate. In CE movement, dissociation and elongation of cells, mediated through the EphB/Dvl2/Daam1 complex and the Dvl2/Daam1/ROCK cascade, respectively, are tightly coupled to achieve orchestrated morphogenetic movement.

Wnt11 regulates the E-cadherin-mediated cohesion of the mesoendoderm progenitors (9). In this process, Rab5, one of the key regulators of early endocytosis and intracellular trafficking, mediates Wnt11 activity. The dynamic modulation of cell cohesion through the interaction of Wnt11 and Rab5 as well as the trafficking of E-cadherin is pivotal for the migration of many cell types. The notochord, however, is less sensitive to changes in Rac5 activity (9). Our data reveal that the noncanonical Wnt cascade regulates this process through endocytosis in a distinctive way, namely the switching of EphB-mediated repulsion and adhesion of cells. Furthermore, our data identifies the direct molecular links, highlighting Daam1, Dvl2, and dynamin as central players that commence the dynamic behaviors of the notochord cells.

In axonal pathfinding, EphBs and Wnts have been identified as guidance molecules for spinal cord commissural axons and corticospinal axons (24, 25). Recently, Wnt3, a classical morphogen, has been identified as a guidance factor, counterbalancing EphB/EphrinB1 activity (26). Although the precise molecular nature of their interaction has not been elucidated, it is evident that a functional link between EphB/EphrinB signaling and the Wnt cascades exists. Our results indicate that Daam1, which is also expressed in the nervous tissues (27), might be involved in this phenomenon, acting at the intersection of two different cascades to organize the correct patterning and pathfinding. It is likely that Daam1 and the noncanonical Wnt pathway may also have a similar involvement in the progression of tumors such as colorectal cancer (28).

Materials and Methods

Antibody and Reagents.

Antibodies against Myc-, Flag-, and HA-tags were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Sigma (St. Louis, MO), and Rockland (Gilbertsville, PA), respectively. Anti-phospho-tyrosine antibody was purchased from Zymed (San Francisco, CA). FM1–43FX and Alexa Fluor 594-conjugated Transferrin (50 μg/ml; Invitrogen, Carlsbad, CA) was used for the incorporation assay as described (29). FM1–43FZ is a derivative of FM4–64 suitable for immunohistochemistry. The anti-Daam1 antibody was prepared by immunizing rabbits with a synthesized peptide corresponding to the 1,051- to 1,078-aa region of the human Daam1 protein. We also used another anti-Daam1 antibody (M05), which was obtained from Abnova (Taipei, Taiwan).

cDNA Probes and in Situ Hybridization.

Zebrafish Daam1a cDNA (IMAGp964D2115Q) was purchased from the RZPD (Berlin, Germany) clone bank (www.rzpd.de). HA-tagged zebrafish NDaam1a (1–417-aa region) and Myc-tagged zebrafish CDaam1a (439–1,079-aa region) were constructed from this cDNA by using RT-PCR techniques. Myc-tagged mouse full-length Daam1 was the kind gift of T. Yamaguchi (National Cancer Institute, Frederick, MD). GFP-RhoB and GFP-RhoB(N19) plasmids were kindly provided by C. Rondanino and G. Apodaca (University of Pittsburgh, Pittsburgh, PA) (20). Mouse EphBs and EphrinBs were kindly provided by M. Tanaka (National Cancer Center Research Institute, Tokyo, Japan) (30). Mouse EphB1 and EphB2 cDNAs were obtained by RT-PCR from mouse cDNAs. Mutant forms of EphB1 were constructed with the PCR techniques and appropriate primers. Mutants of mouse Dvl2 (FL-Dvl2, MT-Dvl2-DIX, -PDZ, -DEP) and Xdd1 were kindly gifted by R. Habas (University of Medicine and Dentistry of New Jersey, Newark, NJ) (8) and S. Y. Sokol (Mount Sinai School of Medicine, New York, NY) (4), respectively. Zebrafish dynamin1 cDNA (IMAGp998N1014600Q) was obtained from the RZPD, and their mutants were constructed by PCR-based techniques. Maintenance of the fish colony and whole-mount in situ hybridization were performed as described (31). pFlh-EGFP was a kind gift of M. E. Halpern (Carnegie Institute, Baltimore, MD) (22).

Immunocytochemistry, Immunoprecipitation, and Western Blotting.

Transfection, immunocytochemistry, and Western blotting of HEK293 cells were performed as described (32). For phalloidin staining, embryos were fixed overnight in 4% paraformaldehyde–PBS solution. After fixation, embryos were lysed for 2 h with PBDT (PBS/1%BSA/0.1% Triton X-100/1% DMSO). Embryos were incubated with primary antibodies overnight and then washed and incubated with a second antibody conjugated with Alexa Fluor 594 or Alexa Fluor 594-conjugated Phalloidin for actin staining. For nuclear staining, cells were incubated with DAPI for 10 min.

MOs or mRNA Injection into Zebrafish Eggs.

MOs were designed and synthesized by Gene Tools (Philomath, OR). Sequences were as follows: zDaam1a: 5′-GGCTCAAGGGATAATGGGAACGAGG; zDaam1a: 5′-GGCTCAAGGGATAATGGGAACG-AGG; zDaam1b: 5′-AGCTATGACCCCCTCTCAAAATGGC; zDaam2: 5′-AGCTGGCAATGCGAACATGGCTTCC; zEphb2: 5′-TGCAGTCGCCGTCGTGGAGTCCATC; zEphb4: 5′-AGCTCCATCGCGGAATCACGAGTGT; zDnm1: 5′-GTAATCAAAATTGTCCTACCGTCAG; and zWnt11: GAAAGTTCCTGTATTCTGTCATGTC. Control MOs had four base mismatches. Oligonucleotides were solubilized in Danieau solution. For in vitro synthesis of mRNAs, linearized plasmids were used as templates in the RiboMAX Large Scale RNA Production system (Promega, Madison, WI). Synthesized mRNAs were purified after treatment with RNase-free DNase and dissolved in nuclease-free water. Injection of MOs or mRNAs and in situ hybridization were performed as described (31).

Abbreviations

CE

convergent extension

MO

morpholino oligonucleotide

PCP

planar cell polarity.