ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 000, 000–000 doi:10.1242/jcs.168989

CORRECTION Loss of Porcupine impairs convergent extension during gastrulation in zebrafish

Qiuhong Chen, Ritsuko Takada and Shinji Takada

There was an error published in J. Cell Sci. 125, 2224–2234.

In Fig. 2, panel R was inadvertently duplicated and displayed as panel S. Panel S has been replaced with the correct panel in the figure reprinted below. There are no changes to the figure legend, which is accurate. This error does not affect the conclusions of the study.

We apologise to the readers for any confusion that this error might have caused.

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Journal of Cell Science JCS168989.3d 22/1/15 10:57:22 The Charlesworth Group, Wakefield +44(0)1924 369598 - Rev 9.0.225/W (Oct 13 2006) 2224 Research Article Loss of Porcupine impairs convergent extension during gastrulation in zebrafish

Qiuhong Chen1, Ritsuko Takada1 and Shinji Takada1,2,* 1Okazaki Institute for Integrative Bioscience and National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan 2Department of Molecular Biomechanics, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi 444-8787, Japan *Author for correspondence ([email protected])

Accepted 9 January 2012 Journal of Cell Science 125, 2224–2234 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.098368

Summary Porcupine (Porcn), an O-acyltransferase located in the endoplasmic reticulum (ER), is required for lipidation of Wnt to enable their trafficking from the ER in mammalian cell culture. However, it is unclear whether Porcn is required for trafficking of all members of the Wnt family. In this study, we investigated the function of Porcn in zebrafish embryos. We identified two zebrafish homologs of porcupine, porcn and porcupine-like (porcn-l). Zebrafish porcn, but not porcn-l, restores secretion of Wnt proteins in porcn-deficient mouse L cells. Morpholino-mediated knockdown of porcn in zebrafish embryos impairs convergence and extension (CE) during gastrulation without changing embryonic patterning. Moreover, porcn interacts genetically with wnt5b and wnt11 in regulating CE. By contrast, porcn-deficient embryos do not exhibit phenotypes caused by failure in canonical Wnt signaling, which is activated by several Wnt ligands, including Wnt3a. Furthermore, expression of regulated by the canonical is not perturbed in knockdown embryos relative to that in controls. Although the trafficking and lipidation of ectopically expressed zebrafish Wnt5b and mouse Wnt5a are impaired in porcn-deficient embryos, those of ectopically expressed Wnt3a are less or not affected. In addition, the secretion of Wnt5a is inhibited by less Porcn inhibitor than that of Wnt3a in HEK293T cells. Thus, a decrease of Porcn activity does not equivalently affect trafficking and lipidation of different Wnt proteins in zebrafish embryos and in cultured mammalian cells.

Key words: Wnt, Secretion, Lipidation, Porcupine, Convergent extension, Zebrafish

Introduction polarity , similar to wingless,inDrosophila and is

Journal of Cell Science Wnt proteins constitute a large family of secreted signal proteins evolutionally conserved from worms to mammals (Siegfried et al., that play roles in embryogenesis and tissue homeostasis. They 1994; Kadowaki et al., 1997; Thorpe et al., 1997; Rocheleau et al., require specific machinery for their secretion; for example, 1997; Tanaka et al., 2000; Hofmann, 2000). Porcn proteins localize Wntless/Evi/Sprinter, a seven-pass membrane , is required to the ER and are required for Wnt trafficking from the ER in for intracellular transport of Wnt proteins (Ba¨nziger et al., 2006; mammalian cells and probably in Drosophila embryos. Porcn Bartscherer et al., 2006; Goodman et al., 2006). After release of overexpression promotes lipid modification of Wnt1 and Wnt3a Wnt proteins into the extracellular space, Wntless/Evi/Sprinter is and causes a steepened Wnt gradient in the chick neural tube, recycled from the plasma membrane back to the Golgi by the suggesting it has an important function in vertebrate embryogenesis retromer complex (Prasad and Clark, 2006; Coudreuse et al., (Galli et al., 2007). In addition to the palmitoleoylation, Wnt 2006; Franch-Marro et al., 2008; Port et al., 2008; Belenkaya proteins are modified with another fatty acid, palmitate (C16:0), at et al., 2008; Yang et al., 2008; Pan et al., 2008). Even before the a cysteine residue (Willert et al., 2003), but it is uncertain whether association with Wntless/Evi/Sprinter, Wnt proteins are modified Porcn is required for this lipidation. with a fatty acid, palmitoleate (C16:1), at a serine residue in the Until now, the role of vertebrate Porcn in vivo has been endoplasmic reticulum (ER) (Takada et al., 2006; Coombs et al., examined in mice and humans. A systemic screening approach 2010). The amino acid sequence around this serine residue is of mouse X- genes using gene-trap technologies highly conserved in the Wnt family, and palmitoleoylation might revealed that Porcn mutant mouse embryos fail to complete therefore occur on most Wnt proteins. The serine residue is gastrulation, but remain in an epiblast-like state, similar to Wnt3 required for secretion of Wnt proteins from mammalian culture mutants (Cox et al., 2010; Biechele et al., 2011). However, cells and in vertebrate embryos (Takada et al., 2006; Galli et al., mouse Porcn is not expressed ubiquitously (Biechele et al., 2011) 2007). However, in the case of Wingless, the Drosophila and it is uncertain whether Porcn is similarly involved in all of ortholog of Wnt1, this serine is not required for secretion from secretion of Wnt proteins. In humans, mutations in PORCN cause Drosophila S2 cells, but for the function of the protein (Franch- focal dermal hypoplasia (FDH), which is an X-linked dominant Marro et al., 2008). Thus, the role of the palmitoleoylation might disorder characterized by patchy hypoplastic skin and differ for different Wnt proteins or in different cellular contexts. malformations of a wide variety of tissues (Wang et al., 2007; Palmitoleoylation requires an enzyme, Porcupine (Porcn), a Grzeschik et al., 2007). However, it is unclear whether all of the member of the family of membrane-bound O-acyltransferases FDH phenotypes are actually caused by abnormalities in Wnt (MBOATs). The Porcn gene was first identified as a segment signaling. And it remains elusive whether vertebrate porcn is Role of Porcn in Wnt secretion 2225

actually required for all Wnt signaling in vivo, or whether such a Porcn does not equivalently affect the trafficking and lipidation requirement is equivalent for all members of the Wnt family. of different Wnt proteins in these embryos. To investigate these questions, we used zebrafish as a model system and examined the effects of defects in Porcn function on Cellular function and developmental expression of Wnt signals in early embryonic stages because the roles of Wnt zebrafish Porcn signals have been precisely characterized in early zebrafish We screened a zebrafish genome database, found two genes embryos. Porcn-deficient embryos specifically exhibited defects encoding proteins structurally similar to Porcn in other species in convergent-extension (CE) movement, which requires non- and tentatively designated them porcn1 and porcn2 (Fig. 1A). To canonical Wnt signaling. Surprisingly, these embryos showed no assess whether these two zebrafish porcn homologs actually sign of reduction in canonical Wnt signaling. Consistent with this encode proteins with Porcn function, we tested their abilities to array of phenotypes, trafficking of zebrafish Wnt5b and mouse restore Wnt3a secretion in a mouse L cell clone in which Wnt5a, but not zebrafish and mouse Wnt3a, was specifically exogenous mouse Wnt3a had been expressed and endogenous impaired in Porcn-deficient embryos. Thus, Porcn is required for Porcn activity had been stably reduced by siRNA (Takada et al., the proper function and trafficking of at least one Wnt protein 2006). Because we found that Wnt5a also required porcn for during early developmental stages of zebrafish, but a decrease of its secretion from L cells (supplementary material Fig. S1), Journal of Cell Science

Fig. 1. Function and expression of zebrafish homologs of Porcupine. (A) Phylogenetic analysis of two porcn-related genes in zebrafish with Porcn genes in other species. The tree was constructed on the basis of percentage identity at the amino acid level. The GenBank accession numbers for the sequences listed above are the following: Drosophila (#AAB18992), Caenorhabditis elegans (#AAC47728), Homo sapiens (NP_982301), Mus musculus (#NP_076127), Xenopus tropicalis (#NP_989365) and Bos taurus (#NP_001094678). The zebrafish Porcupine protein sequences are based on the sequence of our clones. (B,C) Myc- tagged Porcn1 (Porcn) but not Porcn2 (Porcn-l) restored the secretion of both Wnt3a and Wnt5a from Porcn-deficient mouse L cells. Porcn-deficient L cells expressing either Wnt3a (B) or Wnt5a (C) were transiently transfected with either control DNA, Myc-tagged zebrafish porcn1 or Myc-tagged zebrafish porcn2,as indicated. The amount of Wnt3a or Wnt5a in the culture medium (secreted Wnts) and that in the cell lysate were examined by western blotting. Expression of Porcn1 and Porcn2 proteins was also monitored. (D–N) Expression of porcn1 (porcn) (D–I) or porcn2 (porcn-l) (J–N) was assessed at the eight-cell stage (D,J), at the shield stage (E,K), at the tail bud stage (F,L), at the ten-somite stage (M), at the 14-somite stage (G), and at 24 h.p.f. (H,I,N). The transverse section through the trunk of embryos stained with porcn1 (porcn) probe indicates expression in the ventral spinal cord (I). All photographs, except that in I, were taken from a lateral view. In E,F,K,L, anterior is top and dorsal is right. n, notochord. 2226 Journal of Cell Science 125 (9)

restoration of Wnt5a secretion was also examined. The transient Reduction in Porcn expression results in morphological expression of Myc-tagged Porcn1, but not that of Porcn2, defects in axis elongation efficiently restored the secretion of both Wnt proteins Next, we examined the role of zebrafish porcn during (Fig. 1B,C), indicating that Porcn1, but not Porcn2, had a embryogenesis by using antisense morpholino-mediated function comparable to that of mouse Porcn in Wnt secretion. knockdown. An antisense morpholino oligo against porcn Thus, we considered that porcn1 is the functional homolog of (porcn MO) was designed to target the sequence around the mouse Porcn, and so we refer to porcn1 and porcn2 as porcn and first methionine codon in porcn. As expected, porcn MO, but not porcupine-like ( porcn-l), respectively, hereafter. a control MO, which was generated by changing five bases in the Whole-mount in situ hybridization analysis revealed that both porcn MO (5 mis control MO), successfully blocked the porcn and porcn-l transcripts were expressed during early expression of porcn–GFP (supplementary material Fig. S3A,B). embryogenesis. Both mRNAs were maternally deposited Injection of 1.5 ng porcn MO into embryos caused a defect in (Fig. 1D,J). Weak and ubiquitous expression of porcn could be axis elongation, a phenotype typically observed in embryos that observed from the eight-cell stage until the tail-bud stage have abnormal CE movement, resulting from a disturbance in (Fig. 1D–F). During mid-somitogenesis, the ventral spinal cord non-canonical Wnt signaling (Jessen et al., 2002; Carreira- and the ventral hindbrain as well as the ventral diencephalons Barbosa et al., 2003; Topczewski et al., 2001; Kilian et al., 2003; were enriched with porcn RNA (Fig. 1G). By 24 hours post Westfall et al., 2003). The morphants specifically exhibited fertilization (h.p.f.), expression of porcn was clearly observed in shortened body axes, laterally expanded notochords and the telencephalon region in addition to ventral regions in the compressed somites (Fig. 2A,B). These defects were apparent neural tube (Fig. 1H,I). By contrast, we note that no obvious in 24 h.p.f. morphant embryos, which displayed a short expression of porcn was observed in the dorsal neural tube, nodulating notochord and neural tube (Fig. 2C,D). Embryos where several wnt genes, including wnt1 and wnt3a are injected with a higher dosage of porcn MO exhibited severer expressed. Expression of porcn-l was ubiquitous, stronger in phenotypes, including a greatly shortened trunk and tail, as well brain regions until 24 h.p.f. (Fig. 1J–N). However, because four as split body axis or unfinished epiboly (Fig. 2E). A significant splicing variants of Porcn have been identified in mouse and number of dead cells was observed in the head and trunk regions chick, we also examined which variants were expressed in of the morphants. However, such cell death was not observed in zebrafish embryos, and detected two of them corresponding to embryos co-injected with the porcn MO and a p53 MO (Fig. 2F), mouse PorcnC and PorcnD (supplementary material Fig. S2). which is known to reduce apoptosis caused by non-specific Journal of Cell Science

Fig. 2. Abnormalities of zebrafish embryos injected with porcn MO or porcn mRNA. (A,B) Dorsal views of seven- somite-stage embryos and (C–E) lateral views of 24 h.p.f. embryos injected with 5mis control MO (C) or porcn MO (D). A porcn MO-injected embryo with severe defects (E) is also indicated. (F) Embryos injected with porcn MO and p53 MO exhibited short body axis, but reduced cell death in the brain. (G) A lateral view of a porcn mRNA-injected embryo at 24 h.p.f. stage. (H–W) Molecular marker analysis indicates that loss or gain of porcn function disrupts CE without changing dorsal–ventral patterning. Embryos injected with either 1.5 ng of 5mis control MO (H,J,M,O,R,U), 1.5 ng of porcn MO (I,K,N,P,S,V) or 100 pg of porcn mRNA (L,Q,T,W) were stained with ntl and hgg1 (H,I), flh and papc (M,N), myoD (J–L,O–Q), chd (R–T) or bmp2b (U–W) probes. Dorsal views of embryos are shown at the tailbud stage (H–N) and eight- somite stage (O–Q), animal views of embryos are shown at the shield stage with dorsal to the right (R–W). Staining patterns in embryos injected with 5mis control MO were identical to those in normal embryos (data not shown). Role of Porcn in Wnt secretion 2227

effects of MO treatment (Robu et al., 2007); therefore, these cell in porcn-deficient embryos at the four-somite stage (Fig. 3A–D). deaths might not be specifically caused by knockdown of porcn. Cells in the control embryos were elongated having a length-to- Similar, but less severe, phenotypes were observed by injecting width ratio (LWR) of 5.160.5 and aligned mediolaterally at an two other non-overlapping morpholinos targeting the 59UTR of average angle of 4.862.4˚ relative to a line perpendicular to the porcn (porcn MO9 and porcn MO0) (supplementary material Fig. A–P embryonic axis (Fig. 3A,C,D). By contrast, cells in the S4A–D). Moreover, co-injection of two of the three porcn MOs porcn-deficient embryos were rounder with an average LWR of (either porcn MO with MO9 or MO9 with MO0) at low dosage 2.860.79 and aligned more randomly at a greater angle resulted in a synergistic increase in the percentage, 91.4% and (11.963˚) (Fig. 3B–D). 81.44% respectively, of embryos with body elongation defects, Furthermore, we examined movement of porcn-defective cells whereas either MO alone caused the phenotype in less than 5% of by transplantation of biotin-labeled mesoderm cells from control embryos at this dosage (supplementary material Fig. S4E–H). or porcn-defective donor embryos into the margin adjacent or Finally, the defect in axis elongation was rescued, although not lateral to the shield of control or porcn-defective host embryos completely, by injection of mouse Porcn mRNA, which did (Fig. 3E–K). Whereas transplanted cells from control embryos not contain the sequence targeted by zebrafish porcn MO moved anteriorly and formed strings of cells in their control hosts (supplementary material Fig. S3C–G). These results indicate (Fig. 3G,J,L), porcn-defective cells moved in a more spreading that defective elongation of the body axis caused by all these fashion in porcn-defective hosts (Fig. 3H,K). Furthermore, porcn MOs was due to specific interference with the function of porcn-defective cells moved in a similar manner to control Porcn. cells when transplanted into control host (Fig. 3I,L). Thus, cells We also designed two non-overlapping MOs, porcn-l MO and in porcn-defective embryos did not move properly during CE porcn-l MO9, which targeted the porcn-l translation start and the movement, and the porcn deficiency affected cells in a cell non- 59UTR sequence, respectively, to investigate the role of porcn-l autonomous manner. during zebrafish embryogenesis. Although porcn-l MO blocked As well as loss of function, gain of function in components the expression of porcn-l–GFP (supplementary material Fig. of the non-canonical Wnt pathway can cause abnormal CE S5A,B), the injection of either MO alone or the combination of phenotype (Kilian et al., 2003; Ungar et al., 1995; Matsui et al., both MOs did not cause an obvious abnormal phenotype, even 2005); therefore, we examined the effect of overexpression of when high doses of MOs were injected (supplementary material Porcn on CE movement. Microinjection of 75–150 pg porcn Fig. S5C; data not shown). We also confirmed that porcn-l MO mRNA into wild-type embryos caused defects in axial elongation did not significantly influence the effect of porcn MOs when (Fig. 2G) and in gene expression (Fig. 2L,Q,T,W) similar to these MOs were injected at various doses (supplementary those of porcn morphants. Taken together, porcn appeared to be material Fig. S5D–G). Furthermore, our evidence to show that required for proper CE movement in a cell non-autonomous porcn MO did not affect the expression of porcn-l GFP manner. (supplementary material Fig. S3A) excluded the possibility that the defects caused by porcn MO were partly due to a cross Genetic interaction of porcn with wnt5b and wnt11 in the targeting of the related gene, porcn-l. Therefore, we considered CE phenotype

Journal of Cell Science that porcn-l did not play a significant role in early zebrafish To determine whether porcn function was associated with Wnt development and that there was no obvious redundancy in function during CE movement, we examined genetic interactions function between porcn and porcn-l. between porcn and wnt5b (pipetail) and wnt11 (silberblick), both of which are known to be required for CE (Kilian et al., 2003; Reduction in Porcn expression disrupts the convergent Heisenberg et al., 2000). Whereas injection of low concentrations and extension movements in a cell non-autonomous of either the porcn MO or wnt5b MO led to no significant defects, manner co-injection of a low concentration of both morpholinos together To precisely examine whether CE movement was impaired in yielded CE defects in 80% of the injected embryos (Fig. 4A–I), porcn-deficient embryos, we analyzed the expression of several indicating that genetic interactions between these two genes mesoderm markers. The expression of ntl was used to mark the could lead to the CE phenotype. Similarly, CE defects were notochord in tailbud-stage embryos. The notochord was observed when low concentrations of porcn MO and wnt11 MO apparently shorter along the anterior–posterior (A–P) axis and were co-injected (Fig. 4J–R). Thus, we concluded that porcn mediolaterally expanded in embryos injected with porcn MO plays a role in concert with wnt5b and wnt11 in mediating CE than in the control embryos (Fig. 2H,I). In addition, broader and movements. shorter paraxial mesoderm in the tailbud stage of porcn MO- injected embryos was confirmed by myoD (Fig. 2J,K,O,P) porcn-deficient embryos exhibit no obvious abnormality in and papc (Fig. 2M,N) expression. By contrast, the expression developmental processes regulated by canonical Wnt of a dorsal marker, chd, and a ventral marker, bmp2b, in early signaling gastrula were apparently normal in porcn-deficient embryos Although porcn deficiency caused severe defect in CE (Fig. 2R,S,U,V), indicating that the dorsal–ventral patterning in movement, it did not result in the phenotypes caused by these embryos was unaffected. These expression patterns, as well deficits in several other Wnt genes. For instance, whereas a as morphological abnormalities, are characteristic of mutants deficit of wnt8 causes anteriorization of neural tissues, as such as pipetail/wnt5b, silberblick/wnt11, knypek and trilobite,in indicated by expanded expression of six3, an eye primordial which the CE movement is impaired (Westfall et al., 2003; Jessen marker, and an anterior shift of the expression of eng2,a et al., 2002; Topczewski et al., 2001). midbrain marker (Lekven et al., 2001; Kim et al., 2002), the We also observed an abnormality in cell intercalation during expression of these marker genes was not altered in porcn- CE when analyzing the shape and orientation of notochord cells deficient embryos (Fig. 5A–C,G–I). Defects in the expression of 2228 Journal of Cell Science 125 (9)

Fig. 3. Abnormal intercalation and movement of porcn-defective cells during CE. (A–D) Cell intercalation of embryos injected with 5mis control MO (A) or porcn MO (B) was examined along with expression of membrane-bound Venus. The shapes of notochord cells of control embryo (A) and porcn-deficient embryo (B) at the four-somite stage are shown. Notochords are indicated by brackets. LWR (length-to-width ratio) of notochord cells (C) and the angle of the long axis of notochord cells relative to the line perpendicular to the embryonic axis (D) at the four-somite stage are represented. Data were analyzed by Student’s t-test. (E–K) Transplantation analysis to examine movement of porcn-defective cells. (E,F) Cell transplantation procedures. Cells transplanted deep into the margin either close to (E) or lateral to (F) the shield are schematically represented. (G–K) Biotin-labeled mesoderm cells from donor embryos were transplanted into host embryos at the shield stage, and the transplanted embryos were fixed at the one-somite stage and stained with papc and pax2.1 probes. Cells derived from embryos injected with 5mis control or porcn MO together with biotin were transplanted deep into the margin close to the shield (G–I) or lateral to the shield (J,K) of host embryos injected with 5mis control or porcn MO. The distance of transplanted cells (G–I) from the embryo midline were measured and represented in (L). First, distances of all transplanted cells from the midline of the embryo were measured and averaged in each embryo. Then, the mean values 6 s.e.m. of three different embryos were summarized in each transplantation and are indicated in each bar. Journal of Cell Science these marker genes were not observed even in embryos co- effects of this deficiency might be limited to specific cells or injected with either of additional porcn-specific MO (porcn MO9) specific stages during development. Alternatively, this deficiency or porcn-l MO with porcn MO (Fig. 5D,E,J,K). Furthermore, the might not affect each member of the Wnt family in the same way. porcn MO did not obviously enhance the phenotypes of wnt8, As a first step to assess these possibilities, we directly compared wnt3a, wnt1 or wnt10b morphants when porcn MO was co- the effect of porcn depletion on the trafficking of several distinct injected with each wnt-specific MO (data not shown). Injection Wnt molecules in their secretory process under the same set of of porcn mRNA at a concentration that induced the CE defect did conditions. Specifically, the trafficking of mouse and zebrafish not cause any obvious abnormality in the expression of the above Wnt3a, both of which can activate the canonical Wnt pathways in marker genes (Fig. 5F,L; data not shown). zebrafish embryos, was examined in epiblast cells of porcn- Because wnt8, wnt3a, wnt1 and wnt10b induce canonical Wnt deficient zebrafish embryos and compared with the trafficking of signaling in many cases, we examined targets of canonical Wnt several Wnt proteins, zebrafish Wnt5b and mouse Wnt5a, that are signaling in porcn-deficient zebrafish embryos during epiboly, known to activate the non-canonical Wnt pathway. We injected when porcn is ubiquitously expressed. Injection of porcn MO did mRNAs encoding EGFP-tagged forms of these Wnt proteins with not reduce the expression of the TOP–dGFP gene (Fig. 5M,N), a porcn MO or a control MO into one-cell-stage embryos. Even reporter of Wnt canonical signaling (Dorsky et al., 2002), nor that with the addition of EGFP tags, Wnt proteins still retained of axin2 (Fig. 5O,P), a target of the canonical Wnt signaling, in biological activity, although this was reduced (supplementary 80% epiboly embryos. Thus, in spite of the CE abnormality, material Fig. S6). In embryos injected with the control MO, Wnt developmental events regulated by canonical Wnt signaling protein was distributed in cell-to-cell junction areas as puncta, appeared normal in porcn-deficient zebrafish embryos at least in indicating that the tagged proteins had been secreted or were in early embryonic stages, suggesting that a deficit of porcn did not the process of secretion, as in the case of authentic Wnt3a affect all the distinct events regulated by different Wnt proteins. observed in Xenopus embryos (Fig. 6A,C,E,G) (Takada et al., 2006). These puncta were not detected in un-injected embryos or Abnormal trafficking of Wnt5b, but not Wnt3a, from porcn- when only secondary antibodies were used to probe the samples, defective zebrafish embryonic cells indicating that the signals were specific (data not shown). One possible explanation for this non-equivalent effect of the Knockdown of porcn resulted in a change in the localization of porcn deficiency on different Wnt-mediated events is that the zebrafish Wnt5b and mouse Wnt5a; most of these Wnt proteins Role of Porcn in Wnt secretion 2229

Fig. 4. porcn acts synergistically with wnt5b or wnt11 in the regulation of CE movement. (A–I) Synergistic interaction of porcn with wnt5b. Live images of embryos injected with 1 ng of 5mis control wnt5b MO (A,C) or wnt5b MO (B,D) along with 0.5 ng of 5mis control porcn MO (A,B), or porcn MO (C,D). (E) The embryos shown in A–D were scored morphologically at 24 h.p.f. for the percentage of CE defects. Three independent experiments were scored and the percentage of normal, mildly or moderately CE defective embryos are indicated. n represents total number of embryos injected for each condition. Embryos injected with the same amounts of MOs in A–D were hybridized with ntl and hgg1 probes are shown in F–I. (J–R) Synergistic interaction of porcn with wnt11. Embryos were injected with 1.5 ng of 5 mis control wnt11 MO (J,L) or wnt11 MO (K,M) along with 0.5 ng of 5mis control porcn MO (J,K), or porcn MO (L,M). The figures shown in J–R correspond to A–I, respectively. Combinatorial injection of suboptimal doses of wnt5b MO

Journal of Cell Science or wnt11 MO and porcn MO resulted in a shorter and wider notochord.

Fig. 5. Canonical Wnt signaling is normal even in porcn-deficient and porcn-overexpressing embryos. (A–L) The anterior–posterior patterning of the brain examined by expression of six3 (A–F) and eng2 (G–L) in embryos injected with 1.5 ng of 5mis control MO (A,G), 2.5 ng each of two wnt8 MOs (B,H) 1.5 ng of porcn MO (C,I), 1.5 ng of porcn MO and 5 ng porcn MO9 (D,J), 1.5 ng porcn MO and 5 ng porcn-l MO (E,K), and with 100 pg porcn mRNA (F,L). All the embryos were stained at the one-somite stage. In double MO injection experiments (D,E,J,K), higher amounts of these MOs caused widespread cell death and embryonic death probably as a result of off-target effects of these MOs. (M,N) TOP–dGFP transgenic embryos were injected with 1.5 ng of 5mis control MO (M) or porcn MO (N) and were stained with the gfp probe at the 80% epiboly stage. (O,P) Embryos were injected with 1.5 ng of 5mis control MO (O) or porcn MO (P) and were stained with the axin2 probe at the 80% epiboly stage. 2230 Journal of Cell Science 125 (9)

Deficiency of Porcupine does not equivalently affect lipidation of different Wnt proteins in zebrafish embryos The result showing that Wnt3a was normally secreted even in porcn-deficient zebrafish embryos led us to speculate that Wnt3a might not require lipidation for its secretion in zebrafish embryos. However, a mutant form of mouse Wnt3a lacking the lipidation (palmitoleoylation) site, was not trafficked into the cell-to-cell junctions but remained in the cells, as in the case of mouse mutant Wnt5a (supplementary material Fig. S7). Thus, contrary to our speculation, mouse Wnt3a seems to require lipidation for its secretion in zebrafish embryos. These results strongly suggest that Wnt3a, but not zebrafish Wnt5b and mouse Wnt5a, is normally lipidated even in porcn- deficient zebrafish embryos. To test this possibility more directly, we compared the hydrophobicity of zebrafish Wnt3a and Wnt5b and mouse Wnt5a in porcn-deficient embryos with that in control embryos by temperature-induced phase separation in Triton X-114 (Fig. 7). First, we confirmed that decrease of the hydrophobicity of a lipidation-defective form of mouse Wnt3a, in which Ser209 was substituted with Ala, was actually detected in this assay (Fig. 7A). In embryos injected with control MO, all Wnt proteins were partitioned into both the aqueous and detergent phases, probably reflecting the pre-palmitoleoylated and the palmitoleoylated forms, respectively, although the ratio of amounts partitioned into the two phases were different among the three Wnt proteins (Fig. 7B–D). Whereas the relative proportion of zebrafish Wnt5b, as well as mouse Wnt5a, in the aqueous fraction was apparently increased in embryos injected with porcn MO (Fig. 7C,D), that of zebrafish Wnt3a was apparently unchanged. Thus, the porcn deficiency increased the unlipidated population of zebrafish Wnt5a and mouse Wnt5b, but not that of Wnt3a.

Secretion of Wnt5a is inhibited by less Porcn inhibitor than

Journal of Cell Science that of Wnt3a in HEK293T cells These results showed that porcn is preferentially required for trafficking and lipidation of a particular Wnt, Wnt5b, in early zebrafish embryos. However, this appeared to be inconsistent with our previous results in mouse L cells, in which the porcn deficiency affected the secretion of both Wnt5a and Wnt3a. Therefore, we performed more precise analyses to examine the effect of porcn deficiency in mammalian culture cells by depressing the level of Porcn activity with a Porcn-specific Fig. 6. Secretion of Wnt proteins in zebrafish embryos. Confocal images of inhibitor, IWP-2. HEK293T cells were transiently transfected localization of EGFP-tagged zebrafish Wnt5b (A,B), mouse Wnt5a (C,D), with mouse Wnt3a or Wnt5a and treated with different zebrafish Wnt3a, (E,F) and mouse Wnt3a (G,H) with membrane markers, concentrations of IWP-2 under the same conditions. Whereas which were ectopically expressed membrane-bound RFP (A–F) or endogenous only 10–20 nM IWP-2 was effective for 50% inhibition of mouse b-catenin (G,H), in epiblast cells of embryos injected with 5mis control MO Wnt5a secretion, mouse Wnt3a required high doses of around (A,C,E,G) or porcn MO (B,D,F,H) are shown. Localization of EGFP-tagged 100–200 nM IWP-2. Thus, the decrease of Porcn activity does Wnt proteins, membrane-bound RFP and endogenous b-catenin was visualized not equivalently affect the secretion of different Wnt proteins in by immunostaining with anti-GFP, anti-RFP and anti-b-catenin antibodies, culture cells, as in the case of their trafficking in early zebrafish respectively. Merged images of Wnts and membrane markers are also presented. embryos.

were not localized to cell-to-cell junctions, but mainly found Discussion within the cytoplasm (Fig. 6B,D). In some cells that expressed In this study, we unexpectedly found that zebrafish embryos more zebrafish Wnt5b, the cytoplasmic signals were condensed defective in porcn exhibited only limited phenotypes caused by as puncta (data not shown). By contrast, neither zebrafish nor deficiency of Wnt signaling. Whereas the CE movements were mouse Wnt3a distribution was changed by depletion of porcn defective in porcn-deficient embryos, these embryos did not (Fig. 6F,H). These results indicate that porcn deficiency affected show any defects specifically caused by a reduction in canonical the trafficking of mouse Wnt5a and zebrafish Wnt5b proteins, but Wnt signaling. Consistent with this result, neither expression of not mouse and zebrafish Wnt3a, in zebrafish epiblast cells. axin2, an endogenous target of canonical Wnt signaling, nor that Role of Porcn in Wnt secretion 2231

Fig. 7. Deficiency of Porcn does not equivalently affect hydrophobicities of different Wnt proteins in zebrafish embryos. (A) Triton X-114 phase-separation analysis indicated an increase in hydrophilicity of the palmitoleoylation-defective form of mouse Wnt3a (S209A) relative to the wild-type form in zebrafish embryos. Zebrafish embryos (n550) injected with 25 pg of the wild-type or S209A forms of mouse Wnt3a mRNA were lysed at the 80% epiboly stage and extracted with Triton X-114. Wnt3a proteins partitioned in the aqueous (Aq) and detergent (T) phases were detected on western blots with antibody against Wnt3a. As a control, partition of PCNA, a soluble nuclear protein, into the in aqueous phase is also shown. (B–D) Knockdown of porcn promoted the partitioning of Wnt5b, Wnt5a into the hydrophilic phase. Embryos (n5100) were injected with 25 pg of FLAG–EGFP-tagged zebrafish wnt3a, wnt5b or mouse Wnt5a mRNA together with either 2.5 ng 5mis control MO or porcn MO as indicated. Embryos were lysed, extracted with Triton X-114 and subjected to western blotting. The blots were probed with antibodies against FLAG and PCNA.

of TOP–dGFP, a reporter of canonical signaling, was reduced in Wnt3a, whereas some particular Wnts require less Porcn for their porcn-deficient embryos. Thus, the porcn MO phenotype looked lipidation. Consistent with this explanation, we found mouse strikingly similar to those phenotypes of pipetail/wnt5b, Wnt5a required less Porcn activity for its secretion than Wnt3a silberblick/wnt11, knypek and trilobite, in which the CE in HEK293T cells. Another possibility is that some other movement is specifically impaired. acyltransferase might specifically catalyze particular types of To gain more insight into the reason why CE movement was Wnt proteins, including Wnt3a. Possible candidates for such an specifically impaired in porcn-deficient embryos, we directly enzyme are members of the MBOAT family of membrane-bound compared trafficking and lipidation of different Wnts in porcn- acyltransferases. In this study, we examined whether a member deficient embryonic cells. A precise comparison of localization of this family, Porcn-l, could have a role in the regulation of Wnt Journal of Cell Science of ectopically expressed Wnt proteins under the same conditions signaling by injecting a specific MO. However, our results did showed that the trafficking of zebrafish Wnt5b and mouse not support this possibility up to now. Wnt5a, but not zebrafish and mouse Wnt3a, was impaired by a Interestingly, we observed that porcn was not ubiquitously reduction in porcn. Although Wnt3a trafficking was unaffected in expressed after the mid-somite stage in zebrafish embryos. Such porcn-deficient embryos, our results suggested that lipidation non-ubiquitous expression of porcn is also observed in mouse was still required for Wnt3a trafficking in zebrafish embryos embryos (Biechele et al., 2011). Of note, in the neural tube of because a Wnt3a mutant that lacked the palmitoleoylation site zebrafish embryos, porcn expression was detected in the ventral was retained within the cells. If this holds true, lipidation of side, but not in the dorsal side, where several wnt genes, Wnt3a should be unaffected by injection of porcn MO. including wnt3a are expressed. This expression pattern of porcn Consistent with this, we found that lipidation of Wnt3a was suggests that some unidentified machinery, including another similar between normal and porcn-deficient embryos. By acyltransferase might be involved in Wnt secretion in the dorsal contrast, lipidation of zebrafish Wnt5b and mouse Wnt5a was neural tube of zebrafish embryos. More extensive analyses in reduced in porcn-deficient embryos. Thus, porcn deficiency future will reveal the precise mechanism of Wnt lipidation in caused by injection of porcn-specific MOs did not equivalently vertebrate embryos. affect all Wnt proteins as examined in epiblast cells in zebrafish In conclusion, we found that trafficking and lipidation of at embryos. Similarly, differences in the effect of porcn deficiency least some Wnt ligands, such as zebrafish Wnt5b and mouse on Wnt5a and Wnt3a secretion were observed in HEK293 cells. Wnt5a, required Porcn activity in early zebrafish, whereas These results suggest that the non-equivalent effect of Porcn on trafficking and/or lipidation of other Wnt ligands, zebrafish and several different Wnt proteins is the reason for the generation of mouse Wnt3a, required less or no Porcn activity. Similarly, the CE limited phenotype of porcn-defective embryos. secretion of mouse Wnt5a required more Porcn activity than that There are several possibilities to explain how porcn deficiency of Wnt3a in mammalian culture cells. This is the first report to non-equivalently affected the trafficking and lipidation of show that a decrease of Porcn does not equivalently affect different Wnt proteins in zebrafish embryos. Given that the trafficking and lipidation of different Wnt proteins. Our findings porcn morphant is not null but hypomorphic, one possible indicate that the machinery required for Wnt protein secretion is explanation is that residual amounts of Porcn in the porcn not as simple as previously predicted; rather, secretion of distinct morphants were sufficient for several Wnt proteins including Wnt proteins appears to be differentially regulated. 2232 Journal of Cell Science 125 (9)

Fig. 8. Secretion of mouse Wnt5a is more susceptible than Wnt3a to Porcupine inhibitor IWP-2. (A,B) HEK293T cells transiently expressing either mouse Wnt3a or Wnt5a were treated with IWP-2 compound at different concentrations as indicated for 48 hours before lysis. Cell culture medium were subjected to western blot analysis to determine levels of secreted Wnts. Standards to enable quantification of secreted Wnts were also loaded (left). The level of Wnt protein in cell lysate was not obviously changed by treatment with IWP-2. (C) Quantification of secreted Wnts in conditioned medium. Western blot images were scanned and Wnt protein bands were quantified using ImageJ software. For each lane, the secreted Wnts level relative to that of cell lysate was calculated and presented as percentage of the control. Results are means 6 s.e.m.

Journal of Cell Science Materials and Methods cells were detected with a Vectastain Kit (Vector Laboratories) to reveal the Zebrafish maintenance biotin labeling. Zebrafish of the TL and India strains were maintained and bred according to standard procedures (Westerfield, 2000). In situ hybridization and notochord cell shape analysis Whole-mount in situ hybridization was carried out as previously described Cloning of zebrafish porcn cDNAs (Westerfield, 2000). Antisense RNA probes were synthesized with digoxigenin By a BLAST analysis with the 9th zebrafish genome assembly released from the RNA labeling kits (Roche Diagnostics). To investigate the shapes of notochord Sanger Institute, we found two genes, porcn and porcn-l, to be related to porcn cells, embryos were injected with 20 pg of membrane-bound Venus mRNA and genes identified in other species. mRNA was harvested from embryos 24 hours 1.5 ng of 5mis control or porcn MO and were then fixed with 4% after fertilization and cDNA was synthesized by reversed transcription using paraformaldehyde (PFA) at the four-somite stage. Immunostaining of mVenus random primers. This cDNA was used to amplify the porcn genes by PCR using was performed as described previously (Westerfield, 2000). The antibody used for the specific primers described in supplementary material Table S1A. Both cDNAs detection was 598 anti-GFP (1:3000, MBL). Length-width-ratio (LWR) of encoded the same type of splicing variants corresponding to PorcnD in mouse notochord cells and the angles of the long axis relative to a line perpendicular (Tanaka et al., 2000; Caricasole et al., 2002). A phylogenetic analysis and tree to the embryonic axis as represented by the notochord were analyzed using Object- construction of porcupine sequences were conducted by using CLUSTALW image software. (http://www.ebi.ac.uk/Tools/es/cgi-bin/clustalw2). Analysis of subcellular protein localization RNA and morpholino antisense oligonucleotide (MO) injection Wnt proteins tagged with FLAG and EGFP (EGFP–Wnts) had the signal peptide of Capped sense RNAs encoding the full-length mouse Porcn, Myc-tagged zebrafish mouse Wnt3a added before these tags. Embryos at the one-cell stage were co- Porcn, m-Venus (Wallingford et al., 2000), truncated forms of zebrafish porcn and injected with an RNA encoding EGFP–Wnt (6.25 pg of zebrafish wnt3a, mouse porcn-l tagged with EGFP, FLAG- and EGFP-tagged zebrafish wnt5b, zebrafish Wnt3a or mouse Wnt5a mRNA was used, but 12.5 pg of zebrafish wnt5b mRNA wnt3a, mouse Wnt5a, and mouse Wnt3a were synthesized using mMessage was used) and 15 pg of membrane-bound RFP with either 2.5 ng of 5mis control Machine system (Ambion). All injections were performed on one-cell stage MO or porcn MO. Embryos were fixed at 80–90% epiboly and anti-GFP, anti-RFP embryos. For the sequences of MOs, see supplementary material Table S1B. or anti-b-catenin immunostaining was performed as previously described (Westerfield, 2000). Images were acquired using a confocal microscope. The Cell transplantation following antibodies were used: 598 anti-GFP (1:3000, MBL), M155-3 anti-RFP For transplantations, donor embryos were injected with a mixture of Rhodamine (1:2000, MBL), #C19200 anti-b-catenin (1:500, Transduction Labs), anti-rabbit and biotin–dextran plus either 1.5 ng of porcn MO or 1.5 ng of 5mis control MO IgG antibody conjugated to Alexa Fluor 488 (1:500, Molecular Probes), and anti- at the one-cell stage. Groups of donor cells were taken from shield-stage embryos mouse IgG antibody conjugated to Alexa Fluor 546 (1:500, Molecular Probes). and transplanted into the deep region (close to the embryonic shield) or into the lateral germ ring of host embryos that were injected with either 5mis control or Cell culture, transfection and western blotting porcn MO as described previously (Heisenberg et al., 2000). Host embryos were For mouse Porcn-knockdown cells, pSilencer 3.0-H1 plasmid, expressing an harvested at the one-somite stage and fixed for in situ hybridization. Transplanted siRNA specific for Porcn, was used to transfect cells that stably expressed and Role of Porcn in Wnt secretion 2233

secreted Wnt3a or Wnt5a as previously described (Takada et al., 2006). Stable Carreira-Barbosa, F., Concha, M. L., Takeuchi, M., Ueno, N., Wilson, S. W. and clones with varying levels of defective mouse Porcn expression were confirmed by Tada, M. (2003). Prickle 1 regulates cell movements during gastrulation and neuronal real-time RT-PCR. migration in zebrafish. Development 130, 4037-4046. Mouse L cells deficient in Porcn expression but stably expressing either Wnt3a Chen, B., Dodge, M. E., Tang, W., Lu, J., Ma, Z., Fan, C. W., Wei, S., Hao, W., or Wnt5a were plated at density of 26104 cells/cm2 and transiently transfected Kilgore, J., Williams, N. S. et al. (2009). Small molecule-mediated disruption of with Myc-tagged porcn, porcn-l or control vector as indicated. Culture Wnt-dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100- supernatants or cell lysates were collected 36 hours after transfection. The final 107. Coombs, G. S., Yu, J., Canning, C. A., Veltri, C. A., Covey, T. M., Cheong, J. K., loading amount of each sample on the SDS-PAGE gel was normalized to cell Utomo, V., Banerjee, N., Zhang, Z. H., Jadulco, R. C. et al. (2010). WLS- number calculated at the harvest time point. Proteins on the SDS-PAGE gel were dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification. blotted to a PVDF membrane and probed with anti-Myc (1:2000, MBL), anti- J. Cell Sci. 123, 3357-3367. Wnt3a or anti-Wnt5a monoclonal antibody as described previously (Takada et al., Coudreuse, D. Y., Roe¨l, G., Betist, M. C., Destre´e, O. and Korswagen, H. C. (2006). 2006). Wnt gradient formation requires retromer function in Wnt-producing cells. Science 312, 921-924. Triton X-114 phase separation Cox, B. J., Vollmer, M., Tamplin, O., Lu, M., Biechele, S., Gertsenstein, M., van Twenty-five pg of the wild-type or a palmitoleoylation-defective (S209A) form Campenhout, C., Floss, T., Ku¨hn, R., Wurst, W. et al. (2010). 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