Wnt 6 Regulates the Epithelialisation Process of the Segmental Plate Mesoderm Leading to Somite Formation

Developmental Biology 271 (2004) 198–209 www.elsevier.com/locate/ydbio

Wnt 6 regulates the epithelialisation process of the segmental plate mesoderm leading to somite formation

Corina Schmidt,a,b Mechthild Stoeckelhuber,a Iain McKinnell,b Reinhard Putz,a Bodo Christ,c and Ketan Patelb,*

a Institute of Anatomy, Ludwigs-Maximilians-University of Munich, D-80336 Munich, Germany b Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU, UK c Institute of Anatomy, Albert-Ludwigs-University of Freiburg, D-79001 Freiburg, Germany Received for publication 31 October 2003, revised 9 February 2004, accepted 1 March 2004

Available online 6 May 2004

Abstract

In higher vertebrates, the paraxial mesoderm undergoes a mesenchymal to epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study, we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. Ectopic expression of Wnt6 leads to sustained expression of markers associated with the epithelial somites and reduced or delayed expression of markers associated with mesenchymally organised somitic tissue. More importantly, we show that Wnt6- producing cells are able to rescue somite formation after ectoderm ablation. Furthermore, injection of Wnt6-producing cells following the isolation of the neural tube/notochord from the segmental plate was able to rescue somite formation at both the structural (epithelialisation) and molecular level, as determined by the expression of marker genes like Paraxis or Pax-3. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists, which result in the segmental plate being unable to form somites. These results show that Wnt6, the only known member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. We propose a model in which the localisation of Wnt6 and its antagonists regulates the process of epithelialisation in the paraxial mesoderm. D 2004 Published by Elsevier Inc.

Keywords: Epithelialisation; Vertebrate; Mesoderm

Introduction part in the formation of the intervertebral discs, intervertebral joints and proximal ribs (Huang et al., 1994, 1996, Somites form as a consequence of a mesenchymal to 2000). The ventromedial part of the somite subsequently epithelial transition of the paraxial mesoderm (Christ and undergoes an epithelial–mesenchymal transition and forms Ordahl, 1995; Christ et al., 1998; Stockdale et al., 2000). the sclerotome, which—together with the cells of the The newly formed somites consist of an epithelial ball somitocoel—gives rise to the vertebral column and ribs. enclosing the somitocoel (Mestres and Hinrichsen, 1976). Development of the sclerotome, marked by the expression The somitocoel consists of mesenchymal cells, which take of Pax-1, is regulated by the notochord and the floor plate of the neural tube (Balling et al., 1996; Ebensperger et al., 1995). The remaining epithelial, dorsolateral part of the somite forms the dermomyotome. The dermomyotome * Corresponding author. Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 OTU, UK. gives rise to the musculature of the body and extremities Fax: +44-207-388-1027. as well as to the dermis of the back (Christ et al., 1977, E-mail address: [email protected] (K. Patel). 1983). The pair-domain transcription factor Pax-3 is an

0012-1606/$ - see front matter D 2004 Published by Elsevier Inc. doi:10.1016/j.ydbio.2004.03.016 C. Schmidt et al. / Developmental Biology 271 (2004) 198–209 199 excellent marker for the dermomyotome. Under the influ- or to activation of existing biochemical cascades. Wnt ence of the neural tube and the overlying surface ectoderm, genes are expressed in numerous embryonic tissues in- Pax-3 is expressed at low levels in the cells of the segmental cluding the neural tube (McMahon, 1992; Nusse and plate. Pax-3 expression is subsequently found throughout Varmus, 1992; Parr et al., 1993) and in the ectoderm the newly formed epithelial somites and later becomes (Fan et al., 1997; Parr et al., 1993; Rodriguez-Niedenfuhr localised to the dermomyotome (Williams and Ordahl, et al., 2003; Schubert et al., 2002) and influence process- 1994). Later, Pax-3 expression is found in the medial and es as diverse as cell differentiation, polarity, migration and lateral part of the dermomyotome, which includes the proliferation. Wnt proteins have been implicated in nu- regions from which muscle progenitor cells of the back merous somitic patterning processes. However, of partic- and the extremities originate (Christ et al., 1974, 1977; ular interest for this study, Wnts have also been Ordahl and Le Douarin, 1992). implicated in mesenchymal to epithelial transformation Cells in specific regions of the epithelially organised processes during embryonic kidney development (Kispert dermomyotome ingress to form the myotome under the et al., 1998). Of all the family members, Wnt6 is an influence of neighbouring structures. In chicken embryos, excellent candidate for regulating the epithelialisation of the cells of the medial myotome express the myogenic somites, since it is the sole known member of the Wnt determination factors Myf-5 and MyoD. These cells form family to be expressed in the surface ectoderm of the the epaxial myotome giving rise to the musculature of the chick. back (Kiefer and Hauschka, 2001). The lateral part of the Wnt proteins not only bind the Frizzled receptors but also myotome is the source of the hypaxial musculature (Denet- to the secreted frizzled related proteins (Sfrps), which are claw and Ordahl, 2000; Denetclaw et al., 1997; Huang et structurally related to the frizzled-receptors but lack a al., 2000; Kiefer and Hauschka, 2001). The differentiation transmembrane domain (Finch et al., 1997; Hoang et al., of the somites into sclerotome, dermomyotome and myo- 1998; Lee et al., 2000; Lescher et al., 1998; Melkonyan et tome formation is regulated by signals emanating from the al., 1997) and thus act as Wnt antagonists. Together with notochord/floor plate, the dorsal neural tube and the other Wnt antagonising proteins, Sfrps have been shown to overlying surface ectoderm. The candidate signalling mol- modulate Wnt activity. (Ladher et al., 2000; Lin et al., 1997; ecules resulting in the compartmentalization of the ventral Moon et al., 1997a,b; Terry et al., 2000). Sfrp2 is of somite region are Noggin (Dockter, 2000; Hirsinger et al., particular interest as it is expressed in the portion of the 1997; Marcelle et al., 1997) and Shh (Christ et al., 1998; somite that undergoes an epithelial to mesenchymal transi- Dietrich et al., 1993; Fan and Tessier-Lavigne, 1994; tion during the formation of the sclerotome. (Lee et al., Koseki et al., 1993; Johnson et al. 1994) emanating from 2000). the notochord, and in the dorsal region different Wnts In this study, we examined the role of Wnt6 on the emanating from the dorsal neural tube and surface ecto- formation of the somites and their subsequent differenti- derm (Fan et al., 1997; Ikeya and Takada, 1998; McMa- ation. Application of Wnt6-producing cells to the caudal hon, 1992; Nusse and Varmus, 1992; Parr et al., 1993; segmental plate lead to the precocious formation of Rodriguez-Niedenfuhr et al., 2003; Wodarz and Nusse, somites. Ablation of ectoderm overlying the segmental 1998). plate prevented somite formation. However, when the At present, we have a far greater understanding of the same protocol was followed together with application of mechanisms regulating the patterning of somites com- Wnt6-producing cells, somite formation was restored. pared to the processes that control the formation of these Overexpression of Wnt6 maintained the somite in an epithelial structures in the first place. In addition, it is of epithelial state which had a profound effect on the interest that the differentiation of the epithelially organ- differentiation of somitic derivatives. Differentiation of ised somitic cells proceeds after they have reverted to a the sclerotome was delayed and the number of migrating mesenchymal state and the influence of tissue organisa- muscle precursors was reduced leading to the develop- tion on the differentiation process has yet to be addressed. ment of limbs with a diminished myogenic content. Experiments have shown that signals originating from the Finally, we also show that overexpression of the Wnt ectoderm overlying the somites or from midline structures antagonist, Sfrp2, inhibits the formation of somites im- are required for the transformation of the mesenchymally plying that Wnts do indeed play a role in the epithelial- organised segmental plate into epithelial somites (Schmidt isation of the segmental plate. et al., 1998, 2001). A potential candidate could be a member of the Wnt family of secreted glycoproteins. The Wnt family consists of at least 17 members in vertebrates. Material and methods Wnt signalling is mediated through the Frizzled family of seven-pass transmembrane receptors (Bhanot et al., 1996; Embryos Borello et al., 1999; He et al., 1997; Cauthen et al., 2001; Chan et al., 1992). A variety of intracellular signalling Fertilized eggs (White Leghorn) were kindly provided pathways can be employed leading to gene transcription by the Institute of Avian Diseases (Ludwigs-Maximilians- 200 C. Schmidt et al. / Developmental Biology 271 (2004) 198–209

University of Munich) and incubated at 38j and 80% Paraxis, full-length clone (see Sosic et al., 1997) was a relative humidity for 2 days up to HH stages 12–14 gift from Professor Eric Olson. MyoD, clone CMD9 full (Hamburger and Hamilton, 1951). 1.5 kb length fragment (gift from Professor Bruce Pat- terson). The embryos were cryosectioned for further Cell lines and culture conditions examination.

Murine Wnt6-expressing NIH 3T3 cells were kindly provided by Seppo Vainio. Lac-Z-transfected control cells Results were kindly provided by Andreas Kispert. The cells were selected in G418 and grown under conditions as described Consequences of ectoderm removal on the epithelialisation by Itaeranta et al. (2002). of segmental plate (14/15)

Operations In all cases, operations were performed at the level of the caudal segmental plate. Ectoderm removal resulted in All operations were performed at the level of the caudal unsegmented paraxial mesoderm (segmental plate).

1. Ectoderm removal and replacement with gold foil (n = 15). Ectoderm destined for ablation was first marked using Nile Blue Sulphate and was non-enzymatically removed using a tungsten needle. 2. Implantation of Wnt6-producing cells beneath the ectoderm or lateral to the paraxial mesoderm (n = 40; 30: short reincubation: 10: long reincubation). Care was taken to apply cells directly under the ectoderm using a micropipette without disturbing contact between unoperated tissues. Importantly, the ectoderm at the level of cell implantation was never damaged. 3. Ectoderm removal, replacement with gold foil and implantation of Wnt6-producing cells beneath the gold foil (n = 26). This was a combination of procedures 1 and 2. 4. Implantation of Wnt6-producing cells beneath the ectoderm and of an impermeable polycarbonate membrane between neural tube/notochord and segmental plate (n = 20). 5. Implantation of Sfrp-2 beads beneath the ectoderm at level of the segmental plate (n = 25). Affigel Blue beads were soaked in Sfrp-2 (R&D Systems, USA) (2 Ag/Al, made in PBS) for a minimum period of 12 h.

A combination of ectoderm removal and implantation of an impermeable barrier was not performed since we have previously shown that this leads to extensive cell death in the paraxial mesoderm (Schmidt et al., 1998).

In situ hybridisation Fig. 1. Ectoderm removal prevents somite formation. Transverse section of a 3-day-old chick embryo after ectoderm removal and replacement by gold foil Embryos were washed in PBS and then fixed over- showing expression of (A) Pax-3, (B) Pax-1 and (C) Myo-D. (A) On the night in 4% paraformaldehyde at 4jC. The antisense operation side, only the medial lip of the dermomyotome (arrowhead) near to RNA probe was labelled with digoxigenin and whole- the dorsal neural tube shows strong Pax-3 expression. dm, dermomyotome; mount in situ hybridisation was performed as described st, sclerotome; wd, wolffian duct; nc, notochord; nt, neural tube; arrow marks the implanted gold foil. (B) The Pax-1 expression on the operation side is not by Nieto et al. (1996). The following probes were used changed in comparison to the control side. The arrowhead marks the medial in this study: Pax-3, 645 bp fragment corresponding to lip of the dermomyotome. (C) Arrowhead marks the weak Paraxis nucleotides 468-1113 (gift from Dr Martin Goulding), expression on the operation side, there is no dermomyotome. C. Schmidt et al. / Developmental Biology 271 (2004) 198–209 201 abolition of somite formation on the operated side. Pax-3 is Ectoderm removal followed by re-incubation resulted in a useful marker since it is expressed at high levels in the a dramatic downregulation in the high levels of Pax-3 epithelialised dorsal compartment of the somite. expression associated with epithelialised dermomyotome

Fig. 2. Wnt6 induces epithelialisation of paraxial mesoderm. (A) Whole-mount in situ hybridisation for Pax-3 of a 3-day-old chicken embryo after implantation of Wnt-6-expressing cells lateral to the segmental plate. Stronger Pax-3 expression is found on the operation side (marked by arrowheads). (B) Transverse section of the embryo shown in A. On the operation side, an epithelial somite is found (marked by an arrowhead) which is Pax-3-positive, on the control side, the somite is differentiated into the dermomyotome (marked by an arrow) and the sclerotome (st). (C) Whole-mount in situ hybridisation for Pax-3 of a 3-day-old chicken embryo after implantation of Wnt-6-expressing cells beneath the ectoderm at level of segmental plate. On the operation side, the somites are smaller (marked by an arrowhead) and Pax-3 expression in the limb bud is reduced (asterisk). (D) Whole-mount in situ hybridisation for Pax-1 of a 3-day-old chicken embryo after implantation of Wnt-6-expressing cells lateral to the segmental plate. On the operation side (arrowheads), the Pax-1 expression is reduced. (E) Transverse section of the embryo shown in D. Pax-1 expression (arrowheads) is markedly reduced on the operation side compared to the control side. (F) Whole-mount in situ hybridisation for Paraxis of a 3-day-old embryo after implantation of Wnt-6-expressing cells lateral to the segmental plate. On the operation side, there are smaller but increased number of somites (arrowheads). Paraxis expression is reduced in the limb bud (asterisk). (G) Transverse section of the embryo shown in F. On the operation side, a somite-like Paraxis-positive structure is found (arrowheads), whereas on the control side, the somite is differentiated into the dermomyotome and sclerotome. (H) Whole-mount in situ hybridisation for Paraxis of a 3-day-old chicken embryo after implantation of Wnt-6-expressing cells beneath the ectoderm at level of segmental plate. The operation side shows two rows of Paraxis-positive somites. (I) Transverse section of the embryo shown in H. On the operation side, two Paraxis-positive epithelial somites are found (asterisk marks implanted Wnt-6-expressing cells). On the control side, the somite is differentiated in dermomyotome and sclerotome. (J) Whole-mount in situ hybridisation for MyoD of a 3-day-old chicken embryo. On the operation side, the somites are smaller, MyoD expression seems more condensed. The MyoD expression itself is retarded on the operation side in comparison to the control side (levels of MyoD expression on operation and control side are marked by black lines). (K) Transverse section of the embryo shown in J. On the operation side, the MyoD expression is more condensed (arrowhead). (L) Whole-mount in situ hybridisation for MyoD of a 3-day-old chicken embryo after implantation of Wnt-6-expressing cells lateral to the segmental plate. The somites on the operation side are smaller (marked by arrowheads). 202 C. Schmidt et al. / Developmental Biology 271 (2004) 198–209 at the site of operation (Fig. 1A). Some Pax-3 expression ed side (Fig. 2J). Subsequently, expression of MyoD was was detected directly especially adjacent to the dorsal initiated. However, the expression domain was smaller than neural tube coinciding to a small region that remained in the unoperated side (Fig. 2L) and this leads to a compact an epithelial state. Examination of a marker of the ventral domain of expression (Fig. 2K). Therefore, overexpression compartment, Pax-1, following ectoderm removal showed of Wnt6 prolongs the epithelial state of the somites. This robust expression of the gene at the site of operation. results in a delay in somite differentiation and decrease of Expression levels of Pax-1 on the operated side were muscle precursor migration signified by a reduction in Pax- similar to those on the control side (Fig. 1B). Paraxis a 3 expression in the limb (Fig. 2C). marker for the epithelialisation of the paraxial mesoderm (Barnes et al., 1997; Burgess et al., 1995; Sosic et al., 1997) was downregulated like Pax-3, at the site of ectoderm removal (Fig. 1C). No evidence of cell death following ectoderm removal could be detected (Schmidt et al., 1998). Therefore, ectoderm removal results in the loss of epithelial organisation of the somites except in a small region directly adjacent to the dorsal neural tube. At the molecular level, removal of the ectoderm leads to the loss of epithelial somite markers, except in the region adjacent to the neural tube. However, this is not accompanied by an upregulation of sclerotome markers.

Effect of Wnt6 on the development of somites (30/30)

Wnt6 is a good candidate as an epithelialising factor originating from the ectoderm, as it is not only expressed in this tissue but in other systems has been shown to confer epithelialising activity (Itaeranta et al., 2002; Kis- pert et al., 1998). We investigated the effect of Wnt6 on the development of somites by implanting Wnt6-protein- expressing cells at the caudal segmental plate level but taking care to leave the ectoderm in place. Implantation of Wnt6-producing cells leads to the strong expression of Pax-3 at the site of operation (Fig. 2A). Site of operation was noted at the end of each procedure. Wnt6-expressing and control cells could be easily identified after fixation since they appear as a white stripe and related to gross phenotype. Transverse sections showed on the unoperated side a differentiated somite with a Pax-3-positive epithelial dermomyotome and a Pax-3-negative mesenchymally organised sclerotome. However on the operated side we detected the presence of an epithelial somite (Fig. 2B). Furthermore, somites on the operated side were smaller compared to those on the unoperated side (Figs. 2A and B). Expression of Pax-3 at the site of operation resembled that found in normally occurring epithelial somites. Wnt 6 Fig. 3. Wnt6 rescues somite formation after ectoderm removal. Transverse expression also caused a decrease in Pax-1 expression section of a 3-day-old chick embryo after ectoderm removal and (Figs. 2D and E). implantation of Wnt-6-expressing cells beneath the gold foil and following reincubation of 18 h showing expression of (A) Pax-3, (B) Paraxis and (C) Expression of Paraxis on the operated side was similar to MyoD. (A) On the operation side, the Wnt6-expressing cells enable the the expression of Pax-3, reminiscent of the predicted ex- epithelialisation of the paraxial mesoderm and initiate strong Pax-3 pression pattern of this gene in epithelial somites (Figs. 2F expression. Arrowheads mark the Pax-3-positive somite on the operation and G). Application of Wnt-6-expressing cells in more side. (B) On the operation side, a somite-expressing Paraxis is found dorsal regions leads to the development of two rows of (arrowhead marks the somite). Note that the gold foil has been removed during processing. (C) On the operation side, no MyoD expression is found epithelial somites (Figs. 2H and I). We subsequently exam- (arrowhead marks the epithelial structure in the paraxial mesoderm induced ined the effect of Wnt6 on the myogenic programme. by the Wnt6-expressing cells). Control side showing normal MyoD Expression of MyoD was transiently delayed on the operat- expression in the myotome (arrow). C. Schmidt et al. / Developmental Biology 271 (2004) 198–209 203

Wnt6 rescues the effect of ectoderm removal (24/26) of epithelial somite formation and high levels of Pax-3 expression, additional implantation of Wnt6-expressing Since Wnt6 maintains epithelialisation of somites, we cells rescues not only the expression of Pax-3, but also investigated whether this protein could mimic the effect of the epithelialisation of the segmental plate (Fig. 3A).Ina the dorsal ectoderm. To this end, we removed the ectoderm similar manner, expression of Paraxis was rescued by and implanted Wnt6-producing cells to the caudal segmen- Wnt6-producing cells (Fig. 3B). MyoD expression was tal plate. Whereas ectoderm removal alone leads to the loss delayed following the dual procedure (Fig. 3C).

Fig. 4. Wnt6 rescues somite formation after medial structure isolation. Whole-mount in situ hybridisation of a 3-day-old chicken embryo after implantation of Wnt-6-expressing cells beneath the ectoderm of the segmental plate and additional implantation of a polycarbonate membrane (marked by asterisks) between neural tube/notochord and segmental plate showing expression of Pax-3 (A, B) and Paraxis (C, D). (A) On the operation side, Pax-3 expression is found near to the implanted cells (arrowheads) and lateral to the implanted polycarbonate membrane (marked by asterisks). (B) Transverse section of the embryo shown in A. On the operation side, Pax-3 expression is found in the paraxial mesoderm (marked by arrowheads) lateral to the polycarbonate membrane (marked by asterisks). (C) On the operation side, Paraxis expression is found lateral to the polycarbonate membrane (marked by arrowheads). (D) Transverse section of the embryo shown in (C). On the operation side, a somite-like structure expressing Paraxis is found lateral to the polycarbonate membrane (arrowheads). Whole-mount in situ hybridisation of a 3-day-old chicken embryo after implantation of Lac-Z (control) cells beneath the ectoderm of the segmental plate and additional implantation of a polycarbonate membrane between neural tube/notochord and segmental plate showing expression of Pax-3 (E, F) and Paraxis (G, H). (E) On the operation side, no epithelial structures and no Pax-3 expression are found (marked by arrowheads). (F) Transverse section of the embryo shown in E. On the operation side, no Pax-3 expression is found lateral to the polycarbonate membrane (arrowheads). (G) On the operation side, no Paraxis expression is found lateral to the membrane. (H) Transverse section of the embryo shown in G. On the operation side, no Paraxis expression is found lateral to the polycarbonate membrane. 204 C. Schmidt et al. / Developmental Biology 271 (2004) 198–209

Wnt6 rescues the effect of medial barrier insertion (19/20) the section of the control side (Fig. 5D) at the same level. These results show that cells that were once maintained in Insertion of an impermeable barrier between the an epithelial state are eventually capable of muscle neural tube/notochord and the caudal segmental plate differentiation. leads to the loss of epithelial somites and the expression of genes including Pax-3 and Paraxis. We investigated Effect of Sfrp-2 on somite development (23/25) whether Wnt6 could rescue the formation of somites and expression of these genes following medial tissue isola- Experiments outlined thus far suggest that Wnts play a tion. Implantation of a medial barrier and Wnt6-produc- role in the epithelialisation process. To investigate this ing cells at the level of the segmental plate led to the possibility, we tested the hypothesis that application of formation of epithelial somites and the expression of Wnt antagonists to the segmental plate should prevent the Pax-3 (Figs. 4A and B) and Paraxis (Figs. 4C and D). epithelialisation of somites. Secreted frizzled related proteins Implantation of a medial barrier and LacZ-expressing (Sfrps) are potent antagonists of Wnt signalling and are cells shows no rescue of either epithelialisation or the expressed in a dynamic manner in the segmental plate and expression of Pax-3 (Figs. 4E and F) or Paraxis (Figs. somites (Hoang et al., 1998; Lee et al., 2000). Implantation 4G and H). There was no evidence of cell death of Affigel beads soaked with Sfrp-2 protein at a concentra- following medial barrier insertion (Schmidt et al., tion of 2 Ag/Al at the level of the caudal segmental plate leads 1998). These experiments show that a signal responsible to the inhibition of epithelialisation of the segmental plate. for the epithelialisation of the somites originates from Either no somites were formed (Figs. 6A and B) or very medial structures. Wnt6 overexpression is able to rescue small somites developed directly in contact with the neural the effect of the loss of medial signalling. tube (Figs. 6C and D). Expression of Paraxis was either completely abolished at the site of Sfrp-2 bead implantation Effect of Wnt6 expression on muscle development (10/10) (Figs. 6A and B) or was found in the small somites adjacent to the neural tube (Figs. 6C and D). However, in both cases, Examination of embryos treated with Wnt6-producing expression of Paraxis as an epithelial marker was reduced at cells with an intact ectoderm leads to a significant the operation site. Implantation of Sfrp-2 beads at level of decrease in the expression of MyoD in both axial (data cranial segmental plate leads to formation of smaller somites not shown) and limb musculature (Fig. 5A) compared to that expressed Pax-3 (Fig. 6E, arrowheads). Implantation of the control site at same level (Fig. 5B). Transverse section Sfrp-2 beads at the caudal segmental plate resulted in the loss of the limb on the operation side shows a significant of the epithelialisation of segmental plate, which displayed a decrease of MyoD expression and the massive loss of the Pax-3 expression pattern similar to its profile in unsegment- ventral and dorsal muscle masses (Fig. 5C) compared to ed paraxial mesoderm. (Fig. 6E, arrows). Robust expression

Fig. 5. Wnt6 leads to decreased limb muscle development. (A) Whole-mount in situ hybridisation for MyoD D of a stage 26 embryo after implantation of Wnt- 6-expressing cells beneath the ectoderm at level of segmental plate. The lower limb of the operation side is shown. The MyoD expression compared to the control side shown in B is markedly reduced. (B) The lower limb of the control side with normal MyoD expression. (C) Transverse section of the limb shown in A. The loss of the myogenic content in both dorsal and ventral muscle mass is evident. (D) Transverse section of the control limb shown in B. C. Schmidt et al. / Developmental Biology 271 (2004) 198–209 205

Fig. 6. The Wnt antagonist Sfrp-2 inhibits somite formation. (A) Whole-mount in situ hybridisation for Paraxis of a 3-day-old chicken embryo after implantation of Sfrp-2 soaked beads at level of caudal segmental plate. (B) Higher magnification of the interested area of A. On the operation side, at level of beads, no somites and no Paraxis expression (arrows) were found (asterisks: beads). (C) Whole-mount in situ hybridisation for Paraxis of a 3-day-old chicken embryo after implantation of Sfrp-2 soaked beads at level of caudal segmental plate. (D) Higher magnification of the interested area of C. On the operation side, at level of beads (asterisks: beads), only a very small band of Paraxis expression is found in direct contact to the neural tube (arrows). (E) Whole-mount in situ hybridisation for Pax-3 of a 3-day-old chicken embryo after implantation of Sfrp-2 soaked beads at level of cranial and at level of caudal segmental plate. On the operation side, at level of cranial beads (asterisks: beads), smaller Pax-3-positive somites are found (arrowheads), at level of caudal beads no somites, but Pax-3 expression is found (arrows). (F) Whole-mount in situ hybridisation for Pax-3 of a 3-day-old chicken embryo after implantation of control beads, with no effect on somite formation. of Pax-3 following Sfrp-2 bead application also indicated genesis is the epithelialisation of the segmentally organised that apoptosis was not induced. Implantation of control paraxial mesoderm. Deletion of numerous genes has shown beads at level of segmental plate has no effect on somite that somitogenesis is not a prerequisite for the differentia- formation (Fig. 6F). tion of somitic derivatives (Burgess et al., 1996; Hrabe de Angelis et al., 1997; Johnson et al., 2001). However, although the ParaxisÀ/À mouse (which fails to form Discussion somites) still generates muscle and axial skeleton, these tissues are disorganised (fused vertebrae, fused ribs and The processes regulating the development and matura- patterning defects of axial musculature, Burgess et al., tion of the somites are being slowly unravelled. Whereas the 1996). Therefore, the process of somitogenesis seems to segmentation of the paraxial mesoderm is driven by intrinsic regulate the precise organisation and growth of tissues that properties (Cooke and Zeeman, 1976; Pourquie, 2000), the allows them to eventually differentiate in an orderly manner. organisation of the same tissue and subsequent differentia- This study has identified Wnt6 originating from the tion is regulated by extrinsic factors originating from dorsal ectoderm as a factor that can regulate the process adjacent tissues (Brand-Saberi et al., 1996a,b; Rong et al., of somite formation. The role of ectodermal Wnt6 identified 1992). There is a clear distinction between segmentation and in this study seems to be similar to the role played by somitogenesis. Segmentation represents formation of repet- Paraxis, that is, they both promote epithelialisation. We itive portions of the paraxial mesoderm, whereas somito- show that removal of ectoderm leads to the loss of Paraxis 206 C. Schmidt et al. / Developmental Biology 271 (2004) 198–209 and that Wnt6 rescues both Paraxis expression and epithe- Wnts can activate several signalling pathways (Dale, lialisation of segmental plate. Paraxis expression is initiated 1998), each with a distinct consequence in terms of cellular in the segmental plate. Therefore, we suggest that Wnt6 development. The two most commonly used are the canon- signalling could either initiate or stabilise the expression of ical and non-canonical pathways. The former involves the Paraxis, facilitating epithelialisation. The above experi- activation of a signalling process that results in transloca- ments show that Wnt6 can influence the epithelialisation tion of h-catenin from the cytoplasm to the nucleus and of the paraxial mesoderm. Since the application of the Wnt leads to the initiation of gene transcription (Gumbiner, antagonist Sfrp-2 interferes with somite epithelialisation, 1995; Huber et al., 1996; Willert and Nusse, 1998). This this demonstrates that Wnts play an active role in this signalling cascade is deployed during tissue patterning. In process. In the cranial segmental plate, a region marked contrast, the non-canonical (or in Drosophila, the Planar by the expression of Paraxis, Sfrp-2 did not totally prevent Cell Polarity-PCP) pathway signals through small GTPases epithelialisation but lead to the formation of small somites. and activates c-Jun-N-terminal kinases to control cell be- The size of the Pax-3 expression domain was reduced but haviour and arrangement (e.g., epithelialisation) rather than levels were normal in the small somites, which were formed identity (Weston and Davis, 2002). However, h-catenin under the influence of Sfrp-2. However, application of Sfrp- does have a role in the non-canonical as well as the 2 to the caudal, non-Paraxis-expressing region of the canonical pathway. E-cadherins are calcium-dependent segmental plate completely inhibited epithelialisation. When transmembrane adhesion molecules found on epithelial somite formation was totally inhibited, Pax-3 expression cells and are linked to the cytoskeleton by h-catenin and levels were still found similar to the expression of this gene form the adherens junction complex—a key feature of following genetic deletion of Paraxis (Burgess et al., 1995). epithelial tissue. The enzyme glycogen synthase kinase 3 The role of promoting epithelialisation performed by Wnt6 activates the proteosome-mediated degradation of free in- seems to be unique amongst this family. We have previously tracellular h-catenin (Pfeifer and Polakis, 2000). However, investigated the role of Wnt 1, Wnt3a and Wnt4 and found translocation of free h-catenin to the plasma membrane, that they were involved in regulating myogenesis (Wagner where it complexes with E-Cadherin and actin filaments, et al., 2000). not only prevents it from being degraded, but also confers In this study, we show that transplantation of cells epithelial characteristics to the cell, without protein trans- expressing Wnt6 beneath the ectoderm overlying the lation or gene transcription (Wiggan and Hamel, 2002). caudal segmental plate leads to an upregulation of Truncation of the h-catenin protein renders it unable to markers associated with epithelial somites and reduced participate in the formation of adherens junctions and leads levels of Pax-1, a gene associated with the mesenchymal to a loss in the intercellular adhesiveness of cells and has compartment of the somites. Furthermore, the expression been implicated in the metastasis process (Oyama et al., of MyoD was greatly reduced as shown in the limb (Figs. 1994). Furthermore, the translocation of h-catenin to the 5A and C). However, there was no evidence of cell loss nucleus suppresses the transcription of E-cadherin, again as the somites appeared free of dying cells. These resulting in decreased cell adhesion (Jamora et al., 2003).In observations suggest Wnt 6 overexpression lead to cells the case of the epithelialisation of the segmental plate, we becoming locked in an epithelial state for an extended suggest that Wnt6 acts to stabilise adherens junctions period. Cells are eventually released from the epithelial possibly by translocating h-catenin to the plasma mem- state to continue their differentiation programme demon- brane and not the nucleus, where it may suppress the strated by the presence of axial skeleton (data not shown) expression of E-Cadherin (Jamora et al., 2003). and muscle in old embryos. Results from this and other studies establish the need to Application of Wnt6-producing cells lead to a reduction develop an epithelial somite (for organisation of differen- in the amount of muscle in the limbs and we were able to tiated derivative) and a requirement for the temporal show a pronounced decrease in the expression of markers regulation of this organisation. If cells are locked into associated with migrating muscle precursors. All limb an epithelial state, then the cells cannot undergo their muscles originate from the somites (Christ et al., 1977). normal programme of differentiation as seen by the Somitic cells migrate as undifferentiated precursors into the delayed onset of genes marking the differentiation of the limb bud, where they execute their myogenic programme sclerotome or myotome. We provide conclusive evidence of development. Since the limb outgrowth programme is that Wnt6, which is expressed in the dorsal ectoderm, can not affected by the implantation of Wnt6-producing cells, substitute for the dorsal ectoderm in its ability to regulate we assume that the expression of the migration-inducing the development of epithelial somites. We also noted that agent Scatter Factor (Brand-Saberi et al., 1996a,b; Dietrich there was a remnant of an epithelial somite at the dorsal et al., 1999; Scaal et al., 1999) is not altered but is medial aspect following ectoderm ablation suggesting that nevertheless not sufficient to induce limb muscle migration. factors others than Wnt6 can initiate epithelialisation. This These results suggest that there must also be a modulation action could be mediated through another member of the of the epithelial organisation of the hypaxial lip to facilitate Wnt protein family since numerous members are the migratory process. expressed in this region. Since epithelial organisation C. Schmidt et al. / Developmental Biology 271 (2004) 198–209 207 was lost following medial barrier implantation, when the References dorsal ectoderm was intact, this would suggest that a signal from medial structures could be responsible for Balling, R., Neubu¨ser, A., Christ, B., 1996. 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Differential expression of the Wnt putative receptors Frizzled ventral portion, it is difficult to envisage how a gradient of during mouse somitogenesis. Mech. Dev. 89, 173–177. Wnt6 could be generated from a relatively flat epithelium Brand-Saberi, B., Wilting, J., Ebensperger, C., Christ, B., 1996a. The formation of somite compartments in the avian embryo. Int. J. Dev. Biol. to solely target this region. We suggest that the medial- 40, 411–420. ventral portion of the somite escapes the epithelialising Brand-Saberi, B., Mueller, T.S., Wilting, J., Christ, B., Birchmeier, C., properties of Wnt6 through the action of antagonists. 1996b. Scatter factor/hepatocyte growth factor (SF/HGF) induces emi- Furthermore, the expression and regulation of the Wnts gration of myogenic cells at interlimb level in vivo. Dev. Biol. 179, and their antagonists could explain the formation of 303–308. Burgess, R., Cserjesi, P., Ligon, K.L., Olson, E.N., 1995. Paraxis: a basic somites. 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