Wnt signaling in somite development

ARTICLE IN PRESS Ann Anat 190 (2008) 208—222

www.elsevier.de/aanat

INVITED REVIEW

Wnt signaling in somite development Poongodi Geetha-Loganathan, Suresh Nimmagadda1, Martin Scaal, Ruijin Huang, Bodo Christ Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, Albertstrasse 17, D-79104 Freiburg, Germany Received 7 December 2007; accepted 10 December 2007

KEYWORDS Wnts; Somite formation; Patterning; Differentiation

Summary During vertebrate embryogenesis, specialized mesodermal structures, called somites, give rise to a variety of mesodermal tissues including skeletal muscles, vertebrae and dermis. Development of the somites is a rhythmic process that involves a series of steps including segmentation of the paraxial mesoderm, epithelialization, somite formation, somite maturation, somite patterning and differentiation of somitic cells into different lineages. Wnt signaling has been found to play crucial roles in multiple steps of somite development. In this review, we present a brief overview of current knowledge on Wnt signaling events during the development of somites and their derivatives. & 2008 Elsevier GmbH. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt signaling during somite formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epithelialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Segmental clock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt signaling and somite patterning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt signaling and somitic cell lineages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The muscle lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other somitic derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

209 210 210 212 212 213 213 215 217 217

Corresponding author. Current address: Department of Oral Health Sciences, Life Sciences Institute, University of British Columbia,

Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. Tel.: +1 604 822 0749; fax: +1 604 822 2316. E-mail addresses: [email protected], [email protected] (P. Geetha-Loganathan). 1 Current address: Department of Oral Health Sciences, Life Sciences Institute, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. 0940-9602/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2007.12.003

ARTICLE IN PRESS Wnt signaling in somite development

Introduction Somites are segmental units of the bilateral paraxial mesoderm (PM) lying lateral to the neural tube and notochord, and medial to the intermediate and lateral plate mesoderm. Dorsally, the somites are covered by surface ectoderm, and ventrally they are closely opposed to the endoderm and the aorta (Christ and Ordahl, 1995). A basal lamina surrounds each newly formed somite, connecting the somite with surrounding structures. Somites are generated sequentially in an anterior to posterior direction. The PM arises by ingression of mesoderm precursor cells through the primitive streak during gastrulation. It forms a uniform band of mesenchymal tissues that flanks the neural tube and the notochord. The PM differs from other compartments of the mesoderm by its ability to undergo segmentation and form somites (Keynes and Stern, 1988; Christ and Ordahl, 1995). The formation of somites is preceded by an epithelia-

209 lization of the segmental plate (SP) mesoderm (Figure 1; Christ et al., 1972a, b). During epithelialization of the SP, the mesenchymal core, giving rise to somitocoel cells, is separated from the outer epithelium forming the wall of the somite (Christ et al., 1972a, b). Somitic maturation is accompanied by a commitment of somite cells to different lineages in response to distinct signals emanating from adjacent tissues. The somites at stages I–III (according to Christ and Ordahl, 1995) are still plastic with respect to their developmental fate. If their cells are experimentally moved to a new position, they differentiate according to their new environment (Aoyama and Asamoto, 1988; Christ et al., 1992; Ordahl and Le Douarin, 1992). The first compartments to be determined are the cranial and caudal halves (Stern et al., 1986). This cranio-caudal decision is made during somite formation (Stern et al., 1988) and is morphologically restricted to the sclerotome originating from the ventral portion of the somite (Christ and

Figure 1. Scanning electron microscopic views of a 2-day embryo (A), an epithelial somite (C) and a matured somite (B) (ao aorta, dm dermomyotome, ds dorsal somite, nt neural tube, nc notochord, sc sclerotome, so somitocoel, sm somatopleure, sp splanchnopleure, vs ventral somite, wd Wolffian duct) (courtesy of Dr. Heinz Ju ¨rgen Jacob, Bochum, Germany).

ARTICLE IN PRESS 210 Ordahl, 1995). The most striking event during early somite maturation is the epithelio-mesenchymal transition of the ventral part, leading to the sclerotome that gives rise to the axial skeleton and the ribs (Trelstad et al., 1967; Duband et al., 1987; Balling et al., 1996). The dorsal part of the somite retains its epithelial organization and is known as the dermomyotome because it contains precursors for both muscle and dermis (Christ and Ordahl, 1995). The differentiation of this epithelium depends on signals emanating from adjacent tissues. The dermomyotome is an epithelial layer, the four edges or lips (medial, lateral, cranial and caudal) of which are inwardly curved. The dermomyotome gives rise to precursors to all of the epaxial (back) and hypaxial (limb and ventral body wall) musculature (Christ et al., 1977, 1978). Epaxial muscle was shown to originate from the medial half and hypaxial muscle from the lateral half of the somite (Ordahl and Le Douarin, 1992). Wnts are signaling molecules regulating various developmental processes, such as proliferation, asymmetric division, patterning and cell fate determination (Wodarz and Nusse, 1998; Peifer and Polakis, 2000; Huelsken and Birchmeier, 2001; Moon et al., 1997) (http://www.stanford.edu/ rnusse/wntwindow.html). They function as signals via either the canonical pathway or at least two different noncanonical pathways (Figure 2; Kuhl et al., 2000; Niehrs et al., 2001; Winklbauer et al., 2001). Wnt/b-catenin signaling mediates b-catenin accumulation in the cytoplasm and translocation into the nucleus, where it forms a complex with the Tcf/Lef transcription factors to regulate the expression of target genes (Wodarz and Nusse, 1998; Peifer and Polakis, 2000; Chan and Struhl, 2002; Bienz and Clevers, 2003). In addition, a ‘non-canonical’ Wnt/Ca2+ pathway leads to the activation of downstream genes involved in patterning (Kuhl et al., 2000). The Wnt/Ca2+ pathway leads to release of intracellular calcium, possibly via G-proteins (Sheldahl et al., 1999; Kuhl et al., 2000). This pathway involves activation of phospholipase C and protein kinase C (PKC). A Wnt/JNK pathway plays a role in cell polarity, asymmetrical cell division and apoptosis (Boutros et al., 1998; Lisovsky et al., 2002; Yamanaka et al., 2002). In this planar cell polarity pathway, frizzled activates JNK and directs asymmetric cytoskeletal organization and coordinated polarization of cells within the plane of epithelial sheets (Boutros et al., 1998; Li et al., 1999). Complex crosstalk between these ‘canonical’ and ‘non-canonical’ pathways may regulate the cellular readout of Wnt signaling (Pandur et al., 2002). The aim of this review is to discuss the involvement of Wnts in somite development.

P. Geetha-Loganathan et al.

Wnt signaling during somite formation Epithelialization The PM at the trunk level consists of somites and, in the caudal portion, the SP that extends from Hensen’s node, or tail bud, to the most recently formed somite. The N-cadherin/catenin complex expressed within the SP is essential in defining and recruiting a population of cells that are incorporated into a single somite. Bellairs et al. (1978) have shown that cell adhesion increases during SP maturation. Since the canonical Wnt signal transduction pathway leads to an accumulation of b-catenin (Hinck et al., 1994) that links the cadherins to the cytoskeleton, it has been suggested that Wnts promote the process of SP epithelialization. b-catenin which, being the central player of canonical Wnt signaling, is an initiator signal for somite formation, and N-cadherin/ b-catenin-mediated adhesion may be an important component in the complex series of events that have been proposed for the initiation and formation of somites (Linask et al., 1998). The Eph family of receptors tyrosin kinases and their ligands, the ephrins, are also assumed to be involved in the regulation of cytoskeletal organization. EphA4 is expressed in the SP in a region in which cells undergo changes in cell shape as a prelude to somite formation and patterning of somite boundaries (Bergemann et al., 1995; Schmidt et al., 2001). Signals from the surface ectoderm are required for the induction of EphA4 which has been suggested to act downstream of Paraxis (Schmidt et al., 2001). Wnt6 is a candidate mediator for contact-dependent inductive activity of the surface ectoderm on the underlying somite epithelium, the cells of which extend filamentous processes through the ECM to contact the ectodermal cells (Fan et al., 1997; Cauthen et al., 2001, Linker et al., 2005, Schmidt et al., 2004). Wnt-3a signaling may play a role in regulating paraxial mesodermal fates, at the expense of neurectodermal fates, within the primitive ectoderm of the gastrulating mouse embryo. In Wnt-3a mutants, cells that have been ingressed through the primitive streak do not migrate laterally but remain under the streak and form an additional neural tube (Yoshikawa et al., 1997). In the cranial half of the SP, cells become polarized with their long axes perpendicular to the surface ectoderm and begin to express Paraxis, a basic helix–loop–helix transcription factor. Paraxis expression is a prerequisite for somite epithelialization (Burgess et al., 1996; Barnes et al., 1997; Sosic et al., 1997). The induction of Paraxis

ARTICLE IN PRESS Wnt signaling in somite development

211

Figure 2. Three Wnt signaling pathways (modified from Huelsken and Behrens, 2002). (A) The Wnt/b-catenin pathway, Wnt signal is transduced through the frizzled receptor (Fz) via dishevelled (Dsh) which represses the axin/glycogen synthase kinase-3b (GSK3b) complex which induces the degradation of b-catenin. Accumulated cytoplasmic b-catenin is translocated to the nucleus where it binds with Tcf/Lef, activating the transcription of its target gene. (B) Another pathway activated in response to Wnt signaling signals via the small GTPases Rho and Cdc42 to c-Jun N-terminal kinase (JNK). (C) Another class of Wnts stimulate the release of intracellular Ca2+, activating protein kinase C (PKC) and Ca2+/ calmodulin-dependent kinase II (CamKII).

expression might be mediated by Wnts because Paraxis expression can be dramatically amplified after implantation of Wnt-1, -3a and -4 secreting cells (Wagner et al., 2000). The Wnt antagonist, Wif-1, is expressed in the PM and its superabundant expression in Xenopus embryos interferes with proper somite formation (Hsieh et al., 1999). Additionally, indirect evidence argues for a role of the Wnt pathway in the process of SP maturation. Frizzled-1, -2 and -7 have been shown to be expressed in the SP and the somites (Cauthen et al., 2001; Linker et al., 2003). It has been shown that the initiation and maintenance of Paraxis expression is dependent on inductive signals from the surface ectoderm (Sosic et al., 1997). Wnt6 is the only Wnt known to be expressed in the ectoderm overlying the cranial SP and early somites

of the chick embryo (Cauthen et al., 2001; Marcelle et al., 2002; Schubert et al., 2002; RodriguezNiedenfuhr et al., 2003; Geetha-Loganathan et al., 2006b), which makes it a likely candidate for playing a role in this process. Wnt6 promotes epithelial organization of the SP mesenchyme (Schmidt et al., 2004). Recently it has been shown that Wnt11 promotes the expression of Paraxis at the expense of Pax-1 which is a sclerotome marker (Geetha-Loganathan et al., 2006b). b-Catenin activity, initiated by Wnt6 through Fz7 and mediated by paraxis, is required for the maintenance of the epithelial organization of somites (Linker et al., 2005). Recently we have shown that Wnt11 expression in the dorsomedial lip (DML) inhibits Wnt6 expression in the ectoderm overlying the medial and central dermomyotome, thus

ARTICLE IN PRESS 212 restricting Wnt6 expression to the lateral ectoderm overlying the ventrolateral lip (VLL.). Both Wnt6 and Wnt11 are epithelialization factors, the DML (via Wnt11) and the VLL (via Wnt6) remain epithelial and contribute to dermomyotomal growth (Geetha-Loganathan et al., 2006b). Taken together, these findings suggest that Wnts promote the process of epithelialization.

Segmental clock Formation of the somites is a rhythmic process that involves an oscillator – the segmentation clock – which acts in the PM. The clock ticks in somite precursors and halts when they reach a specific maturation stage defined as the wave front. This process converts the temporal oscillations into the periodic spatial pattern of somite boundaries. In vertebrates, genes of the Notch–Delta signaling pathway, including the Notch-responsive HES transcription factors, the Notch ligand Delta-like (Dll) and the Notch antagonist lunatic fringe (Lnfg), have been found to cycle during somitogenesis (Pourquie, 2003). Wnt signaling is involved in the process of oscillation in combination with other signaling molecules such as Notch and FGFs. The inhibitor of Wnt signaling Axin2 has been identified as a cycling gene (Aulehla et al., 2003). In the mouse, axin2 is expressed in a dynamic sequence similar to, but out of phase with that of the Notchrelated cyclic genes. Axin2 is directly regulated by Wnt signaling and could participate in the establishment of an auto-regulatory negative feedback loop involved in its periodic expression. Axin2 oscillations persist in Notch pathway mutants, whereas both axin2 and lunatic fringe oscillations are disrupted in wnt3a mutants, indicating that Wnt signaling acts upstream to the Notch-regulated cyclic genes (Pourquie, 2003). Therefore, in the mouse, the segmentation clock appears to be composed of a Wnt-based regulatory loop entraining a series of Notch-based loops. Aside from the Axin2 feedback mechanism, it had been shown that many other negative regulators of the Wnt/ b-catenin pathway are also direct target genes, and that the Wnt/b-catenin pathway is capable of generating oscillatory behavior independently of external factors (Aulehla and Herrmann, 2004; Wawra et al., 2007). In addition, inactivation of the Wnt inhibitors Sfrp1 and Sfrp2 perturbed the oscillating expression of Nkd1, an oscillator gene activated downstream of Wnt signaling in the PSM (Ishikawa et al., 2004). Wnt3A has recently been proposed to play a role similar to that of FGF8 by establishing a

P. Geetha-Loganathan et al. gradient controlling segmentation in the PSM (Aulehla et al., 2003; Rodriguez-Gonzalez et al., 2007). A reduction in Wnt3a expression in mice carrying a hypomorphic allele of Wnt3a results in alterations in vertebrate identity and oscillating Notch activity (Greco et al., 1996; Ikeya and Takada, 2001; Aulehla et al., 2003). Wnt3a, via the downstream transcription factors LEF1/TCF, has been suggested to directly regulate Dll1, thus providing a molecular link between the Wnt and Notch signaling pathways in somitogenesis (Hofmann et al., 2004; Galceran et al., 2004).

Wnt signaling and somite patterning The formation of the dermomyotome is a result of the dorsoventral patterning of the somite which is controlled by an antagonistic action of ventralizing and dorsalizing signals originating from adjacent tissues and tranversing the ECM that surrounds the somite. Dorsalizing signals originate from the dorsal part of the neural tube and the surface ectoderm (Spence et al., 1996). In the absence of these structures, the epithelial organization of the dorsal somite compartment is lost and Pax3 expression is down-regulated. Ectopically expressed Wnt1 in the presomitic mesoderm (PSM) of chick embryos completely represses sclerotomal markers and enhances and expands expression of dermomyotomal markers (Capdevila et al., 1998). Delivery of an activated form of b-catenin to somitic mesoderm mimics the effect of Wnt1. Rashbass et al. (1994) have shown that Wnt8 can act as a dorsalizing signal. In Xenopus embryos, injection of XWnt-8 mRNA into the vegetal pole of oocytes, or into ventral blastomeres of early cleaving embryos, can produce a duplicated embryonic axis and an alteration in the fate of ventral cells, such that they differentiate into dorsal rather than ventral mesoderm (Smith and Harland, 1991; Sokol et al., 1991). Wnt/b-catenin function activates somite-specific expression of Gli2 which becomes restricted to the myotome in the dorsal somite during the initiation of somite (Borycki et al., 1998, 2000). Fan et al. (1997) have shown that cells expressing Wnt-1, -3a, -4 and -6 can induce and maintain the expression of the dermomyotome markers Pax-3, -7 and Sim1 in tissue culture. In vivo ectopically implanted Wnt- 1, -3a and -4 secreting cells alter the process of dorsoventral somite compartmentalization (Wagner et al., 2000). Implantation of Wnt-secreting cells ventrally, between neural tube/notochord and epithelial somites of chicken embryos, leads to an enlarged dorsal somite compartment at the expense of the

ARTICLE IN PRESS Wnt signaling in somite development ventral compartment. This shift of the border between the muscle- and the skeleton-forming areas was identified by an increase in the Pax3 expression domain and a complete loss of Pax1 expression. In mouse embryos lacking both Wnt-1 and-3a the medial part of the dermomyotome is not formed and the expression of the lateral dermomyotome marker gene, Sim1, is expanded medially, indicating that different members of the Wnt family are required to control the mediolateral pattern of the dermomyotome (Olivera-Martinez et al., 2004). Recently, it has been shown that Wnt1, 3a, 4 and 6 proteins can upregulate and expand the expression of Pax3 and Pax7 within the dorsal somite. Additionally, Wnt6 can mimic the effect of the dorsal ectoderm in maintaining Pax3 and Pax7 expression (Otto et al., 2006). Somitogenesis is a principal component of anterior–posterior (AP) growth of the embryo. Members of the Wnt signaling pathway have been implicated in regulating AP patterning. Wnt3a, involved in the control of positional information along the body axis (Ikeya and Takada, 2001), is also required for the elongation of the body axis (Greco et al., 1996; Takada et al., 1994; Aulehla et al., 2003). Thus, Wnt3a appears to control and integrate all three processes, body axis elongation, allocation of positional information and segmentation. Mouse embryos having a point mutation in Lrp6, which encodes a Wnt co-receptor, show defects in the establishment of the AP somite compartmentalization and the formation of segment borders which leads to vertebral malformations (Kokubu et al., 2004). Satoh et al. (2006) have shown that Sfrp1 and Sfrp2 are required for AP axis elongation and somitogenesis in the thoracic region during mouse embryogenesis. A shortened thoracic region appears to be the consequence of AP axis reduction and incomplete somite segmentation. Aberrant somite segmentation is associated with altered oscillations of Notch signaling, providing a link between the Wnt and Notch signaling pathways in AP patterning. In Zebrafish and Xenopus embryos, Wnt8 and Tbx6, acting by a positive regulatory loop, are involved in the specification of posterior mesoderm and thus function in the same cascade to regulate different aspects of AP patterning (Uchiyama et al., 2001; Szeto and Kimelman, 2004). In Amphioxus, AmphiWnt3, AmphiWnt5 and AmphiWnt6 are each expressed in a specific subregion of the tail bud, tentatively suggesting that a combinatorial code of developmental gene expression may help generate specific tissues during posterior elongation and somitogenesis (Schubert et al., 2001).

213 The dermomyotome is patterned along the mediolateral axis into medial, central and lateral compartments, which contain progenitors of epaxial muscle, dermis and hypaxial muscle, respectively. The surface ectoderm can induce formation and/or maintenance of the dermomyotome (Fan and Tessier-Lavigne, 1994; Spence et al., 1996). Wnt6 is expressed throughout the entire ectoderm at the SP and epithelial somite levels, whereas its expression becomes restricted to the lateral ectoderm as the somite matures (Rodriguez-Niedenfuhr et al., 2003; Geetha-Loganathan et al., 2006b). We showed that Wnt11 is a mesoderm-intrinsic epithelialization factor that, upon induction by the neural tube (Marcelle et al., 1997), maintains the epithelial state of the DML while restricting Wnt6 expression to the ectoderm overlying the VLL (Geetha-Loganathan et al., 2006b). It is not known whether Wnt11 is furthermore required for the cell movements during myotomal cell recruitment in the DML. Thus, Wnt11 and Wnt6 maintain the epithelial nature of DML and VLL, respectively, allowing the central domain of the dermomyotome to deepithelialize to form dermis and muscle (Gros et al., 2005), which suggests a role of Wnt signaling in patterning the dermomyotome along its mediolateral axis. Wnt3a is expressed in the node and is required for LR asymmetry and segmentation. Wnt3a activates the Delta/Notch pathway to regulate perinodal expression of the left determinant Nodal, while simultaneously controlling the segmentation clock and the molecular oscillations of the Wnt/ b-catenin and Notch pathways (Nakaya et al., 2005). Thus, Wnt3a links the segmentation clock and AP axis elongation with key left-determining events, suggesting that Wnt3a is an integral component of the trunk organizer. Wnt3a also regulates Nodal expression via Dll1. Activation of Nodal in the lateral aspects of the node establishes an axis of Nodal expression that is perpendicular to the AP axis, leading to the orthogonal orientation of the LR axis (Galceran et al., 2004; Hofmann et al., 2004; Krebs et al., 2003; Raya et al., 2003).

Wnt signaling and somitic cell lineages The muscle lineage Skeletal muscle precursor cells originate from the dermomyotome (Christ and Ordahl, 1995). It has been demonstrated that myogenic differentiation within the somite is governed by positive and

ARTICLE IN PRESS 214 negative signals from surrounding tissues (Borycki et al., 1998; Hirsinger et al., 1997; Johnson et al., 1994; Marcelle et al., 1997; Munsterberg et al., 1995; Pourquie ´ et al., 1996; Pownall et al., 1996; Reshef et al., 1998; Stern et al., 1995, 1997). Known myogenic signals are derived from both axial midline structures (neural tube, floor plate and notochord; Buffinger and Stockdale, 1994; Munsterberg and Lassar, 1995; Pownall et al., 1996; Stern and Hauschka, 1995) and from lateral plate mesoderm (Pourquie et al., 1995). Precursors of the epaxial myotome reside within the medial half of the dermomyotomal epithelium. The process of epaxial muscle cell differentiation requires signals from axial structures. It has been shown that Wnt -1, -3a and -4 are expressed in the dorsal half of the neural tube at the time when epaxial myogenesis begins (Parr et al., 1993; Hollyday et al., 1995; Cauthen et al., 2001) inducing somitic myogenesis (Munsterberg et al., 1995). In contrast, Wnt-7a and -7b, which do not induce somitic myogenesis in vitro, are expressed mainly in the ventral half of the neural tube (Parr et al., 1993). In explants of PSM from mouse embryos, Wnt1, produced in the dorsal neural tube, induces myogenesis through the preferential activation of Myf5, whereas Wnt7a or Wnt6, produced in the dorsal ectoderm, preferentially activate MyoD (Tajbakhsh et al., 1998). Over-expression of Wnt -1, -3a and -4 in the avian embryo at the level of the epithelial somites results in a considerable enlargement of the epaxial muscle domain at the expense of the sclerotome (Wagner et al., 2000). The shift of the border between the muscleforming and skeletal-forming compartments is accompanied by an up-regulation of Paraxis, Pax3, MyoD and Desmin in the premuscular compartment, and a complete down-regulation of the sclerotome marker Pax1. Knockout studies performed in the mouse further demonstrate that Wnt1 and Wnt-3a are required for the establishment of the medial compartment of the dermomyotome. The loss of the medial dermomyotome in these mice is accompanied by a reduction in the levels of Pax-3 and Myf-5 transcripts as well as a disorganized myotome (Ikeya and Takada, 1998; Galli et al., 2004). Schmidt et al. (2000) have shown that a number of genes known to act downstream to Wnt, i.e., Fz1, b-catenin and Lef1, are expressed in a dynamic pattern during somitogenesis. b-catenin and Lef1 transcripts become restricted to the medial somite, high levels of expression emerging in the DML of the dermomyotome as it forms during somitogenesis. In the mouse, expression of Fz1 is restricted to the medial edge of newly formed somites in a pattern similar to that

P. Geetha-Loganathan et al. observed for b-catenin and Lef. Wnt-1 signaling from the neural tube is necessary for both upregulation of b-catenin expression and activation of MyoD expression in the epaxial myotome (Borello et al., 1999). At the onset of somitogenesis, the activation of Gli1-mediated signals together with the Wnt/b-catenin pathway leads to the correct activation of Myf5 transcription in the epaxial domain. Recently it has been shown that in muscle progenitor cells in the epaxial domain of newly formed somites, Wnt signaling is transduced through the canonical b-catenin pathway, mediated by at least two Frizzled receptors, Fz1 and/or Fz6 (Borello et al., 2006). Wnt proteins have an additional effect on epaxial myogenesis by modulating Shh signal transduction through the regulation of Gli2 and Gli3, genes encoding zinc finger transcription factors acting as effectors of Shh signaling in responding cells (Borycki et al., 2000). Lee et al. (2000) have shown that Shh up-regulates not only its own receptor, Ptc1, but also Sfrp2 which reduces the dermomyotome-inducing activity of Wnt1 and -4. Myf5 has been shown to be a direct target of Wnt/ b-catenin signaling, its full activation requiring a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells (Borello et al., 2006). Epaxial myogenesis requires BMP4 antagonizing signaling (Hirsinger et al., 1997). BMP4 is expressed in the LPM and tends to lateralize the dermomyotome (Pourquie et al., 1995). The lateralization of the medial part of the DM is prevented by Noggin expression in the DML in response to Wnt1 (Hirsinger et al., 1997; Marcelle et al., 1997; Reshef et al., 1998; Geetha-Loganathan et al., 2006b; Nimmagadda et al., 2007). In the Wnt1/Wnt3a double mutant mouse, Noggin and Wnt11 expression is lost and the medial dermomyotome is lacking. The requirement of Wnt proteins for muscle development has been confirmed by transplacental delivery of the Wnt antagonist FrzB1 in the mouse (Borello et al., 1999). Over-expression of FrzB1 causes a marked reduction in myogenesis in the embryo, and several genes downstream of the Wnt signaling pathway, such as En1, Noggin and Myf5 are down-regulated. Wnt5b is expressed in cells of the dorsomedial part of the somite and Wnt11 in cells of the DML of the dermomyotome from which the epaxial myotomal cells arise (Marcelle et al., 1997; Cauthen et al., 2001; Geetha-Loganathan et al., 2006b). In the chick, the Wnt antagonist Sfrp2 has been shown to be expressed in the DML and VLL of the dermomyotome, where it may promote proliferation and prevent precocious differentiation of myogenic

ARTICLE IN PRESS Wnt signaling in somite development dermomyotome cells (Ladher et al., 2000). Thus, myotome formation, which is known to be dependent on both the neural tube (Christ et al., 1992; Rong et al., 1992; Bober et al., 1994) and notochord (Goulding and Paquette, 1994), may be cued specifically by the combined action of Wnt family members in the dorsal half of the neural tube and Shh expressed by the ventral midline tissues. Hypaxial muscle precursors delaminate from the VLL of the dermomyotome and migrate to peripheral target sites, including limbs, the tongue, the diaphragm, whereas at the interlimb level, muscles of the ventrolateral body wall form by ventrolateral expansion of the hypaxial myotomal sheets. Unlike epaxial myogenesis, early steps of hypaxial myogenesis do not require Shh signaling (Rong et al., 1992). It has been shown that the surface ectoderm is required to form the hypaxial dermomyotome and is either necessary to maintain or initiate the expression of several dermomyotomal markers (Kenny-Mobbs and Thorogood, 1987; Fan and Tessier-Lavigne, 1994; Kuratani et al., 1994; Schmidt et al., 2001). Ectodermal signals suggested to be involved in the specification and differentiation of lateral dermomyotomal cells include Wnt -3, -4, -5a, -6, -7a, -7b (Parr et al., 1993; Roelink, 1996; Dietrich et al., 1998). In explants of mouse PM the activation of hypaxial myogenesis could be mimicked by Wnt7a (and to a minor extent also by other Wnts such as Wnt4 and Wnt5a), which can replace the surface ectoderm in activating hypaxial myogenesis (Borello et al., 1999). Further, Wnt7a and Wnt6 produced in the dorsal ectoderm preferentially activate MyoD (Cossu et al., 1996; Tajbakhsh et al., 1998), which can occur in the absence of Myf5/Mrf4. In the chick embryo, Wnt6 is expressed in the surface ectoderm immediately adjacent to the Pax3 expression domain in the dorsal somite (Cauthen et al., 2001; GeethaLoganathan et al., 2006b). It has been shown that Wnt6 specifically induces the formation of muscle cells by correspondingly inducing Myf5-dependent and inhibiting MyoD-dependent myogenic pathways during limb myogenesis (Geetha-Loganathan et al., 2005, 2006a). It has not been determined whether the specification of hypaxial muscle precursor cells takes place within the dermomyotome. Naked Cuticle (Nkd1) that acts as an intracellular switch to promote the non-canonical pathway at the expense of non-canonical Wnt signaling has been shown to be positively regulated by Wnt signaling from the neural tube (Schmidt et al., 2006). It is expressed in the region in which cells undergo movements (especially in DM lips) during somite development, suggesting that non-canonical Wnt signaling plays a part in myotome formation.

215 Recently, in explants of PSM, it has been shown that MyoD expression can be activated in the absence of an active Wnt/b-catenin pathway and of Myf5 (Brunelli et al., 2007). Expression of MyoD is Pax3 dependent, and Pax3 transcriptional activity is modulated by PKC, by direct or indirect phosphorylation of the factor itself or putative co-factors. Thus, epaxial and hypaxial myogenesis seem to be regulated by b-catenin/canonical and PKC/noncanonical Wnt pathways, respectively. In mouse explant cultures, adenylyl cyclase signaling via PKA and its target transcription factor CREB have been shown to be important for Wnt-directed myogenic gene expression (Chen et al., 2005). CREB mutants have defects in the medial and lateral edges as well as in the myotome proper, indicating the influence of Wnt signaling on both epaxial and hypaxial myogenesis.

Other somitic derivatives During somite maturation, the ventral portion undergoes an epithelio-mesenchymal transition forming the sclerotome. During this process, Pax3, which is initially expressed throughout the PM, becomes restricted to the dorsal part of the somite (Goulding and Paquette, 1994), while Pax1 and Pax9 are expressed in the ventral portion, which is designated to form sclerotome. The sclerotome gives rise to various mesodermal structures including the vertebral column and ribs (Christ et al., 2004). In the chick, it has been shown that ectopic implantation of various Wntsecreting cells (Wnt -1, -3a, -4, -6, -11) resulted in an expansion of the dorsal somite compartment (shown by upregulation of dorsal marker expression) at the expense of the ventral compartment in which Pax1 expression is lost (Wagner et al., 2000; Schmidt et al., 2004; Geetha-Loganathan et al., 2006b). Further, Sfrp2 is expressed in the ventral somite (Terry et al., 2000) as well as in the neural tube and dermomyotomal lips which counteracts the effect of dorsalizing Wnt signals to induce myogenesis (Ladher et al., 2000). Similarly, carboxypeptidase Z, which binds to Wnt4, can upregulate the expression of Pax3 in the hypaxial dermomyotome and downregulate Pax1 in cells originally fated to form scapula and ribs (Moeller et al., 2003). Thus neural tube and ectodermal Wnts negatively regulate Pax1 expression and chondrogenic differentiation. Zeng et al. (1997) have shown that the mouse Fused locus encodes Axin, an inhibitor of the Wnt/beta-catenin signaling pathway affects vertebral segmentation with rib malformation.

ARTICLE IN PRESS 216 The Wnt pathway has been found to be implicated in the control of somitogenesis (Aulehla et al., 2003; Hamblet et al., 2002) and of bone mass in humans and mice (Boyden et al., 2002; Gong et al., 2001; Kato et al., 2002; Little et al., 2002). Positional cloning of the gene responsible for osteoporosis pseudoglioma syndrome, an autosomal recessive disorder in humans, revealed that loss-offunction mutations in LRP5 (receptor of Wnt) lead to a low bone mass phenotype (osteoporosis) (Gong et al., 2001). Lrp6 has shown to be one of the key genetic components for the pathogenesis of vertebral segmentation defects and of osteoporosis in humans (Kokubu et al., 2004). The homozygous ringelschwanz (rs) phenotype in the mouse is caused by a point mutation in Lrp6, mutants showing a characteristic form of vertebral malformations, similar to dysmorphologies in individuals suffering from spondylocostal dysostosis. Marker expression studies suggests that Lrp6 is required for the crosstalk between the Wnt and Notch–Delta signaling pathways during somitogenesis. Thus, Wnt signaling plays a significant role during somite segmentation and skeletal development. Inter-specific grafting experiments using the quail-chick marker system showed that the dermis of the back is derived from the dermomyotome (Christ et al., 1983; Christ and Ordahl, 1995). Several Wnts such as Wnt-3a, -5a, -7a, -10a, -10b, -11; a number of genes acting downstream to Wnt such as Fz1, b- catenin and Lef1; as well as antagonists of the Wnt signaling pathway such as Dkk1 are expressed in dermal cells and have been implicated to play a role in dermal development (St-Jacques et al., 1998; Millar et al., 1999; Kishimoto et al., 2000; Reddy et al., 2001). Kishimoto et al. (2000) have shown that Wnt3a and Wnt7a can act as inductive signals to maintain the dermal papillae. Wnt7a is expressed in the placode epithelium and can induce new placode formation in association with Msx1 (Chuong et al., 1996). In mouse embryos, Wnt10b is expressed in the epidermis of the hair buds (St-Jacques et al., 1998), suggesting a role in dermal papilla induction. Wnt1 has been shown to initiate Wnt11 expression in the DML, which itself has been implicated not only in myotomal but also in dorsal dermal development (Marcelle et al., 1997; Tanda et al., 1995). Grafting of Wnt1-producing cells in place of excised axial organs (neural tube plus notochord) specifically restores the expression of Wnt11 in the medial somite and, strikingly, the formation of a dense dorsal dermis (OliveraMartinez et al., 2004). In both chick and mouse embryos, Wnt11 is expressed in the DML of the

P. Geetha-Loganathan et al. dermomyotome and during later stages in the dense dermis (Tanda et al., 1995; Marcelle et al., 1997; Olivera-Martinez et al., 2004; Geetha-Loganathan, et al., 2006b). Expression of Lef1 was observed in ectodermal placodes as well as dermal papillae, and ectopic expression of Lef1 can induce ectopic hair formation (Zhou et al., 1995; Kratochwil et al., 1996). Further, Lef-1 null mice display a reduction in follicle numbers and lack whiskers, thereby demonstrating a role for Wnt signaling in hair development (van Genderen et al., 1994). In mouse embryos, over-expression of Dkk1 resulted in complete failure of placode formation (Andl et al., 2002), indicating that activation of Wnt signaling in the skin precedes, and is required for, the initiation of hair follicle placode formation. In mouse embryos, it has been shown that Wnt/ b-catenin signaling specifies the dermomyotomal cells to select the dorsal dermal fate (Atit et al., 2006). When the essential Wnt transducer b-catenin is mutated in En1 cells, it results in the loss of Dermo1-expressing dorsal dermal progenitors and dermis. Conversely, when b-catenin is activated in En1 cells, it induces Dermo1 expression in all cells of the En1 domain and disrupts muscle gene expression. Finally, endothelial precursor cells also reside in the somite. Wilting et al. (1994) have shown that all compartments of the epithelial somite give rise to angioblasts. However, the expression of vascular endothelial growth factor receptor II (VEGFR2/ Quek1) is expressed in the lateral portion of the somite. VEGFR-2-deficient mice failed to form yolksac blood islands and lacked organized blood vessel formation in the embryo proper (Shalaby et al., 1995). Thus, embryonic blood vessel formation depends on this receptor. Recently we have studied the regulation of VEGFR2 during quail somite development, where we showed that the induction of Quek1 expression and endothelial cell formation in the somites are mediated by a cooperative action of BMP4, FGF8, Wnt1 and Wnt3a (Nimmagadda et al., 2005, 2007). It remains to be elucidated whether they act in the same pathway or in different parallel pathways to induce VEGFR2. Implantation of Wnt-1/-3a-secreting cells induces Quek1 expression and endothelial cell formation in the somite, whereas over-expression of Sfrp-2 results in down-regulation of Quek1 expression and a reduction in the number of endothelial cells in the somite, suggesting the role of Wnt signaling in blood vessel development. Schemes show the expression of members of the Wnt signaling pathway during somite development (Figure 3). In conclusion, Wnt signaling is a key player in a multitude of developmental steps during somite

ARTICLE IN PRESS Wnt signaling in somite development

217 supported by the Deutsche Forschungsgemeinschaft (SFB-592, A1 to B.C. and M.S.) and the European Network of Excellence, MYORES (to B.C. and M.S.).

References

Figure 3. Schematic composite of expression patterns of members of the Wnt signaling pathway. (A) Representation of composite expression patterns in the epithelial somite of the chick embryo. Punctuate coloring represents areas of overlap. (B) Representation of composite expression patterns in the mature somite. Abbreviations: EC, ectoderm; LPM, lateral mesoderm; IM, intermediate mesoderm; S, somite; NT, neural tube; NC, notochord; DM, dermomyotome; MY, myotome; SC, sclerotome.

formation, maturation, specification and differentiation. Here, we present but a brief overview of recent advances in understanding of the interplay between various Wnt ligands, receptors, and downstream regulators participates in establishing diversity from a simple structure, the somite. The remaining challenge is to understand how somite cells interpret this plethora of Wnt signals, and how they link it to other signaling pathways to build the vertebrate body.

Acknowledgments We would like to thank Sara Hosseini for helping us in typing the manuscript. This study was

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