Role of Wnt-6 in limb myogenesis
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- Geetha-Loganathan, P., Nimmagadda, S., Huang, R. et al. Anat Embryol (2006) 211: 183. doi:10.1007/s00429-005-0069-6
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Cells from the ventrolateral lip of the dermomyotome at limb levels undergo epithelio-mesenchymal transition and migrate as individual and undifferentiated cells into the limb buds. The precursor cells are under the influence of various signaling factors in the limb. Dorsal and ventral ectoderm influences various aspects of limb development. In addition to our previous studies, we investigated the influence of ectoderm and Wnt-6 on somitic cells in the limb bud. We show that in the absence of ectoderm the precursor cells never form muscle cells but differentiate into endothelial cells. In addition, we show that Wnt-6 that is secreted from the ectoderm influences the precursor cells to form muscle even in the absence of ectoderm. This indicates that Wnt-6 is an ectodermal signal that induces somite-derived progenitor cells to form muscle cells during limb development.
KeywordsQuail–Chick chimeraMyogenic cellsEndothelial cellsEctodermWnt-6
The development of vertebrate limbs is a complex multi-step process which involves specification of the early limb field, directed outgrowth, and patterning to establish the final three dimensional structure of the limb. Precursors of limb musculature are provided by the lateral portion of somites at the level of the limb buds (Christ et al. 1974, 1977; Chevallier et al. 1977; Ordahl and Le Douarin 1992). The limb muscle precursor cells have been described to migrate from the dermomyotome as single cells into the limb bud (Jacob et al. 1978; Solursh et al. 1987). Their migration is initiated by an interaction between the signaling protein scatter factor/hepatocyte growth factor (SF/HGF) and its receptor c-met. The latter, a transmembrane tyrosine kinase receptor, is expressed in the ventrolateral edge of the dermomyotomes (Bladt et al. 1995).
In the limb, development of musculature proceeds in several steps including migration of progenitor cells, homing into the prospective muscle regions, myogenic specification, and differentiation to functional muscle. All of these steps have to be temporally and spatially coordinated and thus require communication between cells, a function which is mediated by specific extracellular signals. It is well known that the dorsal and ventral limb ectoderm influence myogenesis (Gasseling and Saunders 1961; Zwilling 1964; Amprino and Amprino-Bonetti 1967; Stark and Searls 1974; Flickinger 1974; MacCabe et al. 1974; Searls 1976; Kosher et al. 1979; Searls and Smith 1982; Bolender et al. 1993; Dietrich et al. 1998). At HH-stage 15–16, Wnt-6 is expressed in a subset of ectodermal cells located at the dorsoventral boundary in the limb field which corresponds to the prospective apical ectodermal ridge (Rodriguez-Niedenführ et al. 2003). From HH-stage 17, Wnt-6 is strongly expressed in both the dorsal and ventral limb ectoderm including the dorsoventral boundary. A role of Wnt-6 in the induction of the Myf5-dependent myogenic pathway in the chick limb has been reported recently (Geetha-Loganathan et al. 2005).
The current view is that ectodermal signaling is necessary for the progenitor cells to differentiate into functional muscle cells. We grafted the lateral portion of quail somites known to be the source of muscle precursor cells to the chick limb bud in the presence or absence of ectoderm (according to the experimental design, see below). To study the role of Wnt-6 on limb muscle formation, we transplanted the graft to the limb bud in the absence of ectoderm but along with Wnt-6 expressing CHO cells. Our results show that cells derived from the graft form muscle cells only in the presence of ectoderm but can form endothelial cells irrespective of ectodermal signaling. Wnt-6 can compensate for the absence of ectoderm and can induce the formation of muscle cells in the limb.
Materials and methods
Preparation of chick embryos
Fertilized chick (Gallus gallus) and quail eggs (Coturnix coturnix japonica) were incubated at 38°C, and the embryos were staged according to Hamburger and Hamilton (1951).
Grafting of lateral portion of the somite to limb bud
Lateral halves of the quail somites were grafted together with the covering ectoderm to the chick limb bud with intact ectoderm (schematically illustrated in Fig. 1a).
Lateral halves of the quail somites were grafted with ectoderm to the chick limb bud having the ectoderm removed (schematically illustrated in Fig. 2a).
Lateral halves of the quail somites without ectoderm were grafted to the chick limb bud also devoid of ectoderm (schematically illustrated in Fig. 3a).
The same experiment as described later was done except the graft was placed along with Wnt-6 expressing CHO cells (schematically illustrated in Fig. 4a).
Immunohistochemical staining of paraffin sections with QH1, a quail endothelial cell-specific monoclonal antibody (Pardanaud et al. 1987; Developmental Studies Hybridoma Bank, DSHB, University of Iowa, Iowa City).
Double staining of paraffin sections with QCPN (anti-quail antibody, DSHB) and polyclonal anti-desmin antibodies (Sigma, Deisenhofen, Germany).
Transplantation of somites from quail to chick limb buds in the presence or absence of ectoderm was used to analyze the influence of ectoderm on the formation of muscle cells in the limb. Operated embryos (80%) developed normally and could be analyzed after a re-incubation period of 5 days. Grafting of somites to the limb bud, both having their ectoderm intact, results in the formation of both endothelial and muscle cells in the limb (Fig. 1b, c). Quail endothelial and muscle cells were seen even after grafting of quail somites with ectoderm to the limb bud devoid of ectoderm (Fig. 2b, c). However, when grafting was done in the absence of ectoderm, only quail endothelial cells were seen and no quail myogenic cells were identified in the limb muscles (Fig. 3b, c). These data prove that somite-derived precursor cells in the limb require ectodermal signaling to form muscle. To identify the signal from the ectoderm responsible for this activity, we hypothesized Wnt-6 may have a positive influence on limb muscle formation. Previous studies have shown that Wnt-6 is expressed in the dorsal and ventral limb ectoderm (Rodriguez-Niedenführ et al. 2003; Loganathan et al. 2005) and that Wnt-6 promotes the Myf5 dependent pathway during limb myogenesis (Geetha-Loganathan et al. 2005). To check the possible influence of Wnt-6 on the precursor cells to form limb muscle, we performed the grafting of somites to the limb bud in the absence of ectoderm but along with Wnt-6 expressing cells. After reincubation numerous quail cells were found to be located in the limb muscles and were identified as myocytes (Fig. 4b). This proves that Wnt-6 from the ectoderm can induce the formation of limb muscle cells.
Previous studies have shown that not all of the cells in the extremities are born within the limb buds, but derive exclusively or mostly from other sources. These include skeletal muscle, blood vascular endothelial cells, and lymphatic endothelial cells, all of which are derived from the somites (Christ et al. 1974, 1993; Le Douarin 1982; Pardanaud and Dieterlen-Lièvre 1993; Wilting et al. 1995). Precursors of limb musculature are provided by the lateral portion of somites at the level of the limb buds (Christ et al. 1974, 1977; Chevallier et al. 1977; Ordahl and Le Douarin 1992). These progenitor cells migrate from the dermomyotome towards the limb. They then aggregate and differentiate into dorsal and ventral premuscular masses within the limb mesenchyme (Christ et al. 1977). In the limb, the cells are subject to various signaling pathways operated by secreted factors controlling limb development. The premyogenic (Pax3 expressing) and early myogenic (MyoD/Myf5 expressing) cells form the premuscular masses, which are dense collections of cells located beneath the subectodermal mesenchyme (Christ and Ordahl 1995; Amthor et al. 1998) suggesting an interaction with the ectoderm. It has also been shown that the dorsal and ventral ectoderm controls the dorsoventral polarity of the limb bud (Pautou and Kieny 1973; MacCabe et al. 1974; Gavin et al. 1990; Dealy et al. 1993; Parr et al. 1993; Parr and McMahon 1995). After limb ectoderm removal, the limb mesoderm cells differentiate into cartilage and connective tissue but not into muscle (Searls and Smith 1982), indicating that surface ectoderm is necessary for limb muscle formation. It has been recently shown by us that expression of myogenic markers requires ectodermal signaling throughout limb development (Geetha-Loganathan et al. 2005). By grafting the lateral portion of quail somites to the chick limb in the presence and absence of ectoderm, we reinvestigated the necessity of the ectoderm for the cells to differentiate into muscle during limb bud development. Somitic cells that have invaded the limb can differentiate into both endothelial as well as muscle cells in the presence of ectoderm, whereas formation of muscle cells were never observed when there was no interaction with the ectoderm. This proves that formation of endothelial cells does not depend on ectodermal signaling whereas the progenitor cells require interaction with the ectoderm to develop into muscle during limb development.
A number of Wnt molecules, have been shown to be implicated in playing specific roles during vertebrate limb development, including induction of the early limb bud, formation and maintenance of a specific ectodermal structure known as the apical ectodermal ridge (AER), outgrowth of the limb, and patterning of the limb bud axes (Dealy et al. 1993; Parr et al. 1993; Kengaku et al. 1998; Galceran et al. 1999; Kawakami et al. 2001). Expression of Wnt-6 is observed at HH-stage 15–16, during the early period of limb bud formation in a subset of ectodermal cells located at the dorsoventral boundary in the limb field (Rodriguez-Niedenführ et al. 2003) and is also expressed in the dorsal and ventral ectoderm throughout limb development (Loganathan et al. 2005). This suggests that Wnt-6 plays a significant role during limb development.
We showed recently that Wnt-6 is the signal from the ectoderm that promotes the expression of myogenic markers (Geetha-Loganathan et al. 2005). In extension to this study, here we studied the influence of Wnt-6 on the precursor cells when they have reached the limb bud. Formation of limb muscle cells was identified when Wnt-6 signaling is present even in the absence of ectoderm proving that Wnt-6 can induce migrated precursor cells to form muscle during limb development. This proves that Wnt-6 is at least one of the signals from the ectoderm that influences the progenitor cells to form muscle during limb development. But it is still unclear whether the precursor cells are specified to form muscle before they migrate to the limb or become specified after reaching the limb bud under the influence of ectodermal signals. Therefore it is subject of future investigation to uncover the time when the migrating precursor cells are specified. To this end, we conclude that Wnt-6 is the signal from the ectoderm responsible for the formation of muscle during chick limb development.
We would like to thank Mrs. L. Koschny, Mrs. M. Schüttoff and Mr. G. Frank for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (SFB-592, A1 to B.C & M.S) and the European Network of Excellence, MYORES (B.C. and M.S.).