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Keywords:

  • chick embryo;
  • dermis;
  • dermomyotome;
  • sclerotome;
  • somite;
  • spinous process

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Somites compartmentalize into a dorsal epithelial dermomyotome and a ventral mesenchymal sclerotome. While sclerotomes give rise to vertebrae and intervertebral discs, dermomyotomes contribute to skeletal muscle and epaxial dermis. Bone morphogenetic protein (BMP)-signals from the lateral mesoderm induce the lateral portion of the dermomyotome to form chondrogenic precursor cells, forming the cartilage of the scapula blade. The fact that BMPs are expressed in the roof plate of the neural tube where they induce cartilage formation led to the question why cells migrating from the medial part of the dermomyotome do not undergo chondrogenic differentiation and do not contribute to the dorsal part of the vertebrae. In the present study, we traced dermomyotomal derivatives by using the quail–chick marker technique. Our study reveals a temporal sequence in the formation of the vertebral cartilage and the midline dermis. The dorsal mesenchyme overlying the roof plate of the neural tube is formed prior to the de-epithelialization of the dermomyotome. Dermomyotomal cells start to migrate medially into the sub-ectodermal space to form the midline dermis after chondrogenesis of the dorsal mesenchyme has occurred. This time delay between chondrogenesis of the dorsal vertebra and dermal formation allows an undisturbed development of these two tissue components within a narrow region of the embryo.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The vertebral column originates from somites, the first visible segments of the embryo (reviewed in Christ & Wilting, 1992; Christ et al. 2007). Each somite is initially a sphere, composed of an epithelial wall enclosing a mesenchymal cell population, the somitocoele cells (Huang et al. 1994, 1996). During maturation, the somite compartmentalizes into a dorsal and a ventral compartment. The dorsal compartment maintains its epithelial structure and is called the dermomyotome, while the ventral compartment forms mesenchymal cells and is called the sclerotome (reviewed in Christ & Ordahl, 1995; Stockdale et al. 2000).

Cells of the dermomyotome are capable of forming cartilaginous structures as well as skeletal muscle and dermis (reviewed in Christ & Ordahl, 1995; Christ & Brand-Saberi, 2002; Huang et al. 2006). Cells from the dermomyotomal edges migrate ventrally to form the myotome, that subsequently gives rise to skeletal muscles of the vertebral column and body wall. At the limb and occipital level, dermomyotomal cells de-epithelialize and migrate into the limb bud and tongue anlagen to form skeletal muscles (Franz et al. 1993; Bober et al. 1994; Zhi et al. 1996; Chevallier et al. 1977; Huang et al. 1999, 2003a; He et al. 2003). Some muscles, such as the superficial neck muscles, most head muscles and the extra-ocular muscles are derived from the lateral plate mesoderm and the prechordal mesoderm, respectively (Wachtler et al. 1984; Wachtler & Jacob, 1986; reviewed in Noden & Francis-West, 2006; Nathan et al. 2008; Harel et al. 2009; Theis et al. 2010). Those cells that migrate dorsally into the sub-ectodermal space give rise to the dermis in the region of the vertebral column (Zhi et al. 1996; Huang et al. 2000a,b,c,d). Another mesenchymal cell population is released from the lateral dermomyotome under influence of the bone morphogenetic protein (BMP)-signalling pathway, and forms the cartilage of the scapula blade in birds (Huang et al. 2000a,b,c,d; Ehehalt et al. 2004; Prols et al. 2004; Valasek et al. 2011; Wang et al. 2005, 2010). In mammals, this cell population contributes to the medial margin of the scapula (Valasek et al. 2010).

The sclerotome gives rise to cartilaginous and ligamental structures of the occipital bone, the vertebrae, the ribs, the intervertebral discs, the ligaments connecting the vertebrae and the tendons of the epaxial muscles (reviewed by Christ et al. 2000, 2004; Huang et al. 2000a,b,c,d, 2003b; Brent et al. 2003). The development of the dorsal vertebra has been shown to differ from that of the ventral vertebra (Monsoro-Burq et al. 1995, 1996; reviewed in Monsoro-Burq & Le Douarin, 2000). Msx1 and Msx2 are expressed in the dorsal mesenchyme, which is located between the roof plate of the neural tube and the surface ectoderm where the dorsal part of the vertebra forms, whereas Pax1 and Pax9 are expressed in the ventral region that gives rise to the body of the vertebra and the intervertebral disc (Takahashi et al. 1992; Monsoro-Burq et al. 1994, 1996; Wallin et al. 1994; Ebensperger et al. 1995; Balling et al. 1996; Peters et al. 1999). BMP-signals regulate the formation of the dorsal and ventral vertebra (Hirsinger et al. 1997). BMP4-signalling from the roof plate initiates Msx gene expression controlling dorsal vertebral cartilage differentiation, while Sonic Hedgehog (Shh) emanating from the notochord inhibits Msx expression and chondrogenesis of the dorsal vertebral elements (Monsoro-Burq et al. 1996; Watanabe & Le Douarin, 1996; Watanabe et al. 1998). The dorsal vertebral elements have been demonstrated to originate from the sclerotome (reviewed in Christ et al. 2000).

Because the medial and lateral dermomyotome is under the influence of BMP-signalling pathways, we asked why cells from the medial part of the dermomyotome do not contribute to the dorsal vertebral cartilage elements. To trace the cells of the medial dermomyotome, we performed transplantations of the medial dermomyotome between quail and chick embryos. We observed that the sclerotome-derived mesenchyme in the region where the spinous processes and neural arches form undergoes differentiation before the epithelial–mesenchymal transition (EMT) of the dermomyotome takes place. No medial dermomyotomal cells were found in the cartilage of the spinous processes or the dorsal neural arches. At later stages medial dermomyotomal cells migrate medially into the sub-ectodermal space to form dermal elements overlying the dorsal portions of the vertebrae.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Embryos

Fertile chick and quail eggs were purchased from a farm in France and from the Institute of Animal Science, University of Bonn. The eggs were incubated at 38 °C in a humidified atmosphere. Chick embryos were staged after Hamburger & Hamilton (1951), and quail embryos were staged after Ainsworth et al. (2010).

Tissue transplantation

The dermomyotomal transplantation was performed as described in previous studies (Huang & Christ, 2000; Wang et al. 2005, 2010). Briefly, the medial part of the dermomyotomal primordium of one somite was removed at the thorax level in chick host embryos of HH-stages 15–16. A corresponding dermomyotomal tissue was isolated from a stage-matched quail embryo and transferred into a chick host. The grafts were implanted into the space where the dermomyotomal part has been removed before. In some cases, the whole dermomyotome was transplanted. The host embryos were reincubated for a period of 1–6 days.

Immunohistochemistry

The host embryos were fixed in Serra fixative and then prepared for paraffin serial sectioning at 7 μm thickness. The sections were stained by immunohistochemistry with the QCPN monoclonal antibody (Developmental Study Hybridoma Bank, Iowa, USA) and the desmin polyclonal antibody (Sigma-Aldrich, Steinheim, Germany), which recognizes an antigenic determinant of the quail cell chromatin and the desmin filament of muscle cells, respectively. The QCPN antibody was revealed by NBT/BCIP (blue), while the desmin antibody was coloured with DAB reaction (brown).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Dorsomedial migration of dermomyotomal cells occurs later than the formation of the dorsal mesenchyme

To determine when cells from the dermomyotome migrate medially to the sub-ectodermal space, we traced cells derived from the medial dermomyotome by analysing the quail–chick chimeras (= 8). The medial dermomyotome from a quail somite of a stages 15–16 embryo was transplanted into a chick embryo of comparable stage from which the medial dermomytome had been removed. The chimeric embryos were analysed by staining for quail nuclei after reincubation. As expected, the myotome in the operated area of 3-day reincubated embryos consisted of quail-derived myogenic progenitor cells (= 4; Fig. 1A). At this time, the dermomyotome had not undergone EMT; so it remained as a layer of epithelial cells, while the space between the dermomyotome and the ectoderm remained cell free. No migrating quail cells were detected medially to the grafted dermomytome. However, many mesenchymal cells were found in the space between the dermomyotome, the roof plate of the neural tube and the surface ectoderm. This mesenchymal cell population was only formed from chick cells. Thus, this mesenchyme must have originated from the dorsomedial sclerotome.

image

Figure 1.  Late migration of dermal cells. The diagram illustrates the transplantation procedure. The medial part of a dermomyotome (blue) together with the surface ectoderm was isolated from a quail embryo (a), while the medial part of a dermomyotome (red) of a chick embryo was removed (b). The quail dermomyotome part (c) was transplanted into the position of the medial dermomyotome part of the chick embryo (d). dm, dermomyotome; n, neural tube; sc, sclerotome. (A) Transverse section of a quail–chick chimera after the transplantation of the medial part of a dermomyotome after 1 day of reincubation. While the dermomyotome (d and red arrows) is made of quail epithelial cells (with blue nuclei), there are some chick mesenchymal cells medial to the dermomytome over the neural tube (nt) and under the surface ectoderm (e). The myotome (m and red arrowheads, brown = desmin) is made of quail cells (with blue nuclei). Some quail endothelial cells (black arrows) can be found in the sclerotome and the dorsal root ganglion (drg, marked with broken line). (B) Transverse section of a quail–chick chimera 2 days after the transplantation of the medial part of the dermomyotome. Except for the medial lip, the grafted medial dermomyotome has de-epithelialized. Mesenchymal cells derived from the grafted quail dermomyotome (blue) now populate the space between the myotome (m) and surface ectoderm (e). Only a few quail cells (black arrows) migrate medially, but are far from the midline. Chick mesenchymal cells form the dorsal mesenchyme (dm) between the neural tube and the surface ectoderm. d, dermomyotome; dm, dorsal mesenchyme; drg, dorsal root ganglion; e, ectoderm; m, myotome; nt, neural tube.

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Two days after transplantation, numerous quail cells had migrated from the grafted medial part of the dermomyotome into the sub-ectodermal space (= 4; Fig. 1B), and had begun to move medially as a sheet of cells just beneath the surface ectoderm. No quail cells derived from the grafted medial dermomyotome could be found in the dorsal mesenchymal anlagen of the spinous processes and dorsal neural arches.

Formation of the midline dermis occurs later than the formation of the dorsal vertebral elements

Chimeric embryos were incubated until chondrogenesis had occurred in the vertebra to determine when the midline dermis is formed by the medial dermomyotome. After reincubation of 4 days, the cartilage of the vertebral body (data not shown), the neural arch and early chondrogenesis of the spinous process could be first seen (= 4; Fig. 2A). The cartilaginous tissue of all parts of the vertebra consisted solely of chick cells. As expected, the epaxial muscles adjacent to the vertebrae were formed from cells of quail origin. Dermal tissue in the epaxial domain was solely of quail origin. Quail cells now populated the sub-ectodermal space between the surface ectoderm and the primordium of the spinous process, but none was found within the spinous process.

image

Figure 2.  Late formation of the midline dermal tissue. (A) Transverse section of a quail–chick chimera 4 days after transplantation of the medial part of the dermomyotome. Migrating quail cells (black arrows, with blue nuclei) reach the midline but are confined to the space just below the ectoderm. Cartilage formation of the neural ach (na) and the spinous process (sp) is ongoing. (B) Transverse section of a chick host 6 days after transplantation. The spinous process (sp) and the neural arch (na) contain only chick cells. The dermis and the epaxial muscle (em) consist of quail cells. The quail dermal cells (black arrows) populate the midline region. d, dermis anlagen; drg, dorsal root ganglion; em, epaxial muscles; na, neural arch; sc, spinal cord; sp, spinous process.

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By 6 days of transplantation, the cartilage of the whole vertebra, from the vertebral body to the spinous process, was well formed (= 6; Fig. 2B). All portions of the vertebra consisted exclusively of chick cells. Dorsal to the spinous process, in a well-defined dermal layer, cells were derived from the transplanted medial dermomytome. The epaxial muscle laterally to the dorsal vertebra was of quail origin. Endothelial cells derived from the grafted medial dermomyotome could be detected in the meningeal tissue around the spinal cord (Fig. 2B). This is in line with previous observations that all somite parts contribute to endothelial cells of which differentiation is controlled by BMP-, Wnt- and Fgf-signalling (Wilting et al. 1995; Nimmagadda et al. 2005, 2007). When a whole dermomyotome, rather than the medial dermomytome, was grafted, the scapula blade was also of quail origin (= 6, data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

These experiments were designed to determine the temporal sequence of the development of the midline dermis and dorsal vertebral elements. Using quail–chick chimeric embryos where the medial dermomytome had been transplanted to replace the medial dermomyotome of chick embryos, we found cells from the medial dermomyotome migrating medially into the sub-ectodermal space overlying the anlagen of the dorsal vertebral elements. It appears that the cells giving rise to the dorsal portions of the vertebra are committed to this fate prior to the EMT of the dermomyotome (Fig. 3).

image

Figure 3.  Schematic illustration of the two-phase model for the midline cartilage and dermis formation. (a) At day 3 of chick embryos, cells from the dorsomedial part of the sclerotome (d) migrate into the region between the neural tube and the surface ectoderm to form dorsal mesenchyme, while the dermomyotome (dm) is still completely epithelial. (b) At day 5 of chick embryonic development, dermal cells (d) migrate into the region between the cartilage-forming mesenchyme of the spinous process (sp) and the surface ectoderm. em, epaxial muscles; hm, hypaxial muscles; m, myotome; nc, notochord; nt, neural tube; sc, spinal cord; scl, sclerotome; vb, vertebral body.

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The underlying mechanism for commitment to chondrogenesis in the vertebra has been well studied. Shh emanating from the notochord–floor plate complex initiates sclerotome formation (Chiang et al. 1996; Teillet et al. 1998). Lack of expression of Shh in Shh-null mice (Chiang et al. 1996) and ablation of the neural tube/notochord complex in chick embryos (Teillet et al. 1998) lead to an absence of the whole vertebra. However, as development proceeds, the formation of the dorsal and ventral elements of the vertebra is controlled by different mechanisms. Pax1, a master gene for sclerotome formation, is restricted to the ventromedial part of the sclerotome. Cells of the dorsomedial and ventrolateral portion do not express Pax1 (reviewed in Christ et al. 2000). Disruption of Pax1 expression affects mainly the ventromedial vertebral elements (Wallin et al. 1994). The dorsal vertebral elements are formed from the dorsal mesenchyme under the control of Msx1 and Msx2, which are induced by BMP and inhibited by Shh-signalling (Takahashi et al. 1992; Monsoro-Burq et al. 1994, 1996; Hirsinger et al. 1997). These observations reveal that Shh influences the sclerotomal development in two different steps. In the first step, Shh induces the formation of the whole sclerotome. In the second step, it promotes the chondrogenic differentiation of the ventral sclerotomal part, while it inhibits the chondrogenic differentiation of the dorsal part. The results reported here also suggest that there is a temporal sequence in the formation of midline dermis and dorsal vertebral elements. In the first step, the sclerotomal cells migrate dorsomedially to the sub-ectodermal space overlying the roof plate of the neural tube; in the second step, the dermomyotomal cells populate the sub-ectodermal space overlying the already developed anlagen of the dorsal vertebral elements (Fig. 3).

Our results suggest that sclerotome cells migrate dorsally around the neural tube much earlier than dermomyotomal cells do. This might be due to the fact that Wnt-6 expressed in the surface ectoderm prevents migration of mesenchymal cells from the dorsal aspect of the epithelial dermomyotome (Moeller et al. 2003; Schmidt et al. 2004; Otto et al. 2006). Though this Wnt-6 expression is downregulated by the matured neural tube, its expression is maintained in the region overlying the dermomyotome (Geetha-Loganathan et al. 2006). While Wnt-6 may retard the migration from the dermomyotome into this region, the migration of cells from the sclerotome to the dorsal position of the neural tube may be guided by BMP-signalling from the roof plate of the neural tube and the surface ectoderm. On the other hand, Noggin expressed in the medial part of the dermomyotome could prevent BMP activation and dermal cell migration. So, while the sequence of migration of the sclerotomal and dermomyotomal cells is essential for an undisturbed formation of the dorsal vertebra and dermis, the regulation of these migration patterns remains poorly understood.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Our dermomyotomal fate maps show a temporal sequence in the formation of midline dermis and dorsal vertebral elements. Dermomyotomal cells start their migration to form the dermis overlying the spinous process after the chondrogenic differentiation of the dorsal vertebral elements has occurred. This time delay between chondrogenesis of the dorsal vertebra and the dermal formation allows an undisturbed development of these two components within a narrow space of the embryo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

We are grateful for the helpful discussion of Dr Ketan Patel. We thank Mrs L. Koschny and Mr G. Frank for excellent technical assistance. We thank Developmental Studies Hybridoma Bank, Iowa City, IA, USA for the QCPN-antibody. This work was supported by grant of the DFG-729/5 and BONFOR to R.H. We thank Dr Frank E. Stockdale for assistance in the editing of this manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References