Commitment of chondrogenic precursors of the avian scapula takes place after epithelial-mesenchymal transition of the dermomyotome
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 2 March 2010
Accepted: 31 August 2010
Published: 31 August 2010
Cells of the epithelially organised dermomyotome are traditionally believed to give rise to skeletal muscle and dermis. We have previously shown that the dermomyotome can undergo epithelial-mesenchymal transition (EMT) and give rise to chondrogenic cells, which go on to form the scapula blade in birds. At present we have little understanding regarding the issue of when the chondrogenic fate of dermomyotomal cells is determined. Using quail-chick grafting experiments, we investigated whether scapula precursor cells are committed to a chondrogenic fate while in an epithelial state or whether commitment is established after EMT.
We show that the hypaxial dermomyotome, which normally forms the scapula, does not generate cartilaginous tissue after it is grafted to the epaxial domain. In contrast engraftment of the epaxial dermomyotome to the hypaxial domain gives rise to scapula-like cartilage. However, the hypaxial sub-ectodermal mesenchyme (SEM), which originates from the hypaxial dermomyotome after EMT, generates cartilaginous elements in the epaxial domain, whereas in reciprocal grafting experiments, the epaxial SEM cannot form cartilage in the hypaxial domain.
We suggest that the epithelial cells of the dermomyotome are not committed to the chondrogenic lineage. Commitment to this lineage occurs after it has undergone EMT to form the sub-ectodermal mesenchyme.
Epithelial and mesenchymal cells differ from each other in several aspects. Epithelial cells are connected to each other by cell surface contacts such as desmosomes and gap junctions, form dense cell layers resting on a basal lamina and exhibiting apical-basal polarity. In contrast, mesenchymal cells have no association with the basal lamina and usually do not possess elaborated adhesion complexes with neighbouring cells, which increase their migratory potential. Under certain physiological or pathological conditions epithelial cells lose their characteristics and undergo morphological changes to convert into mesenchymal cells, a biological process known as epithelial-mesenchymal transition (EMT) . The process of EMT is reversible and the opposite mechanism converts mesenchymal cells into epithelia (mesenchymal-to-epithelial transition, MET).
EMT and MET play key roles not only during early embryonic development, for example during implantation of the embryo into the uterus, gastrulation and de-lamination of neural crest cells, but also during later developmental stages such as in somite development and organogenesis . Somites are balls of epithelial cells, which arise from the paraxial mesoderm (mesenchyme) by means of MET. Later, the ventral half of each somite undergoes EMT to give rise to the sclerotome , while the dorsal half remains epithelial and forms the dermomyotome, source of the skeletal muscle, dermis , scapula blade in birds  and medial scapula border in mammals .
The avian dermomyotome in specific axial regions undergoes EMT to form sub-ectodermal mesenchyme which gives rise to scapula . This process is controlled by signals from the ectoderm and the lateral plate mesoderm [7, 8]. Furthermore only cells originating from the hypaxial but not the epaxial dermomyotome give rise to scapula . It is still unknown when the fate of chondrogenic precursor cells is determined. To this purpose we performed quail-chick grafting experiments exchanging the epithelial or mesenchymal tissues between epaxial and hypaxial domains. Our results show that chondrogenic progenitor commitment takes place after the formation of sub-ectodermal mesenchyme.
Fertilized White Leghorn chick eggs (Gallus gallus) and the Japanese quail (Coturnix coturnix) were purchased from a local breeder and were incubated at 37.8°C and 80% relative humidity. The embryos were staged according to Hamburger and Hamilton . The quail embryos were staged according to Ainsworth et al (2010) .
Chick embryos at stages from HH-14 to HH-20 were fixed in 3.5% glutaraldehyde and 3.5% paraformaldehyde at 4°C overnight. After dehydration in an ethanol series (30%-100%), embryos were embedded in Epon at 60°C. 0.75 μm transverse semi-thin sections were made through the region of somite 20/21 and stained with methylene blue.
The following grafting experiments from quail to chick embryos were performed at the level of somite 20/21:
(1) Transplantation of the epaxial dermomyotome with overlying ectoderm to hypaxial position at HH-16 (n = 24).
(2) Transplantation of the hypaxial dermomyotome with overlying ectoderm to epaxial position at HH-16 (n = 14).
(3) Transplantation of the epaxial SEM with overlying ectoderm to hypaxial position at HH- 20 (n = 24).
(4) Transplantation of the hypaxial SEM with overlying ectoderm to epaxial position at HH- 20 (n = 22).
All transplantations were performed with stage-matched quail and chick embryos. During transplantation, tissue of chick embryos was removed first, and then the desired quail tissue was isolated and transferred to the prepared position in the chick embryos. After a re-incubation periods of 5 hours to 6 days, the chimeras were harvested for further analysis using in situ hybridization, skeletal staining and immunohistochemistry. All embryos were younger than 10 days. Therefore, ethical approval of the experiments was not needed.
Whole-mount in situ hybridization
Skeletal morphology was established by staining embryos for 24 hours with 0.015% Alcian blue in 80% ethanol and 20% acetic acid. The embryos were dehydrated in 100% ethanol for 24 h and then made transparent in 100% methylsalicylate .
After skeletal staining, specimens were embedded in paraffin and transverse sections were taken through the operated region. Quail cells were detected with monoclonal QCPN-antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). A polyclonal anti-desmin-antibody (Sigma, Deisenhofen, Germany) was used to identify muscle cells. Secondary antibodies and colour reactions have previously been described . On sections desmin appears in brown and the quail nuclei in blue. In some samples, only QCPN-antibody was used and stained with DAB-colour reaction. Using this protocol, quail nuclei appear brown. Finally, sections were counterstained with nuclear-fast-red (Sigma, Deisenhofen, Germany).
Determining the time-window for EMT of scapula-forming dermomyotomes
Assessment of the experimental manipulations
Epaxial epithelial dermomyotome grafted to the hypaxial position gives rise to chondrocytes
In our previous study , the hypaxial dermomyotomal epithelium was homotopically grafted into the hypaxial domain. This experiment can serve as a control for this study. In most cases, the host embryo displayed a thin scapula segment in the operated region. In this scapula, quail cartilage tissue was found. This indicated that the manipulation can influence the scapula morphogenesis. However, the differentiation programme of the grafted dermomyotomal cells was not altered.
We conclude that in the new environment, the epaxial dermomyotome acquires the potential to form hypaxial derivatives, such as scapula.
Hypaxial epithelial dermomyotome grafted to the epaxial position does not form chondrocytes
This observation confirms that scapular precursors in the dermomyotome are not committed to the chondrogenic lineage. They do not form cartilage but develop according to local cues when placed in the epaxial domain.
Epaxial SEM grafted to the hypaxial domain cannot generate chondrocytes
To test to the robustness of our transplantation process, quail SEM was grafted from the hypaxial domain of a quail donor into the same domain of a chick host (n = 6). In 4 out of 6 chimeras, quail cells were found in the scapula cartilage (Additional file 1). In 2 out of 6 samples, a segment of the scapula was missing and few quail cells could be seen. These results indicate that the manipulation may result in malformation in case of failure of engraftment. However, the homotopically grafted quail cells do give rise to cartilage tissue.
Our experiments demonstrate that normal scapula development is disturbed after replacement of hypaxial SEM with epaxial SEM.
Hypaxial SEM grafted to the epaxial domain generates chondrocytes
In the present study we aimed to identify when dermomyotomal cells are committed to the formation of cartilage. Our results, based on a series of quail-chick transplantation experiments, show that these precursors are not committed as long as they are situated in the dermomyotome, but are committed to form cartilage after going through EMT to generate the sub-ectodermal mesenchyme (SEM).
Developmental plasticity of newly formed epithelial somites has been demonstrated in previous studies. Experiments involving the dorsal-ventral rotation of epithelial somites have shown that cells develop according to their new position [16, 17]. These results demonstrate that cells of the epithelial somites lack intrinsic patterning information. Instead, their commitment relies on information imparted by local signalling centres. The results presented here provide another example for the plasticity of the epithelial somitic compartment, the dermomyotome. The fact that hypaxial dermomyotome grafted into epaxial position forms muscles instead of scapular cartilage strongly suggests that hypaxial dermomyotome is not committed at this stage, and is competent to generate epaxial derivatives in the new environment. This led us to the hypothesis that epaxial dermomyotome will differentiate into scapula when grafted to a hypaxial position. Generation of cartilaginous elements made up of transplanted quail donor cells proved this hypothesis to be correct. We further investigated whether scapula precursors are committed to their chondrogenic fate once they form the SEM. Hypaxial SEM grafted into an epaxial position generated cartilage, whereas the reciprocal transplantation of epaxial SEM into the hypaxial domain did not produce chondrocytes. This demonstrates that chondrogenic scapular precursor cells are committed once SEM has formed as a consequence of EMT of the epithelial dermomyotome.
Our results showing the demise of the donor ectoderm and its replacement by host ectoderm, followed by normal dermomyotome development, suggest that regenerated ectoderm develops characteristics associated to its normal position along the medio-lateral axis. In a previous study, we showed that signals from the overlying ectoderm are essential for scapula development . The overlying ectoderm probably functions in two phases. In the early stages, it is responsible for maintaining the epithelial structure of the dermomyotome by secreting Wnt6. Wnt6 signal from the ectoderm, which may act in the chick via canonical and non-canonical pathways during neural crest development , maintains the epithelial structure of the dermomyotome. Thereby it regulates the expression of the epithelial marker paraxis . In the later stages, decrease of Wnt6 in ectoderm  facilitates EMT of the dermomyotome, as a consequence of which the scapular precursors migrate to the sub-ectodermal space to get committed to the chondrogenic lineage by local cues. Disturbed EMT in dermomyotome caused by a prolongated epithelial status may result in a defect scapula. This has been suggested by the observation that overexpression of carboxypeptidase Z (CPZ), a secreted enzyme which promotes Wnt signalling, results in up-regulation of the Wnt-induced epithelial marker Pax3 in the hypaxial dermomyotome, down-regulation of Pax1 in the scapular anlage and loss of scapula . The EMT of the scapular precursor cells in dermomyotome and their commitment in the sub-ectodermal space might be two independent processes. We suggest that cells undergoing EMT are able to respond to pro-chondrogenic signalling which they were unable to do in the epithelial state. It is possible that alterations of the cell membrane, especially modifications of receptors and cell adhesion molecules change not only epithelial cell characteristics but also initiate gene transcription, which may permit cells to respond to local cues. This can be exemplified by the dual role of β-Catenin during EMT. β-Catenin in cell membranes is associated with cell-cell contact in both normal epithelium and non-invasive tumours. On the other hand, in cells undergoing EMT, β-Catenin is translocated from the cell membrane to the nucleus and functions together with the DNA-binding factor TCF as a transcriptional activator [22, 23]. The somitic scapular precursor cells express Pax1, which has first been identified as a sclerotomal marker gene [24–26]. In response to notochord-derived sonic hedgehog, Pax1 is expressed in the ventral half of the somites shortly before their de-epithelialization , thereby regulating development of the vertebral column . In contrast, in the scapular precursor cells Pax1 is activated a considerable time after initiation of EMT of the dermomyotome. Pax1 expression can be seen in the scapular anlagen only from HH-26 onward. Our transplantations of SEM were carried out at HH-20 and generated Pax1-positive cells when hypaxial SEM was grafted into an epaxial position. Our results show that commitment of scapular precursor cells is controlled by genes other than Pax1.
Local cues for commitment of the scapular precursor cells may come from the neighbouring structures like myotome and somatopleure. FGF signaling from myotome increases the proliferation of chondrogenic precursor cells in sclerotome, but has no dramatic effect on dermomyotome . Furthermore, FGF signalling probably is not involved in commitment of the scapula precursor cells since inhibition of FGF receptor-1 does not affect scapula blade development . Since scapula precursor cells in SEM lie adjacent to the lateral somatopleure and somatopleure-derived BMP signalling is required for scapula development , we propose that scapular precursor cells get committed in the sub-ectodermal space by BMP signalling from the lateral somatopleure.
We show that in the epithelial dermomyotome cells are not committed to the chondrogenic lineage. Commitment to chondrogenic cell fate occurs after the formation of sub-ectodermal mesenchyme.
We are grateful to Mr. G. Frank, Mrs. E. Gimbel, Mrs. L. Koschny, Mr. F. Ludewig, Mrs. U. Pein and Mrs. Ch. Zelent for their excellent technical assistance. This work was supported by grant of the Deutsche Forschungsgemeinschaft (Hu729/2) to R.H., and the Graduate Program of Baden-Wüttemberg to B.W.
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