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Retrograde migration of pectoral girdle muscle precursors depends on CXCR4/SDF-1 signaling

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Abstract

In vertebrates, muscles of the pectoral girdle connect the forelimbs with the thorax. During development, the myogenic precursor cells migrate from the somites into the limb buds. Whereas most of the myogenic precursors remain in the limb bud to form the forelimb muscles, several cells migrate back toward the trunk to give rise to the superficial pectoral girdle muscles, such as the large pectoral muscle, the latissimus dorsi and the deltoid. Recently, this developing mode has been referred to as the “In–Out” mechanism. The present study focuses on the mechanisms of the “In–Out” migration during formation of the pectoral girdle muscles. Combining in ovo electroporation, tissue slice-cultures and confocal laser scanning microscopy, we visualize live in detail the retrograde migration of myogenic precursors from the forelimb bud into the trunk region by live imaging. Furthermore, we present for the first time evidence for the involvement of the chemokine receptor CXCR4 and its ligand SDF-1 during these processes. After microsurgical implantations of CXCR4 inhibitor beads in the proximal forelimb region of chicken embryos, we demonstrate with the aid of in situ hybridization and live-cell imaging that CXCR4/SDF-1 signaling is crucial for the retrograde migration of pectoral girdle muscle precursors. Moreover, we analyzed the MyoD expression in CXCR4-mutant mouse embryos and observed a considerable decrease in pectoral girdle musculature. We thus demonstrate the importance of the CXCR4/SDF-1 axis for the pectoral girdle muscle formation in avians and mammals.

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Acknowledgments

The authors thank Prof. Dr. Ruijin Huang. M. Masyuk especially thanks FoRUM (RUB) for financial support by providing a dissertation scholarship. The authors further acknowledge S. Wulf, R. Houmany, and especially A. Lodwig for excellent technical assistance as well as A. Lenz and A. Conrad for secretarial work. This work was supported by MYORES project (511978) funded by the EU´s Sixth Framework Programme.

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Authors and Affiliations

  1. Medical Faculty, Department of Anatomy and Molecular Embryology, Ruhr-University Bochum, Universitätsstraße 150, 44801, Bochum, Germany

    Maryna Masyuk, Aisha Abduelmula, Gabriela Morosan-Puopolo, Rizwan Rehimi, Nargis Khalida, Faisal Yusuf, Carsten Theiss & Beate Brand-Saberi

  2. Department of Cytology, Institute of Anatomy, Ruhr-University Bochum, Universitätsstrasse 150, 44801, Bochum, Germany

    Carsten Theiss

  3. Medical Faculty, Institute of Anatomy, University of Leipzig, Liebigstr. 13, 04103, Leipzig, Germany

    Veysel Ödemis & Jürgen Engele

  4. Faculty of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Carl-von-Ossietzky-Str. 9-11, 26129, Oldenburg, Germany

    Veysel Ödemis

  5. Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-Surugadai 2-3-10, Chiyoda-ku, Tokyo, 101-0062, Japan

    Hirokazu Tamamura

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  1. Maryna Masyuk
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418_2014_1237_MOESM1_ESM.tif

Fig. S1. CXCR4 inhibitor TN14003 does not cause cell death. CXCR4-inhibitor-soaked beads were implanted in the dorsal proximal forelimb region of HH23 chicken embryos. After reincubation up to stage HH26, TUNEL assay on transverse sections through the forelimb region was performed. (A,B) No increased apoptosis was discernible in the area around the CXCR4 inhibitor bead, while single physiologically apoptotic cells (red) were detectable in various locations of the specimen. (C,D) Tissue sections treated with bovine pancreas DNase I that cleaves the DNA-revealed apoptotic cells (red) over the entire specimen. (B,D) are higher magnification views of the bead areas in (A,C). Scale bars: 100 µm. (TIFF 6132 kb)

Movie 1. Live-cell imaging of the retrograde migration during the retrograde migration of pectoral girdle muscle precursors. The movie relates to Fig. 2. Using in ovo electroporation of the ventrolateral dermomyotome of HH14 chicken embryos, premyogenic precursor cells were labeled with Tol2-EGFP construct. After reincubation up to stage HH26, slice cultures of the forelimb region were prepared. With the aid of confocal laser scanning microscopy, time-lapse movies that capture the retrograde migration of myogenic precursors from the forelimb bud toward the trunk were recorded. The myogenic precursor cells (green) are not restricted to the forelimb bud, but two groups of EGFP-labeled precursors are also visible in the dorsal and ventral pectoral girdle region, respectively. Individual cells actively migrate from the forelimb bud toward the trunk. This retrograde migration represents the “Out”-phase of the “In–Out” mechanism deployed during formation of pectoral girdle muscles. During monitoring the cells reveal a high proliferation rate. Scale bar: 100 µm. (MP4 2426 kb)

Movie 2. CXCR4 inhibitor TN14003 affects the retrograde migration of pectoral girdle muscle precursors. The movie relates to Fig. 5. Using in ovo electroporation of the ventrolateral dermomyotome of HH14 chicken embryos, premyogenic precursor cells were labeled with Tol2-EGFP construct. After reincubation up to stage HH23, acrylic beads soaked in CXCR4 inhibitor TN14003 were implanted into the dorsal proximal forelimb. After reincubation up to stage HH26, slice cultures of the forelimb region were prepared. With the aid of confocal laser scanning microscopy, time-lapse movies were recorded. The myogenic precursor cells (green) are visible in the dorsal and ventral forelimb region. The EGFP-labeled myogenic precursors in the dorsal forelimb show highly intensive movements during the entire recording period, but do not succeed in penetrating the area with the CXCR4 inhibitor solution. The retrograde migration of myogenic precursors required for the formation of pectoral girdle muscles is thus affected by the CXCR4 inhibitor TN14003. Scale bar: 100 µm. (MP4 2693 kb)

Movie 3. The acrylic bead does not mechanically affect the retrograde migration of pectoral girdle muscle precursors. The movie relates to Fig. 6. Using in ovo electroporation of the ventrolateral dermomyotome of HH14 chicken embryos, premyogenic precursor cells were labeled with Tol2-EGFP construct. After reincubation up to stage HH23, acrylic beads soaked in CXCR4 inhibitor TN14003 were implanted into the dorsal proximal forelimb. After reincubation up to stage HH26, slice cultures of the forelimb region were prepared. With the aid of confocal laser scanning microscopy, time-lapse movies were recorded. The myogenic precursor cells (green) are visible in the dorsal and ventral forelimb region. During the entire monitoring period, the EGFP-labeled myogenic precursors in the vicinity of the PBS-soaked acrylic bead reveal active motility. We can observe an individual progenitor cell actively migrating in the retrograde migration toward the trunk and passing across the PBS bead without any difficulties. Thus, the acrylic bead does not present a mechanical obstacle for the retrograde migration of the myogenic precursor cell. Scale bar: 100 µm. (MP4 2304 kb)

Movie 4. Tol2-EGFP-transfected cells are not affected by electroporation. The ventrolateral dermomyotome of HH14 chicken embryos was transfected with the Tol2-EGFP construct using electroporation. After three days of reincubation, slice cultures of the forelimb region were prepared. After another four days of reincubation, a movie was recorded using a fluorescent microscope. Contractile and viable EGFP-labeled fibers are detectable, demonstrating that the physiological development of myogenic precursor cells is not adversely affected by in ovo electroporation technique. (MP4 2393 kb)

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Masyuk, M., Abduelmula, A., Morosan-Puopolo, G. et al. Retrograde migration of pectoral girdle muscle precursors depends on CXCR4/SDF-1 signaling. Histochem Cell Biol 142, 473–488 (2014). https://doi.org/10.1007/s00418-014-1237-7

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