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Dr. Wilfred Denetclaw
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| The first expression of skeletal muscle in the vertebrate embryo is the myotome, contained in transitory anatomical structures called somites, and arising from the paraxial mesoderm. Somites are easily recognized as paired ball-like structures straddling both sides of the neural tube in the early developing chicken embryo. Each newly formed somite is signaled by its surrounding environment causing it to compartmentalize into a dorsal cap epithelium, called the dermomyotome, where progenitor cells for skeletal muscle and dermis are found, and into a ventral mesenchyme, called the sclerotome, where progenitor cells for cartilage and bone are found. Although the somite produces the sclerotome, myotome and dermatome germ layers, the mechanisms by which molecular signals (for example, sonic hedgehog) and cellular activity (proliferation, growth, migration, and differentiation) lead to differential cell-lineages and ultimately to the formation of bone, muscle and skin tissues is poorly understood. My present research has focused on the earliest stages of somitic myotome formation in the chicken embryo, a representative animal model of higher vertebrate species. The somite dermomyotome is iontophoretically micro-injected with fluorescent lipophilic vital dyes (diI and diO) and after overnight to 2 days of embryo growth, dye-labeled precursor-product relationships are assessed by laser scanning confocal microscopy. This work has resulted in a determination of (1.) the location of myotome precursor cells for the epaxial (dorsal back muscle) and hypaxial (ventral body wall, abdomen, diaphragm and limb muscles) myotomes; (2.) the growth directions of nascent epaxial and hypaxial myotome fibers; (3.) the relative birthdates of myotome fibers within the epaxial and hypaxial myotome layer; and (4.) the maximum growth rates for epaxial and hypaxial myotomes. We also show that the formation of increasingly deeper layers of myotome fibers occurs by appositional growth such that superficial fibers are older than fibers located deeper in the myotome layer and that myotome development in both epaxial and hypaxial domains is directly correlated with and coupled to a corresponding expansion of the overlying dermomyotome epithelium. These findings have produced a widely accepted mechanistic model for the formation of the myotome that now for the first time takes into account cellular proliferation and morphogenetic cellular movement patterns in the somite. For my future work at San Francisco State University, I am proposing the following investigations. (1.) It is not yet understood how the medio-lateral expansion of the dermomyotome or the dorsomedial-ward growth of the epaxial and ventrolateral-ward growth of the hypaxial myotomes are related. The expansion of the dermomyotome dorsomedial and ventrolateral areas may involve active cellular proliferation and growth directions may be directly indicated by the orientation of the cell metaphase plate as has been recently shown to occur in the elongation of the avian primitive streak. This can be determined by Feulgen staining of embryo whole mounts cleared with benzyl benzoate:benzyl alcohol solution and confocal microscopy. (2.) The mediolateral expansion of the dermomyotome may be assisted by a convergent-extension mechanistic process of myotomal growth shown by nascent myotomal myocytes following their deposition from the dermomyotome into the myotome layer. This process of early myotomal growth can be directly viewed by confocal microscopy by using Bodipy-FL ceramide or BCECF vital dyes either alone or in conjunction with diI microinjection and embryo tissue culture. (3.) It is not clear how epithelial cells from the dermomyotome translocate subjacently into the myotome layer for muscle differentiation. It has been shown previously that the orientation of the metaphase plate in cells of the brain neuroepithelium is able to predict the proliferative or differentiative states of dividing neuroblasts. The dermomyotome is like the neuroepithelium in terms of being epithelial and having a restricted region for cellular division (both occur at the apical ends of the epithelial layer). Using nucleic acid stains like Syto11 and somite cross-sections in tissue culture, it is possible to monitor the dermomyotome for both cell translocation studies and for monitoring cell division outcomes to determine if mitotic division planes predict a proliferative or differentiative fate for dermomyotome cells. I propose to use the techniques of embryo microsurgery and tissue culture, fluorescent dye microinjection and immunohistochemistry, and confocal microscopy with digital image processing and analysis to probe further into the growth and expansion dynamics of the somite dermomyotome during early epaxial and hypaxial myotome formation in the chicken embryo and to investigate cell biological events such as epithelial-mesenchymal transitional growth, convergence-extension growth, and nascent myocyte elongation as dermomyotome cells undergo growth and muscle differentiation. This research can accommodate several undergraduate and graduate students in the MBRS/MARC/Bridges Programs at SFSU.
Last modified July 10, 2002 |
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