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| Aaron Sarvet, Undergrad | |
| Rosemarie Doris, Lab Manager | |
| Stefanie Windner, visiting graduate student | |
| Sara Patterson, Postdoc | |
| Nathan Bird, Postdoc |
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What are the cellular and molecular mechanisms that lead to the development of distinct cell identities during development? What is the embryonic source of dividing muscle precursor cells? How are differentiation and proliferation regulated in muscle precursor cells? What are the cellular behaviors that lead to the development of tissue form? |
Our lab is addressing these broad questions by studying the development of zebrafish skeletal muscle. We are working on muscle development because muscle is a very abundant and easily accessible tissue, and also because diseases of muscle development are debilitating and common childhood diseases. We work on zebrafish because they are readily accessible for experimental manipulations throughout development and because a genetic approach to studying development is feasible in this vertebrate.
Vertebrate muscle precursors derive from a transient embryonic tissue known as the dermomyotome. The dermomyotome contains proliferating cells We have recently demonstrated that embryos of species in all known vertebrate classes contain a dermomyotome (Devoto, et al., 2006; Stellabotte and Devoto, 2007). We have extensively characterized the origin and the fate of zebrafish dermomyotome (Stellabotte, et al., 2007), and examined the development of the dermomyotome into muscle fibers.
Muscle fibers in most vertebrates can be broadly classified as either slow or fast. Slow muscle fibers contract slowly and they fatigue slowly; in fish, slow muscle is used for steady cruising through the water. Fast muscle fibers contract fast and fatigue fast; they are used to power rapid escape responses, such as when a predator appears.
This cross section of a 24 hour old zebrafish embryo was stained with three antibodies to identify muscle fiber types. A monolayer of embryonic slow muscle fibers (yellowish green) is on the surface of the myotome, surrounding the deeper fast muscle fibers (red). All muscle nuclei are teal. Other nuclei in the embryo are labeled blue.
Our long-term goal is to understand how cell-cell interactions regulate the relative positions and proliferation of distinct skeletal muscle cell types during development. Within the trunk, two cell types, adaxial and lateral presomitic, can be unambiguously identified in the presomitic mesoderm. Adaxial cells are large, cuboidal cells adjacent to the notochord which will develop into slow muscle fibers, while lateral presomitic cells are smaller and more irregular cells in the segmental plate which will give rise to both fast muscle and sclerotome (Devoto et al., 1996). One or more members of the hedgehog (Hh) gene family triggers specification of two different types of slow muscle fibers: muscle pioneers and non-muscle pioneers (Du, et al., 1997). Finally, ectopic expression of a member of the BMP4 gene family in the notochord inhibits development of the muscle pioneer class of slow muscle fibers, without affecting development of either the non-muscle pioneer slow muscle fibers or the fast muscle fibers (Du, et al., 1997).
The role of Hh in embryonic slow muscle development
Zebrafish slow muscle development offers a very powerful system in which to identify new genes involved in Hedgehog signaling, and to test the function of known genes. We have tested whether Hh is required for slow muscle development in two ways. First, we have shown that pharmacological elimination of Hh signaling blocks the development of embryonic slow muscle fibers. Second, we have begun characterizing a new gene that we have named slow-muscle-omitted (smo). smo mutant embryos have a nearly complete loss of slow muscle fibers. smo function is required in muscle precursors for them to respond to signaling from the notochord, and thus this gene is a good candidate for genes involved in notochord signaling. We have proposed that smo is the zebrafish homologue of the Hh receptor complex protein Smoothened, and that smu is required for all Hh signal transduction. See Barresi, et al., 2000, for more information.We have also shown that all other genes which are required for Hh or midline signaling are required for slow muscle development (Stickney, et al., 2000).
Muscle growth, larval development, and fiber type identity
After these initial 20 muscle fibers per somite have developed, there is a tremendous growth of the myotome, the adult zebrafish has at least 400 slow muscle fibers in each myotome. We are beginning to characterize this growth. We have shown that new slow muscle fibers are added beginning at about 24h of development, very shortly after embryonic slow muscle fibers form. Surprisingly, these added fibers develop independently of Hh signaling, and independently of the presence of embryonic slow muscle fibers (Barresi, et al., 2001).We have proposed that the dermomyotome is an ancient and conserved structure that evolved prior to the last common ancestor of all vertebrates (Devoto, et al., 2006). We suspect that it has played a very important role in the evolution of vertebrate morphology. We recently demonstrated the existence of the dermomyotome by showing that cells on the external surface of the embryonic myotome differentiate into muscle fibers during the earliest period of myotome growth (Stellabotte, et al., 2007). These cells express the transcription factor Pax7. The zebrafish dermomyotome, like that of amniotes, is directly regulated by Hh signaling (Feng, et al, 2006). See Stellabotte and Devoto, 2007 for a review of the teleost dermomyotome.
Other aspects of somite patterning
In addition to the myotome, the somite gives rise to the vertebrae, the ribs and (in chick) the scapula. These tissues all develop from the sclerotome, which is a relatively small portion of the somite in zebrafish. We are examining the factors that regulate sclerotome development in zebrafish. Our working hypothesis is that initial sclerotome development is not dependent on midline signaling, but that sclerotome maintenance is.
- muscular dystrophy strikes 1 in 4,000 boys in the U.S.
- heart disease accounts for over one-third of all deaths in the U.S.
- 150,000 babies are born with birth defects each year in the U.S.
- cancer accounts for one-quarter of all deaths in the U.S.
Our research into muscle fiber type development in zebrafish has been funded very generously by The Donaghue Foundation, The March of Dimes, and the National Institutes of Health. Our current funding is through NIH grants 5R01HD044929-04 (Selecting for novel Hedgehog signaling mutations), and 2R01HD037509-06A2 (Development of muscle fiber type identity).
At its most fundamental, our research is aimed at understanding why and how cells do what they do. How do they "decide" whether to divide or become specialized? How do cells decide how often to divide? How do they decide which of several possible specializations to acquire? Answers to these questions are necessary for designing appropriate and effective treatments for all of the above diseases.
More specifically, if we can understand how muscle development is triggered, and how specific types of muscle fibers are formed, then we can start to intelligently design therapies that will not only trigger muscle regeneration, but also trigger the appropriate specializations in regenerated tissues that are critical for recovery from heart disease and muscular dystrophy.
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Stephen H. Devoto |
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