Skeletal muscle groups located in the neck region have evolved along with the bone and cartilage to control the rotation of the skull, swallowing, respiration, and vocalization. Vocalization defects (dysphonia) and swallowing defects (dysphagia) have been implicated in several congenital diseases. Laryngeal, tongue, tracheal, diaphragm, and cervical muscles are derived from the occipital and cervical somites. However, little is known about the embryonic events that lead to the development of these muscles. Dr. Rawls has used a muscle-specific-lacZ transgene to perform a detailed study of myotome formation in this region of the mouse embryo. His analysis revealed that myogenesis is initiated at the occipitocervical boundary and progresses both rostrally and caudally. Myotome formation as described by the site of myoblast entry into the myotome and the direction of myocyte elongation occurs in a mirror image on either side of the boundary. This indicates that early events in myogenesis are regulated by positional information along the rostrocaudal axis. Members of the Hox 3 gene family are expressed in somites at the occipitocervical boundary. He found that compound Hox 3 mutant neonatal mice exhibit muscle defects in the intrinsic laryngeal muscles, pharyngeal constrictor muscles, and deep cervical muscles. In this application, he proposes to further investigate the regional differences in the origin of myogenic cells and examine the role of Hox 3 paralogs in regulating muscle development in the neck.
The specific aims are: Specific Aim 1. Examine the cellular origins of the premyogenic cells in the occipital and cervical somites in the mouse embryo. The contribution of cells from discrete domains of the dermomyotome to the myotome of somites O3-C4 will be identified using a microlaser surgery system that links confocal imaging to a multiphoton laser. Small groups of cells will be ablated and the embryos cultured for 8 hrs. The impact of cell ablation will be assessed by comparing the degree of myotome formation in the ablated somite versus the contralateral somite. Specific Aim 2. Examine the role of the Hox 3 paralogs in regulating the pattern of primary myotome formation in C1-3. The axial differences in myotome formation predict that global axial patterning signals are controlling early events in myogenesis. Preliminary studies have revealed a reduction and in some cases a loss of muscles derived from the occipital and cervical somites in Hoxa3-/-/Hoxb3-/- neonates. We will examine the role of the Hox 3 genes in regulating the differences in the occipital and cervical somites. The +1565Myogenin-lacZ transgene will be bred into Hox 3 mutant backgrounds (Hoxa3-/-, Hoxd3-/- and Hoxa3-/-/Hoxd3-/-). Embryos will be examined for lacZ-expressing myoblasts between embryonic day 8.5 and 9.5 when the primary myotome is formed in the occipitocervical region. Specific Aim 3. Examine the role of Hox 3 on the patterning of cervical, laryngeal, and glossal muscle development. He will examine the developmental events associated with Hox 3 gene regulation of muscles of the neck in Hox 3 mutant embryos carrying the +1565Myogenin-lacZ transgene. Embryonic development of muscles in the occipitocervical region will be examined between embryonic day 10.5 and birth by staining for the transgene. This will allow him to identify the role of the Hox 3 genes in this process.
These studies will contribute to our understanding of the regulation of myotome formation, muscle patterning. He will also examine the contribution of Hox genes to establishing regional positional information during muscle formation. This work will support the training of two graduate students and several undergraduate students in the area of developmental genetics .
