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In terrestrial mammals, hearing starts with the perception of acoustic pressure by the tympanic membrane. Vibrations in this membrane are then transduced into the inner ear by the ossicle chain of the middle ear, composed of the malleus, incus and stapes. The proper connection of the ossicle chain with the tympanic membrane, provided by the insertion of the manubrium of the malleus into the eardrum, is essential for the functionality of the hearing apparatus. We describe here the mechanisms regulating the development of the manubrium and its integration into the tympanic membrane. We show that the external acoustic meatus (EAM), which eventually forms the outer epithelium of the tympanic membrane, plays an essential role in this developmental process. Histological and expression analyses indicate that the manubrium develops close to the EAM with a similar temporal sequence. In addition, when the middle ear ossicles are allowed to develop in vitro under conditions that do not support further EAM development, the manubrium develops only up to the stage of its induction at the time of explantation. Moreover, genetically or teratogenically derived alterations in the EAM also have an effect on manubrial development. Finally, we show that the EAM is the source of two quite opposite activities, one that induces chondrogenesis and another that represses it. The combination of these two activities results in the proper positioning of the manubrium.

BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.

Goosecoid (gsc) is an evolutionarily conserved homeobox gene expressed in the gastrula organizer region of a variety of vertebrate embryos, including zebrafish, Xenopus, chicken and mouse. To understand the role of gsc during mouse embryogenesis, we generated gsc-null mice by gene targeting in embryonic stem cells. Surprisingly, gsc-null embryos gastrulated and formed the primary body axes; gsc-null mice were born alive but died soon after birth with numerous craniofacial defects. In addition, rib fusions and sternum abnormalities were detected that varied depending upon the genetic background. Transplantation experiments suggest that the ovary does not provide gsc function to rescue gastrulation defects. These results demonstrate that gsc is not essential for organizer activity in the mouse but is required later during embryogenesis for craniofacial and rib cage development.

The tympanic membrane in mammals is a trilaminar structure formed by the apposition of two epithelial cell layers, along with an intervening layer of cells derived from pharyngeal arch mesenchyme. One epithelial layer is contributed by the external acoustic meatus, a derivative of the first pharyngeal cleft. The other epithelial layer is contributed by the tubotympanic recess, a derivative of the first pharyngeal pouch. We demonstrate here an absolute correlation between formation of the external acoustic meatus and formation of the tympanic ring, a first arch-derived membrane bone that anchors the tympanic membrane. Experimental loss of the tympanic ring by retinoic acid treatment, or duplication of the ring in Hoxa-2 null mutant embryos, resulted in corresponding alterations in formation of the external acoustic meatus. We suggest that the tympanic ring primordium induces formation and morphogenesis of the external acoustic meatus, and that expression of the Hoxa-2 and goosecoid genes may be involved in regulating the formation and morphogenesis of these structures.

In Hoxa-2(-/- )embryos, the normal skeletal elements of the second branchial arch are replaced by a duplicated set of first arch elements. We show here that Hoxa-2 directs proper skeletal formation in the second arch by preventing chondrogenesis and intramembranous ossification. In normal embryos, Hoxa-2 is expressed throughout the second arch mesenchyme, but is excluded from the chondrogenic condensations. In the absence of Hoxa-2, chondrogenesis is activated ectopically within the rostral Hoxa-2 expression domain to form the mutant set of cartilages. In Hoxa-2(-/- )embryos the Sox9 expression domain is shifted into the normal Hoxa-2 domain. Misexpression of Sox9 in this area produces a phenotype resembling that of the Hoxa-2 mutants. These results indicate that Hoxa-2 acts at early stages of the chondrogenic pathway, upstream of Sox9 induction. We also show that Hoxa-2 inhibits dermal bone formation when misexpressed in its precursors. Furthermore, molecular analyses indicate that Cbfa1 is upregulated in the second branchial arches of Hoxa-2 mutant embryos suggesting that prevention of Cbfa1 induction might mediate Hoxa-2 inhibition of dermal bone formation during normal second arch development. The implications of these results on the patterning of the branchial area are discussed.

Avian reovirus S1133 specifies at least 10 primary translation products, eight of which are present in the viral particle and two of which are nonstructural proteins. In the work presented here, we studied the covalent modifications undergone by these translation products in the infected cell. The structural polypeptide mu2 was shown to be intracellularly modified by both myristoylation and proteolysis. The site-specific cleavage of mu2 yielded a large carboxy-terminal fragment and a myristoylated approximately 5,500-Mr peptide corresponding to the amino terminus. Both mu2 and its cleavage products were found to be structural components of the reovirion. Most avian reovirus proteins were found to be glycosylated and to have a blocking group at the amino terminus. In contrast to the mammalian reovirus system, none of the avian reovirus polypeptides was found to incorporate phosphorus during infection. Our results add to current understanding of the similarities and differences between avian and mammalian reoviruses.

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.

Previous reports have suggested that avian reovirus S1133 fails to replicate in mouse L cells. In this article, we report that replication does occur under certain culture conditions. The avian reovirus was found to grow in mouse L cells at pH 6.4 and 7.2 but not at pH 8.2. Culture medium with a basic pH directly inhibited viral transcription and genome replication. As a result, viral protein synthesis was also affected. At permissive pH levels, avian reovirus grew better in monolayers than in suspension cultures of L cells because of the influence of cell attachment status on viral macromolecular synthesis. Our results not only show that avian reovirus can replicate in mouse L cells but also help to explain why it did not in previous studies.