p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength.

During muscle development, the p75(NTR) is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75(NTR)-deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75(NTR) was detected on myoblasts but the high affinity NGF receptor, trk A, was not co-expressed with p75(NTR). In vitro, proliferating C2C12 and primary myoblasts co-expressed the p75(NTR) and MyoD, but immunofluorescent analysis of primary myoblasts and RT-PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75(NTR) in neurons, p75(NTR)-positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I-kappaB and AKT, the cells contained increased levels of both phospho-proteins, both of which promote cell survival. By contrast, neurotrophin-treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75(NTR) mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle.

Developmental expression patterns of Beta-ig (betaIG-H3) and its function as a cell adhesion protein.

Beta-ig is a secretory protein embodied by fasciclin I-like repeats containing sequences that might bind integrins and glycosaminoglycans in vivo. Expression of Beta-ig is responsive to Transforming Growth Factor-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating Beta-ig as an ECM adhesive protein of developmental processes. The spatiotemporal distribution of Beta-ig during various stages of murine development was examined and its ability to support adhesion of various cell types assessed. In situ hybridization of mouse embryos (E12.5-E18.5) indicated a prominent, distinct expression pattern for Beta-ig message in connective tissue. Beta-ig transcripts were abundantly expressed during mesenchymal cell condensation in areas of axial, craniofacial and appendicular primordial cartilage from E12.5-E14.5. Beginning at E15.5, Beta-ig transcripts appeared in collagen-rich tissues, including dura mater and corneal stroma. During E16.5-E18.5, Beta-ig transcripts were observed in proliferating chondrocytes and areas of endochondral ossification in joint and articular cartilage formation. Connective tissues expressed Beta-ig transcripts within the nasal septum and surrounding cartilage primordia, and in the pericardium, optic cup, kidney, ovary, esophagus, diaphragm, bronchi, trachea and corneal epithelium, and during cardiac valve formation. These patterns of expression indicate that Beta-ig may be involved in tissue morphogenesis. Cells derived from mesenchyme attached onto a substratum comprised of purified recombinant Beta-ig. Taken together, the results indicate that Beta-ig is expressed principally in collagen-rich tissues where it may interact with cells and ECM molecules, perhaps playing a role in tissue morphogenesis.

A model experimental system for monitoring changes in sensory neuron phenotype evoked by tooth injury.

The dental pulp is a favorable model for studies of interactions between nociceptive sensory neurons and their peripheral target tissues. In the present study, we retrogradely labeled pulpal afferent neurons with an improved method that permits monitoring of changes in neuronal phenotype in response to controlled tooth injuries. The capacity of retrograde neuronal tracers to diffuse through dentinal tubules was exploited, thereby avoiding the severe injury to the pulp associated with previous tracer application methods. The strategy was to apply the durable fluorescent tracer, Fluoro-gold (FG), to exposed dentin in the floor of shallow cavities in molars, in order to pre-label pulpal neurons in trigeminal ganglia of young adult Sprague-Dawley rats. A high percentage of pupal afferent neurons were retrogradely labeled by application of FG to exposed dentin and the FG fluorescent signal persisted in most labeled neurons for at least 8 weeks. Following tracer application to dentin, the pulp tissue appeared normal histologically, with the exception that a layer of reactive dentin was deposited at the pulp-dentin border beneath the shallow cavities. Assessment of expression of calcitonin gene-related peptide (CGRP) and brain derived neurotrophic factor (BDNF) indicated that pulpal neurons remained in a quiescent, baseline condition cytochemically following application of tracer to cavities in dentin and upregulation of these markers could be detected in neurons that projected to teeth that received a test injury subsequent to tracer application. Thus, labeling of trigeminal neurons via dentinal tubules provides the basis for a useful model for precisely assessing properties of pulpal afferents in both quiescent and activated states.

Diverse dependencies of developing Merkel innervation on the trkA and both full-length and truncated isoforms of trkC.

This study demonstrates that innervation dependent on two different neurotrophin tyrosine kinase (trk) receptors can form the same types of sensory endings (Merkel endings) in the same target (Merkel cells of vibrissa follicles). Some endings transiently express trkA during their initial development, whereas others express trkC throughout their development. Consequently, elimination of kinase domains of either trkA or trkC each result in a partial loss of Merkel endings, whereas absence of kinase domains of both receptors results in a total loss. At the onset of Merkel ending development, at least one kinase-lacking trkC isoform is transiently expressed on all the follicle cells, while neurotrophin 3 is transiently expressed only in the cells at the middle third of the follicle where the Merkel endings and cells develop. This transient non-neuronal expression of truncated trkC is essential for development of any Merkel endings, whereas some Merkel endings and cells still begin to develop in the absence of neurotrophin 3. Therefore, truncated trkC plays a more important role in the development of this innervation than kinase forms of trkA or trkC or of NT3, the only known ligand for trkC receptors.