Sequential interactions of fibroblast growth factor-2, brain-derived neurotrophic factor, neurotrophin-3, and their receptors define critical periods in the development of cochlear ganglion cells.

We studied the interactions of neurotrophin-3 (NT3) with brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF-2), and their effects on tyrosine kinase C (TrkC) expression during cochlear ganglion development. Otocysts were explanted from white leghorn chicken embryos at stages when the neuronal precursors normally start to migrate. Cultures were fed with various combinations of NT3, BDNF, and FGF-2. NT3 appeared to have a greater effect on neurite outgrowth than on migration and was enhanced by BDNF. The results from in situ hybridization and immunostaining for TrkC receptor revealed up-regulation of the mRNA and protein by combining NT-3 and BDNF. NT-3 combined with FGF-2 produced down-regulation of receptor. Neutralizing antibody to NT3 had an inhibitory effect on neuronal development, suggesting that endogenous NT3 is normally active during the period examined. The findings suggest an interactive role of NT3 in early neuronal development. The trophic synergism of NT3 and BDNF may result from up-regulation of TrkC. This hypothesis is consistent with immunostaining in the embryonic basilar papilla, which localized TrkC to the initial axonal invasion sites. While the growth factors each produce particular trophic effects, the interactions of these factors define a critical sequence of developmental events based on modulation of receptor expression.

Role for basic fibroblast growth factor (FGF-2) in tyrosine kinase (TrkB) expression in the early development and innervation of the auditory receptor: in vitro and in situ studies.

A previous study showed that basic fibroblast growth factor (FGF-2) promotes the effects of brain-derived neurotrophic factor (BDNF) on migration and neurite outgrowth from the cochleovestibular ganglion (CVG). This suggests that FGF-2 may up-regulate the receptor for BDNF. Thus we have examined TrkB expression during CVG formation and otic innervation in vitro and in the chicken embryo using immunohistochemistry. Following anatomical staging according to Hamburger-Hamilton, results were compared with mRNA expression in vitro using in situ hybridization. In the embryo at stage 16 (E2+) clusters of either lightly stained or immunonegative cells occurred within the otocyst and among those migrating to the CVG. By stage 22 (E3.5), immunostaining was concentrated in the CVG perikarya and invaded the processes growing into the otic epithelium but not into the rhombencephalon. Subsequently TrkB expression decreased in the perikarya and became localized in the leading processes of the fibers invading the epithelium and in the structures participating in synapse formation with the hair cells. In vitro there was moderate immunostaining and modest in situ hybridization for trkB in the neuroblasts migrating from the otocyst under control conditions. In contrast, neuroblasts previously exposed to FGF-2 exhibited accelerated migration and differentiation, with increased trkB mRNA expression. Morphological differentiation was associated with more intense immunostaining of processes than cell bodies. Evidently TrkB shifts its expression sequentially from sites engaged in migration, ganglion cell differentiation, axonal outgrowth, epithelial innervation, and synapse formation. FGF-2 may promote the role of BDNF in these developmental events by upregulating the TrkB receptor.

Innervation and target tissue interactions differentially regulate acetylcholine receptor subunit mRNA levels in developing neurons in situ.

Neurons engage in two distinct types of cell-cell interactions: they receive innervation and establish synapses on target tissues. Regulatory events that influence synapse formation and function on developing neurons are largely undefined. We show here that nicotinic acetylcholine receptor (AChR) subunit transcript levels are differentially regulated by innervation and target tissue interactions in developing chick ciliary ganglion neurons in situ. Using ganglia that have developed in the absence of pre- or postganglionic tissues and quantitative RT-PCR, we demonstrate that alpha 3 and beta 4 transcript levels are increased by innervation and target tissue interactions. In contrast, alpha 5 transcript levels are increased by innervation, but target tissues have little effect. Whole-cell ACh-induced currents, used to estimate the number of functional AChRs, change in correlation with alpha 3 and beta 4, but not alpha 5, transcript levels. A model is proposed in which the changes in AChR subunit expression regulate levels of synaptic activity, which is a critical determinant of synapse stabilization and elimination, and neuronal cell death.