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 C. L. Brumwell, W. A. Hossain, D. K. Morest, 1 and P. Bernd* Department of Anatomy and Center for Neurological Sciences, University of Connecticut Health Center, Farmington, Connecticut 06030-3405; and *Department of Anatomy and Cell Biology, SUNY Health Science Center, Brooklyn, New York 11203 Received June 9, 1999; accepted November 1, 1999 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), immunostain- ing was concentrated in the CVG perikarya and in- vaded 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 invad- ing 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 in- creased trkB mRNA expression. Morphological differ- entiation was associated with more intense immuno- staining of processes than cell bodies. Evidently TrkB shifts its expression sequentially from sites engaged in migration, ganglion cell differentiation, axonal out- growth, epithelial innervation, and synapse formation. FGF-2 may promote the role of BDNF in these develop- mental events by upregulating the TrkB receptor.  2000 Academic Press Key Words: chicken embryo; brain-derived neuro- trophic factor; tissue culture; immunohistochemistry; in situ hybridization. INTRODUCTION We are studying the development of the sensory neurons and the sensory epithelium of the inner ear, with a particular interest in early differentiation and innervation. In order to study neurogenesis under controlled conditions we have developed a culture system based on explantation of the otocyst at a stage when the cochleovestibular ganglion (CVG) neuro- blasts are beginning to migrate. After migration, the neuroblasts continue their differentiation in vitro. They are physically separate from, but adjacent to, the otocyst, where they may interact in their subsequent development. The developmental sequence of events has been previously documented in situ (Fig. 1) (6, 16, 51, 52) and in vitro (7, 24, 25), and the morphological features have been correlated with the neuronal mark- ers (6, 24, 25, 50). In our culture system we have shown previously that the morphogenetic changes observed in vitro duplicate those occurring in the embryo in many respects, including their sequence and time of appear- ance. The neuroblasts that migrate from the otocyst in vitro exhibit the characteristic bipolar morphology, large perikarya, elongated processes, and spindle shape that distinguish CVG neurons from Schwann cells and other nonneuronal precursors. During development neuronal survival, migration, and differentiation can be influenced by a variety of signals and cellular interactions that are mediated by cell-surface molecules and diffusible trophic factors. Basic fibroblast growth factor (FGF-2), a known mito- gen, also acts as a neurotrophic factor, based on its ability to support the survival, migration, and differen- tiation of neurons of various brain regions (15, 34, 35, 39, 43, 46, 48, 49), including the auditory system (24). Neurotrophic factors, such as NGF, brain-derived neu- rotrophic factor (BDNF), and NT3, may also play an 1 To whom correspondence should be addressed at Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030-3405. Fax: (860) 679-1274. E-mail: kent@neuron.uchc.edu. Experimental Neurology 162, 121–145 (2000) doi:10.1006/exnr.2000.7317, available online at http://www.idealibrary.com on 121 0014-4886/00 $35.00 Copyright  2000 by Academic Press All rights of reproduction in any form reserved.