Expression of a voltage-dependent potassium channel protein (Kv3.1) in the embryonic development of the auditory system.

The present study traces the development of a voltage-dependent potassium channel protein (Kv3.1) in the avian homologue of the cochlear nucleus, in the cochleovestibular ganglion, and in the otic epithelium from early developmental stages until near hatching. Immunohistochemistry with antibodies to the carboxy terminus (recognizing the Kv3.1b splice variant) and to the amino terminus (recognizing either form of Kv3.1) was used on Hamburger-Hamilton-staged chicken embryos. There were three periods in the relative levels of immunostaining in these regions. Early (E2-6), when precursor cells proliferate, migrate, and form axons, there was staining when using either antibody. In the middle period (E6-11), marked by hair cell differentiation, dendritic growth, and early synapse formation, staining levels decreased. In the late period (E11-19), when auditory function begins, staining increased rapidly, especially for Kv3.1b. Early Kv3.1 expression occurs in neuronal and hair cell precursors before they differentiate or function. Later, in the otic epithelium, a high level of Kv3.1 in cilia may precede or coincide with the onset of hair cell function. In neurons, some features of its localization correlate with axon outgrowth and synapse formation, others with the onset of neural activity and function.

Fibroblast growth factors (FGF-1, FGF-2) promote migration and neurite growth of mouse cochlear ganglion cells in vitro: immunohistochemistry and antibody perturbation.

To study the effect of FGF in the early development of the sensory neurons of the auditory system, we established a culture preparation of ganglionic neuroblasts engaged in migration and process outgrowth. The presumed anlage of the cochlear ganglion was dissected from E11 otocysts, just as the neuronal precursors were migrating. The cultures were divided into 4 groups and supplemented for 7-9 days with either hrFGF-1 or hrFGF-2 or both or with defined medium only (control group). Measurements of the increase in explant growth, neuroblast migration, and neurite outgrowth were made by time-lapse imaging techniques in living cultures. Either FGF-1 or FGF-2 alone stimulated early migration and outgrowth of the ganglion cells by 5-10x. The effect of combining FGF-1 and FGF-2 was greater than either alone, but less than additive, consistent with a shared receptor. BrdU labeling confirmed that the effect was on migration, not on proliferation. Adding a neutralizing antibody for FGF-2 to the cultures inhibited migration and neurite outgrowth, suggesting an endogenous FGF-2 activity in these functions. Immunocytochemical observations in vitro and in situ with antibodies to FGF-1, FGF-2, or FGF receptor (R1) demonstrated immunopositive staining of the migrating ganglionic neuroblasts, their processes, and growth cones at corresponding stages (E13). Also non-neuronal cells, hair cells, and Schwann cells (in situ) expressed FGF-1 and FGF-2. Evidently both FGF-1 and FGF-2 play important roles in the migration and initial differentiation of cochlear ganglion neurons in the mouse.

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.

Critical periods of basic fibroblast growth factor and brain-derived neurotrophic factor in the development of the chicken cochleovestibular ganglion in vitro.

The temporal roles of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) in the development of sensory neurons have been studied in a cell culture preparation which models normal embryonic inner ear development (normocytic). Previous studies showed that FGF-2 stimulated migration and differentiation of ganglion cells for the first 2 days in vitro, but after 5 days led to degeneration, implicating other factors in their later development. To see if BDNF could be such a factor, otocysts were explanted from white leghorn embryos at the time when ganglion cell precursors normally start migrating from the otic epithelium. Cultures were grown in a defined medium, either with or without human recombinant FGF-2 for 2 days or with BDNF. On Day 3, FGF-2 was replaced either with BDNF in defined medium or with defined medium only. Measurements of neuroblast migration and neurite outgrowth were made by time-lapse imaging in living cultures. In cultures receiving BDNF on Day 3, cell migration and neurite outgrowth from the explant increased for more than 3 weeks but not in cultures receiving only defined medium from Day 3. Cultures did not survive more than 3-4 days when receiving either BDNF in defined medium or defined medium alone from the first day. A neutralizing antibody to BDNF inhibited neuronal migration and neurite outgrowth, and it also blocked the effects of exogenous BDNF. BDNF did not enhance the effects of FGF-2 by interacting with it. These experiments defined a temporal sequence in which FGF-2 acts early in development, while BDNF affects a later stage.

Basic fibroblast growth factor (FGF-2) affects development of acoustico-vestibular neurons in the chick embryo brain in vitro.

The effects of basic fibroblast growth factor (FGF-2) on presumptive auditory and vestibular neurons from the medulla were studied in primary cell cultures. The part of the rhombic lip that forms nucleus magnocellularis (homologue of the mammalian anteroventral cochlear nucleus) was explanted from white leghorn chicken embryos at Hamburger-Hamilton stage 28 (E5.5), the time when precursors of the magnocellularis bushy cells migrate and begin to differentiate in situ. In vitro the neuroblasts migrated onto 2-D substrates of purified collagen, differentiated, and expressed neuronal markers. One-half of the cultures were supplemented with human recombinant FGF-2 (10 ng/ml daily) for 5-7 days; the others, with fetal bovine serum. FGF-2 more than doubled the length of neurite outgrowth during the first 3 day treatment compared to serum, but the number of migrating neuroblasts was unaffected. Although neurites attained greater lengths in FGF-2, they usually degenerated after 4-5 days; in serum their growth continued for several weeks. Differentiation of neuronal structure, including axons and dendrites, began within 1-2 days in bFGF but required at least 5-7 days in serum. Histochemical observations in vitro and in situ with antibodies to FGF receptor demonstrated immunopositive patches on acoustico-vestibular neuroblasts at stage 28, when they are migrating and first forming their axons. The findings suggest that FGF-2 stimulates neurite outgrowth in the cochlear and vestibular nuclei. FGF-2 may accelerate cell death by overstimulating neuroblasts, but other factors are needed to sustain their further development.

Basic fibroblast growth factor (FGF-2) affects development of acoustico-vestibular neurons in the chick embryo brain in vitro.

The effects of basic fibroblast growth factor (FGF-2) on presumptive auditory and vestibular neurons from the medulla were studied in primary cell cultures. The part of the rhombic lip that forms nucleus magnocellularis (homologue of the mammalian anteroventral cochlear nucleus) was explanted from white leghorn chicken embryos at Hamburger-Hamilton stage 28 (E5.5), the time when precursors of the magnocellularis bushy cells migrate and begin to differentiate in situ. In vitro the neuroblasts migrated onto 2-D substrates of purified collagen, differentiated, and expressed neuronal markers. One-half of the cultures were supplemented with human recombinant FGF-2 (10 ng/ml daily) for 5-7 days; the others, with fetal bovine serum. FGF-2 more than doubled the length of neurite outgrowth during the first 3 day treatment compared to serum, but the number of migrating neuroblasts was unaffected. Although neurites attained greater lengths in FGF-2, they usually degenerated after 4-5 days; in serum their growth continued for several weeks. Differentiation of neuronal structure, including axons and dendrites, began within 1-2 days in bFGF but required at least 5-7 days in serum. Histochemical observations in vitro and in situ with antibodies to FGF receptor demonstrated immunopositive patches on acoustico-vestibular neuroblasts at stage 28, when they are migrating and first forming their axons. The findings suggest that FGF-2 stimulates neurite outgrowth in the cochlear and vestibular nuclei. FGF-2 may accelerate cell death by overstimulating neuroblasts, but other factors are needed to sustain their further development.

Basic fibroblast growth factor affects neuronal migration and differentiation in normotypic cell cultures from the cochleovestibular ganglion of the chick embryo.

To study the role of basic fibroblast growth factor (FGF-2) in the development of sensory neurons, the cochleovestibular ganglion of the chicken embryo provides a well-characterized structure. This permits use of morphological markers in a cell culture preparation comparable to the normal embryo (normocytic). Otocysts were explanted from white leghorn embryos at Hamburger-Hamilton Stages 14-16, when ganglion cell precursors normally start migrating from the otic epithelium. The cultures were supplemented with either fetal bovine serum or human recombinant FGF-2 (in defined medium or serum) for 2 or 5 days. FGF-2 increased explant growth, neuroblast migration, and neurite outgrowth 2- to 10-fold in the first 2 days. Neuronal morphology appeared within 2-3 days with FGF-2 but required at least 4-5 days with serum. FGF-2 in defined medium stimulated early migration and differentiation, but without serum led to degeneration after 5 days. In serum, growth was later and slower but continued for at least 3 weeks. When explants were cultured in serum with a neutralizing antibody to FGF-2, but no FGF added, neuroblast migration and elongation were decreased by 2- to 4-fold, compared to serum alone. Immunocytochemistry demonstrated FGF receptor sites on the migrating ganglionic neuroblasts, on their processes and growth cones, and in the incipient ganglion and otic epithelium at Stages 15-17, both in the embryo and in vitro. The findings suggest that FGF-2 stimulates early migration and differentiation of ganglion cells by activating the receptors of neuroblasts or their precursors in the embryonic otocyst. However, other factors must sustain their later development.