Class III β‐tubulin expression in sensory and nonsensory regions of the developing avian inner ear

Abstract

A previous study showed that class III beta-tubulin, a widely used neuron-specific marker, is expressed in mature and regenerating hair cells but not the support cells of the avian inner ear. We investigated the expression of this marker in the developing avian inner ear. We found that class III beta-tubulin is not neuron-specific in the avian embryo, but appears to accumulate in neuronal cell types, including hair cells, about the time of their differentiation. In the developing inner ear, some degree of class III beta-tubulin immunoreactivity is found in all regions of the otic epithelium from its formation as the otic placode (stage 10 [embryonic day, E1.5]) until about stage 21 (E3.5), when the prospective tegmentum vasculosum begins to lose its staining. By stage 35 (E8-9), most of the nonsensory epithelia have lost their class III beta-tubulin staining, leaving distinct regions of staining between the morphological compartments of the inner ear. Concurrent with the loss of staining from nonsensory regions, the hair cells of the sensory epithelia accumulate class III beta-tubulin, whereas the supporting cells decrease their staining. We also observed a similar pattern of development in another hair cell organ, the paratympanic organ. Double labeling with the 275 kD hair cell antigen (HCA) indicated that the majority of hair cells identifiable with class III beta-tubulin are HCA-positive. Additionally, presumptive hair cells were identified which were not within defined sensory epithelia. Our findings show that class III beta-tubulin can be used as an early marker for hair cell differentiation in all hair cell sensory epithelia in the chicken.

Development of otolith receptors in Japanese quail

“…In addition to neural tracers, nerve fibers and calyx afferent terminals were labeled with an antibody against class III β-tubulin (Tuj1). Tuj1 is a neuron specific marker that has been well characterized for visualization properties in vestibular tissue (Perry et al, 2003; Molea et al, 1999). As shown in Figure 6, afferent innervation of the vestibular sensory epithelia began early, coincident with the receptor hair cell differentiation.…”

Expression of class III Semaphorins and their receptors in the developing chicken (Gallus gallus) inner ear

“…We also confirmed the expression pattern of Nrp1 using RNAscope® in situs (Nrp1 probe Cat #501181). Tuj1 has been previously used to label auditory ganglion neurites and immature HCs in the chicken embryo (Molea, Stone, & Rubel, ). Fluorophore conjugated antibodies, Alexa Fluor 568 donkey antigoat IgG and Alexa Fluor 647 donkey antimouse IgG were used to detect the primary antibodies.…”

Roles of fibroblast growth factor 2 during innervation of the avian inner ear

“…1998). Briefly, after fixation in 4% paraformaldehyde (PFA) and inhibition of endogenous peroxidase, cells were blocked with 10% goat serum and incubated with the supernatant from the A2B5 hybridoma (diluted 1 : 1 in PBS) or the class III β‐tubulin (TuJ1) antibody (diluted 1 : 500; Molea et al . 1999) overnight.…”

“…The A2B5 monoclonal antibody recognizes a cell surface glycolipid on chicken neurons (Vogel and Weston 1988; purchased from the American Type Culture Collection, Manassas, VA, USA). The TuJ1 antibody stains postmitotic neurons of the chicken inner ear (Molea et al . 1999).…”

“…After 2 days cells were fixed with 4% PFA in phosphate‐buffered saline (PBS) for 30 min, washed in PBS and incubated in Tris‐buffered saline (TBS; 50 m m Tris‐HCl pH 7.5, 150 m m NaCl) containing 0.1% Triton X‐100 (TST) and 1% fetal bovine serum (FBS) for 10 min at room temperature. The FGF‐2 antibody (diluted 1 : 200; Dono and Zeller 1994), the TuJ1 antibody (diluted 1 : 500; Molea et al . 1999) or the FGFR‐1 antibody (clone 19B2, diluted 1 : 200, Upstate Biotechnology) were added in TST and 1% FBS.…”