Biotinidase reveals the morphogenetic sequence in cochlea and cochlear nucleus of mice

Abstract

Hearing loss affects children with biotinidase deficiency, an inherited metabolic disorder in the recycling of biotin. The deficit appears shortly after birth during development of the auditory system. Using a mouse model, we sought to discover where and when biotinidase is expressed in the normal development of the cochlea and cochlear nucleus. In the process, we reconstructed the normal morphogenetic sequences of the constituent cells. Immunolabeling for biotinidase was localized to neurons and other cells of the adult and immature mouse, including the embryonic precursors of these regions dating from the stage of the otocyst. Its distribution was compared to the particular morphological changes occurring at each developmental stage. Biotinidase was localized in cells and their processes at the critical stages in their proliferation, migration, structural differentiation, and innervation, covering the entire span of their development. The prevalence of immunostaining peaked in the adult animal, including hair cells and ganglion cells of the cochlea and neurons of the cochlear nucleus. The findings suggest that biotinidase plays a role in the normal development of the auditory system. Besides the pattern of localization of biotinidase, this study provides the first systematic account of each developmental stage in a mammalian auditory system.

Introduction

Individuals with biotinidase deficiency, an autosomal, recessively inherited disorder, exhibit auditory, neurological, and cutaneous symptoms if they are not treated with pharmacological doses of oral biotin (Wolf et al., 1985). Most symptomatic children with biotinidase deficiency develop sensorineural hearing loss (Heller et al., 2002, Wolf et al., 2002). These symptoms can be prevented if biotin treatment is initiated early or ameliorated if the symptoms have already appeared. Biotin is the coenzyme for four carboxylases in humans (Wolf, 2001). Biotin holocarboxylase synthetase catalyzes the covalent binding of biotin to these apocarboxylases to form holocarboxylases. These carboxylases are subsequently proteolytically degraded, resulting in biocytin (biotin linked through an amide bond to the ε-lysine of biotin).

Earlier studies have shown that biotin-deficient adult rats may exhibit differences in hearing, as indicated by their auditory brain stem responses (Heard et al., 1989, Rybak et al., 1991). Subsequently, Heller et al. (2002) localized biotinidase in a number of regions in the adult central nervous system, including some of the auditory and vestibular structures of 129svj mice. These studies suggest the possibility of using the mouse as a model for the study of hearing development and of hearing loss in biotinidase deficiency. To this end, the present study addresses the localization of biotinidase in the mouse during the normal development of the auditory system. A monoclonal antibody made to homogeneously purified human biotinidase was used (Hart et al., 1992). We used this antibody to make detailed observations of the specific cell types of the cochlear nucleus (CN), cochlear ganglion (CG), and the organ of Corti (OC) in the adult mouse and during its development. The stages of mouse development selected for study represent key times in the proliferation, migration, differentiation and eventual maturation of the hair cells, support cells, CG cells, and CN neurons.

In the course of tracing the developmental stages, it became apparent that biotinidase immunostaining served to visualize many, if not most of the processes of the cells at each developmental stage. Thus an unexpected benefit of this study was the opportunity to reconstruct much of the normal morphogenetic sequence of the constituent cells of the CG and their targets in the OC and CN. Previously a more or less systematic reconstruction of the structural changes that characterize the key transitions between developmental stages had been available in the chicken (see Morest and Cotanche, 2004, Book and Morest, 1990) but not in any mammal, despite a number of publications on different stages of development in various species. Traditionally such an analysis would depend on the use of the Golgi methods, which are tedious and very time consuming. Thus the present findings have allowed us to provide a summary of the normal development of the first three levels in the auditory pathway and to discover exactly where in the sequence biotinidase is localized.