Date of Award

8-1-1985

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Abstract

The purpose of the present study was to investigate the development of the otic placode/vesicle and the concurrent synthesis of an associated cell surface coat material (SCM) in the chick embryo. This was accomplished by means of precipitation of the glycoconjugate constituents of the SCM with cetylpyridinium chloride (CPC) and subsequent observation by scanning electron microscopy (SEM). Cryofix- ation and freeze-substitution were utilized to validate the results of CPC precipitation. In addition, specific sugar moieties present within the SCM were characterized, in part, using fluorescein isothiocyanate (FITC)-conjugated lectins.

Embryos utilized for SEM were incubated for 45-56 hours (stages 12-16), fixed in 2% glutaraldehyde with or without the addition of CPC and processed for conventional SEM. Additional embryos were cryofixed in liquid nitrogen cooled Freon 22 with or without prior aldehyde fixation and freeze-substituted in ethanol. Specimens were then warmed to room temperature, critical point dried and observed by SEM.

Embryos used for light microscopy (LM) were cryofixed and freeze-substituted prior to aldehyde fixation, brought to room temperature and embedded in paraffin. Preselected sections through the otic piacode/vesicle were labeled with FITC-conjugated lectins. The latter included: concanavalin A (Con A), wheat-germ agglutinin (WGA) and soybean agglutinin (SBA).

At 45-48 hours (stage 12) the otic placode appeared as a depression within the surface ectoderm. By 43-52 hours (stage 13) the placode had continued to invaginate and formed a distinct pit. Closure of the deepened otic vesicle proceeded between 50-56 hours (stages 15-16) as evidenced by alteration in shape and reduction in size of the associated aperture.

Embryos of all ages revealed a flocculent precipitate over the surface ectoderm which was particularly abundant in association with the otic piacode/vesicle when exposed to CPC. Specimens which were processed by freeze-substitution yielded a comparable precipitate. The otic placode/vesicle labeled positively with all lectins, the binding affinity of which followed the decreasing order: WGA>Con A>SBA. Differences in lectin binding between the surface ectoderm and the otic placode/vesicle were not apparent due to the low resolution and diffuse label given by fluorescein. The data clearly indicated that otic placode invagination was accompanied by the synthesis of copious amounts of SCM rich in glycoconjugates.

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