Masters Theses

Date of Award

12-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Zoology

Major Professor

Jeff MaCabe

Committee Members

Shivers, Kennedy

Abstract

During embryonic development of the chick, several regions of programmed cell death (PCD) occur in the wing bud including the anterior necrotic zone (ANZ), the opaque patch (OP), the posterior necrotic zone (PNZ) and the interdigital necrotic zones (INZ). These regions also undergo cell death in organ culture and are rescued by both basic fibroblast growth factor (FGF-2; a growth factor known to be present in the developing wing bud) and cycloheximide (CHX; an inhibitor of protein synthesis). In this study we examined in vitro cell death in tissues from areas of the chick wing bud not normally programmed to die during development.

Regions of stage 21 chick limb bud were excised and placed in organ culture. These regions were comprised of tissue which contained the opaque patch or the posterior necrotic zone or tissue just anterior or posterior to the opaque patch. This study focused on the anterior and posterior tissues and occasionally included the other regions for comparative purposes. The 51Cr release assay was utilized to quantify the amount of cell death occurring in these tissues cultured in media with or without various supplements. Tissues from anterior and posterior to the opaque patch were inspected by transmission electron microscopy for cellular morphology and debris after culture in base medium or in medium supplemented with 200ng/ml FGF-2 or 10ug/ml CHX. DNA from these two regions of tissue was analyzed by gel electrophoresis to assess whether fragmentation of DNA had occurred during the culture conditions employed for the preceding microscopy.

The results of this study illustrate that anterior and posterior regions of tissue which elude the PCD process in the developing chick wing bud are susceptible to apoptotic fate in organ culture. Production of oligonucleosomal-sized fragments of DNA in organ culture indicates that this death is apoptosis. Inhibition of cellular death and DNA fragmentation by an inhibitor of protein synthesis (CHX) further supports this conclusion. The occurrence of apoptosis rather than necrosis in these tissues in organ culture suggests that developmental cell death occurring in some regions of the chick limb bud mesenchyme may result from lack of access to a certain factor or factors. In addition, evidence from the 51Cr release assay and transmission electron micrographs indicates that the apoptosis observed in this study is inhibited by FGF-2. FGF-2 is known to be expressed in the chick limb bud. Cell proliferation and inhibition of apoptosis in these tissues in vivo may, in part, be attributable to FGF-2 present in the developing limb bud.

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