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Recipient of the 2002 Outstanding Master's Thesis Award - Second Place.

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Open Access Thesis

Keywords

Neural crest; Valproic acid--Physiological effect;

Abstract

All vertebrate species contain a transient ectodermal structure termed the neural crest from which a specific subpopulation of migratory cells, the neural crest cells (NCCs), is derived. Following an epithelial-mesenchymal transition, NCCs actively migrate throughout the developing embryo. Precise temporal and spatial cellular migration is essential for the formation of a diverse array of neural crest derived tissues and structures. Valproic acid (VP A) is a unique anti-convulsant drug that can produce craniofacial and neural tube defects in exposed infants. Alterations of NCC migration, proliferation, and cytoskeletal composition have been proposed as mechanisms of VP A's teratogenicity. To investigate this drug's effects on NCCs, trunk neural fold explants were microdissected from chick embryos and cultured in the presence of 0.75-3.0 mM VPA. Computer assisted video image analysis, BrdU proliferation assays, and immunostaining with antibodies to F-actin and N-cadherin (A-CAM) were employed to investigate NCC behavior during VPA exposure. Overall cell area and width parameters were decreased compared to controls for all VPA-treated groups. Perimeter, length, elongation index, and migration velocity were not consistently changed from controls. Dynamic change in area and dynamic change in perimeter appeared to be negatively correlated with VPA concentration. This suggests inhibited cellular motile activity in the presence of VPA. VPA exposure increased the number of explants exhibiting a continuous epithelial layer of cells extending from the periphery of the attached explants, indicating a disturbed ability of the NCCs to complete the epithelial-mesenchymal transition necessary for proper migration. Proliferation assays revealed that 2.0 mM VPA significantly lowered BrdU incorporation by 11.2% compared to controls. BrdU incorporation was inhibited 100% at 3.0 mM VPA. lmmunostaining analysis of VPA exposed explants revealed A-CAM positive cell-cell junctions within attached epithelial sheets. Independent NCCs within the same cultures were not positive for A-CAM. This suggests VPA altered A-CAM regulation, inhibiting the ability of NCCs to break free of their adherens junctions. F-actin staining revealed dose-dependant decreases in the staining intensity of stress fibers and cortical areas of independent NCCs. In a small proportion of 2.0 mM VPA exposed NCCs there was an irregular, globular distribution of intense (bright) staining not seen in any other concentrations. This pattern was also seen in many rounded cells at this concentration. Within explant attached epithelial sheets, only cortical areas of NCCs were stained positively. Stress fiber staining was absent. Alteration of neural tube integrity and NCC motile capabilities through increased cell-cell adhesion, and lowered or terminated cell proliferation could promote the development of NTDs such as spina bifida. Other VPA induced defects might consequently be produced through lowered number of NCCs available to contribute to neural crest derivatives.

Year of Submission

2001

Year of Award

2002 Award

Degree Name

Master of Science

Department

Department of Biology

First Advisor

Darrell Wiens, Chair, Thesis Committee

Comments

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Date Original

5-2001

Object Description

1 PDF file (ix, 123 pages)

Language

en

File Format

application/pdf

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