PhD, Faculty of Health Sciences, University of the Witwatersrand / While ethanol is recognised beyond doubt as a teratogen to the unborn fetus, research nevertheless continues in order to understand its mode of action and its effects at the cellular level. The present study aimed to investigate the effect of an acute dose of ethanol on cranial morphology and morphometry in mouse fetuses, as well as on the morphology, migration and the expression of cell migration related genes in cultured chick cranial neural crest cells (cNCCs). Thirteen pregnant C57/BL mice were orally administered with 0.03ml/g of 25% (v/v) ethanol daily on gestational days (GD) 6, 7 and 8. Ten control animals received an identical dose of saline. On GD 18, all mice dams were killed and their fetuses were removed. Fetal morphological observations and crown-rump lengths were evaluated as were mean litter size, survival rate, birth weight and cranial dimensions. Cranial neural crest cells (cNCCs) were cultured from Potchefstroom koek koek stages 8-10 (HH) chick embryo neural tubes either in culture medium (DMEM) to which 0.2%, 0.3% and 0.4% ethanol (v/v) respectively, was added (treated) or in DMEM only (controls). Whole-mount HNK-1 immunocytochemistry was performed on treated and control chick embryos, as was an assay for caspase-dependent apoptosis. Photographs were taken of the cultures and the distance which the neural crest cells migrated from the neural tube at 24 and 48 hrs post-culture was measured. 24-hr time-lapse video microscopy recordings were also made to analyse the migration of the neural crest cells. Rhodamine-phalloidin immunocytochemistry for the actin cytoskeleton and scanning electron microscopy of surface ultrastructure were performed on migrating treated and non-treated cNCCs, as were proliferation assays and quantitative PCR of cNCCs‟ β-actin, Rac 1, Rho B and slug genes. There was a statistically significant increase in fetal reabsorption as well as a significantly reduced fetal survival rate observed in newborn mice fetuses that had been exposed to ethanol in utero compared to control fetuses. Ethanol-exposed mice showed a number of abnormalities, which were not significantly increased over vi controls (p>0.5). Birth weight, crown-rump length and mandibular length were also not significantly different in treated fetuses compared to controls (p>0.5). Treated (0.3%) chick cNCCs migrated over a significantly increased distance at both 24hrs and 48hrs compared to controls (p<0.05) in the axes of migration that were studied. The migratory distances of cNCCs derived from embryonic stage 9 (HH) were markedly affected by treatment with alcohol. The actin cytoskeleton of treated cNCCs showed disorganisation and loss of focal adhesion contacts while Rac 1, Rho B and slug genes were either up-regulated or down-regulated depending on the ethanol dose and duration of treatment. Ethanol promotes significant proliferation in cNCCs and may affect their migration by altering the expression of migration-linked genes and the arrangement of the actin cytoskeleton.

Neural crest cells arise during neurulation, a process that occurs during the third week of embryogenesis. These diverse cells then divide into various subtypes including cranial neural crest cells and cardiac neural crest cells. Each of these subtypes gives rise to a wide range of features throughout the fetus. While these cells are extremely diverse, they are also incredibly sensitive to their surrounding environment. Many maternal conditions affect neural crest cell division and migration, but maternal alcohol consumption and hyperglycemia due to gestational diabetes will be discussed in detail, with special attention paid to tissues that derive from cranial neural crest cells. While the initial mechanisms of the pathology vary for both of these conditions, what is remarkable is that they ultimately cause effects in similar ways. Both mechanisms lead to the creation of reactive oxygen species, which in turn trigger apoptotic pathways. Neural crest cell death causes a variety of congenital anomalies in fetuses, including craniofacial defects and cardiac outflow tract defects. Treatment options that have been researched in both conditions also vary, but are based on similar principles. Antioxidant therapies reduce the production of reactive oxygen species, thus reducing the severity of the anomalies affecting the fetus during development. Both maternal alcohol consumption and gestational diabetes are important public health concerns, and their management is of utmost priority in society. By decreasing the rates of women who consume alcohol during pregnancy, and managing gestational diabetes in those at highest risk, the rates of fetal congenital defects could be decreased.

No description available.

The migration of the cranial neural crest is an essential part of cranio-facial development in every vertebrate embryo. The cranial neural crest (CNC) is a transient population of cells that forms the lateral border of the anterior neural plate. In the tailbud stage Xenopus embryo, the neural crest cells delaminate from the neural tube, and undergo a large-scale migration from the dorsal to ventral region of the embryo. The CNC travels along distinct pathways, and populates specific regions of the embryos face. Once the CNC ceases migrating, it differentiates into a variety of tissues that are essential for cranio-facial structure and function. Some of these tissues include bones, muscle, cartilage, and ganglia. The CNC receives a concert of signals from neighboring tissues during and after CNC migration as well as signals transmitted among CNC cells, which act together to determine the fate of each CNC cell. Therefore, the proper migration of the CNC is an essential part of cranio-facial development. What molecules are important for the process of CNC migration? As one might imagine, a milieu of different molecules and interactions are essential for this complicated embryological process to occur. The work presented in this dissertation will focus on the role of a cell adhesion molecule that is important for Xenopus CNC migration. Typically, the amount of cell adhesion decreases within tissues undergoing migration. This behavior is essential to allow fluidity within the tissue as it moves. However, cell adhesions are fundamental for cell migration to occur because the moving cells need a platform on which to mechanically propel themselves. These interactions can occur between the migrating cell and extracellular matrix molecules (ECM), or can happen between cells. The cranial neural crest utilizes both cell-ECM and cell-cell interactions during the process of migration. The amount of cell adhesion mediated by either of these mechanisms will depend on where the cell is located within the CNC. Cells located at the periphery of the CNC tissue, which is surrounded by a matrix of ECM, will have more cell-ECM interactions. Cells located deeper in the CNC tissue, where there is little ECM, will rely more on cell-cell interactions. The work presented in this thesis focuses on a cell-cell adhesion molecule that is part of the cadherin superfamily of molecules. With this in mind, these studies should be descriptive of the environment within the CNC, and to a less degree the environment between the CNC and the surrounding tissues. The work presented in this dissertation will focus on cadherin-11, which is a classical cadherin that is specifically expressed in the cranial neural crest during its migration. How does cadherin-11 function in the CNC during this process? The work presented here suggests that the main role of cadherin-11 in the CNC is to perform as a cell adhesion molecule. However, too much cell adhesion is inhibitory to migration. In this respect, many of the studies described in this work indicate that cadherin-11 mediated cell adhesion is tightly regulated during CNC migration. Here I show that cadherin-11 is extracellularly processed by ADAM metalloproteases, ADAM9 and ADAM13, which removes the adhesive domain of cadherin-11. This extracellular cleavage event occurs throughout CNC migration, and is likely the main mechanism that regulates cadherin-11 mediated cell adhesion. Cleavage of cadherin-11 by ADAMs does not seem to affect its ability to interact with cytoplasmic binding partners, â-catenin and p120-catenin. This observation supports the idea that the “purpose” of cadherin-11 cleavage is to regulate cell adhesion, and not to induce (cell autonomous) signaling events. Additionally, the secreted extracellular domain of cadherin-11 (EC1-3) retains biological activity. This fragment can bind to a number of cell surface molecules in tissue culture including full-length cadherin-11 and specific members of the ADAM family. This observation suggests that EC1-3 may interact with full-length cadherin-11 molecules in vivo, and inhibit cadherin-11 mediated cell adhesion during CNC migration. EC1-3 can rescue CNC migration in embryos that overexpress cadherin-11, further supporting this hypothesis. Many of the above observations have been published in my first-author paper entitled “Extracellular processing of cadherin-11 by ADAM metalloproteases is essential for Xenopus cranial neural crest migration” published in the journal Molecular Biology of the Cell in 2009. Some of the unpublished work in this dissertation further focuses on how EC1-3 effects CNC migration in an ex vivo environment. During these studies, the observation was made that overexpression of EC1-3 in a cranial neural crest explant produces abnormal directional movement. In these experiments, it appeared as though certain regions of the CNC explant were “attracting” other regions of the explant. The preliminary studies described in chapter IV are aimed at answering the question; does EC1-3 attract migrating CNC cells? Here, we generated a Matlab program in order to effectively quantify the amount of directional movement of CNC explants presented with a source of EC1-3. In addition to quantifying cell directionality, this program can also decipher between cells moving with random or directed motion, and measure the velocity of cell migration within certain coordinates. Therefore, this program should be useful other ex vivo studies that require the observation of these features. To conclude, the work presented in this dissertation suggests that the role of cadherin-11 during cranial neural crest migration is predominately based on the adhesive function. In order for CNC migration to proceed, the amount of cadherin-11 mediated cell-cell adhesion is tightly regulated throughout this process. These cell-cell interactions are likely important for “sheet” and “branch” migration where CNC cells maintain a lot of cell-cell cohesion.

No description available.