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Student Number : 9502128Y - MSc dissertation - School of Anatomical Sciences - Faculty of Science / Retinoic acid, the active metabolite of Vitamin A is known to play a major role in embryonic growth and differentiation during development. It has been shown that either excess or deficiency of retinoic acid during embryogenesis can be teratogenic. In order to study the teratogenic effects of retinoic acid, the aim of the present study was therefore to investigate the effect of all-trans retinoic acid on the migration and fate of neural crest cells in vitro and in vivo. In addition, the study investigated the effect of retinoic acid on the cytoskeletal elements of neural crest cells and on Rac and Rho, two members of the Rho family of GTPases. The neural tubes containing neural crest cells of quail embryos were removed at cranial levels and cultured on fibronectin as a substrate. The neural tubes were cultured in either Dulbecco’s minimal essential medium (DMEM) or in DMEM+Dimethylsulphoxide (DMSO) as controls. In order to test the effect of retinoic acid, the neural tubes were cultured in 10-5M all-trans retinoic acid (RA) which was reconstituted in DMSO. The distance of migration of the cultured quail neural crest cells was measured and compared between the controls and the experimentals. To study the effect of RA on the cell actin cytoskeleton in vitro, cultured neural crest cells were stained with rhodamine phalloidin. In addition, following 24 hours of culture, the quail neural crest cells were brought into suspension and micro-injected into 36 hour-old chick hosts. While the migration of neural crest cells was extensive in the control cultures in vitro, migration was inhibited in the retinoic acid-treated neural crest cells. In addition, retinoic-acid treated neural crest cells showed pigmentation and neuronal processes earlier than did the control neural crest cells. Retinoic acid-treated neural crest cells showed a disarray of the cytoskeletal elements as they were devoid of stress fibres and focal adhesions. In addition, retinoic acid appears to decrease the expression of Rac and Rho of cultured quail neural crest cells. Following micro-injection of cultured control and RA-treated quail neural crest into the cranial region of chick hosts, the control cells populated the beak area, whereas the retinoic acid-treated quail neural crest cells migrated to the retina of the eye, a region they normally do not populate. These results suggest that retinoic acid disturbs the migration of neural crest cells. It appears to do this by affecting the cytoskeletal elements of neural crest cells and the genes that are involved in forming these elements.

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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.

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No / The development of hair follicle organ culture techniques is a significant milestone in cutaneous biology research. The hair follicle, or more accurately the "pilo-sebaceous unit", encapsulates all the important physiologic processes found in the human body; controlled cell growth/death, interactions between cells of different histologic type, cell differentiation and migration, and hormone responsitivity to name a few. Thus, the value of the hair follicle as a model for biological scientific research goes way beyond its scope for cutaneous biology or dermatology alone. Indeed, the recent and dramatic upturn in interest in hair follicle biology has focused principally on the pursuit of two of biology's holy grails; post-embryonic morphogenesis and control of cyclical tissue activity. The hair follicle organ culture model, pioneered by Philpott and colleagues, ushered in an exceptionally accessible way to assess how cells of epithelial (e.g., keratinocytes), mesenchymal (e.g., fibroblasts), and neuroectodermal (e.g., melanocytes) origin interact in a three-dimensional manner. Moreover, this assay system allows us to assess how various natural and pharmacologic agents affect complex tissues for growth modulation. In this article, I focus on the culture of the human hair follicle mini-organ, discussing both the practical issues involved and some possible research applications of this assay.