Proper embryonic patterning requires precise spatio-temporal regulation of retinoic acid (RA) activity. Morphogenesis can be regulated at the level of RA distribution, mainly via its synthesis and catabolism by the RALDH and CYP26 enzymes respectively, and at the level of RA-mediated transcription through activation of its cognate nuclear receptor, the retinoic acid receptors (RARs) α, β, and γ. Loss of Cyp26b1 leads to increased local levels of RA in tissues such as the limb and craniofacial structures, and results in neonatal lethality. Visible gross phenotypic defects in neonates include phocomelia (shortening of the limbs), adactyly (missing digits), micrognathia (shortened lower jaw), and open eyes at birth. In addition, these embryos exhibit cleft palate and have a paucity of vibrissal (whisker) and pelage (hair) follicles. We have previously shown that ablating the gene encoding RARγ in a Cyp26a1-null background was able to rescue the caudal abnormalities associated with improper RA exposure in these embryos by limiting aberrant RA signalling, and thus rescuing expression domains of target genes involved in caudal development. I show here that ablating Rarg in a Cyp26b1-null background is able to partially rescue the defects associated with loss of CYP26B1. These include a reduction in the severity of limb defects, rescued vibrissae, fused eyelids, and recovered aspects of axial skeletal development. This compound-null murine model illustrates that RARγ plays a specific role in transducing the RA signal within tissues that are affected by the loss of CYP26B1. Further molecular analysis of the pathways responsible for directing limb bud outgrowth and eyelid fusion provided insight into pathways regulated by RARγ in these rescued tissues. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-04-01 15:38:52.05

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Desmosomes are intercellular junctions that anchor intermediate filaments to the sites of intercellular contacts. They are critical for maintaining the integrity of tissues that experience constant mechanical and structural stresses, like the skin and heart. Perturbation of desmosomal adhesion can lead to devastating epidermal and myocardial diseases. However, little is known about the regulators of desmosomes and the role of desmosomes in cell signalling events. Recent work has suggested that iASPP, an inhibitor of the p53 family of proteins, localises at the intercalated discs where desmosomes reside. However, its role at the desmosomes has remained elusive. Thus, in this thesis, it was investigated whether iASPP is a dual function protein that links desmosome adhesion to gene expression and if desmosome-related diseases develop in the absence of iASPP. iASPP was found to be a novel regulator of desmosomes, co-localising with them by physically interacting with the desmosomal components desmoplakin and K5 intermediate filaments. Loss of iASPP resulted in increased phosphorylation and solubilisation of desmoplakin, leading to the formation of K5 aggregates. This culminated in disrupted intercellular adhesion and enhanced cellular migration. Consistent with the role of iASPP in the maintenance of desmosomal adhesion integrity, focal palmoplantar keratoderma was observed in iASPP-deficient mice — a disorder often associated with desmosome dysfunction. This was accompanied by disrupted intracellular signalling, as exemplified by the disrupted expression of differentiation markers; an increase in the thickness of cell layers expressing differentiation marker K1 was noted, and K5 and K6 cells were ectopically expressed throughout the diseased palmoplantar epidermis. Impaired intercellular adhesion and migration had consequences for wound healing, as iASPP-deficient mice exhibited delayed wound closure. Furthermore, defects in eyelid closure in iASPP-deficient mice were found to be due to increased apoptosis. The localisation of apoptotic cells at the leading edge of the eyelid epidermis implied that apoptosis might have occurred due to a loss of cell-matrix or cell-cell contact, i.e. anoikis. Taken together, these results suggest that iASPP is involved in pathological (palmoplantar keratoderma), physiological (wound healing) and developmental processes (embryonic eyelid closure) through its regulation of desmosomes and their dynamics. Therefore, iASPP represents a new candidate gene in cutaneous disorders and could be implicated in a variety of epidermal and myocardial diseases.