The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and β-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of β-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that β-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of β-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of β-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

No description available.

No description available.

Similarities between the acanthomatous epulis and ameloblastomas resulted in debate regarding the nature and origin of the acanthomatous epulis found in dogs. In an attempt to elucidate the origin and character of the acanthomatous epulides, this study aimed to find suitable cell markers to identify odontogenic epithelium versus oral epithelium in developing dog teeth in order to use in future research on the pathogenesis and pathology of odontogenic neoplasms in dogs. As specific markers for odontogenic epithelium have not been described in dog tissue, proposed markers of odontogenic epithelium of human and rat tissues were tested on developing dog teeth. Keratin 14, keratin 19, amelogenin, p75 neurotrophin receptor and calretinin have been proposed as markers for inner enamel epithelium and/or ameloblasts in human and rat tissue and was therefore included in this study. Keratin 14 and keratin 19 can not be regarded as specific markers of odontogenic epithelium as various other types of epithelium also stained positive with these markers. Amelogenin could be a promising marker to distinguish between odontogenic tumours and non-odontogenic tumours as it was only detected in odontogenic tissues in this study. However, amelogenin has also been observed in other tissues in dogs and rats, and therefore further studies on this protein will be needed to elucidate the expression profile of amelogenin in odontogenic versus non-odontogenic tissues in dogs. p75 Neurotrophin receptor expression was restricted to certain regions of the inner enamel epithelium and no staining was observed in other epithelial cells. It therefore seems to be a promising marker to differentiate between odontogenic and non-odontogenic epithelium, but the widespread staining observed in the mesenchymal tissue makes differentiation between odontogenic and non-odontogenic stromal elements impossible. Calretinin staining was observed in the alveolar epithelial cells directly overlying the developing tooth germ, proposed as the oral epithelium where the dental lamina takes origin from, as well as the dental laminae and Serres rests. No staining was observed in the rest of the oral epithelium and it can therefore be proposed that calretinin could be a useful marker to distinguish between odontogenic and non-odontogenic epithelial cells. In light of the results found in this study on foetal tissue, the expression profile may be different in adult tissue. Odontogenic tumours in adult dogs may originate from remnants of odontogenic tissue like Serres rests and Malassez rests. It is therefore proposed that this study be repeated on adult dog tissue with specific reference to Serres rests, Malassez rests and the associated gingiva Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Oral Pathology and Oral Biology / unrestricted

Expression and interaction of desmosomal components and keratins provide stable cell cohesion and protect the epidermis against various types of stress. The differentiation-specific isotype composition of the keratin cytoskeleton and desmosomes is regarded as major determinant of adhesive strength. However, the functional significance of individual keratins for the composition and adhesion of desmosomes has not been addressed in full. To overcome keratin redundancy following deletion of individual keratin genes, the entire type II or type I keratin cluster was deleted, resulting in the absence of keratin filaments in epidermal keratinocytes. The comparison of mouse keratinocyte cell lines lacking all keratins or re-expressing distinct keratin isotypes provides an excellent model to examine keratin contribution to the formation and stability of desmosomes. In support with the reported phenotype in vivo, desmosomes assemble in the absence of keratins but are endocytosed at accelerated rates. The internalization of desmosomes is regulated by PKCα-mediated desmoplakin phosphorylation, rendering epithelial sheets highly susceptible to mechanical stress in cell culture. Re-expression of the keratin pair K5/K14, inhibition of PKCα activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. The data support a model whereby K5/K14 sequesters RACK1, which can bind PKCα and thereby limits DP phosphorylation, promoting desmosome stability/maintenance and intercellular adhesive strength. To investigate the isotype-specific function of keratins, the respective contribution of K5/K14 or K6/K17 to desmosome adhesion, upon their stable re-expression in keratinocytes lacking all keratins was analyzed. This revealed that K5/K14 support stable desmosomes, whereas expression of “wound healing” keratins K6/K17 induce PKCα-mediated desmosome disassembly and subsequent destabilization of epithelial sheets accompanied by faster wound closure. Furthermore, analysis of adherens junctions and actin organization in keratin-free keratinocytes demonstrated a role of keratins in reorganization of the actin cytoskeleton and maturation of adherens junctions. This study identified a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for wound healing, tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.

No description available.

The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and ¿-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of ¿-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that ¿-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of ¿-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of ¿-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

The eyes of snakes and most geckos, as well as a number of other disparate squamate taxa, are shielded beneath a layer of transparent integument referred to as the “reptilian spectacle.” Derived from the embryonic fusion of palpebral tissues, the spectacle contains a number of specializations of the skin to benefit vision while still allowing it to function as the primary barrier to the environment. For example, in nearly all species that possess it, it is markedly thinned compared to the surrounding integument and its keratinized scale is optically transparent. While the spectacle may thus seem ideally adapted to vision in allowing the eyes to be always unoccluded, it does have a few drawbacks. One such drawback is its vascularity, the implications of which are still not fully understood, but are explored herein. As no recent synthesis exists of the body of knowledge on reptilian spectacles, the first chapter of this thesis consists of a review of spectacle anatomy, physiology, adaptive significance and evolution to help put into context the following chapters that present original research. The second chapter describes the dynamics of blood flow through the spectacle vasculature of colubrid snakes, demonstrating three main points: (1) that the spectacle vasculature exhibits cycles of regular dilation and constriction, (2) that the visual perception of a threat induces vasoconstriction of its vessels, and (3) that spectacle vessels remain dilated throughout the renewal phase. The implications of these points are discussed. The third chapter describes the spectral transmittance of the shed spectacle scale, the only keratinized structure in the animal kingdom to contribute to the dioptric apparatus of the eye, as well as its thickness. Spectacle scale transmittance and thickness was found to differ dramatically between snakes and geckos and found in snakes to vary between families. The adaptive significance of the observed variation is discussed. The fourth chapter describes biochemical analyses of the shed spectacle scales of snakes and geckos and compares their composition to other scales in the integument. Spectacle scales were found to differ significantly from other scales in their keratin composition, and gecko spectacle scales in particular were found to lack ß keratin, that hard corneous protein thought to be common to all reptile scales. The concluding chapter will discuss where this research has brought the state of our knowledge on the spectacle and offers thoughts on potentially useful avenues for further research.

The eyes of snakes and most geckos, as well as a number of other disparate squamate taxa, are shielded beneath a layer of transparent integument referred to as the “reptilian spectacle.” Derived from the embryonic fusion of palpebral tissues, the spectacle contains a number of specializations of the skin to benefit vision while still allowing it to function as the primary barrier to the environment. For example, in nearly all species that possess it, it is markedly thinned compared to the surrounding integument and its keratinized scale is optically transparent. While the spectacle may thus seem ideally adapted to vision in allowing the eyes to be always unoccluded, it does have a few drawbacks. One such drawback is its vascularity, the implications of which are still not fully understood, but are explored herein. As no recent synthesis exists of the body of knowledge on reptilian spectacles, the first chapter of this thesis consists of a review of spectacle anatomy, physiology, adaptive significance and evolution to help put into context the following chapters that present original research. The second chapter describes the dynamics of blood flow through the spectacle vasculature of colubrid snakes, demonstrating three main points: (1) that the spectacle vasculature exhibits cycles of regular dilation and constriction, (2) that the visual perception of a threat induces vasoconstriction of its vessels, and (3) that spectacle vessels remain dilated throughout the renewal phase. The implications of these points are discussed. The third chapter describes the spectral transmittance of the shed spectacle scale, the only keratinized structure in the animal kingdom to contribute to the dioptric apparatus of the eye, as well as its thickness. Spectacle scale transmittance and thickness was found to differ dramatically between snakes and geckos and found in snakes to vary between families. The adaptive significance of the observed variation is discussed. The fourth chapter describes biochemical analyses of the shed spectacle scales of snakes and geckos and compares their composition to other scales in the integument. Spectacle scales were found to differ significantly from other scales in their keratin composition, and gecko spectacle scales in particular were found to lack ß keratin, that hard corneous protein thought to be common to all reptile scales. The concluding chapter will discuss where this research has brought the state of our knowledge on the spectacle and offers thoughts on potentially useful avenues for further research.

A candidate gene approach was used to identify potential genetic markers associated with wool quality traits including mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVD), prickle factor, curvature, yellowness, brightness, staple strength, staple length, yield, greasy fleece weight (GFW) and clean fleece weight (CFW). Inheritance of potential genetic markers was studied in two half-sib Merino families and assessed for association with the wool quality traits. The sire for one of the half-sib families is referred to as MV144-58-00, and wool measurements from its progeny were taken at 12 (n = 131), 24 (n =128) and 36 (n = 37) months of age. The sire for the second half-sib family is referred to as Stoneyhurst, and wool measurements from its progeny (n = 35) were taken at 12 months of age. Genes that code for the keratin intermediate-filament proteins (KRTs) (KRT1.2, KRT2.10) and the keratin intermediate-filament-associated proteins (KAPs) (KAPl.1, KAPl.3, KAP3.2, KAP6.1, KAP 7, KAP8) were targeted for this investigation, along with the beta 3-adrenergic receptor (ADRB3) gene and microsatellites BfMS and OarFCB193. Polymerase chain reaction (PCR) was used to amplify specific DNA fragments from each locus and PCR- single strand conformational polymorphism (PCR-SSCP) analysis was used to detect polymorphism within the half-sib families for all the loci, except for the KAP1.1 gene, where length polymorphism was detected using agarose gel electrophoresis. Only the loci that were heterozygous for the sire (KAP1.1, KAP1.3, KRT1.2, ADRB3, KAP8) and hence were informative, were genotyped in the progeny. The total number of alleles observed at the KAP1.1, KAP1.3, KRT1.2, KAP8 and the ADRB3 loci were four, ten, six, five and six, respectively. Analysis of each of the informative loci revealed allelic associations with various wool traits. In the MV144-58-00 (genotypes KAP1.1 AB; KAP1.3 BD; KRT1.2 AB; ADRB3 CE) half-sib, inheritance of the KAP1.1 A allele was associated with a higher yield at 24 months of age (P = 0.037). This trend also observed at 36 months of age (P = 0.078). At 12 months of age, the KAP1.1 A allele tended to be associated with increased staple length (P = 0.08). At 36 months of age, the inheritance of the KAP1.1 B allele tended towards being associated with whiter wool (P = 0.080). The MV144-58-00 KAP1.3 D allele tended to be associated with increased yield at 24 and 36 months of age (P = 0.091 and 0.059, respectively), and with lower FDSD at 12 months of age (P = 0.055). The sire KAP1.3 B allele was associated with whiter wool colour at 36 months of age (P = 0.045). The inheritance of the MV144-58-00 KR T1.2 B allele was associated with or tended to be associated with a smaller FDSD (P = 0.040), an increase in staple strength (P = 0.025) and an increase in GFW (P = 0.069) at 12 months of age. At 24 months of age, the KR T1.2 B allele tended to be associated with increased yield (P = 0.057). At 36 months of age, the KRTl.2 A allele was associated with whiter wool (P = 0.019) and tended to be associated with increased crimp within the wool fibre (P = 0.089). In the Stoneyhurst (genotypes KAP1.1 BC; KAP1.3 CJ; KRT1.2 DE; ADRB3 CE) half-sib, inheritance of the KAP1.1 B allele was associated with longer staple length (P = 0.018) and a decrease in wool brightness (P = 0.039). In contrast, KAP1.1 C allele was associated with lowest staple length (P = 0.018) and brighter wool colour (P = 0.039). Associations observed with the inheritance of Stoneyhurst KAP 1.1 alleles were similar to the inheritance ofKAPl.3 alleles. Stoneyhurst KAP1.3 J allele was associated with longer staple length (P = 0.017) and a decrease in wool brightness (P = 0.010). In contrast, KAP1.3 C allele was associated with lowest staple length (P = 0.017) and brighter wool colour (P = 0.010). The Stoneyhurst KRT12 D allele was associated with longer staple length and a decrease in wool brightness (P = 0.033). In contrast, KRT1.2 E allele was associated with lowest staple length (P = 0.033) and brighter wool colour (P = 0.022). Sire alleles at the ADRB3 gene locus were associated with variation in staple strength (P = 0.025) for MV144-58-00's progeny, and with variation in yield (P = 0.023) for Stoneyhurst's progeny. The results obtained in this thesis are consistent with KAP1.1, KAP1.3 and KRT1.2 being clustered on one chromosome because both sires in this study passed on two major KAP1.1-KAP1.3-KRT1.2 haplotypes to their progeny, and the associations with wool traits were very similar for all the three loci. The major sire derived KAP1.1 – KAP1.3 - KRT1.2 haplotypes observed within the MV144-58-00 half-sib were: BBA (frequency of 43.4%; n = 43) and ADB (frequency of 44.4%; n = 44). Other minor haplotypes observed were: ADA (frequency of 4.0%; n = 4); BDA (frequency of 2.0%; n = 2); BBB (frequency of 3.0%; n = 3) and BDB (frequency of 3.0%; n = 3). In the Stoneyhurst half-sib, major sire-derived KAP 1.1 - KAP 1.3 - KR Tl.2 haplotypes observed were CCE (frequency of 53.1 %; n = 17) and BJD (frequency of 40.6%; n = 13). The minor haplotype BJE (frequency of 6.3%; n = 2) was also observed. Statistical analyses within the MVI44-58-00 half-sib showed that KAP1.1 AKAP1.3 D - KRT1.2 B haplotype was associated with increased yield (P = 0.023) and tended towards whiter wool colour (P = 0.059), smaller FDSD (P = 0.081) and stronger staple strength (P = 0.092). In the Stoneyhurst half-sib, the KAP1.1 B - KAP1.3 J - KRT1.2 D haplotype was associated with longer staple length (P = 0.010), while the KAP1.1 C - KAP1.3 C - KRT1.2 E haplotype showed a strong trend with increased wool brightness (P = 0.096). Result from this study indicated that the keratin genes on chromosome 11 are recombining relatively frequently at recombination "hotspots". A high rate of recombination among loci that impact on wool traits would make breeding for consistent wool quality very difficult. The results presented in this thesis suggest that genes coding for the KRTs and KAPs have the potential to impact on wool quality. KAP1.1, KAP1.3 and KRT1.2 could potentially be exploited in gene marker-assisted selection programmes within the wool industry to select for animals with increased staple length, 'increased staple strength, higher yield and brighter wool. This study was however limited to two half-sib families, and further investigation is required.