published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Paediatrics / Master / Master of Philosophy

The extracellular matrix is a vital component of the cardiovascular system, in that, it not only provides structural support but also plays a critical role in the maintenance of cellular stability. One of the major components of the vascular matrix is elastin, which confers vessels with the specialized property of stretch and recoil. Elastin deficiency has been implicated in many vascular diseases and determined experimentally to be a negative regulator of smooth muscle cell proliferation. In zebrafish, two elastin genes have been identified, which are actively expressed during development. Based on this finding, protein production and spatial localization for the two elastin proteins was studied by immunohistochemistry with specific antibodies. Results revealed a global distribution for elastin 1 in the ventral aorta and swim bladder, whereas elastin 2 was preferentially localized to the bulbus arteriosus indicating a possible specialized function of elastin 2 in this structure. This observation, and the unique physiological property of this structure, suggests a possible reason for the preservation of both elastin genes during evolution. / In the second part of this study, elastin-null mice were studied to uncover the impact of the loss of elastin on the expression of other elastic fiber-associated proteins. The expression of fibrillin-1, the major component of microfibrils, was not altered in the absence of elastin, implying that elastin is not necessary for the formation of microfibrils. On the other hand, both fibulin-2 and -5 were upregulated in the absence of elastin, suggesting that expression of these genes are controlled by elastin. Overall, this study highlights the importance of elastin in evolution, as well as its potential role in the regulation of expression of other matrix molecules.

The splotch (sp) mutation has been identified as a mutation in the paired box gene, Pax-3. Heterozygous mice carrying this mutation are phenotypically normal, with the exception of a white spot on their bellies. Homozygous embryos do not live to birth, and suffer from a wide range of developmental defects, all of which result from delayed neural tube closure, or inadequate neural crest cell migration. Most notably, homozygotes have an increased rate of spina bifida with or without exencephaly. Retinoic acid (RA), which has been shown to be very important in the development of a number of systems, was shown to cause a selective mortality of the homozygous splotch embryos when administered maternally at day 9 p.c. (Moase and Trasler, 1987). Since cellular retinoic acid binding protein (CRABP) is localized to the tissues which are affected by both the splotch gene, and retinoic acid teratogenesis, its expression patterns in the developing splotch embryo were examined. It was found that the distribution of CRABP mRNA is normal, but its expression levels are excessive in splotch homozygous day 9 mouse embryos.

No description available.

The striped-mouse, Rhabdomys pumilio, is widely distributed throughout southern Africa within a variety of habitats and rainfall regimes. It is found at sea level in the Eastern and Western Cape regions and at altitudes above 2700 m in the Drakensberg mountains. The attraction of R.pumilio to cultivated land and crops has resulted in extensive damage to plants and cultivated crops. A study of the genetic variation between populations of R.pumilio from different regions of southern Africa was undertaken by protein electrophoresis and randomly amplified polymorphic DNA using the polymerase chain reaction (PCR-RAPD). A cytogenetic study was also undertaken. The mean heterozygosity (H=0.074) for R.pumilio was more than twice that estimated for mammals (H=0.036) while the mean percent polymorphism (P=16.1%) was only slightly higher than the mean percent polymorphism obtained for mammals (P=14.7%). The highest heterozygosities were recorded in the Potchefstroom (H=0 .145) and Zimbabwe (H=0 .118) samples and the lowest mean heterozygosity was recorded in the peninsular Western Cape (H=0. 032). A mean Fst value of 0.459 was obtained, suggesting a high degree of genetic differentiation between the samples of R.pumilio but the negative Fis (-0.01) value emphasized that R.pumilio retained an outbreeding population structure. The similarity coefficient between the samples of R.pumilio using PCR-RAPD's ranged between 0.471 and 0.853 and substantiated the argument for genetic divergence between the samples of R.pumilio. An isolation by distance model for the population genetic structure of R.pumilio was supported by the allozymes (r=0.58, p<0.00l) and PCR-RAPD's (0.75, p<0.00l). Temperature and rainfall also had an influence on the allelic frequency distribution of certain loci of R.pumilio. Rogers (1972) genetic similarity varied between 0.796 and 0.988 while the values for Nei's (1978) unbiased genetic distance varied between 0.000 and 0.189 for the different samples of R.pumilio. Subgrouping of the KwaZulu-Natal samples, the peninsular Western Cape and Eastern Cape samples of R.pumilio was evident with the allozymes. With the PCR-RAPD' s the Zimbabwe sample showed the least similarity to the other samples with a KwaZulu-Natal/Potchefstroom subgroup separating from the less well defined Eastern Cape and Western Cape subgroup. Cytogenetic studies of specimens of R.pumilio from some of the localities in southern Africa revealed a chromosomal number of 2n=48 , while the Potchefstroom and Zimbabwe specimens displayed a chromosomal number of 2n=46. Homology in G-and C-banding was recorded. The allozymes, PCR-RAPD's and chromosomal studies suggested subspecies status for the Zimbabwe population of R.pumilio. The Potchefstroom sample displayed a greater genetic similarity to the remaining South African samples of R.pumilio than the Zimbabwe samples and therefore could not be considered for subspecies status. Although the South African samples of R.pumilio displayed a certain degree of genetic divergence, it was insufficient to warrant subspecies status although evolution in this direction was suggested. / Thesis (Ph.D.)-University of Natal, Durban, 1999.

Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.

No description available.

No description available.

No description available.