Bad Heart Formation oolitic ironstone is the largest resources of iron in western Canada. During this study, 45 new sections from outcrop, trench and drill holes were mapped, and 325 samples were collected for petrographic and geochemical analysis. The objective of the first paper is to refine the previously published stratigraphic model based on the new data. The second paper deals with geochemistry and discuss genesis of ooids and source of iron in oolitic ironstone. The textures of the Bad Heart Formation ironstone suggest the ooids formed in-place in a relatively shallow, wave-agitated, oxygenated marine environment with repetitive growth of the ooids in water column. There are two possible source of iron in the ooids. Some geochemical data indicate it is continental sedimentary, but it is also possible that the iron sourced from sub-sea hydrothermal or meteoric vents, similar to recent iron deposits at Paint Pots in Kootenay National Park.

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Tube formation, or tubulogenesis, is an elaborate form of epithelial morphogenesis that includes processes such as cell migration and cell shape changes. The embryonic Drosophila heart, or dorsal vessel, is an excellent model of tubulogenesis and more specifically the signaling mechanisms required for cell migration and lumen formation. Similar to vertebrate heart formation, Drosophila heart tubulogenesis begins with the collective migration of cardioblasts that meet at the midline and adhere at specialised junctions, enclosing a lumen between them. Roundabout, and its ligand Slit, are required to restrict cell-to-cell adhesions to the junctional domains of contralateral cardioblasts, as well as maintain the integrity of the lumen. The localisation patterns of Robo, and other luminal cell surface receptors important for lumen formation are significantly modified throughout heart formation. Initial receptor expression is broadly distributed over the cardioblast surface. Receptors are then relocalised to specific cell surface domains by late embryonic development. The mechanisms by which Robo and other cell surface receptors are localised have yet to be determined. Endocytosis is a promising mechanism by which cell surface receptors are targeted and trafficked to cell surface domains. Specifically, vesicular trafficking proteins, such as Rab GTPases, are molecular switches that regulate endocytic events. Here, we investigated the roles of Rab5, Rab11, and Sec6 during heart formation. Of these, only Rab5, a regulator of the early endosome, was required for lumen formation. Particularly, gain of function, loss of function, and overexpression of rab5 resulted in reduced lumen phenotype, characterised by lumen pockets rather than a continuous lumen along the anterior-posterior axis. Perturbed Rab5 function also resulted in the mislocalisation of Robo at the basal domain. Live imaging showed that expression of rab5 dominant negative, constitutively active, and overexpression constructs did not perturb apical membrane motility of migrating cardioblasts in the developing heart. / Thesis / Master of Science (MSc)

Establishment of the biochemical and molecular nature of cardiac development is essential for us to understand the relationship between genetic and morphological aspects of heart formation. The molecular mechanisms that underly heart development are still not clearly defined. To address this issue we have used two approaches to identify genes involved in early chick cardiac development. Differential display previously conducted in our laboratory led to the identification of two gene fragments differentially expressed in the heart that are further described in this thesis. The full-length cDNA sequence of both eukaryotic translation initiation factor-2b (eIF-2b) and NADH cytochrome b5 reductase (b5R) were isolated using library screening. The upreglation of these genes during heart development is expected given the heart is the first functional organ to form in vertebrates and protein synthesis and cell metabolism at this stage of development is maximal. Limitations in the differential display approach led to the development and optimisation of a subtractive hybridisation approach for use with small amounts of cells or tissue. To focus on cardiac gene expression during the initial phases of heart development, subtractive hybridization was performed between the cardiogenic lateral plate mesoderm of Hamburger and Hamilton stage 4 embryos and the heart primordia of stage 9 embryos. Of the 87 independent clones identified by this procedure, 59 matched known sequences with high homology, 25 matched unknown expressed sequence tag (EST) sequences with high homology, and 3 did not match any known sequence on the database. Known genes isolated included those involved in transcription, translation, cell signalling, RNA processing, and energy production. Two of these genes, high mobility group phosphoprotein A2 (HMGA2) and C1-20C, an unknown gene, were chosen for further characterisation. The role of each gene in early chick heart development and indeed development in general, was addressed using techniques such as in situ hybridisation, transfection analysis, in ovo electroporation and RNAi. HMGA2 is a nuclear phosphoprotein commonly referred to as an architectural transcription factor due to its ability to modulate DNA conformation. In keeping with this function, HMGA2/GFP fusion protein was shown to localise to the nucleus and in particular, the nucleolus. In situ hybridisation analysis suggested a role for HMGA2 in heart and somite development. HMGA2 expression was first detected at HH stage 5 in the lateral plate mesoderm, a region synonymous with cells specified to the cardiac fate. HMGA2 was also strongly expressed in the presomitic segmental plate mesoderm and as somites developed from the segmental plate mesoderm, the expression of HMGA2 showed an increasingly more restricted domain corresponding to the level of maturation of the somite. Restriction of HMGA2 expression was first detected in the dorsal region of the epithelial somite, then the dorsomedial lip of the dermomyotome, and finally the migrating epaxial myotome cells. The novel intronless gene, C1-20C, predicts a protein of 148 amino acids containing a putative zinc finger binding domain and prenyl binding motif. Zinc binding assays showed that the zinc finger domain of C1-20C/MBP fusion protein bound over six times the quantity of zinc compared to MBP alone, although not in a 1:1 stoichiometric molar ratio. C1-20C/GFP fusion protein was shown to localise to as yet unidentified intracellular cytoplasmic vesicular compartments. These compartments did not colocalise with the endosome/lysosome pathway, aparently ruling out a role for C1-20C in protein trafficking, recycling or degradation. Expression of C1-20C in the chick embryo suggests a possible role in heart and notochord development and preliminary results using siRNA suggest that C1-20C is involved in normal heart looping.