by Vernon Rowland and Robert Blumenthal ; Yvonne M. Homsy, writer-editor. / "First published as volume 12, no. 1, March 1974, of the Neurosciences Research Program bulletin." Includes index. / Bibliography: p. 154-187.

Studies on new arthropod models are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, most insect embryos exhibit the short or intermediate-germ type and become enveloped by extensive extraembryonic membranes. The genetic basis of these processes has been the focus of active research in several insects, especially Tribolium castaneum. The processes in question are very dynamic, however, and to study them in depth we require advanced tools for fluorescent labelling of live embryos. In my work, I have used a transient method for strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labelling the chromatin, membrane, cytoskeleton or combinations thereof. I have used several of these new live imaging tools to study the process of cellularisation in Tribolium, and I found that it is strikingly different to what is seen in Drosophila. I was also able to define the stage when cellularisation is complete, a key piece of information that has been unknown until now. Lastly, I carried out extensive live imaging of embryo condensation and extraembryonic tissue formation in both wildtype embryos, and embryos in which caudal gene function was disrupted by RNA interference. Using this approach, I was able to describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. As well as uncovering several of the cellular mechanisms underlying condensation, I have proposed testable hypotheses for other aspects of embryo formation. The work presented in this thesis will serve as a foundation for future studies on cellularisation and tissue morphogenesis in Tribolium. Furthermore, the live imaging method, the fluorescent labelling constructs, and the analysis I carried out should be easily adaptable to other non-model arthropod species.

Morphogenesis in walled organisms represents a highly controlled process by which the variability of shapes arises through changes in the structure and mechanics of the cell wall. Despite taking different evolutionary paths, land plants and some brown algae exhibit great developmental and morphological similarities. In two brown algal model systems: the Sargassum muticum apex and the Fucus serratus embryo, I have used a combination of imaging techniques, growth analyses, surgical and pharmacological treatments, as well as molecular, biochemical and mechanical approaches to characterise the growth patterns and the cell wall contribution to shape change. To understand how the adult algal body is formed, I examined the branching strategy (phyllotaxis) in S. muticum. My results suggest that in S. muticum the spiral phyllotactic pattern and the apical cell division pattern are not linked. The phytohormone auxin and the biochemical changes of the cell wall do not seem to be correlated with the bud outgrowth, contrary to observations in plants. In summary, these results suggest Sargassum convergently developed a distinct growth mechanism with similar shape outcome as observed in plants. This dissertation is one of the first attempts to explore cell wall mechanics in brown algal development and its correlation with underlying cell wall biochemistry utilising the Fucus embryo as a known system. The results suggest a correlation between the wall mechanics and alginate biochemistry with the growing and non-growing regions of the embryo. In addition, altering cell wall deposition or composition has a strong effect on embryo rhizoid elongation and is, in certain cases, accompanied by significant increase in cell wall stiffness and reduction of alginate epitopes. Furthermore, preliminary results exploring transcriptomic changes during development indicate differential expression of particular alginate biosynthesis enzymes (mannuronan C5 epimerases) during development, suggesting alginate conformational modifications might be stage specific. These results contribute to the current knowledge addressing the importance of cell walls in brown algal development using novel tools and approaches. Understanding developmental processes in brown algae will provide a better insight how similar morphogenetic traits are established using different body-building mechanisms.

Upon amputation, zebrafish innately regenerate lost or damaged bone by precisely positioning injury-induced, lineage-restricted osteoblast progenitors (pObs). While substantial progress has been made in identifying the cellular and molecular mechanisms underlying this fascinating process, the cell-specific function of these pathways is poorly understood. Understanding how molecular signals initiate osteoblast dedifferentiation, balance progenitor renewal and re-differentiation, and control bone shape during regeneration are of paramount importance for developing human therapies. We show that fin amputation induces a Wnt/β-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts to generate proliferative Runx2+ pObs. Localized Wnt/β-catenin signaling maintains this progenitor population towards the distal tip of the regenerative blastema. As they become proximally displaced, pObs upregulate sp7 and subsequently mature into re-epithelialized Runx2-/sp7+ osteoblasts that extend pre-existing bone. Autocrine Bone Morphogenetic Protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. Previous studies have implicated Hedgehog/Smoothened (Hh/Smo) signaling in controlling the re-establishment of stereotypically branched bony rays during fin regeneration. Using a photoconvertible patched2 reporter, we resolve active Hh/Smo output to a narrow distal regenerate zone comprising pObs and neighboring migratory basal epidermal cells. Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which instead uses non-canonical signaling to support bone maturation. Using high-resolution imaging and BMS-833923, a uniquely effective Smo inhibitor, we show that Shha/Smo promotes branching by escorting pObs into split groups that mirror transiently divided clusters of Shha-expressing epidermis. Epidermal cellular protrusions directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. These intimate interactions progressively generate physically separated pOb pools that then regenerate independently to collectively re-form a now branched bone. Our studies elucidate a signaling network model that provides a conceptual framework to understand innate bone repair and regeneration mechanisms and rationally design regenerative therapeutics. This dissertation includes previously published co-authored material. / 10000-01-01

During animal development, epithelial tissues undergo morphogenesis in order to build tissues, organs and body structure. A key driving force in epithelial morphogenesis is cell rearrangement, which results from the remodelling of cell-cell contacts. In epithelia, cell-cell contacts are connected via vertices, where 3 or more cells meet. It is unknown whether cell vertices play an active role in regulating cell rearrangement, however recent work from a number of model systems has suggested that cell vertices are important sites for sensing and regulating tissue tension and providing geometric cues for morphogenetic cell behaviours. I have used the Drosophila model system to investigate the behaviour and role of vertices in epithelial morphogenesis, using as an entry point a newly discovered marker of epithelial vertices, the Immunoglobulin-superfamily domain protein Sidekick. I have characterised the localisation of Sidekick tagged with YFP in a number of Drosophila epithelial tissues and found that in most epithelial tissues, Sidekick-YFP localises to vertices at the level of adherens junctions. Using super-resolution microscopy, I investigated the localisation of Sidekick-YFP during germband extension in the early embryo and found that Sidekick-YFP forms string-like structures at cell vertices at the level of adherens junctions, indicating that large multi-protein complexes containing Sidekick define a novel cortical domain in Drosophila epithelia. Through characterisation of a null allele of sidekick in Drosophila early embryos using both manual and automated large-scale quantitative analysis, I have found that tissue geometry, cell adhesion and morphogenetic cell behaviours are abnormal in the absence of sidekick. Together, my work shows that Sidekick at apical vertices is a novel regulator of epithelial morphogenesis.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

Candida albicans is one of the most commonly identified nosocomially acquired pathogens. This organism has a number of virulence traits including production of degrading enzymes, the ability to undergo phenotypic switching, and can rapidly undergo morphogenic switch from a blastospore (yeast) phase to that of a hyphal state. Interest in C. albicans morphogenic regulation has been the focus of a large number of studies, which have characterised transcriptional modulators of these morphologies. Recently, C. albicans has been shown to regulate its morphogenic shift through changes in cell density. It was observed that C. albicans inoculated at cell densities below 106 cells ml -1 under conditions which favour hyphal morphogenesis (pH 7.5, 37°C), will germinate to form hyphae. However, if cells densities are greater than 106 cells ml-1, little germination will occur and cells will maintain yeast morphology. The basis for this cell-density-dependent control of morphogenesis is similar to that which is seen with bacterial cells regulating their activities via quorum sensing (QS). A number of molecules have been identified which affect the ability of C. albicans to undergo the yeast-to-hyphal shift, and three compounds have been demonstrated to be quorum-sensing molecules. The scope of this review is to bring to light what is now understood about QS in C. albicans and address the roles of these molecules in relation to virulence in the host and potential roles in cross-kingdom interactions.

No description available.

No description available.

No description available.