Faculty of Biological and Environmental Sciences

Double homeobox 4 (DUX4) is a transcription factor normally repressed in somatic cells but expressed in early human embryo and involved in embryonic genome activation. Before transcription starts from its own genome, the embryo uses maternally stored transcripts and proteins, which undergo selective degradation as development progresses. DUX4 is transcribed from a repetitive region called D4Z4, along with another gene, DBE-T, that encodes a long non-coding RNA involved in the de-repression of DUX4. DBE-T partially overlaps with the D4Z4 region and shows some sequence similarity with DUX4. DUX4 regulation in early embryo is currently not well understood. The aim of this study was to use updated RNA in situ hybridization technology (RNAscopeTM) to observe DUX4 transcripts in doxycycline-inducible DUX4-TetOn human embryonic stem cells and ovarian samples. DUX4 RNA target probes were first validated in doxycycline-treated DUX4-TetOn human embryonic stem cells. Doxycycline-treated cells express DUX4 protein as observed by immunofluorescence staining. After confirming that the DUX4 target probes were visible in DUX4-expressing stem cells, the same probes were used in human ovarian tissue samples. DUX4 probes showed signals in both somatic granulosa cells and oocytes in primordial and primary follicles in the ovarian samples. However, due to the sequence similarity between DUX4 and DBE-T, there is possible cross-detection of DBE-T with the DUX4 target probes. It is not possible to say with confidence whether the detected signal is derived from DUX4 or DBE-T. These results indicate that D4Z4-related transcription activity occurs in the immature oocytes and further studies are needed to determine which transcripts are expressed in the oocyte.

Antigen presenting dendritic cells (DC) are important mediators between innate and adaptive immunity. They constantly patrol for foreign and self-antigens in peripheral tissues. Capture of an antigen initiates activation cascade in DCs resulting in the upregulation of chemokine receptor CCR7. Endothelial chemokine CCL21, ligand of CCR7, guides activated DCs from the peripheral tissues to the vicinity of the lymphatic vessels (LV) and drives the subsequent transmigration into LV lumen. From LV lumen, DCs are transported to lymph nodes where they activate T cells by presenting captured antigens to them. Despite the importance of DC migration from peripheral tissues to lymph nodes, the molecular mechanisms underlying the guidance of DCs to the transmigration sites on lymphatic endothelium are poorly understood. In this thesis I asked whether DCs have preferential transmigration sites on the lymphatic endothelium and if lymphatic endothelial chemokine CCL21 guides DCs to the transmigration sites. To seek answers to the proposed questions, I employed primary cell co-culture model where wild-type or CCR7-/- mouse bone marrow derived DCs were placed on top of the primary human lymphatic endothelial cell (LEC) monolayer overexpressing CCL21. The DC migration and transmigration events on the LEC monolayer were live imaged on epifluorescence wide field microscope and analysed with image analysis tools (ImageJ and IMARIS). Prior to transmigration wild-type DCs probed the monolayer with their dendrites and polarised them towards the LEC monolayer at the transmigration sites. Majority of wild-type DCs arrested and transmigrated at multicellular junctions (junctions where more than two cells meet). To investigate the role of CCL21 in DC transmigration, I compared the arrest site distributions and migration patterns of wild-type and CCR7-/- DCs on lymphatic endothelium. Again, majority of wild-type DCs found multicellular junctions whereas the arrest sites of CCR7-/- DCs were distributed evenly between multicellular, bicellular (junction where two cells meet) and non-junctional sites. Moreover, the wild-type DCs had straighter migratory paths to the arrest sites, and they were less prone of subsequent detachment than CCR7-/- DCs. In conclusion, in this thesis I define multicellular junctions, for the first time, as preferential DC transmigration sites across lymphatic endothelium. In addition, I show that lymphatic endothelial chemokine CCL21 guides DCs on the LEC monolayer to the multicellular junctions and thus promotes the identification of preferential transmigration sites by DCs. Future studies should focus on characterization of multicellular junctions of lymphatic endothelium and validation studies in vivo. Taken together, these findings improve the understanding of molecular mechanisms in DC transmigration across lymphatic endothelium and give a platform for development of improved vaccines and cancer therapies.

Fluctuating asymmetry is a consequence of developmental instability. It results from environmental and genetic factors and shows how well an individual is able to withstand external factors during development, such as temperature, pollutants or inbreeding. The heritability of this trait is not fully understood. In this study, the color patterns of the cichlid species Chindongo demasoni were used to investigate the heritability of fluctuating asymmetry. Although most wild type C. demasoni have symmetrical color patterns, some individuals exhibit varying degrees of color pattern asymmetry. Individuals with asymmetric bar patterns were crossed to assess the extent of asymmetry in their offspring. The results show that the offspring of asymmetric crosses have a higher bar number and greater asymmetry compared to the parent population. There was no significant difference between the left and right sides of the fish, indicating that body side was not a factor in the asymmetry observed in individuals. In addition, bar thickness decreased as bar number increased, possibly due to spatial constraints and pigment cell interactions. These results indicate that fluctuating asymmetry in bar patterns is heritable, there is an interaction between bar number and thickness in cichlid color patterns and that different features of the color pattern exhibit different levels of robustness. These findings contribute to our understanding of the developmental instability and heritability of asymmetric traits and provide a foundation for future research on genetic and environmental influences on color pattern formation in cichlids and other species.