Design and implementation of transgenic tools to visualise cell cycle progression in mammalian development
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Ford, Matthew Jonathan
Cell cycle progression is the series of steps a cell has to take in order to duplicate its DNA and produce two daughter cells. Correct spatial and temporal coordination of the cell cycle is key for the normal development of any organ or tissue and is stringently controlled during embryogenesis and homeostasis. Misregulation of cell cycle progression is causal in many developmental disorders and diseases such as microcephaly and cancer. Fucci (Fluorescent Ubiquitination based Cell Cycle Indicator) is a system that allows for the visualisation of cell cycle progression by the use of two differently coloured fluorescent probes whose abundance is regulated reciprocally during the cell cycle. The probes contain the E3 ligase recognition domains of Cdt1 and Geminin fused to the fluorophores mCherry (red fluorescence) and mVenus (yellow fluorescence) respectively. Cells are therefore labelled red during G1, yellow in the G1/S transition and green during late S/G2 and M phases of the cell cycle. In order to study development and tissue homoeostasis a Fucci expressing mouse line was developed however this has several key limitations: First, the two Fucci probes are expressed from separate loci complicating mouse colony maintenance. Second, the constructs were not inducible, making it impossible to follow cell cycle progression in specific cell lineages and third the mice were generated by random transgenesis which is prone to silencing and can exhibit variation in expression between different tissues. Here I have characterised an improved version of the original Fucci system known as Fucci2a designed by Dr Richard Mort (University of Edinburgh) to overcome these limitations. The Fucci2a genetic construct contains both Fucci probes fused with the Thosea asigna virus self-cleaving peptide sequence T2A. This allows expression of both probes as a single bicistronic mRNA with subsequent cleavage by ribosomal ‘skipping’ during translation to yield separate proteins. A Fucci2a mouse (R26Fucc2aR) was generated by homologous recombination into the ROSA26 locus using the strong, ubiquitous CAG promoter to drive expression and incorporating a floxed-Neo stop cassette. This allows tissue specific activation by Cre recombinase when combined with a second Cre expressing mouse line. Building on the bicistronic Fucci2a technology I have gone on to develop and characterise four new tricistronic reporter constructs which allow for the dual visualisation of cell cycle progression with apoptosis, cytokinesis and ciliogenesis. In each case an additional fluorescent probe was added to the original Fucci2a construct separated by the self-cleaving peptide P2A and the construct characterised in 3T3 stable cell lines. The combination of a dual cilia and cell cycle reporter construct proved fruitful and I have gone on to investigate the relationship between cell cycle progression and ciliogenesis in 3T3 cells and have generated and characterised the R26Arl13b-Fucci2aR mouse line. I have also illustrated the utility of the R26Fucci2aR mouse for generating quantitative data in development research in two development situations; melanocyte development and lung branching morphogenesis. Melanocytes are specialised melanin producing cells responsible for the pigmentation of the hair, skin and eyes. Their precursors, melanoblasts, are derived from the neural crest where they migrate and proliferate before becoming localised to hair follicles and their study provides a good model for understanding the development of other neural crest derived lineages such as the peripheral nervous system. Using time-lapse imaging of ex vivo skin cultures in which melanoblasts are labelled with the Fucci probes I have characterised melanoblast migration and proliferation. In addition, I have shown that Kit signalling, which is necessary for melanoblast migration and survival, controls melanoblast proliferation in a density dependent manner and that melanoblast migration is more persistent in S/G2/M phases of the cell cycle. Lung branching morphogenesis requires constant proliferation at the apical tip of a growing epithelial branch. Loss of epithelial symmetry through an unidentified mechanism (requiring BMP, FgF10, Shh and Wnt signalling) within a branch is required to initiate branching either latterly from the side of a elongating branch by domain branching or by bifurcation of the tip. In the final section of this thesis I performed a comparative analysis of the behaviour of the developing lung epithelium using proliferative status (Fucci2a expression) to categorise each cell. Using a combination of live imaging and immunohistochemistry I have identified a transition zone 100-150μm from the tip of the branching lung epithelium where epithelial cells become stationary and drop out of the cell cycle corresponding with the onset of proximal bronchial progenitor marker Sox2. A comparative gene expression analysis of the proliferating and non-proliferating regions using Fucci2a to distinguish them has eluded to several interesting genes which could influence branching morphogenesis during lung development.