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
