Transcriptional Signatures of Single Cell Lineages
The goal of the project is to develop tools to study gene expression using a combination of High Throughput Imaging and live cell ﬂuorescence. This will be based on action of the MSX1 regulatory modules and their role in the fate of mesenchymal stem cells, using C2C12 mouse cells as the model cell.
To follow rapid changes in gene expression, a destabilised ﬂuorescent reporter needs to be used. During the ﬁrst year I created a stem cell line expressing Venus Yellow Fluorescent Protein under control of the MSX1 promotor with 4 identiﬁed regulatory regions.
Current systems have shortcomings in segmentation, tracking. The challenge is to create a ﬂexible but easy to use system to identify and analyse the cell data. Different image analysis methods have been developed to track individual cells, build lineages and follow ﬂuorescence intensity during an experiment.
Tools have been developed for statistical analysis and visualisation of the data. These will be further extended to examine other changes in the population. This will include examining any heterogeneity in the cell populations caused by differentiation.
The software has been released as LineageTracker and is compatible with Windows, OSX and Linux running Java and ImageJ.
Recording the Cell Cycle
The LineageTracker software has been used to analyze the cell cycle via FUCCI fluorescent markers, which indicate the cell's passage through the G1 and S-G2-M phases of the cycle.
Intensities of the FUCCI markers following cell division. Fluorescence intensity following cell division for the two daughter cells in figure 6. The two FUCCI channels have been shown for an entire cell cycle. The G1 signal (red) increases gradually following mitosis, then decreases following a rise in S-G2-M signal (green) (From Downey et. al., PLoS One, 2011)
Colour changes during the cell cycle indicated by FUCCI markers. The overlap in the red and green fluorescence (transition between G1 and S phase) is shown. (Adapted from Downey et. al., PLoS One, 2011)