The segmentation clock is a molecular oscillator that drives the cyclic gene expression required for regulating the timing of somitogenesis in the presomitic mesoderm (PSM) during early embryogenesis. Somitogenesis is a key process during the early development of an embryo resulting in the formation of segments (the somites) that go on to form the bones and muscle of the skeleton. Functional disturbance of key genes required for segmentation clock gene expression leads to developmental diseases such as congenital scoliosis as well as several cancers.
For dynamic clock gene expression three levels of regulation are relevant: transcriptional control, post transcriptional regulation (every step from splicing to RNA stability) and protein degradation. A complex system of transcriptional activation and negative feedback loops is emerging, however, current knowledge of the post transcriptional mechanisms that control oscillatory clock gene expression is limited.
This project will systematically identify the post transcriptional mechanisms that regulate oscillatory clock gene expression, using a model system of human induced pluripotent stem (iPS) cells differentiated into PSM cells. The project will involve analysis of ribosomal association and poly(A) tail length changes as well as protein and RNA levels to investigate translational efficiency and mRNA stability of the clock genes and will establish which sequence elements in clock gene mRNAs determine post transcriptional regulation and what factors are interacting with these elements during the clock cycle. Regulators at the post transcriptional level include RNA binding proteins, microRNAs and long noncoding RNAs. The project will establish the function of the newly identified post transcriptional regulators in regulating clock gene expression and somitogenesis. For this the iPS-PSM differentiation assay will be utilised as well as the generation of gastruloids (3D structures formed from stem cells that form somite like structures). The project will improve the understanding of developmental disorders such as scoliosis and cancers linked to misexpression of clock gene pathways.
This project will incorporate a wide variety of techniques, such as culture and differentiation of stem cells and the generation of gastruloids, CRISPR Cas9 modification of stem cells, several Next Gen Seq and Mass Spectrometry approaches as well as the subsequent analysis of these large data sets, bioinformatics, a variety of molecular biology, biochemistry and developmental biology techniques, including advanced microscopy imaging approaches. Professor Sally Lowell uses gastruloids and iPS to PSM cell differentiation assays routinely in her lab. The Dale lab also uses the iPS to PSM cell differentiation assay routinely.
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