The Notch pathway is crucial to vertebrate development. Aberrant levels of Notch underlies many diseases and cancers. is a developmental process in which Notch plays an essential role. When Notch is inhibited, are not formed. Moreover, extending NICD half-life decreases the rate of somite formation.
Unlike most major pathways, Notch does not rely on intermediate mediators. Following ligand activation, the intracellular part of the Notch receptor (NICD) is cleaved, into the nucleus and interacts with transcription complexes turning on gene expression. NICD is then rapidly degraded. Both in development and disease contexts this pathway has been shown to be exquisitely sensitive to NICD levels.
We that transcriptional output of specific cohorts of Notch target genes is regulated by different NICD levels thus directing the wide range of subsequent cell /fate decisions that receiving cells adopt in response to Notch activation.
To test this hypothesis, we will move from the traditional mouse embryo model to engineer human induced pluripotent stem cells () that drive low or high Notch expression and monitor their differentiation potential, on two derivatives the mesoderm (PSM), which gives rise to the , and caudal neural tube which both derive from an intermediate progenitor (NMP) cell type.
We will generate a cell line with differential levels of NICD to discern the effects of differential levels of NICD on stem cell differentiation. This cell line will be generated using CrisprCas9 technology using dCas9-SAM and dCas9-KRAB system to increase or decrease endogenous levels of the Notch receptor respectively. Using established protocols for differentiation of cells we will determine the differentiation dynamics in our assays. To the effects of varying NICD levels we will use a PSM reporter cell line, western blots and qPCR by looking at several key genes that turn on/off during the time course of differentiation. We will also assess the impact of varying NICD levels on global transcription, both intensity of gene expression and differential target gene expression. Lastly, we will use a recently developed CrisprCas9 targeted cell line, with a non- serine to alanine point mutation, that extends NICD half-life. Modulating NICD stability in this way we can in parallel compare ) the time over which the stem cells adopt different cell fates, ii) the choice of cell /or iii) the target genes the stem cells turn on following changes in NICD levels brought about by either NICD production or NICD degradation. Our findings will lead to a better understanding of the molecular basis of Notch and its impact on the physiology of stem cells.
Recent work from the lab can be found in the following references:
Carrieri, F.A., Murray, P.J., , D., Ferris, M.A., Davies, P., Dale, J.K (2019) CDK1 and CDK2 regulate NICD1 turnover and periodicity of the segmentation clock. EMBO Reports. 20, 4, p. 1-22 22 p., e46436 /10.15252/embr.201846436 PMID 31267714 Read Article
Murray, P., Carrieri, F.A., Dale, J.K. (2019) Cell cycle regulation of oscillations yields coupling of growth and from in a computational model of the mesoderm. Journal of Theoretical Biology. /10.1016/j.jtbi.2019.05.006 PMID 31121170 Read Article
, G., Bone, R. A., Dale, J. K., et al. (2015) A balance of positive and negative regulators determines the pace of the segmentation clock. Elife 4: e05842. /10.7554/eLife.05842 PMCID 4601006 PMID 26357015 Read Article