Decoding signalling crosstalks using microfluidics and engineered human pluripotent stem cells

Uncovering the molecular logic allowing cells to integrate multiple signalling cues is of paramount importance to understand how cells commit to distinct functional states during embryonic development, normal tissue function, regeneration and disease.

In the context of human embryonic development, the spatio-temporal dynamics of WNT and NODAL signalling combinatorically dictates the fate of pluripotent cells. We recently evidenced a novel complex interaction between these two ubiquitous pathways across two different time scales (Robles-Garcia et al 2024):

-While it was previously known that WNT signalling induces NODAL ligand expression, we uncovered that WNT dosage dictates the downstream temporal dynamics of NODAL production which in turn dictates cell fates.

-We showed that WNT signalling induces a rapid and transient activation of SMAD2/3 (the intracellular effector of the NODAL pathway) even before NODAL is expressed, suggesting that WNT can directly activate SMAD2/3 in the absence of NODAL.

-Finally, we observed that NODAL inhibition potentiates WNT signalling activity, suggesting that NODAL signalling exerts negative feedback on WNT.   

The molecular mechanisms underlying this crosstalk are unknown, and how distinct signalling dynamics define specific transcriptional changes resulting in cell fate decision at the single-cell level remain to be determined.

This PhD project aims to address this important biological question using a multidisciplinary approach:

The supervisory team established a high-throughput automated microfluidic platform featuring 160 chambers that can be seeded with co-cultures of user-defined composition, generates up to 16 time-varying microenvironments, and supports post-experiment retrieval of the cells for downstream analyses (Caringella G. et al, In Press). The project will leverage this powerful system to:

1) Characterise the cells response to dynamically changing biochemical stimuli using fluorescence video-microscopy and reporter cell lines enabling real-time monitoring of the WNT-NODAL crosstalk, at the molecular level.

2) Analyse the downstream transcriptional changes associated with distinct temporal signalling regimes of WNT and NODAL.

3) Establish novel mechanistic models of the interaction between the two pathways.

Signalling pathways are in principle druggable targets but their complexity and context-dependent kinetics require a systems and control-theoretic approach to elaborate context-aware medical interventions. This project will develop insights and new tools to make a major step towards this goal.

This project is suitable for a candidate with a background in biomedical engineering or in any relevant biological discipline. You will join an enthusiastic and multidisciplinary team to gain training in state-of-the-art techniques in human pluripotent stem cell culture, microfabrication and cell biology.