TONES - Tuning Notch and mechanics to engineer cell fate

Cell patterning integrates chemical and physical cues to establish complex tissue architectures which are key to organ function.

Thecochlea, the best human example of strict tissue architecture, mediates hearing via sensory hair cells and supporting cells arranged ina highly defined pattern.

Preliminary data from the host lab shows that the Notch ligand Jag1 dose-dependently regulates bothcochlear cell fate and placement, with a previously undescribed ectopic cell type emerging with profound loss of Jag1.I hypothesize that Notch dosage instructs cell fate and cellular positioning through differentiation and cell mechanics.

I will gobeyond the state-of-the-art with in utero injections to genetically manipulate and lineage trace cochlear cells (developed by host lab),a dose-dependent mouse model for the Notch disorder Alagille Syndrome (Jag1 mutant established by host lab), combined with liveimaging of cochlear explants (our collaborators), and my expertise in imaging and mathematical modelling.To investigate clonal relations and pathway activity as a function of Jag1 dosage, I will analyze single cell RNA sequencing data fromJag+/+, Jag1+/Ndr and Jag1Ndr/Ndr cochlea transduced with lineage-tracing barcodes during embryogenesis.

To visualize cochleardevelopment as a function of Jag1 dosage, I will design a genetically encoded Notch activity reporter, transduce it into Jag+/+, Jag1+/Ndr and Jag1Ndr/Ndr mice, and perform live imaging of cochlear explants to quantify tissue and signaling dynamics. To fine-tuneJag1 dosage in cochlea, I will modulate Jag1 with shRNAs, and apply the read-outs described above.

Finally, I will integrate theknowledge from these experiments into a hybrid mathematical model by incorporating Jag1 dosage as a variable.Understanding how cell fate and positioning is achieved in inner ear will lay the foundation for strategies to control patterning anddifferentiation, providing insights for hearing loss treatment.