UNIL principal investigator
Prof. Ludovic Telley, Faculty of Biology and Medicine
ULB principal investigator
Dr Jérôme Bonnefont, Faculty of Medicine
Our research proposal aims at understanding the molecular mechanisms underlying the generation of different cortical neuron populations and we postulate that, as for motor neuron specification, at least part of this diversity relies on stochastic cell fate decisions driven by transcriptional bursting/stochastic processes.
To investigate this, we propose to develop the proof of concept of a new methodology allowing the simultaneous comparison of total transcriptional abundance and ongoing transcriptional bursts.
Apical progenitors and its progeny will be labelled and tracked using a high temporal resolution approach called “Flashtag”. Specifically, we will perform CFSE in utero ventricular injection in E12 and E15. Presumptive somatosensory area will be collected after 1, 3, 6, 24 hours post FlashTag in order to cover sequential stages of specification. The cells will be then FAC-sort as single cell pools prompt to generate dedicated single-cell library allowing quantification/comparison of mRNA level and active transcriptional burst. Interestingly this transcriptional burst can be estimated by measuring the recruitment of RNA-pol II (phosphorylated on Ser 2) on the chromatin using a recently developed protocol called CUT&Tag. We propose to adapt this protocol in order to combine it with the high throughput 10X Genomics single cell platform. To do so we will modify the current multiome protocol from 10X Genomics (which allow simultaneous coverage of ATAC and mRNA profiling) in order to allow the simultaneous quantification of RNA-pol II (phosphorylated on Ser 2) recruitment and mRNA total production.
With this pilot experiment, we expect to define, at a cellular resolution, patterns of gene bursting allowing to precisely identify the diversity of committed progenitor subpopulations at two embryonic time points of neurogenesis known to lead to the generation of different neuron populations with specific morphological, laminar and hodological properties.
- 10.2021-01.2022 collection of the first samples at ULB, library preparation/sequencing at UNIL
- 02.2022-05.2022 bioinformatic analysis
- 06.2022-09.2022 collection of complete sample series at ULB, library preparation/sequencing at UNIL
- 10.2022-01.2023 bioinformatic analysis