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Molecular characterization of in vitro specification towards neural fate from primitive streak cells

Russo, Luigi

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Since their discovery, mouse embryonic stem cells (mESCs) constitute an invaluable system to investigate complex developmental biology processes, such as germ layer specification. In my PhD project, I used fluorescent reporter mESC lines to explore the signaling network underlying fate decisions from Primitive Streak (PS) formation to neuroectoderm induction. I first defined culture conditions allowing the generation of cells transcriptionally resembling the PS and relying on the same signaling as their in vivo counterparts. I successfully discriminated the individual contributions of the Wnt and Nodal/TGF-β pathways in this context, the first starting the PS program, and the second enabling to proceed towards the endoderm fate while repressing neuroectoderm formation. Using a Wnt reporter line, I identified cells with active canonical Wnt signaling during PS-like differentiation and characterized by a posterior PS and mesoderm transcriptome. I additionally showed that neural progenitors arise in these conditions, with identity spanning the entire anteroposterior axis, and I uncovered their putative inductive stimuli. A Retinoic Acid (RA) sensor line, generated ad hoc, showed active RA signaling in the PS-like population. Intriguingly, the fraction of neural progenitors obtained was dependent on the activation state of RA signaling. The combination of RA perturbation and Wnt or TGF-β inhibition highlighted the involvement of RA signaling in other mechanisms of neural induction. Finally, I mimicked the phenotype of the RA synthesis-defective mice in my sensor line, observing no RA reporter expression in PS-like differentiation, and a seemingly unaffected specification of neuroectoderm cells. However, the neural fate acquisition in the RA synthesis-defective cells was still sensitive to repression of RA receptors, and providing vitamin A led most of the cells towards the neural lineage, and reactivated the RA reporter. This proves a residual ability of these cells to synthesize RA, undermining a central argument against a role of RA in neural induction by the PS. In conclusion, in this project I established a manipulatable system, suitable for investigating processes going from the primitive streak formation to the acquisition of the neuroectoderm fate. The system provided convincing arguments about the necessity to reevaluate the role of retinoids in neuroectoderm specification by the PS.

Document type: Dissertation
Supervisor: Neveu, Dr. Pierre
Place of Publication: Heidelberg
Date of thesis defense: 3 December 2020
Date Deposited: 14 Dec 2020 12:08
Date: 2021
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > European Molecular Biology Laboratory (EMBL)
DDC-classification: 570 Life sciences
Uncontrolled Keywords: stem cells; primitive streak; neural induction; retinoic acid
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