Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS


SYSTEMATICS Berichtzusammenfassung

Project ID: 322936
Gefördert unter: FP7-IDEAS-ERC
Land: France

Mid-Term Report Summary - SYSTEMATICS (Dynamics and Homeostasis of Germinal Zones in the Adult Vertebrate Brain)

The SyStematics project aims to provide an integrated view of the molecular and cellular rules driving neural stem cell (NSC) pools homeostasis in the vertebrate brain. It relies on a unique experimental system, the zebrafish adult pallium (dorsal telencephalon). This model was brought by the team at the forefront of adult NSC research for its enrichment in NSCs and its amenability to dynamic (live) analyses bridging the single cell and population scales, for the first time in vivo in an intact vertebrate. Towards this goal, the SyStematics projects focuses on a key parameter of adult NSC pools homeostasis, namely the maintenance of a tight balance between NSC quiescence and activation.
At the single cell level, we identified two major pathways promoting adult NSC quiescence in the pallium in vivo: Notch3 signalling (Alunni et al., 2013) and microRNA-9. To identify Notch3 targets in this process, we generated a notch3 mutant and profiled the transcriptome of NSCs in mutants and wildtype siblings. One candidate Notch3 target, selectively expressed in adult NSCs and also recovered from a parallel RNAseq profiling of quiescent versus activated NSCs, is currently being functionally validated in vivo. We further showed that miR-9 and Notch3 pathways cooperate, as miR-9 appears to potentiate Notch3 activity. Interestingly, miR-9 acts through an unconventional, nuclear mechanism, and we are currently taking approaches to identify its nuclear targets.
At the NSC population level, we are using Cre-lox-mediated fate tracing to mathematically describe how the clonal behaviour of individual NSCs accounts for the maintenance of the pallial NSC population under physiological conditions. We have worked out the conditions for sparse induction of recombination within the adult NSC pool, and have produced clones that were then chased between 1 and 8 months. These clones are currently being analysed for their composition, size and location on whole-mount cleared brain preparations to model population behaviour. As a comparison, the clonal behaviour of NSCs from embryo to adult is now also recorded, after we identified the embryonic progenitor populations at the origin of adult pallial NSCs (Dirian et al., 2014).
To link molecular pathways and NSC population behaviour, a major achievement of the SyStematics project to date has been, in collaboration with E. Beaurepaire (Ecole Polytechnique, Palaiseau, France), the development of a powerful live imaging technique permitting to record NSCs in their endogenous niche. We used the dual recording of fluorescence and harmonic signals through the head of adult transparent fish expressing fluorescent reporters in NSCs, revealing skin and skull structures and the underlying brain and NSCs in a completely non-invasive manner. We obtained single cell resolution over a field of more than 1000 cells, and harmonics recording provided landmarks for spatial alignment over weeks (Dray et al., 2015). We are now using this technique to mathematically describe the spatio-temporal profile of individual NSC activation within the NSC population. We are also constructing reporters of endogenous Notch3 activity to test the coincidence of this activation profile with fluctuations of Notch signalling.
Together, this understanding of how single cell and populational NSC maintenance mechanisms interact under physiological conditions will serve as a framework to address, in a second part of the project, pathological situations where adult NSCs are lost or amplified.

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