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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - EDeN (Ependymal cell Development: New insight into neurological diseases)

Teaser

Summary

New neurons are continuously produced from progenitor cells in restricted locations in the adult mammalian brain . These neurons can serve as a source of new cells for regenerative therapies in pathological conditions. Our proposal addressed the mechanisms by which the adult neurogenic niche is set-up in the mouse brain. The niche is mainly composed of two types of glial cells : neural stem cells (astrocytes) and post-mitotic multiciliated ependymal cells, which are organized as pinwheels along the lateral ventricular walls.
The overall objectives are develop a new line of research aimed at understanding the cellular and molecular mechanisms involved in ependymal cell development in the mouse brain. We are using a multidisciplinary approach involving mouse molecular genetics, biophysical approaches, ex-vivo culture systems and advanced live cell imaging to execute the following three interconnected research projects:

1- Identify the mechanisms that specify RGC into ependymal cells;
2- Decipher the mechanisms of centriole amplification in ependymal cell progenitors;
3- Determine how developing ependymal cells contribute to ventricular morphogenesis and adult neurogenesis.

Work performed

Our research projects focused on three main questions:

1- Identify the mechanisms that specify RGC into ependymal cells;

Radial glial cells (RCG) are self-renewing progenitor cells that give rise to neurons and glia during embryonic development. Throughout neurogenesis, these cells contact the cerebral ventricles and bear a primary cilium. Using conditional mutants, we showed that the primary cilia of radial glia determine the size of the surface of their ventricular apical domain through regulation of the mTORC1 pathway. In cilium-less mutants, the orientation of the mitotic spindle in radial glia is also significantly perturbed and associated with an increased number of basal progenitors. The enlarged apical domain of RGC leads to dilatation of the brain ventricles during late embryonic stages (ventriculomegaly), which initiates hydrocephalus during postnatal stages. These phenotypes can all be significantly rescued by treatment with the mTORC1 inhibitor rapamycin. These results suggest that primary cilia regulate ventricle morphogenesis by acting as a brake on the mTORC1 pathway. This opens new avenues for the diagnosis and treatment of hydrocephalus.

2- Decipher the mechanisms of centriole amplification in ependymal cell progenitors;

Mitosis is driven by a regulatory network centred on the activity of the Cdk1-APC/C axis. This network spatiotemporally couples centriole and chromosome dynamics during mitosis for accurate cell division. Multiciliated cells are post mitotic epithelial cells that amplify up to several hundred centrioles to nucleate an equal number of motile cilia required for vital fluid transport. We combined single cell live imaging with pharmacological targeting of mitotic regulators and show that the Cdk1-APC/C axis along with its mitotic regulatory network is transiently activated in post-mitotic progenitors of multiciliated cells to drive the spatiotemporal progression of centriole amplification. Fine-tuning of the mitotic machinery controls centriole number and maturation through checkpoint-like phase transitions necessary for subsequent motile ciliation. Moreover, we demonstrated that deregulating the balance of mitosis activity is sufficient to restore the coupling of centriole and chromosome dynamics and to drive developing multiciliated cells into mitosis. Thus, these data establish a new framework for the study of multiciliated development and associated life-threatening pathologies. The unexpected capacity of differentiating progenitors with amplified centrioles to undergo mitosis introduces a new perspective for the assessment of cancer initiating events in ciliated epithelia.

3- Determine how developing ependymal cells contribute to ventricular morphogenesis and adult neurogenesis.

As a first step toward the dynamic analysis of apical surface pattern, we have developed bioinformatic tools to convert 3D image stacks into 2D images. Three-dimensional fluorescence microscopy followed by image processing is routinely used to study biological objects at various scales such as cells and tissue. However, maximum intensity projection, the most broadly used rendering tool, extracts a discontinuous layer of voxels, obviously creating important artifacts and possibly misleading interpretation. Here we propose smooth manifold extraction, an algorithm that produces a continuous focused 2D extraction from a 3D volume, hence preserving local spatial relationships. We demonstrate the usefulness of our approach by applying it to various biological applications using confocal and wide-field microscopy 3D image stacks. We provide a parameter-free ImageJ/Fiji plugin that allows 2D visualization and interpretation of 3D image stacks with maximum accuracy.

We also studied the mechanisms of ependymal cilia maintenance in the adult brain. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centriole

Final results

1- We have set-up some multidisciplinary experiments to identify the mechanisms that specify a progenitor cell toward the ependymal or adult neural stem cells, respectively. We are expecting to identify the precise spatiotemporal events that lead to these differentiation process, together with the molecular cascades driving these choices.

2- We are analyzing RNASequencing experiments to identify the molecular cascade that lead to centriole amplification in ependymal progenitor cells.

3- We will determine how developing ependymal cells contribute to ventricular morphogenesis and adult neurogenesis through the systematic analysis of brain ventricular walls labeled with specific antibodies to cell junctions, cilia, basal bodies and deuterosomes.