Periodic Reporting for period 4 - EDeN (Ependymal cell Development: New insight into neurological diseases)
Okres sprawozdawczy: 2020-06-01 do 2021-11-30
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.
We used 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.
The overall objectives have been fulfilled. Our results will be useful for the society because NSCs provide important therapeutic potentials and our work will be of great interest to researchers across multiple disciplines: development, neuroscience, cell biology.
We used 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.
The overall objectives have been fulfilled. Our results will be useful for the society because NSCs provide important therapeutic potentials and our work will be of great interest to researchers across multiple disciplines: development, neuroscience, cell biology.
Our research project aimed at understanding how glial cells develop and integrate into the mammalian brain. My team is composed of 10 scientists (permanent researchers, students, post-docs and engineers). Our fundamental research has major exploitations for therapeutic treatments of neurodevelopmental diseases such as microcephaly and hydrocephaly, to name a few.
More specifically, we studied three main questions:
1- Identify the mechanisms that specify RGC into ependymal cells;
1-1 mTORC1 signaling and primary cilia are required for brain ventricle morphogenesis (Foerster et al., Development 2017).
We show 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. 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.
1-2 Exon junction complex dependent mRNA localization is linked to centrosome organization during ciliogenesis (Kwon et al., Nature Communications 2021).
Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle.Here we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development.
1-3 Adult neural stem cells and multiciliated ependymal cells share a common lineage regulated by the geminin family members (Ortiz-Alvarez et al., Neuron 2019 & Ortiz-Alvarez et al., in revision).
Adult neural stem cells are sister cells to ependymal cells, whereas most ependymal cells arise from the terminal symmetric divisions of the lineage. We show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, arrests the cell cycle due to the activation of the p53-p21 pathway, which contributes to centriole amplification.
2- Decipher the mechanisms of centriole amplification in ependymal cell progenitors;
2-1 Calibrated mitotic oscillator drives motile ciliogenesis (Al Jord et al., Science 2017)
Mitosis is driven by a regulatory network centred on the activity of the Cdk1-APC/C axis. 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.
2-2 Massive centriole production can occur in the absence of deuterosomes in multiciliated cells (Mercey et al., Nature Cell Biology 2019 & Mercey et al., Sci Rep 2019)
We show that MCCs lacking deuterosomes amplify the correct number of centrioles with normal step-wise kinetics. This is achieved through a massive production of centrioles on the surface and in the vicinity of parent centrioles. These data show that the centriole number is set independently of their nucleation platforms and suggest that massive centriole production in MCCs is a robust process that can self-organize.
3- Determine how developing ependymal cells contribute to ventricular morphogenesis and adult neurogenesis.
3-1 Smooth 2D manifold extraction from 3D image stack (Shihavuddin et al., Nature Communications 2017).
We have developed bioinformatic tools to convert 3D image stacks into 2D images. 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.
3-2 Ependymal cilia beating induces an actin network to protect centrioles against shear stress (Mahuzier et al., Nature Communications 2018)
We show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. In conclusion, cilia beating controls the apical actin network around centrioles.
3-3 Entrainment of mammalian motile cilia in the brain with hydrodynamic forces (Pellicciotta et al., PNAS 2020).
We have also studied the entrainment of cilia beat in multiciliated cells from brain ventricles. Combining experiments and simulations, we studied how cilia from brain tissue align their beating direction. We subjected cilia to a broad range of shear stresses, similar to the fluid flow that cilia themselves generate, in a microfluidic setup.
More specifically, we studied three main questions:
1- Identify the mechanisms that specify RGC into ependymal cells;
1-1 mTORC1 signaling and primary cilia are required for brain ventricle morphogenesis (Foerster et al., Development 2017).
We show 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. 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.
1-2 Exon junction complex dependent mRNA localization is linked to centrosome organization during ciliogenesis (Kwon et al., Nature Communications 2021).
Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle.Here we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development.
1-3 Adult neural stem cells and multiciliated ependymal cells share a common lineage regulated by the geminin family members (Ortiz-Alvarez et al., Neuron 2019 & Ortiz-Alvarez et al., in revision).
Adult neural stem cells are sister cells to ependymal cells, whereas most ependymal cells arise from the terminal symmetric divisions of the lineage. We show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, arrests the cell cycle due to the activation of the p53-p21 pathway, which contributes to centriole amplification.
2- Decipher the mechanisms of centriole amplification in ependymal cell progenitors;
2-1 Calibrated mitotic oscillator drives motile ciliogenesis (Al Jord et al., Science 2017)
Mitosis is driven by a regulatory network centred on the activity of the Cdk1-APC/C axis. 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.
2-2 Massive centriole production can occur in the absence of deuterosomes in multiciliated cells (Mercey et al., Nature Cell Biology 2019 & Mercey et al., Sci Rep 2019)
We show that MCCs lacking deuterosomes amplify the correct number of centrioles with normal step-wise kinetics. This is achieved through a massive production of centrioles on the surface and in the vicinity of parent centrioles. These data show that the centriole number is set independently of their nucleation platforms and suggest that massive centriole production in MCCs is a robust process that can self-organize.
3- Determine how developing ependymal cells contribute to ventricular morphogenesis and adult neurogenesis.
3-1 Smooth 2D manifold extraction from 3D image stack (Shihavuddin et al., Nature Communications 2017).
We have developed bioinformatic tools to convert 3D image stacks into 2D images. 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.
3-2 Ependymal cilia beating induces an actin network to protect centrioles against shear stress (Mahuzier et al., Nature Communications 2018)
We show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. In conclusion, cilia beating controls the apical actin network around centrioles.
3-3 Entrainment of mammalian motile cilia in the brain with hydrodynamic forces (Pellicciotta et al., PNAS 2020).
We have also studied the entrainment of cilia beat in multiciliated cells from brain ventricles. Combining experiments and simulations, we studied how cilia from brain tissue align their beating direction. We subjected cilia to a broad range of shear stresses, similar to the fluid flow that cilia themselves generate, in a microfluidic setup.
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 have analyzed RNASequencing experiments to identify the molecular cascade that lead to centriole amplification in ependymal progenitor cells and identified the molecular mechanisms controlling ependymal cell specification.
3- We have determined 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.
2- We have analyzed RNASequencing experiments to identify the molecular cascade that lead to centriole amplification in ependymal progenitor cells and identified the molecular mechanisms controlling ependymal cell specification.
3- We have determined 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.