Biologie cellulaire de la neurogenèse des mammifères

The neocortex is the center for higher cognitive functions such as language and decision-making. During its embryonic development, neural stem cells known as radial glial (RG) progenitor cells generate all neurons, astrocytes and oligodendrocytes of the neocortex. Two types of RG cells co-exist, with different relative proportions across species. Apical RG (aRG) cells are common to all mammals and localize in the ventricular zone (VZ). They are epithelial cells, anchored to the apical surface through adherens junctions. aRG cells generate neurons indirectly, through the production of an intermediate progenitor, and later on switch to gliogenesis. Basal RG (bRG) cells derive from aRG cells but have delaminated from the neuroepithelium. As such, they are non-epithelial and localize more basally, in the outer sub-ventricular zone (OSVZ). bRG cells undergo a unique form of migration, called mitotic somal translocation (MST), during which their soma very rapidly translocates just before cytokinesis. The abundance of bRG cells varies greatly across species. They are for example very rare in mouse, where they moreover may have limited proliferative properties. They are particularly abundant in certain primates, and especially in humans, where they are believed to account for their massive neocortex size expansion.

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

The Zika virus outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remained unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. In collaboration with the group of Nathalie Pardigon at institut Pasteur, we developed an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4) (Brault et al, EBioMed, 2016). We showed that this tissue was able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis revealed a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displayed a very different tropism of infection, with a bias towards neurons. We further showed that ZIKV infection, but not WNV infection, impaired cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work established a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and revealed specific preferential infection of neural stem cells by ZIKV.

ZIKV infects RG progenitors preferentially. E16 mouse cortical slice infected with Zika virus (ZIKV) shows preferential infection of the elongated RG cells.
Image belongs to Team BAFFET

Brault JB, Khou C, Basset J, Coquand L, …, Pardigon N, Baffet AD. (2016) Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex. EBioMedicine 10, 71-76.

CAMSAPs organize an acentrosomal microtubule network from basal varicosities in radial glial cells.

A high number of genetic mutations associated with cortical malformations are found in genes coding for microtubule-related factors. Accordingly, microtubules play a variety of functions in RG cells, including cell division and polarized transport. A large number of microtubule regulators linked to disease have been studies in the past but surprisingly, the organization of the microtubule cytoskeleton I these cells remained unknown. This was mostly due to the challenge of performing subcellular live imaging within this thick tissue. We therefore developed a reliable method to perform high speed and high-resolution live imaging within brain slices, which we have been using for several studies. Using the method, we indentified the organization of the microtubule cytoskeleton in RG cells (Coquand et al, JCB, 2021). We showed that microtubules in the apical process uniformly emanate from the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identified acentrosomal microtubule organizing centers localized in varicosities of the basal fiber and showed that CAMSAP family members accumulate in these varicosities where they control microtubule growth. Double knockdown of CAMSAP1 & 2 led to a destabilization of the entire basal process. Finally, using live imaging of human fetal cortex, we revealed that this organization is conserved in basal radial glial cells, the progenitor cell population associated with human brain size expansion.

(Left) Live imaging of CAMSAP3-GFP and EB3-mCherry in mouse aRG cell basal process, and corresponding kymograph.
(Right) The microtubule network of the apical process of aRG cells is unipolar, with the minus ends concentrated apically, and the plus ends growing in the basal direction. The mother centriole serves as a template for the primary cilium, which extends into the ventricle. A second ring-like microtubule network is present at the apical endfoot (PMID: 32238932). In the basal process of both aRG and bRG cells, the microtubule network in bipolar. Microtubules are organized from varicosities, via CAMSAP 1 & 2, which anchor the minus ends. 
 

Team BAFFET

Coquand L.*, Victoria G. S.*, Tata A., Brault J.B., Guimiot F., Fraisier V., Baffet AD (2021) CAMSAPs organize an acentrosomal microtubule network from basal varicosities in radial glial cells. J Cell Biol. 2;220(8):e202003151

RAB6 and dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination

            The development of fast subcellular live imaging within thick samples enabled us to address the long-standing question of how polarized trafficking is regulated in aRG cells. In particular, we revealed how transmembrane cargos are transported from the perinuclear area, where the Endoplasmic reticulum and Golgi are localized, to the apical surface of these cells (Brault et al, Embo Reports, 2022). Post-Golgi trafficking had been extensively studied in 2D non-polarized cells, and had been shown to be dependent on plus end-directed kinesins. Using live imaging of post-Golgi RAB6+ vesicles, we showed here that apical trafficking was dependent on the minus end directed dynein and its partner LIS1, indicating that in polarized epithelial cells such as aRG cells, the polarity of motors is completely inverted. In RG cells, we showed that the RAB6-dynein-LIS1 pathway was required for transport to the apical surface of Crumbs, a major regulator of epithelial polarity. Genetic ablation of LIS1 or double knock-out of RAB6A and brain-specific RAB6B (that we generated for this study), all led to an impairment of Crumbs localization at the apical surface and, as a consequence, to a loss of apical junctions. This led to a delamination of aRG cells, observed using live imaging. Detached aRG cells were aberrantly localized basally and continued to proliferate. This work therefore highlights an important molecular mechanism for the delamination of aRG cells, which in human is at the basis of the production of bRG cells.

Post-Golgi apical transport of Crumbs is driven by dynein in aRG cells
© Team BAFFET

Brault J.B., Bardin S., Lampic M., Carpentieri J.A., Coquand L., Penisson M., Lachuer H., Victoria G.S., Baloul S., El Marjou F., Boncompain G., Miserey-Lenkei S., Belvindrah R., Fraisier V, Francis F., Perez F., Goud B., Baffet AD (2022) RAB6 and dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination. EMBO Rep. Aug 18:e54605
 

Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly

            A large number of genetic causes for cortical malformations remain undiscovered. We have established a close collaboration with Nadia Bahi-Buisson (PU-PH, Necker-Enfants maladies & Imagine Institute) who works on identifying novel mutations using the latest developments in omics methods. Our goal is to identify the molecular functions of identified factors during neocortex development, and the basis of the pathological alterations. We identified human mutations leading to severe microcephaly in WDR81, a gene coding for a regulator of endosomal maturation (Cavallin et al., Brain, 2017). To identify the function of WDR81 in the developing neocortex, we generated a KO mouse, which displayed microcephaly and altered neuronal positioning, phenocopying the human disease (Carpentieri et al, Nat Comm, 2022). Mechanistically, we showed that WDR81 loss of function alters endosomal trafficking of the EGF receptor, leading to reduced MAPK activation, reduced RG proliferation and, as a consequence, microcephaly. As microcephaly is largely linked to apoptotic cell death, this was a striking example of reduced brain size being a direct consequence of progenitor proliferation defects. Interestingly, megalencephaly (enlarged brain) has been described to arise from overproliferation of the progenitor pool. Accordingly, we were able to rescue the WDR81^-/- proliferation defects with a megalencephaly-causing mutated form of Cyclin D2, indicating that microcephaly and megalencephaly can be viewed as two sides of the same coin.

WDR81 KO mice display reduced brain size and altered neuronal positioning
© Team BAFFET

Carpentieri JA, Di Cicco A, Andreau D, Del Maestro L, El Marjou F, Coquand L, Bahi-Buisson N, Brault JB, Baffet AD. (2022) Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly. Nat Commun. Jan 10;13(1):16

A cell fate decision map reveals abundant direct neurogenesis in the human developing neocortex.

Recent advances in genomic methods have shed light on the cellular diversity of the human neocortex, but how it arises during development is unclear. Cell diversity is a consequence of cell fate decisions that occur at the level of individual RG cells, but no methods were available to identify and quantitatively map them. To address this, we first developed methods to cultivate human cerebral organoids and human fetal tissue coming from medical pregnancy terminations. We have developed an approach that consists in live imaging cerebral organoids or human fetal tissue for 48 hours and, following fixation and immunostaining, to identify the daughter cells of dividing bRG cells in order to probe for their fate. This semi-automated live/fixed correlative microscopy method is very robust and allowed us to quantitatively probe for cell fate decision in the human developing neocortex (Coquand et al, BioRxiv, 2022). We demonstrated that a) fate decision are very conserved between organoids and fetal tissue, b) bRG cells have a high self-amplification potential at the stages investigated, c) unlike mouse aRG cells, bRG cells undergo frequent direct neurogenesis, bypassing the generation of intermediate progenitor, and d) upon asymmetric cell division, Notch is specifically activated in the bRG daughter, but this is not caused by the asymmetric inheritance of the long basal process, as previously hypothesized.

A semi-automated correlative imaging method to identify cell fate decisions in cerebral organoids.
©Team BAFFET

Coquand L, Macé A-S, Farcy S, Brunet AvalosC, Di Cicco A, Lampic M, Bessières B, Attie-Bittach T, Fraisier V, Guimiot F, Baffet AD (2022) A cell fate decision map reveals abundant direct neurogenesis in the human developing neocortex.

BioRxiv https://doi.org/10.1101/2022.02.01.478661

Projects         

            The pattern of cell fate decisions taken by individual progenitors cannot be inferred from their clonal output. These successive branch points are critical regulatory hubs that will define the future cellular composition of the brain and, as such, are likely to vary substantially in time, space, evolution, and in pathological contexts. Our global objective is to precisely measure these fate decisions in the human developing neocortex, along the space-time axes, using imaging approaches. We will test how these variations are influenced by the cellular microenvironment, through non-cell autonomous mechanisms. Moreover, we aim to compare these features with chimpanzees, our closest living relative, and identify the human-specific features of cell fate decisions that underlie difference in cellular composition and neocortex size.

Aim1: A space-time map of cell fate decisions in the human developing neocortex. We aim to establish a high-resolution space-time cell fate decision map in the human developing neocortex, both in fetal tissue and cerebral organoids. We will analyze how fate decisions evolve over time by establishing maps at multiple key stages, and in space, along the apico-basal axis of the neocortex.

Aim 2: Influence of the microenvironment on cell fate decision plasticity. Cell autonomous and non-cell autonomous mechanisms were reported to govern progenitor cell output variations during development. Here, we will probe for the contribution of the microenvironment in cell fate decision control. We will test how fate decisions are affected by microenvironment changes in time, by performing heterochronic RG transplantations, and in space, by altering bRG cell position in the OSVZ.

Aim 3: Identification of factors associated with non-cell autonomous fate decisions. Non-cell autonomous reprogramming can be due to a combination of signaling coming from the extracellular space and from the surrounding cells. To discriminate between these two elements, we will perform heterochronic RG grafting experiments into decellularized cortical slices. We will establish the proteome and RG transcriptome of cortical tissue in time and space, and correlate these data with our detailed space-time maps of cell fate decisions, in order to identify and characterize factors associated with each division mode.

Aim 4: Investigating how cell fate decision variations underlie human brain size expansion. Increased brain size in humans, as compared to other primates, is believed to support the development of more complex neural architectures and underlie higher cognitive functions. Using human and chimpanzee cerebral organoids, we will generate quantitative maps of cell fate decisions through development in these two closely-related species. We will probe how the microenvironment may underlie some of the observed differences by performing inter-species transplantation experiments.