Integrin signalling regulates the expansion of neuroepithelial progenitors and neurogenesis via Wnt7a and Decorin

Development of the cerebral cortex requires regulation of proliferation and differentiation of neural stem cells and a diverse range of progenitors. Recent work suggests a role for extracellular matrix (ECM) and the major family of ECM receptors, the integrins. Here we show that enhancing integrin beta-1 signalling, by expressing a constitutively active integrin beta-1 (CA*β1) in the embryonic chick mesencephalon, enhances neurogenesis and increases the number of mitotic cells dividing away from the ventricular surface, analogous to sub-apical progenitors in mouse. Only non-integrin-expressing neighbouring cells (lacking CA*β1) contributed to the increased neurogenesis. Transcriptome analysis reveals upregulation of Wnt7a within the CA*β1 cells and upregulation of the ECM protein Decorin in the neighbouring non-expressing cells. Experiments using inhibitors in explant models and genetic knock-downs in vivo reveal an integrin-Wnt7a-Decorin pathway that promotes proliferation and differentiation of neuroepithelial cells, and identify Decorin as a novel neurogenic factor in the central nervous system

Ontogenesis of glial diversity in the cerebellum as revealed through quantitative in vivo clonal analyses

Trabajo presentado en GLIA 2019: XIV European Meeting on Glial Cells in Health and disease en Oporto el 10 de Julio de 2019.In the cerebellum, astrocytes (AS) and oligodendrocytes (OLs) are characterized by a peculiar phenotypic heterogeneity, closely related to specific functional features. Nevertheless, the ontogenesis of this glial diversity remains poorly understood. By combining in vivo clonal analyses employing StarTrack plasmids and Confetti mice withproliferation studies, we have recently demonstrated that the major cerebellar AS types derive from embryonic and postnatal progenitors with diverse lineage potentials. Moreover, AS heterogeneity appeared to emerge according to an unprecedented and remarkably orderly developmental program, likely driven by deterministic components as suggested by in silico simulations. Similar StarTrack clonal analyses are now being applied to elucidate the origins, clonal relationships, and spatial distribution of cerebellar OLs born at distinct developmental stages. While AS clones showed a time-dependent allocation first to the hemispheres and then to the vermis, preliminary data indicate that OL clones are not medio-laterally compartmentalised, being capable of populating either of the two regions independently from their developmental origin. This suggests that the two progenitor pools, originally spatially segregated, also follow distinct guidance mechanisms to populate different cerebellar territories within the same developmental time window. On the other side, both AS and OLs displayed a similar behaviour along the antero-posterior axis: cells belonging to distinct clones rarely intermingled and, rather, clustered to populate discrete cerebellar lobules, thereby suggesting that each AS or OL clone is involved in the functional maturation and/or maintenance of specific neural circuit compartments, with poor contributions from others. Last, similarly to AS clones characterized by stereotyped architectures and recurrent modularities, immunohistochemical and morphological analyses of the cells strikingly highlighted that OL clones have a constant relative contribution of immature and mature cells, in favour of the mature part. While the modularity in AS clones was demonstrated to reflect layer-specific dynamics of postnatal proliferation/differentiation, what determines the equilibrium between immature and post-mitotic cells in OL clones is unknown. It will be interesting to investigate whether it reflects intrinsic properties of the progenitors themselves or it depends upon extrinsic cues, such as the surrounding axonal availability

Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex

The link between cortical precursors G1 duration (TG1) and their mode of division remains a major unresolved issue of potential importance for regulating corticogenesis. Here, we induced a 25% reduction in TG1 in mouse cortical precursors via forced expression of cyclin D1 and cyclin E1. We found that in utero electroporation-mediated gene transfer transfects a cohort of synchronously cycling precursors, necessitating alternative methods of measuring cell-cycle phases to those classical used. TG1 reduction promotes cell-cycle reentry at the expense of differentiation and increases the self-renewal capacities of Pax6 precursors as well as of Tbr2 basal precursors (BPs). A population level analysis reveals sequential and lineage-specific effects, showing that TG1 reduction: (i) promotes Pax6 self-renewing proliferative divisions before promoting divisions wherein Pax6 precursors generate Tbr2 BPs and (ii) promotes self-renewing proliferative divisions of Tbr2 precursors at the expense of neurogenesis, thus leading to an amplification of the BPs pool in the subventricular zone and the dispersed mitotic compartment of the intermediate zone. These results point to the G1 mode of division relationship as an essential control mechanism of corticogenesis. This is further supported by long-term studies showing that TG1 reduction results in cytoarchitectural modifications including supernumerary supragranular neuron production. Modeling confirms that the TG1-induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. These findings also have implications for understanding the mechanisms that have contributed to brain enlargement and complexity during evolution

Dynamic behaviour of human neuroepithelial cells in the developing forebrain

To understand how diverse progenitor cells contribute to human neocortex development, we examined forebrain progenitor behaviour using timelapse imaging. Here we find that cell cycle dynamics of human neuroepithelial (NE) cells differ from radial glial (RG) cells in both primary tissue and in stem cell-derived organoids. NE cells undergoing proliferative, symmetric divisions retract their basal processes, and both daughter cells regrow a new process following cytokinesis. The mitotic retraction of the basal process is recapitulated by NE cells in cerebral organoids generated from human-induced pluripotent stem cells. In contrast, RG cells undergoing vertical cleavage retain their basal fibres throughout mitosis, both in primary tissue and in older organoids. Our findings highlight developmentally regulated changes in mitotic behaviour that may relate to the role of RG cells to provide a stable scaffold for neuronal migration, and suggest that the transition in mitotic dynamics can be studied in organoid models

Glycine receptors control the generation of projection neurons in the developing cerebral cortex

The development of the cerebral cortex requires coordinated regulation of proliferation, specification, migration and differentiation of cortical progenitors into functionally integrated neurons. The completion of the neurogenic program requires a dynamic interplay between cell intrinsic regulators and extrinsic cues, such as growth factor and neurotransmitters. We previously demonstrated a role for extrasynaptic glycine receptors (GlyRs) containing the α2 subunit in cerebral cortical neurogenesis, revealing that endogenous GlyR activation promotes interneuron migration in the developing cortical wall. The proliferative compartment of the cortex comprises apical progenitors that give birth to neurons directly or indirectly through the generation of basal progenitors, which serve as amplification step to generate the bulk of cortical neurons. The present work shows that genetic inactivation of Glra2, the gene coding the α2 subunit of GlyRs, disrupts dorsal cortical progenitor homeostasis with an impaired capability of apical progenitors to generate basal progenitors. This defect results in an overall reduction of projection neurons that settle in upper or deep layers of the cerebral cortex. Overall, the depletion of cortical neurons observed in Glra2-knockout embryos leads to moderate microcephaly in newborn Glra2-knockout mice. Taken together, our findings support a contribution of GlyR α2 to early processes in cerebral cortical neurogenesis that are required later for the proper development of cortical circuits