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.
©Team BAFFET
|
A semi-automated correlative imaging method to identify cell fate decisions in cerebral organoids. A. Schematic representation of correlative microscopy pipeline. B. Step-by-step protocol for semi-automated correlative microscopy. (1) bRG cells are live imaged for 48 hours. (2) 4X brightfield images containing the video coordinates are assembled. (3) Organoid slices are fixed, immunostained for SOX2, EOMES and NEUN and imaged. (4) Images are automatically segmented to outline slices from live and fixed samples. (5) Slice contours are automatically paired based on shape and area and (6) aligned (including a horizontal flip if needed). (7) Video fields of view are automatically annotated on the immunostaining images. (8) Regions of interest are re-imaged at higher resolution 40X and cells from live and fixed samples are manually matched. C. Live/fixed correlative analysis of a dividing bRG cell generating a self-renewing bRG daughter and a differentiating IP daughter. |
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
©Team BAFFET