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

Laure Coquand, Anne-Sophie Macé, Sarah Farcy, Clarisse Brunet Avalos, Amandine Di Cicco, Marusa Lampic, Betina Bessières, Tania Attie-Bitach, Vincent Fraisier, Fabien Guimiot, Alexandre Baffet

Preprint posted on February 02, 2022 https://www.biorxiv.org/content/10.1101/2022.02.01.478661v1

In the spotlight: How human neural progenitors were caught red-handed

Selected by Juan Moriano

Background

During human neocortical development, two transient zones contain neural progenitor cells that are responsible for the generation of excitatory neurons: the ventricular zone (VZ), located close to the ventricles (or apically), and the subventricular zone (SVZ), above (basal to) the VZ and below other transient regions and the neocortical plate1. The SVZ is divided into two subregions, the outermost part of which (the outer SVZ, oSVZ), is particularly enlarged in our species. This means the SVZ has received special attention in evolutionary studies. The human oSVZ is abundantly populated by progenitor cells named basal radial glia cells (bRGCs), a cell type thought to be key for the neocorticalization observed in primates and, most prominently, in humans. Hence, the investigation of the mechanisms governing the generation, behaviour and division of bRGCs is fundamental to understanding human brain development, evolution and disease2–6.

In this preprint, Coquand and colleagues studied the behavior of bRGCs and the fate of their daughter cells. To do this, the researchers developed a correlative microscopy method consisting of live-imaging of progenitor cells virally transfected with green fluorescent protein (GFP) followed by the fixation and immunostaining with cell fate markers (see Figure 1). The identification of the cells after these two steps is challenging due to the cellular complexity of the slices, multiple images recorded in parallel, and the random movement of the slices during immunofluorescence. The authors addressed these challenges using a contour-based approach to correctly identify the cells across images from live and fixed samples. A detailed documentation of this image processing strategy is provided in the Supplementary Material of the preprint.

Coquand and colleagues applied this method to stage-matched human primary tissue (gestational weeks 14 to 18) and dorsal forebrain organoids (8-10 weeks), recording around one thousand cell divisions and quantitatively characterizing the cell divisions of bRGCs and the resulting progeny (see Figure 1).

Figure 1. Schematic representation of: neural progenitors present in the developing neocortex and the correlative microscopy-based method of this study (figures taken from Figures 1 and 2 of the preprint). CP: Cortical plate; SVZ: Subventricular zone; VZ: Ventricular zone; aRG: apical radial glia; IP: Intermediate progenitor cells; bRG: basal radial glia.

Main findings

First, the authors identified different morphotypes of bRGCs by immunostaining mitotic cells with the specific marker phospho-Vimentin and labelling non-mitotic cells with the cytoplasmic green fluorescent protein (GFP), staining with SOX2 (progenitor marker), EOMES (IP marker) and NEUN (neuron marker). bRGCs (SOX2-positive, EOMES-negative and NEUN-negative) were classified in four types, according to the number of processes: 1) one apical process, 2) one basal process, 3) two processes -one apical and another basal-, and 4) no processes. Similar results were obtained for stage-matched human fetal tissues and cerebral organoids, although, for the latter, the identification of apical/basal processes was impeded by the distribution of these cells between organoid lumens. Most cells – around 80% of the total – had at least one process.

  • A map of bRG cell-fate decisions. Coquand and colleagues first applied their correlative microscopy-based method to cerebral organoids, analyzing a total of 444 dividing bRGCs. These cells were found to mostly perform proliferative divisions, giving rise to two other bRGCs (~60% of total divisions). For neurogenic divisions, the majority were self-renewing, that is, giving rise to another bRGCs and either an IP or a neuron (~60% of neurogenic divisions), while a relevant proportion were self-consuming (generating either two IPCs or two neurons; ~40% of neurogenic divisions).

    For human fetal tissue, 522 human basal radial glial cells were profiled and similar results were obtained. Most of the divisions were proliferative (~70%), while for the 30% of neurogenic divisions, ~60% were self-renewing and 40% self-consuming. The authors also found that IPs showed a high proliferative capacity with 85.7% of divisions being symmetric proliferative. The authors concluded that organoid bRGCs largely recapitulate the progenitor behavior observed in human primary tissue, and that bRGCs show high proliferative potential with abundant direct neurogenesis (bRG to neurons).

Figure 2. A quantification of the modes of divisions of bRGCs and resulting progeny in the fetal human neocortex (image taken from Figure 4 of the preprint).

  • Division modes dependent of progenitor location. Compared to other primates, the SVZ is very large in humans. To examine putative differences in progenitor cell behavior depending on their location along the SVZ apicobasal axis, the researchers compared the bRGCs by measuring the distance to the apical surface at the time of cytokinesis. While they found similar proportions of modes of divisions (proliferative vs neurogenic) between those cells closer to the apical surface and those located more basally, the production of EOMES-positive cells were frequently located apically while direct neuron production was found more abundantly in more basal locations of the SVZ.

  • Basal process inheritance. To investigate the role of bRGC processes on daughter cell fate, the authors focused on asymmetric divisions (leading to bRG and either an IP or a neuron). In both cerebral organoids and human fetal tissues, there was no relationship between bRGC process inheritance and cell fate acquisition (e.g. in fetal tissue, 52.4% of cells inheriting the basal process remain as bRG, and 48.6% differentiated).

    In mouse apical RGC, however, process inheritance has been reported to correlate with the fate of the daughter cells. Coquand and colleagues therefore decided to study NOTCH signaling as a key player in cell fate acquisition. In both organoids and fetal samples, authors found that HES1 (selected as a readout of NOTCH signaling) was never found in differentiating cells (EOMES+ or NEUN+ cells). HES1 was then linked to the self-maintenance of bRG. The authors also noticed that there was no correlation between process inheritance and HES1 expression (half of cells expressing HES1 inherited the basal process and the other half not). The authors propose that the differences between apical and basal RGCs may therefore be due to the different microenviroments they are exposed to.

Why I chose this preprint

This study, from Alexandre Baffet’s lab at Institut Curie in Paris, provides a detailed map of basal radial glia cell divisions during corticogenesis based on an innovative microscopy-based analysis of both human primary samples – which are scarcely available -, and dorsal forebrain organoids. The methodology developed in this study will be a great asset for future studies of brain development -and other fields -, widening the range of questions researchers can now try to answer quantitatively.

Questions

1) Could this – already powerful – methodology be further scaled to profile a higher number of cells and analyze more cell subtypes?

2) The expression of fate markers in the newly differentiating cells was reported to be very rapid (1-2 hours after mother cell division). Under your approach, would it be possible to detect the expression of some markers before cell division, as an instance of priming?

3) Does the distribution of the different morphotypes of bRGCs vary along the apicobasal axis of the SVZ? Could you spot any difference in the proportion of division modes among the bRGC morphotypes?

References

1. Kostović, I. & Judaš, M. Embryonic and Fetal Development of the Human Cerebral Cortex. In Brain Mapping (ed. Toga, A. W.) 167–175 (Academic Press, 2015). doi:10.1016/B978-0-12-397025-1.00193-7.

2. Lui, J. H., Hansen, D. V. & Kriegstein, A. R. Development and Evolution of the Human Neocortex. Cell 146, 18–36 (2011).

3. Betizeau, M. et al. Precursor Diversity and Complexity of Lineage Relationships in the Outer Subventricular Zone of the Primate. Neuron 80, 442–457 (2013).

4. Dehay, C., Kennedy, H. & Kosik, K. S. The Outer Subventricular Zone and Primate-Specific Cortical Complexification. Neuron 85, (2015).

5. Kalebic, N. & Huttner, W. B. Basal Progenitor Morphology and Neocortex Evolution. Trends in Neurosciences 43, 843–853 (2020).

6. Ossola, C. & Kalebic, N. Roots of the Malformations of Cortical Development in the Cell Biology of Neural Progenitor Cells. Frontiers in Neuroscience 15, (2022).

Tags: brain development, microscopy

Posted on: 4th April 2022

doi: https://doi.org/10.1242/prelights.31735

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Author's response

Alexandre Baffet shared

1) Could this – already powerful – methodology be further scaled to profile a higher number of cells and analyze more cell subtypes?

Absolutely, and this is currently ongoing in the lab. We are measuring how these fate decisions vary in time, and are probing for more cell type markers (neuronal and glial subtypes). Multiplex imaging methods, that allow to probe for more markers within the same sample, are particularly relevant in this context.

2) The expression of fate markers in the newly differentiating cells was reported to be very rapid (1-2 hours after mother cell division). Under your approach, would it be possible to detect the expression of some markers before cell division, as an instance of priming?

Accessing the expression of certain “priming” markers prior to division would indeed be valuable. This cannot be achieved through immunostaining, as these experiments are only relevant in conjunction with the fate of the daughter cells. One way to address this would be to generate fluorescent reporter lines under the control of specific promoters, observable through live imaging.

3) Does the distribution of the different morphotypes of bRGCs vary along the apicobasal axis of the SVZ? Could you spot any difference in the proportion of division modes among the bRGC morphotypes?

We often get this question lately and should probably include this data in the final manuscript. No, we do not detect any different behavior based on morphology, and morphologies appear equally represented all along the apico-basal axis.

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