Cerebral organoids as tools to study forebrain development: the role of Pax6
Item statusRestricted Access
Embargo end date29/06/2020
The cerebral cortex forms the major part of the dorsal telencephalon and contains a highly diverse population of neurons that form complex neuronal networks, which is key in higher cognitive functions. Despite its complexity, recent findings have shown that this highly organised cytoarchitecture can be recapitulated spatio-temporally in a 3D in vitro model known as cerebral organoids. This provides a new platform to investigate corticogenesis in a more accessible and controlled environment. However, as mechanisms underlying the development of cerebral organoids are much less understood, the accuracy of this model as a tool in investigating cortical development remains an unanswered question. To address this question, this study investigates the cerebral organoid system by removing Pax6, one of the crucial transcription factors involved in early corticogenesis. This study aims to compare phenotypes of Pax6-/- mouse cerebral organoids to the established phenotypes of Pax6-/- mouse embryonic cortices, which will provide insights to whether the roles of Pax6 in cerebral organoids accurately match with its function in vivo. Using an established 3D culture protocol that generates robust neuroepithelial-like (NE) structures, we found that Pax6-/- cerebral organoids show precocious differentiation, with similar characteristics to Pax6-/- embryonic cortices, such as an increase of abventricular mitotic cells and alteration in the labelling index of cells in S-phase. These results suggest a consistent role for Pax6 in cortical development in both 3D in vitro and in vivo models. Recent studies have shown that Pax6 controls proliferation of cortical progenitors by repressing cell cycle genes. In order to examine Pax6 control in the cell cycle of cerebral organoids, Pax6 conditional knockout (cKO) embryonic stem (ES) cell lines were derived from tamoxifen-inducible Pax6 cKO mice. This enabled the generation of cerebral organoids that can be interrogated by acute deletion of Pax6 in the cortex-specific region. Using a combination of cumulative EdU labelling and mosaic analysis, we found that acute deletion of Pax6 in cerebral organoids does not cause significant changes in the total cell cycle time (Tc) of the cortical progenitors, consistent with the phenotypes of cortical progenitors in the caudo-medial region of Pax6 cKO mice. This may also suggests that Pax6 is cell non-autonomously required in cortical proliferation of organoids. In addition, we found that the proliferation rate of cortical progenitors in control organoids is slower than embryonic cortical progenitors. Overall, this study indicates that cell proliferation in cerebral organoids requires Pax6. However, the roles of Pax6 in cell cycle regulation in this system remain unclear, due to the complex and context-specific functions of Pax6.