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Investigating the role of ZEB2 in the establishment of neuroepithelial architecture and axon tract formation

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Giandomenico, Stefano Luca 


A bioinformatic screen of comparative genomic and transcriptomic datasets identified the transcription factor ZEB2 as a putative regulator of brain size. In this work we show that in both human brain organoids and mouse embryos ZEB2 is expressed in telencephalic neuroepithelial cells (NECs) before the switch to radial glia (RGCs). By establishing a human embryonic stem cell (hESC) model of ZEB2 heterozygous loss-of-function we show that this gene modulates the changes in cell-cell contacts at the transition from NECs to RGCs. Upon partial loss of ZEB2, changes in cell adhesion are mirrored by changes in tissue architecture, including thin elongated neuroepithelial buds with densely packed cells. We demonstrate that the secreted growth factor FGF2 is a positive regulator of ZEB2, which in turn suppresses FGF2 expression, thus establishing a link between ZEB2 and a known regulator of NEC proliferation. Preliminary gain-of-function (GOF) experiments confirm ZEB2 as a neurogenic driver and pharmacological rescue by dual SMAD inhibition suggests that ZEB2 may be acting by inhibiting BMP and TGFβ at the transition from NECs to RGCs. In an attempt to model also later aspects of the ZEB2 mutant phenotype we adapt air-liquid interface culture to cerebral organoids. Air-liquid interface cerebral organoids (ALI-COs) develop thick axon tracts with distinct morphologies and hodologies; including long-range projection within and away from the organoid, growth cone turning, decussation and dynamics typical of pioneer and follower axons. Single-cell RNA sequencing on ALI-COs reveals a wide array of cortical cell types and retrograde tracing demonstrates that the tracts established have distinct and accurate molecular identities. ALI-COs develop active neuronal networks and escaping tracts can innervate mouse spinal cord explants and evoke paraspinal muscle contractions. Overall, we establish a novel culture paradigm that allows in vitro modeling of axon guidance and network establishment. Lastly, we demonstrate that ALI-COs can be used to study the ultrastructure of navigating axons by cryo-electron tomography.





Lancaster, Madeline Alden


ZEB2, ALI-CO, Cerebral organoids


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge