Rand, D. | bioRxiv Science

bioRxiv2026

Minimal essential requirements for neural tube self-organisation

The reliable generation of diverse cell types in precise proportions is essential for the formation of functional tissues during embryonic development. Three-dimensional organoid models derived from pluripotent stem cells (PSCs) provide powerful systems for identifying principles governing tissue self-organisation. Neural tube organoids (NTOs), initiated from single PSCs with a pulse of retinoic acid (RA), self-organise into structures containing floorplate cells that secrete SHH morphogen to pattern adjacent neural tissue. Yet, how the initial cellular diversity arises and how appropriate cell-type proportions are allocated has remained unclear. Here, using time-resolved single-cell transcriptomics, quantitative immunofluorescence, and dynamical systems modelling we show that RA triggers a transient co-expression state of the transcription factors PAX6 and FOXA2 from which cells asynchronously resolve into two opposing fates: PAX6 neural precursors and FOXA2 floorplate precursors. PAX6 and FOXA2 are both necessary and sufficient to reconstitute self-organisation, establishing these transcription factors as key determinants of emergent tissue pattern. Rather than operating as a simple feed-forward system in which cells are guided solely by RA, feedback between the alternative cell fates, mediated by BMP signalling from floorplate precursors, determines cell type proportions and ensures reproducible cell-type diversity in each NTO. This dual expression state was also identified in mouse embryos, demonstrating how in vitro models inform in vivo biology. These findings establish a general design strategy - symmetry breaking through opposing fate determinants coupled to proportioning via signal feedback control - that may operate broadly across developmental contexts to generate tissues with predictable cellular compositions.

developmental biology↗

bioRxiv2020

Endothelial iron homeostasis regulates BBB integrity via the HIF2α-Ve-cadherin pathway

The blood-brain barrier (BBB) serves as the guardian of the CNS, tightly regulating the movement of ions, molecules, and cells between the circulatory system and brain. This barrier is critical in maintaining brain homeostasis, allowing proper neuronal function and protecting the brain from injury and disease. Chronic and acute exposure to various chemicals lead to BBB breakdown through pathways that are also affected in neurological diseases. Therefore, we have created an in-vitro BBB injury model to gain a better understanding of the mechanisms controlling BBB integrity. This model exposes a co-culture of human stem-cell derived brain-like endothelial cells (BLEC) and brain pericytes that mimic the BBB, to the organophosphate paraoxon. This exposure results in rapid lipid peroxidation, initiating a ferroptosis-like process and leading to endothelium cell toxicity. Mitochondrial ROS formation (MRF) and increase in mitochondrial membrane permeability (MMP), which occur 8 - 10 h post paraoxon-induced injury, also trigger apoptotic cell death. Yet, these processes do not directly result in damage to barrier functionality since blocking them does not reverse the increased permeability. Looking for a crucial pathway affecting barrier functionality we analyzed the iron homeostasis in our model since the iron chelator, Desferal(C) (DFO) rescued endothelial cell viability. Upon BBB insult, the liable iron pool (LIP) is rapidly increased, preventing the increased expression of the stress related hypoxia-induced factor 2 (HIF2) transcription factor. This results in a decrease in surface expression of the adherens junction and permeability master regulator protein, Ve-cadherin, ultimately damaging BBB integrity. Unlike the apoptosis inhibitor ZVAD that rescues BLEC from cell toxicity, yet exacerbates damage to the barrier functionality, DFO significantly decreases MRF and apoptosis subsequent to PX exposure, while also rescuing barrier integrity by inhibiting the liable iron pool increase, inducing HIF2 expression and preventing the degradation of Ve-cadherin on the cell surface. Moreover, the novel nitroxide JP4-039 significantly rescues both injury-induced endothelium cell toxicity and barrier functionality. Collectively, we have elucidated the cellular processes initiated by chemical injury to the endothelium barrier that result in cell toxicity; yet, inhibiting these processes does not necessarily protect BBB integrity which is regulated by the iron mediated HIF2 - Ve-Cadherin axis. DFO protects BBB integrity by inhibiting the injury-induced deregulation of this axis. Additionally, we have discovered a novel compound, JP4-039, that inhibits both damage to endothelium functionality and cell toxicity. Elucidating a regulatory pathway that maintains BBB integrity and discovering both a novel and an FDA approved compound that interfere with this pathway elucidates a potential therapeutic approach to protect the BBB degradation that is evident in many neurological diseases.

neuroscience↗