Investigating the role of PAX6 during neurodevelopment using human stem cell-derived brain organoids
Creators: name: Marshall, Helen
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: PAX6
neurodevelopment
human stem cell-derived brain organoids
human stem cells (hiPSCs)
extracellular matrix (ECM)
Matrigel-free protocol
PAX6⁺/⁺ organoids
PAX6⁻/⁻ organoids
Gene Ontology
Matrigel

description: PAX6 is an evolutionarily conserved transcription factor which acts as a master regulator in the development of the eyes and brain. People with heterozygous mutations in PAX6 suffer from varying levels of optical defects (aniridia) and present with minor brain abnormalities, including thinning of parts of the cerebral cortex. In the few compound heterozygous cases recorded in humans, loss of functional PAX6 results in severe morphological brain defects, and only one patient survived into childhood. The role of PAX6 in human neurodevelopment remains understudied due to the practical and ethical limitations of studying human embryonic processes. To explore the function of PAX6 in the earliest stages of neurodevelopment, human stem cells (hiPSCs) were used to generate cortical organoids which are a powerful humans-specific method of studying developmental processes. The initial experiment compared PAX6⁻/⁻ and PAX6⁺/⁺ organoids in different extracellular matrix (ECM) conditions throughout the first 30 days of culture. This time course experiment revealed a significant impact of exogenous ECM addition (in the form of Matrigel) on growth. The Matrigel-free protocol resulted in significantly smaller PAX6⁺/⁺ organoids but did not impact the growth of PAX6⁻/⁻ organoids. PAX6 potentially suppressed growth in response to a lack of exogenous ECM. At the end of the standard Matrigel-supplemented protocol, there was no significant difference in the size of PAX6⁻/⁻ and PAX6⁺/⁺ organoids. Specific periods of development within the first 30 days of organoid growth were then investigated in more detail. At the neural induction-like period of organoid development, where organoids began forming neuroepithelium (day 10 of the protocol), bulk RNA sequencing was performed. Gene Ontology analysis revealed several dysregulated terms, including cerebral cortex regionalisation, axon guidance, fate specification and cell adhesion. In addition, differential gene expression analysis uncovered over 40 dysregulated cell adhesion-related genes including CDH8, CDH23, and CLDN11. Dysregulation of several neural tube closure-associated genes were also found, which included GHRL2, CDH1 and CLDN4. Building on Gene Ontology results, the potential link between PAX6 and neural tube closure was investigated histologically by analysing the transition of open neuroepithelial structures to closed rosettes. There was no difference in rosette formation between PAX6⁺/⁺ and PAX6⁻/⁻ organoids, both of which underwent efficient closure of rosettes within 24 hours of Matrigel embedding. Despite the dysregulation of neural tube closure-associated genes, defects relating to neural tube closure were not evident histologically. Finally, the impact of PAX6 and the extracellular matrix on dorsoventral regionalisation was investigated. Organoids were taken at days 20 and 30 of development when dorsal TBR2 and ventral DLX2 positive cells were present. At day 20, omitting embedding from the protocol had a significant negative impact on the capacity of PAX6⁺/⁺ organoids to form dorsal cells, in contrast to PAX6⁻/⁻ organoids which developed comparable levels of TBR2 regardless of embedding. This effect persisted to day 30 of culture. Ventralisation of PAX6⁻/⁻ organoids was evident at day 20 and increased at day 30. These results indicated that the reduced size of PAX6⁺/⁺ organoids in the initial time course experiment could be due to restricted development by PAX6 under low ECM conditions. These findings suggest that PAX6 may regulate several processes in the early stages of development, from neural induction to regionalisation. In addition, a new link between PAX6 and the ECM has been highlighted, which may contribute to regulation of development. Organoids are a powerful tool to investigate stages of human development which would otherwise be impossible, and this thesis raises interesting questions for future research.