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This thesis investigates the mechanisms of gene expression regulation in the human brain, and in particular the role of transcriptional regulation implicated in intellectual disability, and the interplay between canonical splicing and back-splicing in the brain. Mutations in Zinc Finger Myeloid, Nervy, and DEAF-1 (MYND) type containing 8 (ZMYND8) are linked to a neurodevelopmental syndrome characterized by intellectual disability (ID). While ZMYND8 is known to play an integral role in the epigenetic regulations important for DNA damage response and cancer biology, its role in the brain is yet uncharacterized. To investigate the potential roles of ZMYND8 in brain development, this thesis assessed the effects of ZMYND8 knock-out (KO) on gene expression and brain organoid development. In line with the known role of ZMYND8 in transcriptional and epigenetic regulations, we observed widespread changes in gene expressions in ZMYND8 KO iPSCs. In brain organoids generated by directed differentiation to the dorsal forebrain-like tissues, ZMYND8 KO led to impaired organoid development, with loss of the layered structure observed in wild-type cortical spheroids. Single-cell RNA-seq analysis of early-stage organoids uncovered accelerated differentiation of certain neuronal cell types and a shift from excitatory to inhibitory neuron identities, suggesting critical roles of ZMYND8 in embryonic and potentially corticogenesis. Both canonical alternative splicing (AS) and back-splicing (BS) occur frequently in the mammalian brain, and events from both processes have been shown to play essential roles during neurodevelopment. Currently identified splicing regulators do not account for all the AS and BS events occurring in the human brain. To address this knowledge gap, this thesis examined and uncovered novel roles of six RNA binding proteins in AS and BS regulations in a system modelling the catecholaminergic neurons. The nature of the interplay between AS and BS in the brain, as well as the crosstalk between the pathways that regulate these two processes, has not been fully characterized. To address this knowledge gap, this thesis examined the nascent and steady-state RNA-seq data of human astrocytes, which uncovered the “direct back-splicing” model being accountable for most of the co-transcriptionally occurring BS events in human astrocytes. It also uncovered that BS occurs predominantly post-transcriptionally and prefers the usage of AS-involved splice sites (ss). This ss-usage preference is further elevated among alternative BS events. This thesis also uncovered evidence suggesting mechanisms unique to BS regulation that promote the coupling of AS-involved ss. Overall, these observations broaden our appreciation of the complexity of BS in relation to ss-usage preference and transcriptional timing, as well as how it compares to that of canonical splicing.

With the increasing global interest in space exploration and the permanent presence ofhumans in Low Earth Orbit (LEO), it is crucial to establish the impact of microgravity exposure on the human body, and specially, the central nervous system (CNS). In collaboration with Space Tango, the Muotri Lab is targeting the microgravity environment by sending cortical brain organoids for 30 day missions to the International Space Station. Brain organoids are tridimensional self-organized structures derived from induced pluripotent or embryonic stem cells that mimic the cellular diversity and the spatial architecture of the developing human brain. We foresee that studies using organoid models on Earth versus LEO will improve our understanding of brain biology and cell-to-cell interactions. This will allow us to assess the impact of the space environment on the human brain and ultimately alleviate the effects of long-term exposure to space in humans. This project requires designing an automated device (CubeLab) capable of replicating organoid culture on board of the ISS. Here, we perform several ground control experiments to define the baseline conditions of organoid growth and survival in order to simplify and improve the design of our CubeLab.