The 15q13.3 microdeletion is a common genetic disorder associated with multiple neurodevelopmental disorders including autism spectrum disorder, epilepsy, and schizophrenia. Patients have diverse clinical presentations, often prompting genetic assays that identify the CNV in the clinic. This late-stage screening leaves a considerable gap in our understanding of the prenatal and prediagnostic developmental impairments in these individuals, providing a barrier to understanding the disease pathobiology. We provide the first investigation into embryonic brain development of individuals with the 15q13.3 microdeletion by generating multiple 3D neural organoid models from the largest clinical cohort in reported literature. We incorporated unguided and guided forebrain organoid models into our multi-transcriptomic phenotyping pipeline to uncover changes in cell type emergence and disruptions to circuit development, all of which had underlying changes to cell adhesion pathways. Specifically, we identified accelerated growth trajectories in 15q13.3del unguided neural organoids and used single cell RNA sequencing to identify changes in radial glia dynamics that affect neurogenesis. We measured changes in the pseudotemporal trajectory of matured unguided neural organoids, and later identified disruptions in synaptic signaling modules amongst the primary constituents to neural circuitry, excitatory and inhibitory neurons. We leveraged dorsal and ventral forebrain organoid models to better assess circuit dynamics, as they faithfully produce the excitatory and inhibitory neurons in the pallium and subpallium, respectively. We then used the entire 15q13.3del cohort and performed bulk RNA sequencing on each tissue type at two timepoints and discovered convergence on transcriptional dysregulation and disruptions to human-specific zinc finger proteins localized to chromosome 19. We also identified cell type-specific vulnerabilities to DNA damage and cell migration amongst the dorsal and ventral organoids, respectively, which was consistent with the excitatory and inhibitory neural subpopulations amongst the unguided neural organoids scRNA Seq, respectively. We then examined neuron migration in a 3D assembloid model by sparsely labeling dorsal-ventral forebrain organoids from multiple genotype-lineage combinations. Light sheet microscopy identified deficits in inhibitory neuron migration and morphology, but not migration distance, suggesting a complex disruption to cortical circuitry. This novel combination of cell type characterization, pathway identification, and circuitry phenotyping provides a novel perspective of how the 15q13.3 deletions impair prenatal development and can be applied to other NDD models to leverage understanding of early disease pathogenesis. / Dissertation / Doctor of Science (PhD) / The development of the human brain is a highly complex and tightly regulated process that requires the participation of multiple cell types throughout development. Disturbances to the emergence, differentiation, or placement of these cell types can cause disruptions and local miswiring of neural circuits, which is often associated with neurodevelopmental disorders (NDDs). The 15q13.3 microdeletion syndrome is a highly complex condition associated with multiple NDDs and has seldom been studied in a human context. To address this, we used stem cells derived from a 15q13.3 microdeletion syndrome cohort and their typically developing familial controls to generate unguided (“whole brain”) and region-specific organoids to investigate early fetal development across time. We used the largest 15q13.3 microdeletion cohort in reported literature to identify shared disruptions in early developmental milestones such as neurogenesis, neural migration, and neural patterning. We identified expansion of specific cell populations, including progenitors that later give rise to mature neurons. Abnormalities persisted in more mature cell populations, including the inhibitory neurons responsible for establishing critical microcircuitry in the human cortex. By generating guided organoids that enrich for excitatory and inhibitory neural populations, we were able to merge the models to form assembloids, where we captured early migratory and morphological deficits in inhibitory neuron populations, which is supported by the multi-transcriptomics experiments performed in both organoid models. This study provides a framework for examining fetal development in a neurodevelopmental disorder context. By using the 15q13.3 microdeletion background, we found novel disruptions in cell type emergence and circuit formation previously unreported in mouse or 2D neuron models, highlighting the utility of the phenotyping platform for disease modeling.

Les mutations somatiques sur le variant d’histone H3.3 et les régulateurs épigénétiques associés à H3.3 ont été identifiés dans 30 % des glioblastomes pédiatriques. Ces mutations sont caractérisées par des substitutions d'acides aminés à des positions spécifiques dans la région N-terminale de l'histone H3.3 telles que la glycine 34 en valine/arginine (G34V/R), l'alanine 29 en proline (A29P), ou une haplo-insuffisance de la protéine Zinc Finger MYND-Type Containing 11 (ZMYND11). ZMYND11 est un co-répresseur de la transcription qui se lie spécifiquement à H3.3K36me3 pour moduler l'activité de l'ARN polymérase II. De plus, il est intéressant de mentionner que l’interaction entre ZMYND11 et H3.3K36me3 est altérée lorsque le résidu G34 est muté en G34V. Récemment, les mutations germinales H3.3G34V, H3.3A29P et ZMYND11 ont été identifiées chez des patients présentant une déficience neurologique. Nous émettons l'hypothèse que les mutants H3.3G34V et H3.3A29P empêchent ZMYND11 de se lier à H3.3K36me3 et pourrait converger mécaniquement avec la perte de fonction de ZMYND11, ce qui perturberait la neurogenèse. À l'aide de la technologie CRISPR Cas9, nous avons généré des modèles mutants isogéniques à partir de cellules souches pluripotentes (iPSC) pour H3F3B-A29P, H3F3B-G34V et ZMYND11-knock-out (KO). Par la suite, nous avons stimulé la différenciation de ces modèles vers des lignées neuronales afin d’identifier si ces mutations affectent la neurogenèse. Enfin, en utilisant des méthodes de séquençage à haut-débit nous avons analysé le profil épigénomique et transcriptomique pour déterminer comment l’interaction entre ZMYND11 et H3K36me3 est perturbée et à quels degrés ces mutations impactent sur les modifications post-traductionnelles des histones. Ce projet permettra de mieux comprendre les fonctions de ZMYND11 sur le remodelage de la chromatine et sa fonction biologique au cours du développement cérébral. / Somatic mutations on the histone 3 variant H3.3 and H3.3-associated chromatin modifiers have been identified in 30% of pediatric high-grade gliomas (pHGG). The mutations are characterized by amino acid substitutions at specific positions within the histone H3.3 tail such as glycine 34 to valine/arginine (G34V/R), alanine 29 to proline (A29P), or haploinsufficiency of the chromatin reader Zinc Finger MYND-Type Containing 11 (ZMYND11). ZMYND11 is a transcriptional co-repressor that specifically reads H3.3K36me3 to modulate RNA polymerase II activity. Notably, binding of ZMYND11 to H3.3K36me3 is altered when G34 residue is mutated to G34V. Recently, germline mutations of H3.3G34V, H3.3A29P, and ZMYND11 have been identified in patients with Intellectual disability. We hypothesize that H3.3 G34V and H3.3A29P mutants impede the binding of ZMYND11 to H3.3K36me3 and may mechanistically converge with ZMYND11 loss-of-function mutation to perturb neurogenesis. Using CRISPR Cas9-mediated gene editing, we will generate isogenic human induced pluripotent stem cell (iPSC) models for H3F3B-A29P, H3F3B-G34V and ZMYND11-KO, and perform in vitro neural differentiation to identify whether specific neural lineages are affected. Next, using epigenomic and transcriptomic profiling we will study whether binding between ZMYND11 and H3K36me3 is disrupted, and the downstream impact on Post-Translational Modifications of histones (PTMs) and transcription. This project will lead to a better understanding of the crucial role of the chromatin reader ZMYND11 on chromatin remodeling and the biological function during neural development.