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.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/29106 |
| Date | January 2023 |
| Creators | Kilpatrick, Savannah |
| Contributors | Singh, Karun, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0026 seconds