Date of Award

January 2026

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Kartik Pattabiraman

Abstract

TRANSCRIPTOMIC CHARACTERIZATION OF REGIONALLY DIFFERENTIATING TRANSIENT CORTICAL CIRCUIT SCAFFOLDSHelen Cai, Matthew Yuen, Rachel Bandler-Cohen, Daniel Doyle, Yuting Liu, and Kartik Pattabiraman. Child Study Center, Yale University, School of Medicine, New Haven, CT. Background/Scientific premise The subplate is a structure present in the brain during mid-fetal development that undergoes rapid expansion and rapid regression. Despite its transient nature, it is critical for appropriate formation of durable circuits between regions of the cortex and the thalamus. Disruption of these durable circuits is thought to be the basis for a broad range of neuropsychiatric disorders with the phenotype of sensory and perceptive disturbances, including autism spectrum disorder and schizophrenia. Postmortem tissue studies of patients with schizophrenia find high numbers of interstitial white matter cells, thought to be remnants of the subplate that have failed to undergo apoptosis and formed aberrant circuits. Several genes associated with autism have previously been found to be enriched in the subplate, with a subset being subplate-specific. Investigation and characterization of the enigmatic subplate will serve as the foundation for understanding appropriate circuit development and developing future therapeutic interventions. Research Aims In this study, we aim to use single-nucleus RNA sequencing of nuclei harvested from the murine subplate during cortical development to gain insight into the transcriptomic and functional heterogeneity of the subplate. Hypothesis We hypothesize that the neurons isolated from the murine subplate harbor a distinct transcriptomic signature uniquely identifiable from other cortical structures. Further, we hypothesize that these neurons form distinct subpopulations with heterogeneous transcriptomic profiles and functional characteristics. Methods To collect tissue samples, a transgenic mouse line was created in which a Cre-dependent green fluorescent reporter is expressed in nuclei destined for the subplate. Pregnant mice were fed tamoxifen at embryonic (E) day 11 to drive Cre recombinase; male and female pups were then harvested at E15 and E18. Pups were sacrificed and microdissected to collect tissue from the dorsal telencephalon. Tissue was dissociated and underwent fluorescence-activated nuclear sorting to divide samples into GFP+ and GFP- samples; pooled samples were then prepared and submitted for single-nucleus RNA sequencing. Data analysis was performed using a combination of standardized bioinformatics pipelines (e.g. using the Seurat workflow) and custom scripts written in R and Python. Results We successfully identify and annotate a subgroup of excitatory neurons which demonstrate high expression of canonical markers used to identify the subplate. These neurons additionally demonstrate high expression of many genes which are significantly upregulated in the subplate as compared to other populations; in this work, we identify 47 high-confidence novel markers of the subplate. Genes which are found to be highly expressed in the subplate are also highly associated with disease risk genes for schizophrenia and ASD, demonstrated with hypergeometric testing. Within subplate cells, we also subcategorize cells as belonging to one of six clusters with unique differentially expressed genes, and find that these demonstrate patterns of gene expression which are restricted to regions within cortical layers. Statement of scientific impact Derangements to normal subplate connectivity and longevity may result in aberrant sensory processing pathways, leading to phenotypes such as hypersensitivity to touch and sound seen in patients with ASD and auditory or visual hallucinations seen in patients with schizophrenia. Here, we describe and characterize cell subtypes within the subplate and identify regional restriction of gene expression. Identification of the functional roles of these cells and the cascade which results in regionalized expression will form the initial steps to understanding the underpinnings of early neuropsychiatric disease.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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