Autism spectrum disorder (ASD) is a neurodevelopmental condition with a strong genetic basis, but how genes contribute to abnormal circuit assembly remains unclear. Studies have reported abnormal timing of key differentiation steps, yet whether this occurs across genetic causes and its impact on circuits is unknown. Longitudinal studies examining in vivo molecular and circuit maturation during development are thus crucial to address these questions. This requires being able to assess the developmental effects of multiple genes on specific cell types in living organisms, which Denis Jabaudon and Rosa Cossart propose to do in this project. Their approach leverages new technologies: in vivo parallel multi-gene disruption (DJ lab) and longitudinal imaging of neuronal activity in developing pups (RC lab). We propose a strategy focusing on the maturation of intratelencephalic (IT) neurons in the mouse neocortex, a cell population affected in ASD. The research program comprises four interconnected aims:

1. Design of AAV libraries for in vivo manipulation of approximately 90 ASD-related genes in parallel (Perturb-seq). High-priority targets will be selected by cross-referencing the SFARI Gene database with a database of cortical cell-type-specific developmental gene expression dynamics (http://www.humous.org). Using CRISPRi technology, Jabaudon and Cossart will create three libraries, each containing gRNAs targeting approximately 30 genes, for delivery into developing embryonic mouse brains.
2. Assessing how parallel perturbation of approximately 90 ASD-related genes in developing mouse cortex affects the timing of developmental transcriptional programs in each case. Using CRISPRi-based gene perturbation (Perturb-seq) and single-cell RNA sequencing at three developmental timepoints (E16.5, E18.5, P8), the team will analyze the impact on molecular differentiation pace using state-of-the-art bioinformatic approaches.
3. Evaluation of gene perturbation effects on the maturation of local circuit integration of neurons in the developing mouse cortex. Jabaudon and Cossart will perform daily in vivo calcium imaging and holographic photostimulation in live mouse pups starting at P8 to track the maturation of single-neuron spontaneous activity (approximately top 30 genes) and measure reciprocal connections between IT neurons and GABAergic neurons (approximately top 10 genes).
4. Assessment of the maturation of long-range sensory input integration onto cells with perturbed gene expression (approximately top 10 genes). Jabaudon and Cossart will track neuronal responses to controlled whisker deflections and analyze the evolution of single-neuron calcium responses across the second postnatal week. Additionally, they will use machine learning approaches to infer mouse behavioral states from neuronal activity and measure the stability of representations across days.

This proposal unites two laboratories with internationally recognized expertise in the molecular (DJ) and circuit (RC) developmental neurobiology to bridge cell type-specific disruption of ASD-related genes with their downstream consequences on the molecular and circuit maturation of cortical neurons. Preliminary data demonstrate the feasibility of this research program, including successful in vivo delivery of dozens of gRNAs simultaneously, complex bioinformatic analysis of corresponding single-cell RNA sequencing data and longitudinal tracking of neural activity throughout early postnatal cortical development.