ABSTRACT Assembly of the brain relies upon spatiotemporally coordinated action of a small number of intracellular signaling pathways, which together govern an astonishingly wide array of processes, ranging from cell differentiation to tissue morphogenesis. How a handful of pathways can provide the precision and specificity required for the emergence of complex tissue structure and function that unfold over long periods of time, such as those of the human brain, has been a long-standing question in developmental biology. Even though landmark studies in developmental genetics, biochemistry and synthetic biology over the last decades have uncovered the working mechanisms of many signaling pathways and downstream genetic circuits, a major challenge that remains is to decipher how cells integrate information transmitted through the concerted action of several signaling factors which then drive region-specific morphogenetic processes in developmentally relevant contexts. Moreover, although the key components of these pathways are highly conserved across species, how they are functionally implemented to enable uniquely complex stratification of regions and cell types during brain development remains unknown, due to lack of functional access to primary tissue, appropriate models and molecular tools. To address this challenge, here we propose a spatiotemporally resolved approach that interfaces the dynamics of multiple signaling pathways over space and time in physiologically relevant systems. Specifically, this proposal will seek to combine several state-of-art techniques (DNA memory devices, multiplex biosensor imaging, spatial transcriptomics and pooled CRISPR screens) with human induced pluripotent stem cell (hiPSC)-derived organoid models, primary human neural progenitors and fetal marmoset brain sections to chart the cell signaling atlas for the developing brain at a resolution that has not previously been accessible. In Aim 1, we will implement in hiPSC lines DNA Typewriter and ENGRAM (DT/E), which together enable prime editing- mediated, temporally ordered insertion of signal-specific barcodes onto the genome to deconvolve the history of signaling dynamics of single cells and correlate them with their cellular and regional identities in unguided human brain organoids. In Aim 2, we will use optically barcoded biosensor imaging to assess the differential recruitment of morphogen-coupled signaling effectors. In Aim 3, using spatial transcriptomics, we will investigate the spatiotemporal dynamics between core developmental signaling and mechanotransduction pathways in specific fetal marmoset brain regions. Then, using 3D printing and pooled CRISPR engineering, we will assess how perturbed mechanotransduction affects regional specification programs in human brain organoids in response to varying degrees of biomechanical challenge. Ultimately, this proposal is designed to have a broad impact on multiple areas of scientific research across basic and translational developmental neurobiology and provide transformative new ways to better understand the signaling landscape underlying brain assembly in health and disease.

PROJECT NARRATIVE How the complexity of the brain emerges during development remains a long-standing yet poorly understood question in developmental biology. With this proposal, we will seek to integrate several cutting-edge molecular and cellular tools, such as DNA memory devices and 3D cell culture models of the developing human brain, to record the developmental signals that govern brain organogenesis at a previously inaccessible resolution and scale. The successful completion of this proposal will provide an unprecedented look into the programs underlying human brain assembly and pave way to novel therapeutic opportunities in tissue repair, cancer, and neurodevelopmental disorders.