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.
