Friday, May 9, 2025
5/9/2025
Dynamic regulatory mechanisms of robust pattern formation in the neural tube - ABSTRACT Dorsoventral (DV) patterning of the vertebrate neural tube has served as a model system for understanding how cells acquire position-dependent cell fates during development. The reigning paradigm for patterning is based on the French flag model, which suggests that cells along the DV axis of the neural tube reside in a gradient of an extracellular morphogen and interpret the concentration of this signal to assume specific fates. This model overlooks the highly dynamic nature of the tissue leading up to neural tube formation, during which cells move large distances through multiple spatiotemporally varying signaling gradients. Our previous work on this grant has revealed critical gaps in the current understanding, highlighting the need for a comprehensive analysis that takes into account the dynamics of the system as well as the role of a multiplexed, noisy signaling environment. Here we seek to address this challenge by taking advantage of the zebrafish model system and leveraging our expertise in high-resolution 4-D microscopy, single-cell transcriptomics, and systems-biological approaches in vivo. Based on our work and recent work from other labs, we hypothesize that cells in the nascent neural tissue perceive multiple time-varying signals as a consequence of complex morphogenetic movements during gastrulation, which are processed by a gene regulatory network (GRN) to initially ‘pre-pattern’ the neural plate. We suggest that pre-patterning integrates with subsequent dynamic signaling in the neural tube to enable precise DV patterning. To investigate this idea, in Aim 1 we will first systematically characterize pre-patterning in the zebrafish neural plate by generating a spatial atlas of transcription factor (TF) expression. We will then analyze whether and how pre-patterned TFs contribute to neural tube fates using fate-mapping approaches as well as a novel in vivo Perturb-seq technique, which combines multiplexed CRISPR/Cas9 perturbation of TFs with single-cell RNAseq for large- scale functional analysis. Finally, guided by mathematical modeling, we will analyze how pre-patterning contributes to the precision of neural tube patterning using perturbation studies. In Aim 2 we will analyze how multiplexed, dynamic signaling in the gastrula leads to neural pre-patterning. Starting with a single-cell level characterization of the signaling landscape of the nascent neural plate, we will combine signal perturbations, TF Perturb-seq, and mathematical modeling to dissect how a neural GRN processes temporally varying signaling to establish a pre-pattern. Together, this work will provide a comprehensive view of neural tube patterning, elucidating how it is accomplished in the early embryo through multiple dynamic signals interacting with organized tissue movements. Furthermore, we expect that the conceptual ideas and approaches developed here will be broadly applicable to understanding robust patterning across developmental systems and organisms.
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