Fine-grained mapping of neural progenitors subtypes in the embryonic brain unveils context-dependent roles of signalling on cell fate decisions

The mammalian brain develops through precisely orchestrated events that transform a simple neural tube into a complex structure. While single-cell RNA sequencing has revealed remarkable cellular heterogeneity, the lack of spatial context limits our understanding of how this diversity relates to functional organization. This thesis addresses this gap by establishing a spatial transcriptomics atlas of the developing mouse brain and using it to investigate fundamental questions about neural development. We generated high-resolution spatial gene expression maps from embryonic day 9.5 to 15.5 using Hybridization-based in situ sequencing across approximately 150 tissue sections. We developed Pointillhist, a graph neural network framework for integrating spatial transcriptomics with single-cell RNA sequencing data. This integration enabled mapping of hundreds of transcriptionally distinct populations at single-cell resolution, revealing stable territorial organization of progenitor populations throughout development. Building on this atlas, we examined how cellular territories relate to functional organization. By integrating spatial proximity and transcriptional similarity identified putative lineage relationships between populations. Analysis of ligand-receptor interactions around key organizer regions uncovered heterogeneous signaling repertoires within organizer subpopulations and corresponding receptor diversity in receiving populations, suggesting how combinatorial signaling contributes to cellular heterogeneity during development. Additionally, correlation analysis between spatial clustering and cytoskeletal gene expression revealed molecular programs underlying the structural organization of progenitors versus neurons. Finally, inspired by traditional approaches to studying morphogen function, we investigated how Sonic Hedgehog signaling shapes regional identity through single-cell analysis of pathway perturbation models. Comparison of telencephalic and midbrain-hindbrain conditional knockouts revealed both population-specific and region-specific responses to signaling ablation. Proliferating progenitors showed distinct molecular adaptations compared to more differentiated populations, with cycling cells exhibiting altered cell cycle dynamics and differentiated cells showing compromised lineage-specific transcriptional networks. In the telencephalon, GABAergic lineage cells showed progressive depletion, while midbrain populations exhibited more moderate reductions with compensatory activation of alternative regulatory pathways. This molecular characterization provides mechanistic insight into the differential vulnerability of neural populations to morphogen perturbation across brain regions. This work establishes a multilevel understanding of brain development, connecting molecular cartography to functional organization. By preserving spatial context while achieving single-cell resolution, we advance our understanding of the establishment of emb