Specification of a rostro-caudal axis in cortical assembloids through a polarized source of FGF8 (scRNA-Seq)

Current organoid protocols are widely used to mechanistically study cortical development, evolution and brain pathologies. While these systems generate all major cortical cell types in histologically distinct compartments, they lack cortical area topography, which is initiated by gradients of signaling molecules released by signaling organizers that establish transcriptionally distinct domains along the rostro-caudal axis of the developing cortex. Here, we engineer human cortical assembloids that combine an organizer-like structure expressing FGF8 with an elongated organoid design to enable the controlled modulation of FGF8 signaling along the longitudinal organoid axis. We demonstrate that individual polarized cortical assembloids (polCA) mount a position-dependent transcriptional program that matches the in vivo rostro-caudal gene expression patterns. Importantly, positional identity is lost in polCA carrying a mutation in the FGFR3 gene associated with temporal lobe malformations and intellectual disability in humans. Similar to in vivo, this program is accompanied by transcriptional divergence of radial glia cells and excitatory neurons and their segregation along the longitudinal axis of individual organoids. Thus, this method reproduces in vivo cortical topography in individual organoids and enables the analysis of area-related phenotypes underlying human disorders. Overall design: In order to explore the position-dependent transcriptional changes in polar cortical asembloids (polCAs), we performed bulk RNA-sequencing analysis on consecutive tissue segments of D60 polCAs (7 replica, 1 is an outlier) and compared to unpatterned controls without FGF8 OrEB. We defined these sections based on their relative distance from the OrEB (when present) as proximal (P), medial (M) and distal (D) to the source. 3 Elongated organoids without OrEB were used to generate three control segments (C1, C2 and C3). Further, FGF3 mutant polCAs (3 replica) were sequenced. To further discern cell type composition, we performed additiona scRNAseq analysis on P-M-D segments from 3 polCAs and C segments from 3 controls at D60