Human cortical mosaicoids reveal impact on spatial organization and progressive loss of cross-genotype functional coupling in MECP2 mosaicism

Abstract

Cellular mosaicism arising from postzygotic mutation or X-chromosome inactivation is a major determinant of phenotypic variability in neurodevelopmental disorders. Rett syndrome, which is caused by mutations in the X-linked gene MECP2, is a striking example in which wild-type and mutant neural cells coexist within the developing brain. However, how MECP2 mosaicism shapes human cortical tissue organization and circuit assembly remains poorly understood. Here, we developed MosaicoidX, a human mosaic organoid platform combining controlled co-seeding of isogenic wild-type and MECP2-mutant hiPS cells with genotype-resolved calcium imaging, computational modeling, and single-cell transcriptomics. FACS-based single-cell seeding enabled reproducible generation of cortical mosaicoids with defined initial genotype ratios. Structural two-photon imaging revealed mutation-dependent spatial organization, with MECP2[V247X] mosaicoids (harboring a truncating mutation) showing pronounced skewing of radial and angular segregation compared with MECP2[R306C] mosaicoids (harboring a missense mutation). MosaicoidSim, a computational model, showed that genotype-dependent adhesion, proliferation, differentiation, and migration parameters can account for the emergence of mosaic spatial patterning. Genotype-resolved two-photon calcium imaging demonstrated asymmetric functional connectivity, in which wild-type neuron pairs formed stronger and more frequent activity-coupled interactions than mutant-mutant or wild-type-mutant pairs. Finally, single-cell RNA-seq combined with ligand-receptor analysis inferred genotype- and cell-type-dependent alterations in synaptic signaling pathways, including GABAergic and neurexin-neuroligin interactions. Together, these results show that MECP2 mosaicism actively shapes organoid development through both cell-autonomous and non-cell-autonomous mechanisms. MosaicoidX provides a general framework for dissecting genetic mosaicism in Rett syndrome and other mosaic neurodevelopmental disorders.