This study investigates the 3D genome reorganization during mouse neural development using ultra-deep Hi-C mapping. The authors found that transcription is correlated with chromatin insulation but is not sufficient for creating topologically associated domains (TADs) boundaries de novo. They observed dynamic interactions among exon-rich gene bodies, enhancer-promoter pairs, and transcription factor (TF) sites. During neural differentiation, contacts between active TADs decreased, while contacts between inactive TADs increased. The Polycomb network was disrupted, and novel TF interactions emerged. Enhancer-promoter contacts were mostly cell-type specific and correlated with gene expression. This work provides insights into the complex dynamics of chromatin interactions during development and highlights the role of multiple factors in shaping genome architecture.This study investigates the 3D genome reorganization during mouse neural development using ultra-deep Hi-C mapping. The authors found that transcription is correlated with chromatin insulation but is not sufficient for creating topologically associated domains (TADs) boundaries de novo. They observed dynamic interactions among exon-rich gene bodies, enhancer-promoter pairs, and transcription factor (TF) sites. During neural differentiation, contacts between active TADs decreased, while contacts between inactive TADs increased. The Polycomb network was disrupted, and novel TF interactions emerged. Enhancer-promoter contacts were mostly cell-type specific and correlated with gene expression. This work provides insights into the complex dynamics of chromatin interactions during development and highlights the role of multiple factors in shaping genome architecture.