Following fertilization in mammals, the chromatin landscape inherited from the two parental genomes and the nuclear organization are extensively reprogrammed. A tight regulation of nuclear organization is important for developmental success. One main nuclear feature is the organization of the chromosomes in discrete and individual nuclear spaces known as chromosome territories (CTs). In culture cells, their arrangements can be constrained depending on their genomic content (e.g., gene density or repeats) or by specific nuclear constrains such as the periphery or the nucleolus. However, during the early steps of mouse embryonic development, much less is known, specifically regarding how and when the two parental genomes intermingle. Here, we describe a three-dimensional fluorescence in situ hybridization (3D-FISH) for chromosome painting (3D-ChromoPaint) optimized to gain understanding in nuclear organization of specific CTs following fertilization. Our approach preserves the nuclear structure, and the acquired images allow full spatial analysis of interphase chromosome positioning and morphology across the cell cycle and during early development. This method will be useful in understanding the dynamics of chromosome repositioning during development as well as the alteration of chromosome territories upon changes in transcriptional status during key developmental steps. This protocol can be adapted to any other species or organoids in culture.
Keywords: 3D DNA FISH; Chromosome territories; Mouse embryo; Nuclear organization.