Allele-specific CRISPR perturbation of the imprinted Dlk1-Dio3 domain reveals regulation of BMP-NOTCH-VEGF signaling in embryonic organogenesis.

The Dlk1-Dio3 imprinted domain is essential for mammalian development, yet its role in coordinating multi-organ embryogenesis is not fully understood. Here we generated four distinct mouse models via CRISPR/Cas9-mediated insertion of a transcriptional termination cassette within the Dlk1-Dio3 domain, encompassing homozygous (HOMO), maternally inherited (MK), paternally inherited (PK), and wild-type alleles (WT), to elucidate parent-of-origin-specific effects. Integrative transcriptomic analyses across multiple scales revealed allele-specific dysregulation of imprinted genes and altered epigenetic stability at the locus. Crucially, scRNA-seq of embryonic day 14.5 (E14.5) liver, heart, and placenta showed that embryos homozygous or maternally targeted for Dlk1-Dio3 inactivation arrest in development due to collapse of a BMP-NOTCH-VEGF signaling network. This manifests as organ-specific pathologies: (i) impaired decidual-trophoblast paracrine communication and aberrant BMP/WNT signaling compromise the maternal-fetal blood barrier, thereby disrupting placental angiogenesis and restricting maternal-fetal nutrient exchange; (ii) fetal liver hematopoietic progenitors display impaired myeloid lineage priming, concomitant with aberrant NOTCH and WNT signaling, leading to failure in hepatocyte differentiation; (iii) cardiac progenitors do not receive sufficient BMP-NOTCH-VEGF cues, yielding immature cardiomyocytes and vascular defects. Collectively, these findings identify the Dlk1-Dio3 imprinted domain as a pivotal integrative hub for cell-type-specific signaling pathways during organogenesis and provide mechanistic insights into how dysregulation of genomic imprinting can precipitate systemic embryonic developmental failure.