Highlights d The Drosophila posterior salivary gland imaginal ring is a transition-zone model d Persistent Notch signaling induces neoplastic transformation in the transition zone d Endogenous inflammatory JAK-STAT and JNK signaling promotes tumorigenesis d JNK-induced MMP1 shapes a weaker ECM structure for advanced tumorigenesis

The human transcription factors OCT4, SOX2, and NANOG form a core signaling network critical for maintaining stem cell pluripotency and self-renewal potential. The spatiotemporal expression dynamics of these pluripotency factors throughout differentiation is unclear, limiting our understanding of stem cell fate decisions. Here, we combined CRISPR/Cas9-mediated gene editing with microraft array technology to generate human embryonic stem cell lines with endogenously tagged fluorophores for OCT4, SOX2, and NANOG. Fluorescence time-lapse imaging revealed that pluripotent stem cells show gastrulation-like patterning without direct chemical induction. Directed differentiation to the three primary germ layers - endoderm, mesoderm, and ectoderm - revealed distinct spatiotemporal patterns of SOX2 and NANOG expression in single cells. Finally, we captured dynamic changes in cell morphology during ectoderm differentiation corresponding to the formation of neural rosettes. This study provides a robust method for generating live-cell reporters in human stem cells and describes the single-cell dynamics of human pluripotency factors during differentiation.

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