Extrusion fountains are hallmarks of chromosome organization emerging upon zygotic genome activation.

The first activation of gene expression during development (zygotic genome activation, ZGA) is accompanied by massive changes in chromosome organization. The connection between these two processes remains unknown. Using Hi-C for zebrafish embryos, we found that chromosome folding starts by establishing "fountains", novel elements of chromosome organization, emerging selectively at enhancers upon ZGA. Using polymer simulations, we demonstrate that fountains can emerge as sites of targeted cohesin loading and require two-sided, yet desynchronized, loop extrusion. Specific loss of fountains upon loss of pioneer transcription factors that drive ZGA reveals a causal connection between enhancer activity and fountain formation. Finally, we show that fountains emerge in early Medaka and Xenopus embryos; moreover, we found cohesin-dependent fountain pattern on enhancers of mouse embryonic stem cells. Taken together, fountains are the first enhancer-specific elements of chromosome organization; they constitute starting points of chromosome folding during early development, likely serving as sites of targeted cohesin loading.