a, Illustration of the dissection strategy of the PROX1-positive region from organoids with representative images. The imaging experiments were repeated with 12 independent samples with similar results. Scale bar, 100 µm. b, Optimization of the organoid culture system, comparing: (1) floating, (2) embedding in Matrigel, (3) embedding in Matrigel and culture in Transwells from day 13 and (4) dissection followed by embedding in Matrigel, and culture in Transwell from day 13. Left, the typical morphology of invaginating or branching organoid. The imaging experiments were repeated with 12 independent samples with similar results. Scale bar, 100 µm. c, Illustration of optimization of in vitro culture system. We compared various culture formats to enhance morphological change, such as invagination and branching morphogenesis, of PROX1-positive HBP domains. At day 7, anterior and posterior gut spheroids were mixed after 24 h of culture. Connected spheroids were transferred into a Matrigel drop or a low-binding culture plate to compare between non-floating and floating conditions during emergence of the HBP domain. Organoids in the Matrigel-embedded group started to express tdTomato at day 11. The tdTomato-positive region was manually dissected under the microscope according to the fluorescent signal and transferred into a Matrigel again or a Transwell to compare the effect of various agonists and antagonists in the medium. d, Morphogenesis of boundary organoids during 2 days from day 13. Imaging experiments were repeated independently three times with similar results. Scale bar, 100 µm.