Intestinal organoids derived from single cells undergo complex crypt-villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Through light-sheet microscopy and large-scale imaging quantification, we demonstrate that crypt formation coincides with stark lumen volume reduction. We develop a 3D biophysical model to computationally screen different mechanical scenarios of crypt morphogenesis. Combining this with live-imaging data and multiple mechanical perturbations, we show that actomyosin-driven crypt apical contraction and villus basal tension work synergistically with lumen volume reduction to drive crypt morphogenesis, and demonstrate the existence of a critical point in differential tensions above which crypt morphology becomes robust to volume changes. Finally, we identified a sodium/glucose cotransporter specific to differentiated enterocytes that modulates lumen volume reduction via cell swelling in villus region. Altogether, our study uncovers the cellular basis of how cell fate modulates osmotic and actomyosin forces to coordinate robust morphogenesis.

Highlights d Bulk actin polymerization waves move through the oocyte from animal to vegetal d An animal-to-vegetal actin gradient triggers animal-directed actin flows d Actin flows drag the ooplasm toward the animal pole via differential friction d Actin comets formed on yolk granules push them toward the vegetal pole

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Intestinal organoids derived from single cells undergo complex crypt-villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Through light-sheet microscopy and mechanical perturbations, we demonstrate that organoid crypt formation coincides with stark lumen volume reduction, which works synergistically with actomyosin-generated crypt apical and villus basal tension to drive morphogenesis. We analyse these mechanical features in a quantitative 3D biophysical model and detect a critical point in actomyosin tensions, above which crypt becomes robust to volume changes. Finally, via single-cell RNA sequencing and pharmacological perturbations, we show that enterocyte-specific expressed sodium/glucose cotransporter modulates lumen volume reduction via promoting cell swelling. Altogether, our study reveals how cell fate-specific changes in osmotic and actomyosin forces coordinate robust organoid morphogenesis. One Sentence Summary:Emergence of region-specific cell fates drive actomyosin patterns and luminal osmotic changes in organoid development.

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