Loss of Paneth cell contact starts a WNT differentiation timer in intestinal crypts

<title>Abstract</title> WNT signaling is pivotal to the renewal of many organs including the intestine. WNT gradients are thought to provide a spatial differentiation cue, and hence control the required spatial patterning of cell types. Testing this mechanism is difficult, however, as current methods are static while the underlying processes are highly dynamic. Here, we show that a ‘timer’ mechanism rather than spatial cues controls the WNT signaling decreases that drive cell differentiation. Using live-cell imaging and single-cell tracking of intestinal organoids, we find that WNT signaling in stem cells decreases immediately after loss of Paneth cell contact. This WNT signaling decrease does not depend on cell growth and division, and precedes differentiation, as shown by subsequent antibody staining and cell type propagation along lineage trees. Cells frequently regain Paneth cell contact, which in turn increases WNT signaling again, showing that differentiation is not progressive but rather repeatedly reset. Our WNT timer mechanism overturns existing spatial gradient models, explains differentiation within the intestinal crypt, and may well be more broadly relevant to WNT-mediated tissue renewal.