Figure 1. Separation of dividing daughter cells during apical cytokinesis underlies intermingling of cell lineages.

(A) Left: Cartoon depicting organoid derivation. Right: Frames from time-lapse imaging of a dividing cell of the secretory lineage (red, Atoh1^CreER; R26^RFP) interspersing with non-secretory cells (green membranes). Arrowheads: dividing cell. Fraction of divisions in which labeled daughters separated is shown on the right panel. (B) Frames from 3D reconstructed SPIM of a secretory cell (red, Atoh1^CreER; R26^RFP) inserting in the cytokinetic furrow of a dividing stem cell (green, Lgr5^DTR-GFP). Arrows: dividing cell. (C) Frames from 3D reconstructed SPIM of a dividing cell of the secretory lineage (Atoh1^CreER; R26^RFP). Arrowheads: dividing cell. (D) Frames from 3D reconstructed SPIM of a secretory cell (red) undergoing a division in which daughter cells do not separate during cytokinesis (top, white arrows indicate daughter cells). Subsequently, these daughter cells become separated by a dividing cell pushing between them (bottom, white arrows indicate daughter cells and yellow arrowhead indicates newly dividing cell inserting between the adjacent daughters). (E) Confocal images of crypts in which cells have been labeled with a stochastic multicolor reporter in vivo (Vil1^CreER; R26^Brainbow2.1) and the positions of progeny analyzed three days after induction of the reporter. Left: sagittal view from 50 µm sections. Right: transverse views from 20 µm sections. Arrowheads indicate interspersed progeny. Progeny can also remain adjacent, as in the organoids, indicated by asterisks. Scale bars, 10 µm.

Figure 1—figure supplement 1. Cell interspersion in intestinal organoids.

(A) Top: Frame from time-lapse imaging of an intestinal organoid in which the stem cells and absorptive cells are labeled (Notch1^CreERT2; R26^RFP). Bottom: Frames from inset showing a dividing cell. Fraction of divisions in which labeled daughters separated is shown on the right panel. (B) Frames from time-lapse SPIM of an intestinal organoid, viewed from the basal surface of the epithelium. A stem cell (green, Lgr5^DTR-GFP) divides and the daughters becoming separated by insertion of a neighboring secretory cell (red, Atoh^CreER; R26^RFP). Arrow points to the stem cell, which displaces apically (out of the plane of view) before reappearing as two separated daughters (arrowheads). (C) Frames from time-lapse imaging of a dividing cell of the secretory lineage (Atoh^CreER; R26^RFP) following >24 hr treatment with the Notch (gamma-secretase) inhibitor, DAPT. Fraction of divisions in which labeled daughters separated is shown on the right panel. (D) Stills of control (left) or DAPT-treated (right) organoids showing accumulation of secretory cells following DAPT treatment for 3 days. E) Frame from time-lapse SPIM of an intestinal organoid, showing apical surface of the secretory daughter cells (Atoh1^CreER; R26^RFP) from Figure 1C. Arrowheads indicate the daughter cells. Scale bars, 10 µm.

Figure 1—video 1. 3D reconstruction of separated daughter cells.

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DOI: 10.7554/eLife.36739.005

Dividing cell of the secretory lineage imaged by SPIM with 40X objectives at 2 min time points. Separated daughters are then rotated toward the viewer.

Figure 1—video 2. Daughters that remain neighbors can become separated by subsequent nearby mitosis.

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DOI: 10.7554/eLife.36739.006

Cell of the secretory lineage (red, Atoh1^CreER; R26^RFP) divides and the daughter cells remain side-by-side. Subsequently, these daughters become separated by a non-secretory cell dividing between them. Imaged by SPIM with 40X objectives at 2 min time points.