Cellular aspect ratio and cell division mechanics underlie the patterning of cell progeny in diverse mammalian epithelia
- University of California, San Francisco, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1 with 3 supplements
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, Atoh1CreER; R26RFP) interspersing with non-secretory cells …
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 (Notch1CreERT2; R26RFP). Bottom: Frames from inset showing a dividing cell. …
Figure 1—video 1
3D reconstruction of separated daughter cells.
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.
Cell of the secretory lineage (red, Atoh1CreER; R26RFP) divides and the daughter cells remain side-by-side. Subsequently, these daughters become separated by a non-secretory cell dividing between …
Figure 2 with 14 supplements
Polarized actin-dependent cell shape changes underlie division-coupled interspersion behaviors.
(A) Frames from time-lapse imaging of cytokinesis in an organoid expressing myosin regulatory light chain (MRLC)-mScarlet. (B) 3D reconstruction from live imaging of a cell dissociated from EB3-GFP …
Figure 2—figure supplement 1
Polarized actin-dependent changes in cell shape during division in intestinal organoids.
(A) 3D reconstruction of immunofluorescence images. Arrow indicates mitotic cell. Arrowhead indicates the corresponding apical footprint. DNA: Hoechst 33342, actin: Alexa488-phalloidin, tight …
Figure 2—video 1
Cytokinesis in the intestinal organoids.
MRLC-mGFP organoids imaged by spinning disc confocal with 60 X objective at 20 s time points.
Figure 2—video 2
Cytokinesis in the intestinal organoids.
MRLC-mScarlet organoids imaged by spinning disc confocal with 60X objective at 20 s time points.
Figure 2—video 3
Cytokinesis in the intestinal organoids.
Rare membrane-GFP cells in organoids generated by stochastic recombination of the R26mTmG reporter as described in Figure 2—figure supplement 1E were imaged by spinning disc with 60X objective at 20 …
Figure 2—video 4
Furrow ingression in dissociated intestinal cells.
3D reconstruction of a single time point from live imaging of mitotic exit in cells dissociated from an EB3-GFP organoid. The reconstruction is rotated away from the viewer. Imaging was performed by …
Figure 2—video 5
Spindle assembly and cell rounding during mitosis.
EB3-GFP organoids imaged from early prophase by spinning disc confocal with 60X objective at 20 s time points.
Figure 2—video 6
Cell rounding during mitosis in intestinal organoids.
Membrane-tomato labeled organoids (R26mTmG in the absence of recombination) imaged by spinning disc confocal with 20X objective at 7 min time points.
Figure 2—video 7
Chromosome movements at mitotic onset in Latrunculin A-treated organoids.
DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.
Figure 2—video 8
Chromosome movements at mitotic onset in nocodazole-treated organoids.
DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points. The cell does not undergo chromosome segregation as it is unable to assemble a mitotic spindle.
Figure 2—video 9
Chromosome movements at mitotic onset in control organoids
DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.
Figure 2—video 10
Cell reinsertion behavior.
A daughter cell within an organoid in which a subset of cells is labeled with membrane-GFP (R26mTmG; Vil1CreER induced at low levels) blebbing and extending protrusions to the basal surface …
Figure 2—video 11
Chromosome movements following induced mitotic exit in STLC-treated organoids.
DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.
Figure 2—video 12
Chromosome movements following induced mitotic exit in STLC and Latrunculin A-treated organoids.
DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.
Figure 2—video 13
Chromosome movements following induced mitotic exit in STLC and nocodazole treated organoids.
DNA labeled with SiR-DNA dye. Imaged by spinning disc confocal with 40X objective at 4 min time points.
Figure 3 with 4 supplements
Cellular aspect ratio is a key parameter for division-coupled interspersion in the intestine and the embryo.
(A) 3D reconstruction of a spherical organoid derived from fetal (E13.5) mouse intestine. DNA: Syto 21 dye, arrowhead: mitotic cell. (B) Frames from 3D reconstructed time-lapse SPIM of chromosome …
Figure 3—figure supplement 1
Epithelia with low aspect ratio.
(A) A slice from a 3D reconstruction of a spherical organoid generated by growth in culture with exogenous Wnt3a, shown in two orientations. DNA: H2B-GFP. Arrowhead indicates mitotic cell. Frames …
Figure 3—video 1
Division in fetal spheroids with a short apical-basal axis.
3D reconstruction from live imaging of a mitotic cell in a fetal spheroid, imaged by SPIM with 40X objectives. DNA labeled with Syto21 dye. A section of the sphere is rotated.
Figure 3—video 2
Division in fetal spheroids.
H2B-mScarlet fetal organoids imaged by SPIM with 40X objectives at 2 min time points. The view from the basal surface is shown.
Figure 3—video 3
Division in the embryonic visceral endoderm.
H2B-GFP E7.5 embryo imaged by SPIM with 40X objectives at 3 min time points. The view from the external surface of the embryo is shown.
Figure 4 with 1 supplement
Model for cell progeny patterning in mammalian epithelia.
Top: Cartoon of the influence of cell height on the relative positioning of cells derived from a given progenitor (magenta cells). Bottom: Model for interspersion of cell progeny in elongated …
Figure 4—figure supplement 1
Daughter cell geometries.
Cartoon comparing daughter cell geometries generated by vertebrate and Drosophila cytokinesis, also see (Higashi et al., 2016).
Videos
Video 1
Secretory cell separation during division.
Cells of the secretory lineage (red, Atoh1CreER; R26RFP) interspersed with non-secretory cells (green membranes) imaged by spinning disc confocal with 20X objective at 3 min time points.
Video 2
Stem cell separation during division by insertion of a secretory cell into the cytokinetic furrow.
Cell of the secretory lineage (red, Atoh1CreER; R26RFP) inserts into the furrow of a dividing stem cell (green, Lgr5DTR-GFP). Imaged by SPIM with 40X objectives at 2 min time points. Second clip …
Video 3
Chromosome movements in intestinal organoids.
H2B-mScarlet labeled organoids were imaged by SPIM using 40X objectives at 2 min time points.
Video 4
Cell separation during division in the embryonic epiblast/primitive ectoderm.
An E7.5 mouse embryo was imaged by SPIM with 40X objectives at 4 min time points. Cells expressing cytoplasmic RFP from a CAGGSCreER; R26Brainbow2.1/+ embryo are shown.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | R26mTmG | Jackson Labs, PMID: 17868096 | MGI: 3716464 | |
| Strain, strain background (Mus musculus) | Vil1Cre-ERT2 | Averil Ma lab, PMID: 15282745 | MGI: 3053826 | |
| Strain, strain background (Mus musculus) | Atoh1CreERT | Jackson labs, PMID: 16958097 | MGI: 3686985 | |
| Strain, strain background (Mus musculus) | R26RFP | Jackson Labs, PMID: 20023653 | MGI: 3809524 | |
| Strain, strain background (Mus musculus) | Lgr5DTR-GFP | de Sauvage Lab (Genentech), PMID: 21927002 | MGI: 5294798 | |
| Strain, strain background (Mus musculus) | C57BL/6J | Jackson Labs | ||
| Strain, strain background (Mus musculus) | R26Brainbow2.1 | Jackson Labs, PMID: 20887898 | MGI: 164644 | |
| Strain, strain background (Mus musculus) | H2B-GFP | Jackson Labs, PMID:15619330 | MGI: 109836 | |
| Strain, strain background (Mus musculus) | Notch1CreERT2(SAT) | PMID: 21991352 | MGI: 5304912 | |
| Antibody | Rabbit anti-ZO-1 | Thermo Fisher | RRID:AB_2533456 | |
| Chemical compound, drug | Alexa 488-Phalloidin | Thermo Fisher | A12379 | |
| Chemical compound, drug | Hoechst 33342 | Molecular Probes | H3570 | |
| Chemical compound, drug | See Table 1 for pharmacological inhibitors | |||
| Software, algorithm | MicroManager | Open Imaging, PMID: 20890901 |
Table 1
Small molecules used in this study.
https://doi.org/10.7554/eLife.36739.033| Molecule | Function | Source | Cat # | Final concentration |
|---|---|---|---|---|
| Nocodazole | Microtubule inhibitor | Calbiochem | 487929 | 5 µM |
| Latrunculin A | F-actin inhibitor | Calbiochem | 428026 | 4 µM |
| SiR DNA | DNA dye | Cytoskeleton Inc | CY-SC007 | 1 µM |
| Verapamil | Efflux pump inhibitor | Cytoskeleton Inc | CY-SC007 | 10 µM |
| MG132 | Proteasome inhibitor | Sigma | ML449 | 10 µM |
| STLC | Eg5 inhibitor | Sigma | 164739 | 10 µM |
| RO-3306 | CDK inhibitor | Calbiochem | 217699 | 10 µM |
| AZ3146 | Mps1 inhibitor | Tocris | 3994 | 2 µM |
| BI2536 | Plk1 inhibitor | Selleck Biochem | S1109 | 10 µM |
| Tamoxifen | Cre-ER inducer (applied for 6–16 hr in culture) | Sigma | T5648 | 1 µM (Atoh1CreER and Notch1CreER) 0.1 µM (Vil1CreER) |
| S -- Blebbistatin | Myosin II inhibitor | Abcam | ab120491 | 200 µM |
| S -- Blebbistatin | Myosin II inhibitor | Cayman | 13013 | 200 µM |
| Y27632 | ROCK inhibitor | Selleck | S1049 | 10 µM |
| DAPT | Gamma-secretase (Notch) inhibitor | Abcam | ab120633 | 50 µM |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36739.034
