Telophase correction refines division orientation in stratified epithelia
- The University of North Carolina, United States
- The University of North Carolina School of Dentistry, United States
- University of California, San Francisco, United States
Figures
Figure 1 with 5 supplements
Telophase reorientation corrects oblique anaphase orientations.
(A) Sagittal sections from E16.5 embryos showing mitotic basal cells at indicated stages. Yellow arrows indicate division axis relative to basement membrane (dashed white line). Apical LGN (red) is …
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Figure 1—source data 1
Original measurements used to generate panels B, C, F, G, H.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Telophase reorientation corrects oblique anaphase orientations.
(A) Cumulative frequency histograms of division angles from fixed sagittal sections organized by mouse background strain: CD1 (left), C57 (middle), and 129 (right) all show randomized division …
Figure 1—video 1
Planar anaphase orientation is fixed.
Z-projection of Krt14H2B-GFP; Rosa26mT/mG epidermal explant taken from an E16.5 embryo imaged in 5 min intervals. The mitotic cell (white dashed outline) orients its division parallel to the …
Figure 1—video 2
Perpendicular anaphase orientation is fixed.
Z-projection of Krt14H2B-GFP; Rosa26mT/mG epidermal explants taken from E16.5 embryos imaged in 5 min intervals. The mitotic cell (white dashed outline) orients its division orthogonal to the …
Figure 1—video 3
Oblique anaphase orientations undergo planar telophase reorientation.
Z-projection of Krt14H2B-GFP; Rosa26mT/mG epidermal explants taken from E16.5 embryos imaged in 5 min intervals. The mitotic cell (white dashed outline) initiates anaphase at an oblique (30–60°) …
Figure 1—video 4
Oblique anaphase divisions display perpendicular correction.
Z-projection of Krt14H2B-GFP; Rosa26mT/mG epidermal explants taken from E16.5 embryos imaged in 5 min intervals. The mitotic cell (white dashed outline) initiates anaphase at an oblique (30–60°) …
Figure 2
LGN mediates perpendicular but not planar telophase correction.
(A) Schematic of modified experimental protocol of live imaging of epidermal explants (see Figure 1D) incorporating lentiviral shRNA transduction to generate mosaic knockdown tissue. Transduced/knock…
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Figure 2—source data 1
Original measurements used to generate panels C, D, E.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig2-data1-v2.xlsx
Figure 3 with 1 supplement
Maintenance of basal contact correlates with planar-directed telophase correction.
(A) (top) z-projection stills from a movie of a mitotic cell as it enters anaphase (t = 0) through 60 min post-anaphase onset, depicting planar telophase correction. Epidermal-dermal boundary shown …
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Figure 3—source data 1
Original measurements used to generate panels B, C, D, F, G.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
A basal endfoot mediates planar telophase correction.
(A) On the x-axis, divisions are grouped by orientation at anaphase onset (φ, planar = 0–30°; oblique = 30–60°; perpendicular = 60–90°) and Gpsm21617 status. The y-axis depicts the proportion of …
Figure 4 with 1 supplement
Vinculin and afadin regulate α-E-catenin conformation and AJ linkage to the actin cytoskeleton.
(A) Stable primary murine keratinocytes cell lines grown in the presence of high (1.5 mM) Ca2+ for 8h form nascent cell-cell adhesions, stained for total α-E-catenin (green); open, "tensile" …
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Figure 4—source data 1
Original measurements used to generate panels B, C, E, G, H, I.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
The α-E-catenin/vinculin/afadin complex demonstrates reciprocal regulation to form mature adherens junctions in vitro.
(A) Quantification of α18 (left) and α-E-catenin (right) fluorescence intensity in 8h Ca2+-shift assays in Scramble and Vcl shRNA expressing primary keratinocytes. (B) Time-course of junction …
Figure 5 with 1 supplement
The α-E-catenin/vinculin/afadin pathway is required for normal division orientation.
(A) Immunofluorescent images taken from E16.5 sagittal sections of wild-type littermate controls (left) or embryos transduced with Ctnna1912 H2B-RFP (right). Epidermal junctional α-E-catenin (green) …
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Figure 5—source data 1
Original measurements used to generate panels D, E, F, G, H, I.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
AJ loss-of-function mutants display errors in division orientation.
(A) Telophase cells marked by Survivin (green), from E16.5 wild-type littermates (left, top), Afdn2711 RFP- (top right), Afdn2711 RFP+ (bottom right), and Vcl2803 RFP+ knockdowns (bottom left). (B) …
Figure 6 with 4 supplements
AJ mutants fail at both planar and perpendicular telophase correction.
(A) Movie stills of Ctnna1912 RFP+ mitotic cell, annotated as in Figure 3A. While the presence of basal contact (open arrowhead) would predict planar correction, this division remains oblique when …
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Figure 6—source data 1
Original measurements used to generate panels B, C, D, E, G, H, I, J.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
AJ loss-of-function mutants display errors in division orientation.
(A) Time-course stills from Ctnna1912 H2B-RFP (red) and mGFP (gray) live imaging experiment demonstrating failed telophase correction. (B) Telophase reorientation (θ-φ) quantification in Ctnna1912 …
Figure 6—video 1
Persistent oblique division in Ctnna1912 knockdown mitotic basal cell.
Z-projection of RFP+ basal cell from Krt14Cre; Rosa26mT/mG epidermal explants transduced with Ctnna1912 shRNA, taken from E16.5 embryos imaged in 5 min intervals. The mitotic cell (white dashed …
Figure 6—video 2
Persistent oblique division in Vcl3466 knockdown mitotic basal cell.
Z-projection of RFP+ basal cell from Krt14Cre; Rosa26mT/mG epidermal explants transduced with Vcl3346 shRNA, taken from E16.5 embryos imaged in 5 min intervals. The mitotic cell (white dashed …
Figure 6—video 3
Persistent oblique division in Afdn2711 knockdown mitotic basal cell.
Z-projection of RFP+ basal cell from Krt14Cre; Rosa26mT/mG epidermal explants transduced with Afdn2711 shRNA, taken from E16.5 embryos imaged in 5 min intervals. The mitotic cell (white dashed …
Figure 7 with 2 supplements
AJ mutants alter division orientation via LGN-independent mechanisms.
(A) Immunostaining for LGN (green) in E16.5 Afdnfl/fl and Afdn-cKO epidermis. LGN localizes at the apical cortex during mitosis regardless of afadin loss. (B) Quantification of LGN radial …
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Figure 7—source data 1
Original measurements used to generate panels B, C, D, F, H, I.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Afdn loss-of-function does not affect functional apicobasal polarity or downstream components of spindle orientation.
(A) Immunostaining of E16.5 interphase cell with Par3 (red). (B) Quantification of Par3 radial fluorescent intensity. Apical accumulation is reduced by afadin knockout via Krt14Cre (Afdn cKO) or …
Figure 7—figure supplement 2
AJ components alter division orientation in an LGN-independent manner.
(A) Cumulative frequency distribution of spindle orientation at metaphase, and division orientation at telophase, in Afdn cKO and Afdnfl/fl control littermates. WT spindle orientation (gray line) is …
Figure 8 with 1 supplement
Planar telophase correction limits precocious and sustained differentiation and biases clones towards symmetric cell divisions.
(A) (top) z-projection stills from a movie of an E14.5 mitotic cell, annotated as in Figure 1E. (B) Cumulative frequency distribution of division angles from live imaging experiments of E14.5 …
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Figure 8—source data 1
Original measurements used to generate panels B, C, F, H , I.
- https://cdn.elifesciences.org/articles/49249/elife-49249-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Failed telophase correction induces precious, sustained hyperstratification without impacting proliferation or delamination.
(A) Quantification of K10+ suprabasal proportion in E14.5 wild-type and Afdn2711 mutants. (B) Mitotic index quantification at E14.5 and E16.5 in wild-type and Afdn2711 mutants. (C) …
Figure 9
Two-step model of division orientation.
Model of OCD in the embryonic epidermis. During stratification, LGN (green) is recruited to the apical cortex in ~50% of mitoses, promoting perpendicular divisions. For OCDs with perpendicular and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or ref. | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | mTmG | Jackson Labs | IMSR Cat# JAX:007576, RRID:IMSR_JAX:007576 | |
| Strain, strain background (Mus musculus) | Krt14Cre | (Dassule et al., 2000) | ||
| Strain, strain background (Mus musculus) | Krt14H2B-GFP | (Tumbar et al., 2004) | ||
| Strain, strain background (Mus musculus) | CD1 | Charles River | IMSR Cat# CRL:022, RRID:IMSR_CRL:022 | New females integrated into colony every ~ 5 years to maintain as outbred strain. |
| Strain, strain background (Mus musculus) | LSL-Confetti | Jackson Labs | IMSR Cat# JAX:013731, RRID:IMSR_JAX:013731 | |
| Strain, strain background (Mus musculus) | Krt14CreER | Jackson Labs | IMSR Cat# JAX:005107, RRID:IMSR_JAX:005107 | |
| Strain, strain background (Mus musculus) | Afdnfl/fl | (Beaudoin et al., 2012) | ||
| Antibody | Guinea-pig polyclonal anti-LGN | (Williams et al., 2011) | (1:500 dilution) | |
| Antibody | Rabbit monoclonal anti-survivin (71G4B7) | Cell Signaling | Cat# 2808, RRID:AB_2063948 | (1:1000 dilution) |
| Antibody | Chicken polyclonal anti-GFP | Abcam | Cat# ab13970, RRID:AB_300798 | (1:2000 dilution) |
| Antibody | Rat monoclonal anti-mCherry (16D7) | Thermo Fisher Scientific | Cat# M11217, RRID:AB_2536611 | (1:2000 dilution) |
| Antibody | Rabbit polyclonal anti-LGN | Millipore | Cat# ABT174 | (1:2000 dilution) |
| Antibody | Rat monoclonal anti-β4 integrin | Thermo-Fisher | BD Biosciences Cat# 553745, RRID:AB_395027 | (1:1000 dilution) |
| Antibody | Rabbit polyclonal anti-Gai3 | EMD Millipore | Millipore Cat# 371726–50 UL, RRID:AB_211897 | (1:500 dilution) |
| Antibody | Goat polyclonal anti-dynactin | Abcam | Abcam Cat# ab11806, RRID:AB_298590 | (1:500 dilution) |
| Antibody | Mouse (IgM) monoclonal anti-NuMA | BD Transduction Labs | BD Biosciences Cat# 610562, RRID:AB_397914 | (1:300 dilution; use Jackson labs Donkey anti-IgM Cy3 conjugated secondary) |
| Antibody | Rabbit polyclonal anti-histone H3, phospho (Ser10) | Millipore | Cat# 06–570, RRID:AB_310177 | (1:1000 dilution) |
| Antibody | Rat monoclonal anti-a-tubulin | EMD Millipore | Millipore Cat# CBL270, RRID:AB_93477 | (1:500 dilution) |
| Antibody | Rabbit polyclonal anti-pericentrin | Covance | Covance Cat# PRB-432C-200, RRID:AB_291635 | (1:500 dilution) |
| Antibody | Rabbit polyclonal anti-cytokeratin 10 (Poly19054) | Bio-Legend | Cat# 905404, RRID:AB_2616955 | (1:1000 dilution) |
| Antibody | Rabbit polyclonal anti-Par3 | EMD Millipore | Millipore Cat# 07–330, RRID:AB_2101325 | (1:500 dilution) |
| Antibody | Rat monoclonal anti-E-cadherin (ECCD-2) | Life Technologies | Thermo Fisher Scientific Cat# 13–1900, RRID:AB_2533005 | (1:1000 dilution) |
| Antibody | Goat polyclonal anti-E-cadherin | R and D systems | R and D Systems Cat# AF748, RRID:AB_355568 | (1:1000 dilution) |
| Antibody | Rabbit polyclonal anti-a-E-catenin | Invitrogen | Thermo Fisher Scientific Cat# 71–1200, RRID:AB_2533974 | (1:300 dilution; tissue sections) |
| Antibody | Rat monoclonal anti-a18 | (Yonemura et al., 2010) | (1:10000 dilution; tissue sections) (1:2000 dilution; wholemounts) | |
| Antibody | Rabbit polyclonal anti-vinculin | Gift from Dr. Keith Burridge | (1:1000 dilution) | |
| Antibody | Mouse monoclonal anti-vinculin | Sigma Aldrich | Sigma-Aldrich Cat# V9131, RRID:AB_477629 | (1:500 dilution) |
| Antibody | Rabbit polyclonal anti-afadin | Sigma Aldrich | Sigma-Aldrich Cat# A0224, RRID:AB_257871 | (1:500 dilution) |
| Antibody | Mouse monoclonal anti-pMLC2 (Ser19) | Cell Signaling | Cell Signaling Technology Cat# 3675, RRID:AB_2250969 | (1:500 dilution) |
| Chemical compound | Phalloidin AF-647 conjugated | Life Technologies | Thermo Fisher Scientific Cat# A22287, RRID:AB_2620155 | (1:500 dilution) |
| Cell line(s) (Mus musculus) | Primary keratinocytes | This publication. | Isolated as described in Materials and methods section. | |
| Chemical compound | Tamoxifen | Sigma-Aldrich | Cat# T5648 | |
| Software | FIJI | Source:https://imagej.net/Fiji Reference: doi:10.1038/nmeth.2019 |
