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Anisotropic stress orients remodelling of mammalian limb bud ectoderm

Abstract

The physical forces that drive morphogenesis are not well characterized in vivo, especially among vertebrates. In the early limb bud, dorsal and ventral ectoderm converge to form the apical ectodermal ridge (AER), although the underlying mechanisms are unclear. By live imaging mouse embryos, we show that prospective AER progenitors intercalate at the dorsoventral boundary and that ectoderm remodels by concomitant cell division and neighbour exchange. Mesodermal expansion and ectodermal tension together generate a dorsoventrally biased stress pattern that orients ectodermal remodelling. Polarized distribution of cortical actin reflects this stress pattern in a β-catenin- and Fgfr2-dependent manner. Intercalation of AER progenitors generates a tensile gradient that reorients resolution of multicellular rosettes on adjacent surfaces, a process facilitated by β-catenin-dependent attachment of cortex to membrane. Therefore, feedback between tissue stress pattern and cell intercalations remodels mammalian ectoderm.

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Figure 1: Cell topology and intercalation of AER progenitors.
Figure 2: Planar polarity of pre-AER ectodermal cells.
Figure 3: Mesodermal growth anisotropically stresses ectoderm during limb initiation.
Figure 4: Cell division precipitates cell neighbour exchange and oriented remodelling of pre-AER 18–22 som. ectoderm.
Figure 5: Tension augments the DV stress pattern and orients rosettes in pre-AER ectoderm.
Figure 6: Ectodermal β-catenin is required to polarize actin and orient cell behaviour in response to stress.
Figure 7: Direct and indirect functions of β-catenin and Fgfr2.

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Acknowledgements

We thank J. Zallen, Y. Bellaïche, C-P. Heisenberg, J. Gros and C-c. Hui for critical review of the manuscript. This work was financially supported by a March of Dimes Birth Defects Foundation Grant 1-FY10-366, and a Canadian Institutes of Health Research Grant MOP-126115 (S.H.).

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Authors and Affiliations

Authors

Contributions

K.L., H.T. and S.H. designed the experiments. H.T., H.L., C.S. and Y.S. performed and analysed AFM experiments. J.W. performed FEM. K.L., H.T., K.S. and S.H. performed and analysed live imaging and immunofluorescence experiments. N.S. performed cell neighbour analysis. K.L., K.S., S.L., J.T.A.B. and I.S. performed and analysed injection experiments. M.D.W. and R.M.H. performed OPT experiments. D.L. analysed cell division plane and rosette remodelling data. S.D. performed measurements of lateral plate ectoderm. S.H. and B.C. performed zebrafish experiments. T.W. provided Crect mice. A-K.H. provided CAG::myr–Venus and Tcf/Lef::H2B–Venus mice. K.L. and R.F-G. performed and analysed laser ablation experiments. R.F-G. provided SIESTA software and MATLAB scripts. S.H. wrote the manuscript.

Corresponding author

Correspondence to Sevan Hopyan.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1

(a) Confocal projection of 20s limb bud visualising pHH3 (red), Tcf/Lef::H2B-Venus (green), and DAPI (blue). Shown are multiple images stitched together. (b) xy sections of limb field in embryos treated with water control (left) or 100 μM NSC23766 (right) visualising actin (red) and DAPI (blue). (ce) xy sections at the DV boundary of initiating limb bud ectoderm visualising Myosin IIB (c), Myosin IIA (d), phospho-Myosin Light Chain-1 (e) (green) and DAPI (blue). (f) Composite confocal projection of 17-somite stage lateral plate visualising DAPI used to measure dimensions of pre-limb lateral plate ectoderm for simulation. (g) Scanning electron micrograph of a custom 10 μm spherical AFM probe. (h) Two-paired Prony series fitting of limb bud viscoelastic properties. (i) Illustration of finite element model of initiating limb bud. Red arrow indicates direction of mesodermal growth, yellow arrowhead indicates fixed support. (j) Percentage of pHH3-positive cells relative to total cells in control versus PEG-injected embryos (n = 3 18–21 som. embryos; p = 0.9871 (Student’s t-test)). (k) xy section away from DV boundary of pre-AER 23 som. limb bud ectoderm visualising Myosin IIB (green) and Actin (red). (l) xy section of the apical plane of 19 som. limb bud visualising actin (red) and myosin (green). (m) Model of 22 som. pre-AER limb bud. Black arrow indicates mesodermal growth, yellow arrowhead indicates fixed support. (n) Model of AER progenitor intercalation at DV boundary. Black arrows indicate direction of ectodermal tension (modelled as pulling force). (o,p) Stresses are linearly proportional to the pulling force applied at the DV midline; (o) 2 pN, (p) 1 pN. (q,s,u) Distance between two vertices attached to cut interface for (q) distal AP versus proximal AP, p = 0.0142, (s) lateral plate AP versus lateral plate DV, p = 0.1381, (u) intact AP versus post-cut AP interfaces, p = 0.0475. (r,t,v) Peak retraction velocities of ablated (r) distal AP versus proximal AP, (t) lateral plate AP versus lateral plate DV, (v) AP versus post-cut AP interfaces interfaces ((r) p = 0.0023, (t) p = 0.1544, (v) p = 0.0488; (qv) n 15 ablations for each of 4 embryos). ((q,r) Student’s t-test with Holm’s correction). Scale bars indicate 5 μm (g), 10 μm (ce,k,l), 20 μm (a,b), 100 μm (f). Error bars indicate SEM.

Supplementary Figure 2

(a) Confocal xy section of initiating limb bud ectoderm visualising Frizzled6 (green) and DAPI (blue). (b) Confocal xy section of initiating limb bud ectoderm visualising Disheveled3 (green) and DAPI (blue). (c,d) Confocal xy section of initiating limb bud ectoderm visualizing Par1 (green) and DAPI (blue) (c) and Par3 (green) and E-cadherin (red) (d). In all images, scale bar indicates 20 μm; anterior is left and ventral is up. (e) Wildtype and (f) Maternal zygotic (MZ) Vangl2−/− 56 hpf zebrafish pectoral fin bud visualising H2B-GFP nuclei (green). (g) Wildtype and aPKC−/− 34 hpf zebrafish embryos. (h) Wildtype and (i) aPKC−/− zebrafish pectoral fin bud. (j) Wildtype and Wnt5b−/− 96 hfp zebrafish embryos. (k) Wildtype and Wnt5b−/− zebrafish pectoral fin bud. Scale bars indicate (20 μm (a,b), 10 μm (c,d), 100 μm (e,f,h,i,k), 250 μm (g,j).

Supplementary Figure 3

(a) Confocal z sections and xy sections of ZEG;Crect limb buds at 17 som. stage (E9.0) (top) and 25 som. stage (E9.5) (bottom) visualising GFP (green) and DAPI (blue). (b) Cartilage (Alcian Blue) and bone (Alizarian Red) staining of E18.5 WT and βcatf/f;Crect mutant forelimb (top panels) and hindlimb (bottom panels) skeletons. (c) Confocal z sections (top panels) and xy sections (bottom panels) of control βcatf/f and βcatf/f ;Crect mutant limb bud ectoderm visualising β-catenin (red) and DAPI (blue). (d) Confocal xy sections of control βcatf/f and βcatf/f;Crect mutant limb bud ectoderm expressing Tcf/Lef::H2B-Venus. Shown are GFP (green) and DAPI (blue). (e) Histogram representing percentage GFP-positive nuclei in control βcatf/f and βcatf/f;Crect mutant limb bud ectodermal cells expressing Tcf/Lef::H2B-Venus (p = 1.7 × 10−5 (Student’s t-test); n = 3 19–25 som. embryos). (f) Confocal projection of control βcatf/f and βcatf/f ;Crect mutant limb bud ectoderm visualizing phospho-histone H3 (red), Caspase3 (green), and DAPI (blue). (g) Histogram representing percentage of pHH3-positive cells in control βcatf/f and βcatf/f;Crect mutant limb bud ectodermal cells (p = 0.8956). (h) Histogram representing number of Caspase3-positive cells in control βcatf/f and βcatf/f ;Crect mutant limb bud ectodermal cells (p = 0.8413). (i) Histogram representing percentage of pHH3-positive cells in control βcatf/f and βcatf/f ;Crect mutant limb bud mesodermal cells (p = 0.9671). (j) Histogram representing number of Caspase3-positive cells in control βcatf/f and βcatf/f ;Crect mutant limb bud mesodermal cells (p = 0.7691). (For gj n = 3 19–21 som. embryos per condition, Student’s t-test) (k) Fgf10 expression in control βcatf/f and βcatf/f;Crect mutant limb buds at 22 som. (E9.25). (l) Fgf10 expression in control βcatf/f and βcatf/f ;Crect mutant limb buds at E10.5. Scale bar indicates 10 μm (a,c,d), 100 μm (f,k,l), 1 mm (b). Error bars indicate SEM. Asterisk indicates P < 0.05.

Supplementary Figure 4

(a) Length-width ratio of control βcatf/f and βcatf/f; Crect mutant limb bud ectodermal cells. (n = 3 19–21 som. embryos per condition; p = 0.0324 (Student’s t-test)) (b) Angle of cell orientation in control βcatf/f and βcatf/f;Crect mutant limb bud ectoderm. Histogram shows distribution of cells grouped into 6 bins of 15° (n = 3 19–21 som. embryos per condition; p = 0.0332 (75–90° bin) (Student’s t-test)). (c) Time series of initiating limb bud ectoderm cells in WT and βcatf/f; Crect mutant embryos expressing R26R::Venus-Actin. (d, e) Representative force curves for control βcatf/f (d) and βcatf/f; Crect (e) mutant AFM experiments. (f) Confocal XY section of control βcatf/f and βcatf/f;Crect mutant limb bud ectoderm visualizing E-cadherin (green) and DAPI (blue). (g) Axin2 expression in embryos that were treated in 6-hour roller culture with DMSO control (top) or IWR-1 (bottom). (h) phospho-Dishevelled 2 (p-Dvl2) is diminished in embryos treated with IWP-2. Uncropped blots in Supplementary Fig. 6. Scale bar indicates 20 μm (c, f), 200 μm (g); anterior is left and ventral is up. Error bars indicate SEM. Asterisk indicates P < 0.05.

Supplementary Figure 5

(a) Confocal Z sections (top panels) and XY sections (bottom panels) of control Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising βcatenin (red) and DAPI (blue). (b) Histogram representing percentage Venus-positive nuclei from Fgfr2f/f and Fgfr2f/f ;Crect mutant limb bud ectodermal cells expressing Tcf/Lef::H2B-Venus (p = 0.0014 (Student’s t-test); WT n = 3, mutant n = 4 20-25 som. embryos). (c) Confocal XY section of Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising phospho-ERK (green) and DAPI (blue). (d) Cartilage (Alcian Blue) and bone (Alizarian Red) staining of E18.5 Fgfr2f/f;Crect mutant forelimb (top) and hindlimb (bottom) skeletons. (e) Histogram representing percentage of pHH3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectodermal cells (p = 0.8731). (f) Histogram representing number of Caspase3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectodermal cells (p = 0.6567). (g) Histogram representing percentage of pHH3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud mesodermal cells (p = 0.9973). (h) Histogram representing number of Caspase3-positive cells in Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud mesodermal cells (p = 0.6032). (For e-h, n = 3 19-21 som. embryos per condition, Student’s t-test) (i) Confocal XY section of Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising E-cadherin (red) and DAPI (blue). (j)Fgf10 expression in Fgfr2f/f; Crect mutant limb buds at E9.5 (left) and E10.5 (right). (k) Length-width ratio of WT and Fgfr2f/f;Crect mutant limb bud ectodermal cells (n = e 19-21 som. embryos per condition; p = 0.0189 (Student’s t-test)). (l) Angle of cell orientation in WT and Fgfr2f/f;Crect mutant limb bud ectoderm. Histogram shows distribution of cells grouped into 6 bins of 15° (n = 3 19-21 som. embryos per condition; p = 0.0412 (75-90° bin) (Student’s t-test)). (m) Confocal XY projection of E10.0 (32 somite stage) Fgfr2f/f;Crect mutant forelimb bud expressing Tcf/Lef::H2B-Venus. (n) Confocal images of Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising CD44 (green) and DAPI (blue). (o) Confocal XY sections Fgfr2f/f and Fgfr2f/f;Crect mutant limb bud ectoderm visualising phospho-βcatenin (red) and DAPI (blue). Scale bar indicates 20 μm (a,c,o), 10 μm (i,n), 50 μm (m), 100 μm (j), 1 mm (d). Error bars indicate SEM. Asterisk indicate p < 0.05.

Supplementary Figure 6 Uncropped Western blot showing reduced levels of p-Dvl2 in IWP-2 treated embryos compared with control DMSO treatment.

(Right) Re-blot of E-cadherin as loading control.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1769 kb)

DV movement of Tcf/Lef::H2B-Venus-positive cells in ventral ectoderm of a 23 som. stage limb bud.

Shown is a lateral view of the posterior half of the limb bud. Anterior is to the left, dorsal is down. Time course is 165 min, shown in 5 fps. Evidence of convergence by intercalation can be seen, as can DV-biased cell division planes. Over a longer time period (nearly 1 day), these cells will progressively crowd just ventral to the DV midline to form the AER. (MOV 184 kb)

Tcf/Lef::H2B-Venus-positive cells at 23 som. stage intercalate just ventral to the dorsoventral boundary (in our estimation).

Anterior is to the left; dorsal is down. Time course is 165 min, shown in 5 fps. (MOV 108 kb)

Low magnifcation time lapse movie demonstrating interdigitation of Tcf/Lef::H2B-Venus-positive cells (precipitated by division of the light blue cell) near the DV midline of a 20 som. stage limb bud.

Anterior is to the left, dorsal is downward, and ventral is upward. Time course is 355 min, shown in 5 fps. (MOV 728 kb)

Wild type 20 som. myr-Venus transgenic embryo with a dorsally biased view of anterior half of limb field (left half) and lateral plate ectoderm (right half).

Polygonal, anisotropic cell shapes dominate, and multiple examples of cell division and cell neighbour exchange are apparent. Anterior is to the right and dorsal is downward with the DV midline 2/3 up from the base of the visible field. (MOV 410 kb)

NSC23766 treatment of 22 som. stage myr-Venus embryo demonstrates transition from elongated cell shapes to more isotropic shapes.

Diminished cell neighbour exchange is also apparent. Time course is 75 min, shown in 5 fps. (MOV 189 kb)

Protrusive membrane activity of ectodermal cells in 22 som. limb bud expressing myr-Venus in a mosaic fashion.

Yellow arrowhead indicates membrane protrusion. To the left, a relatively dark cell intercalates among two bright cells. Anterior is to the left and dorsal is downward. Time course is 180 min, shown in 5 fps. (MOV 538 kb)

Relaxation of principal stress vectors during transient 300 s viscoelastic simulation for 17 som. stage limb bud initiation based on mesodermal growth.

All principal stresses are relaxed until the material property reaches the asymptote. Same relaxation applies to the stress field (not shown). (MOV 142 kb)

Daughter cells (blue) in mouse embryonic ectoderm (20 som.) frequently sever the interface between them immediately following division and precipitate neighbour exchange.

Anterior is left; dorsal, down. Time course is 149 min, shown in 5 fps. (MOV 117 kb)

A daughter cell (blue) intercalates between neighbouring cells following division (20 som.).

Anterior is left; dorsal, down. Time course is 149 min, shown in 5 fps. (MOV 853 kb)

A daughter cell (pink) precipitates rosette formation by intercalating between cells and contributes to the central apex (20 som.).

Anterior is left; dorsal, down. Time course is 83 min, shown in 5 fps. (MOV 105 kb)

A large multicellular rosette resolves into two rows of cells by dissolving its central apex and generating new intercellular junctions (20 som.).

Anterior is left; dorsal, down. Time course is 123 min, shown in 5 fps. (MOV 248 kb)

41556_2015_BFncb3156_MOESM54_ESM.mov

Relaxation of the stress field during transient 300 s viscoelastic simulation for 22 som. stage pre-AER limb bud due to mesodermal growth. (MOV 48 kb)

41556_2015_BFncb3156_MOESM55_ESM.mov

Relaxation of the stress field during transient 300 s viscoelastic simulation for 22 som. stage pre-AER limb bud due to an ectodermal pulling force at the DV midline. (MOV 75 kb)

Precise laser ablation of a single AP interface (that is parallel to the DV axis) results in relatively fast initial recoil of adjacent apices along the DV axis (21 som.).

Anterior is left. Time course is 132 sec, shown in 5 fps. (MOV 402 kb)

Precise laser ablation of a single DV interface (that is parallel to the AP axis) results in relatively slow initial recoil of adjacent apices along the AP axis (21 som.).

Dorsal is left. Time course is 132 sec, shown in 5 fps. (MOV 400 kb)

Laser ablation of multiple interfaces between the coloured rosette and the prospective AER (to the right) results in resolution of the rosette along the AP axis (up/down) rather than toward the AER (21 som.).

Anterior is up; dorsal is left. Time course is 192 sec, shown in 5 fps. (MOV 417 kb)

βcatf/f;Crect mutant daughter cells maintain a long interface following division. Anterior is left; dorsal, down (20 som.).

Time course is 135 min, shown in 5 fps. (MOV 358 kb)

βcatf/f;Crect mutant rosette resolves with minimal angular change of its long axis.

Anterior is left; dorsal, down (20 som.). Time course is 140 min, shown in 5 fps. (MOV 146 kb)

Live R26R::Venus-Actin reporter in vivo shows oscillatory cortical contractions in wild type embryo.

Anterior is left; dorsal, down. Time course is 120 min, shown in 5 fps. (MOV 271 kb)

Live R26R::Venus-Actin reporter in vivo shows diminished amplitude and rate of change of oscillatory cortical contractions in βcatf/f;Crect mutant embryo (21 som.).

Anterior is left; dorsal, down. Time course is 120 min, shown in 5 fps. (MOV 168 kb)

Tcf/Lef::H2B-Venus positive cells in Fgfr2f/f;Crect mutant limb bud ectoderm move along the DV axis, albeit less efficiently than in WT (21 som.).

Anterior is left; dorsal, down. Time course is 120 min, shown in 5 fps. (MOV 681 kb)

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Lau, K., Tao, H., Liu, H. et al. Anisotropic stress orients remodelling of mammalian limb bud ectoderm. Nat Cell Biol 17, 569–579 (2015). https://doi.org/10.1038/ncb3156

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