Identification of the corneo-limbal boundary in the WT and <i>Pax6^+/−</i> mouse ocular surface epithelium.

<p>CD31 (red) and keratin 19 (green) double immunofluorescence staining in the ocular surface of (<b>A, B</b>) WT and (<b>C, D</b>) <i>Pax6^+/−</i> mice. Images (B) and (D) are higher magnifications of the areas outlined in (A) and (C) respectively. Both CD31-positive blood vessels and keratin 19-positive epithelial cells are restricted to the conjunctiva and limbus in WT eyes but they both extend into the corneal epithelium in <i>Pax6^+/−</i> mice. Abbreviations: L: Limbus; Co: Cornea; Cj: Conjunctiva. Red immunofluorescence: CD31; green: keratin 19; blue: TO-PRO3 iodide nuclear counterstain. Scale bars are 1 mm (A,C) and 0.1 mm (B,D).</p

Association of BrdU label-retaining cells with blood vessels in <i>Pax6^+/−</i> corneas.

<p>Double immunofluorescent detection of BrdU (yellow) and CD31-positive blood vessels (red) in WT and <i>Pax6^+/−</i> mice. (<b>A</b>) Flattened WT cornea showing BrdU-positive, label-retaining cells (LRCs) and CD31-positive blood vessels in the limbus. (<b>B</b>) Flattened <i>Pax6^+/−</i> cornea demonstrating CD31-positive blood vessels extending from the limbus into the cornea and BrdU LRCs in the cornea. (<b>C, D</b>) Montages of flattened <i>Pax6^+/−</i> corneas showing CD31-positive blood vessels and BrdU LRCs even in the central cornea. Arrows show blood vessels with adjacent BrdU-positive cells (LRCs). For demonstration purposes the counterstain channel was deactivated. Abbreviations: L: Limbus; Co: Cornea; Yellow immunofluorescence: BrdU. Red immunofluorescence: CD31; Blue: TO-PRO3 iodide counterstain. Scale bars are 100 µm.</p

Distributions of BrdU-positive cells in different regions of WT and <i>Pax6^+/−</i> corneal epithelia.

<p>The mean (±95% CI) BrdU labelling indices are shown separately for the peripheral (P), intermediate (I) and central (C) regions of the cornea for chase periods of 4 hours (A, D & G), 1 day (B, E & H) and 3 days (C, F & I). (<b>A–C</b>) BrdU basal labelling index (BrdU positive basal cells as a percentage of total basal cells). (<b>D–F</b>) BrdU suprabasal labelling index (BrdU positive suprabasal cells as a percentage of total suprabasal cells). (<b>G–I</b>) Adjusted suprabasal BrdU labelling index (BrdU positive suprabasal cells as a percentage of total basal cells). In most cases statistical comparisons were made by 2-way analyses of variance (ANOVAs) of log-transformed data followed by pairwise Bonferroni post-hoc tests and separate linear regression analyses for WT and <i>Pax6^+/−</i> genotypes. Non-parametric Kruskal-Wallis (KW) tests followed by pairwise Dunn’s multiple comparison tests were used for D and G because there were many zero values and the log-transformed data were not normally distributed. Significant differences for the 2-way ANOVAs and linear regressions (or Kruskal-Wallis tests) are shown in each panel. The only two significant pairwise post-hoc tests between genotypes are shown by asterisks over the two bars compared (central region in E and H). The post-hoc tests between regions are not shown on the histograms. For WT corneas, only the post-hoc test between regions P vs. C in B was significant (<i>P</i><0.05). For <i>Pax6^+/−</i> corneas, post-hoc tests between pairs of regions were significant for P vs. C in A (<i>P</i><0.05), B (<i>P</i><0.05), E (<i>P</i><0.001) and H (<i>P</i><0.001), and for P vs. I in B (<i>P</i><0.05) and H (<i>P</i><0.05). Abbreviations: LI, labelling index; WT, wild-type; NS, not significant; *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; ****<i>P</i><0.0001. 6–12 eyes per group as shown in Fig. 2E–G.</p

Percentage of all the labelled cells that are in the suprabasal layers 24 hours after BrdU injection.

<p><i>Abbreviations:</i> ASLI = adjusted suprabasal labelling index; BLI = basal labelling index; SLI = suprabasal labelling index. <i>P</i>-value is for <i>t</i>-test with Welch’s correction for unequal variances.</p

Acute BrdU labelling of WT and <i>Pax6^+/−</i> corneal epithelia.

<p>(<b>A, B</b>) BrdU immunohistochemistry 4 hours after BrdU injection of (A) WT and (B) heterozygous <i>Pax6^+/−</i> mice. (<b>C, D)</b> BrdU immunohistochemistry 24 hours after BrdU injection of (C) WT and (D) <i>Pax6^+/−</i> mice. BrdU-positive nuclei in the corneal epithelium appear dark. (<b>E–G</b>) The mean (±95% CI) BrdU labelling indices for mid-sections are shown for chase periods of 4 hours (4 h) to 14 days (14 d). (<b>E</b>) BrdU basal labelling index (BrdU positive basal cells as a percentage of total basal cells). (<b>F</b>) BrdU suprabasal labelling index (BrdU positive suprabasal cells as a percentage of total suprabasal cells). (<b>G</b>) Adjusted suprabasal BrdU labelling index (BrdU positive suprabasal cells as a percentage of total basal cells). Results for 2-way analyses of variance (ANOVAs) for log transformed data are shown. Where genotype differences were significant overall, pairwise comparisons were made between genotypes for each time point using Bonferroni post-hoc tests (significant differences are shown by asterisks). Separate 1-way ANOVAs and Bonferroni post-hoc tests for each genotype showed that the frequencies of BrdU-positive cells increased in the suprabasal layers from 4 h to 3 days (<i>P</i><0.001 for both WT and <i>Pax6^+/−</i> in E & F) and then declined from 3 to 14 days (<i>P</i><0.001 for both WT and <i>Pax6^+/−</i> in E & F). Abbreviations: LI, labelling index; NS, not significant; **<i>P</i><0.01; ***<i>P</i><0.001; ****<i>P</i><0.0001. 6–12 eyes per group as shown within or above the bars.</p

Alternative hypotheses to explain the reduction in corrected stripe numbers in <i>Pax6^+/−</i> mosaic corneas.

<p><b>(A–D) Normal development:</b> The developing mosaic surface ectoderm (A) comprises two genetically marked cell populations (shown as blue and yellow hexagons) and the future corneal epithelium is shown as a disk with the limbus at its periphery. LESCs are specified (shown as stars in B) from a pool of cells in the surface ectoderm and become active postnatally (C). Centripetal movement of daughter TACs forms radial stripe patterns in the adult corneal epithelium (D). <b>(E–H) Hypothesis 1– reduced </b><b><i>Pax6^+/−</i></b><b> LESC numbers:</b> If fewer LESCs are specified in the <i>Pax6^+/−</i> ocular surface (F) then individual clones of corneal epithelial cells produced by each LESC will colonise a larger sector of the cornea, forming fewer, wider stripes (H). A similar result is expected if normal numbers of LESCs are specified but some fail to survive. <b>(I–L) Hypothesis 2– reduced cell mixing during </b><b><i>Pax6^+/−</i></b><b> development:</b> If there is less mixing of the two genetically marked cell populations in the <i>Pax6^+/−</i> mosaic surface ectoderm during development, cells will be grouped into larger coherent clones to form a coarse-grained mosaic pattern (I). There is a higher probability that two adjacent stem cells belong to the same population (e.g. adjacent blue stem cells in J) so wider stripes are produced. Although, in this case, the distribution of LESCs around the circumference will be non-random, the distribution of LESC clones should still be random. The corrected stripe number described in the text is expected to be proportional to the number of LESC clones but the number of LESCs per clone may vary as shown (e.g. compare H and L).</p

Identification of label-retaining cells in the limbal region of the WT and <i>Pax6^+/−</i> ocular surface epithelium.

<p>(<b>A</b>) Diagram showing the 8 radial cuts in a corneal button, shaded to represent the cornea (lightest), limbus (intermediate) and conjunctiva (darkest). The rectangles show the location of the sampling boxes (one per sector but not to scale). (<b>B</b>) Rectangular 340×200 µm sampling box (yellow outline), used to count LRCs, superimposed on the limbal region of an image of BrdU-labelled nuclei (red) counterstained with TO-PRO3 iodide (blue) in a whole mount flattened corneal button with associated conjunctival tissue. (<b>C–F</b>) Examples of BrdU label-retaining cells in the limbal region after 1-week BrdU exposure and 10 week chase period in (<b>C</b>) 15-week old WT, (<b>D</b>) 15-week old <i>Pax6^+/−</i>, (<b>E</b>) 30-week old WT and (<b>F</b>) 30-week old <i>Pax6^+/−</i>. Pixel resolution: 1024×1024. Abbreviations: Cj: Conjunctiva; L: Limbus; Co: Cornea. Red immunofluorescence: BrdU; Blue: TO-PRO3 iodide counterstain. Scale bars are 100 µm.</p

Dependence of mesenchyme-only patterning on restricted TGFβ availability.

<p>(A) Western blot detection of phospho-SMAD2, total SMAD2 and γ-tubulin in E13.5 skin cultures treated with recombinant transforming growth factor (TGF) β2 (100 ng/ml), the TGFβ receptor inhibitor LY2109761 (25 μM,) or both agents for 8 h. (B) Effects of TGFβ2 supplementation and LY2109761 on normal and mesenchyme-only patterning. Condensates are slow to appear in LY2109761, and expression of the placode marker <i>Dkk4</i> expands through the epidermis. Mesenchyme-only patterning in FGF^HiBMP^Lo conditions is abolished upon either suppression or augmentation of TGFβ signalling. Scale bars: 250 μm. (C) Whole-mount in situ hybridisation (top panel) and corresponding transverse section (bottom panel) detecting spatial arrangement of <i>Tgfb2</i> expression in E14.5 mouse embryos. Expression is most intense at sites of dermal condensate formation. Scale bars: top panel = 1 mm, bottom panel = 50 μm. (D) At E13.5, phospho-SMAD2 immunofluorescence detects signal throughout the dermal mesenchyme (De.) and epidermis (Ep.), with this signal becoming intensified in the nascent dermal condensate at E14.5 (arrowhead). Epidermis is demarcated by dotted lines. Scale bar: 25 μm. (E) Dermal mesenchymal cell attraction (arrows) to sources of TGFβ2. Images of bovine serum albumin (BSA) control and TGFβ2 loaded beads placed on E12.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin for 48 h. Scale bars: 250 μm.</p

Cell behaviours underlying mesenchymal self-organisation.

<p>(A) Time-lapse images showing dermal condensate formation in FGF^HiBMP^Lo conditions. Scale bar: 50 μm. (B) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue) under FGF^HiBMP^Lo conditions. Tracking was halted on cell entry. Plots showing (C) the mean Euclidean angle and (D) the mean level of persistence of condensate and intercondensate cells for 360-minute windows relative to time of entry into the condensate. From 6 h before entry, the condensate-bound cells show oriented and persistent movement under FGF^HiBMP^Lo conditions. Error bars represent SEM (condensate cells <i>n</i> = 17, 21, and 25 and intercondensate <i>n</i> = 91, 104, and 108 for 12, 6, and 0 h before entry, respectively). Statistical significance was calculated using a Kruskal–Wallis test followed by Mann–Whitney U tests with Bonferroni’s correction (***<i>p</i> < 0.001). (E) Comparison between per track summaries of condensate (Cond.) and intercondensate (Int.) cells under control or FGF^HiBMP^Lo conditions for (top) accumulated velocity, (middle) Euclidian velocity, and (bottom) persistence. Statistical significance was calculated using a Kruskal–Wallis test followed by Mann–Whitney U tests with Bonferroni’s correction (*<i>p</i> < 0.05, ***<i>p</i> < 0.001). Error bars represent SEM (control intercondensate <i>n</i> = 292, control condensate <i>n</i> = 28, FGF^HiBMP^Lo intercondensate <i>n</i> = 137 and FGF^HiBMP^Lo condensate <i>n</i> = 33). Raw tracking data for (B–E) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s014" target="_blank">S2 Data</a>. (F) Particle image velocimetry analysis of normal and FGF^HiBMP^Lo condensate formation over 30 h. Coloured tracks show very local cell movement in control conditions but a much broader field of recruitment for the mesenchyme-only patterned condensates. Colour scale shows track length. Scale bar: 100 μm. (G) Simulation of boundary effects on patterning in chemotactic aggregation-driven patterning. (H) Experimental test of pattern behaviours. Distinct pattern behaviours at tissue edges. Under control conditions, primordia align along the edge. FGF^HiBMP^Lo condensates align with but form at a distance from boundaries introduced in skin explants prior to pattern formation. White dotted lines indicate the boundary. Magenta dotted lines indicate the extent of the patterned region where dermal condensates form. Scale bar: 250 μm.</p

Dermal condensate formation occurs after epidermal patterning through local cell attraction.

<p>(A) Single frames from time-lapse sequences of E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explant culture captured by confocal microscopy. Dashed circles indicate ultimate condensate location. Scale bar: 50 μm. (B) Analysis of tracked cells showing the probability of joining the dermal condensate based upon initial location relative to its centre. Two hundred and forty individual cells were tracked across 8 condensates from 4 independent skins. (C) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue). Tracking was halted on cell entry. (D) Plots showing the mean Euclidean angle (top) and mean level of persistence (bottom) of condensate-entering and intercondensate cells for 6-h windows relative to time of entry into the condensate. Error bars represent SEM (condensate cells <i>n</i> = 9, 14, and 20 and intercondensate <i>n</i> = 263, 245, and 197 for 12, 6, and 0 h before entry, respectively). Statistical significance was calculated using a Kruskal–Wallis test (<i>p</i> < 0.0001 and <i>p</i> < 0.001 for angle and persistence, respectively) followed by Mann–Whitney U tests with Bonferroni’s correction (**<i>p</i> < 0.01). The raw numerical tracking data (for B, C, and D) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s014" target="_blank">S2 Data</a>. (E) Detection of a molecular prepattern prior to dermal condensate formation. TCF/Lef::H2B-GFP skin explants were fixed at intermediate stages of pattern formation, imaged to detect GFP, and <i>Dkk4</i> expression determined in the same skin sample. Asterisk represents an area where <i>Dkk4</i>-positive foci are present but corresponding dermal condensates are absent. Scale bar: 500 μm. (F) Time-lapse images of E12.75 TCF/Lef::H2B-GFP dorsal skin explants cultured with recombinant fibroblast growth factor (FGF) 9- or bovine serum albumin (BSA)-loaded beads. Cells accumulate around FGF9-loaded beads. Scale bar: 250 μm.</p