BMP destabilises mesenchymal aggregates.

<p>(A) Epidermis and dermis isolated from E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explants cultured with LDN193189 (LDN) (10 μM) or fibroblast growth factor (FGF) 9 (1 μg/ml) for 27 h, counterstained with propidium iodide (PI) and imaged using confocal microscopy. Scale bar: 100 μm. (B) Number of condensates per square mm, (C) mean condensate area, and (D) intercondensate cell density measured in E13.5 TCF/Lef::H2B-GFP skin explants cultured with either LDN or FGF. Error bars represent SEM from 5 independent experiments. Significant difference was calculated using a Student paired <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). (E) E13.75 TCF/Lef::H2B-GFP skin explants (with pre-existing dermal condensates) were treated with bone morphogenetic protein (BMP) 4 for 72 h. Skins were counterstained with 4’6-Diamidino-2-phenylindole dihydrochloride (DAPI) and confocal imaged for GFP at 72 h. Scale bars: 100 μm. (F) Quantification of individual condensate area following BMP supplementation. Error bars represent SEM from at least 3 independent experiments. Significant difference was tested using a paired Student <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). The raw numerical values (for B, C, D, and F) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s015" target="_blank">S3 Data</a>.</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

Mesenchymal nuclei and Collagen-I align with mechanical strain.

(A) Plot showing the percentage decrease in anterior-posterior (A-P) and dorsal-ventral (Lat) length of mouse skin explants in a 30 min period (n = 5) when suspended freely in culture medium. (B) Images of E13.5 TCF/Lef::H2B-GFP mouse skin explants before and after a lateral stretch (upper panels) or a stretch along the anterior-posterior (A-P) axis of the embryo (lower panels). White dashed arrows show direction of stretch. (C) Nucleus orientation angle for relaxed (n = 3; upper), lateral stretched (n = 4; middle), and A-P stretched (n = 3; lower) skins. (D) Alignment score of nucleus orientation angle from relaxed (n = 3), laterally stretched (n = 4), and A-P stretched (n = 3) skins. (E) Single planes from confocal imaging of Collagen-I immunofluorescence in E13.5 TCF/Lef::H2B-GFP skin explants in stretched states. Dashed white line indicates direction of stretch. (F) Alignment scores of Collagen fibres from skin samples shown in E. The raw numerical values for A, C, D, and F can be found in S4 Data. (G) Single planes from confocal imaging of an A-P stretched E7 membrane GFP (mGFP) chicken skin. Daughter nucleus pairs are connected by white lines, showing coherent angles of mitosis aligned with applied tension. Error bars represent SEM. Scale bar in B = 2 mm; scale bar in E = 100 μm; scale bar in G = 50 μm. (TIF)</p

TGFβ enhances cell attraction to focal FGF sources.

<p>(A, B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of E13.5 or E13.75 (with condensates) skins treated with transforming growth factor (TGF) β2, fibroblast growth factor (FGF) 9, or bone morphogenetic protein (BMP) 4 for 8 or 24 h, respectively, followed by assessment of transcript abundance. TGFβ2 upregulates expression of genes associated with cell movement and the extracellular matrix. Statistical significance from control was calculated using a Student <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). Error bars represent SEM from at least 3 independent experiments. (C) Cell aggregation at FGF9 beads in E12.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explants. TGFβ2 (100 ng/ml) or LY2109761 (25 μM) is present in the culture medium as indicated. TGFβ2 enhances aggregation at FGF9 beads, while LY2109761 suppresses cell accumulation. (D) FGF9 presence in culture medium does not detectably increase cell recruitment to TGFβ2 beads. (E, F) Quantification of areas of high cell density around FGF9- or TGFβ2-coated beads under conditions as indicated. Statistical significance was calculated using Student <i>t</i> tests (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). Error bars represent SEM of at least 3 independent experiments. Scale bars: 250 μm. The raw numerical values (for A, B, E, and F) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s016" target="_blank">S4 Data</a>.</p

Time-lapse imaging of cells dividing within a dermal condensate in TCF/Lef::H2B-GFP mouse skin.

Confocal time-lapse imaging of a cultured E13.5 TCF/Lef::H2B-GFP skin explant. Magenta and cyan dots highlight nuclei that have divided within a condensate, magenta and cyan circles indicate the point at which cells divide. In contrast to the general dermal mesenchyme, dividing cells within dermal condensates produce daughters that remain in close proximity to one another. (AVI)</p

Newly born mesenchymal cells have increased probability of dermal condensate entry.

(A) Timing and proportion of tracked dividing and non-dividing cells entering the condensates as a cumulative percentage. Vertical dashed lines and asterisks indicate time windows and significance levels for a Fisher’s exact test (S1 Table). Time 0 is the point of mitosis for dividing cells. Cells from 0 to 180 min post-mitosis have an increased rate of condensate entry compared to all other cells (*p p p n = 8 from 4 independent embryos, mean number of dividing cells tracked per video = 42, non-dividing cells tracked per video = 50. (B) Averaged spatially dependent variance (variogram) in mitosis angles. Approximately 70% of the variance in the mitosis angle occurs within a distance of 0 to 60 μm from the condensate (Monte Carlo probability p n = 5 from 5 independent embryos, mean number of mitosis angles plotted per video n = 622. (C) Upper: Cell division angles relative to their nearest condensate for a single representative time-lapse sequence. Black lines indicate direction of mitosis, green lines connect division events to the condensate centre, from which angles relative to the condensate were calculated. Lower: heat map of the cell division angles relative to nearest dermal condensate for the same dataset (black circle = centre). The coordinate system used is indicated in the top left-hand corner. The raw mitosis angle and follicle position data for B and C can be found in S2 Data. (D) Schematic of the agent-based model of mesenchymal cell division composed of a mitotic jump (left panel) displacing the daughters in diametrically opposite directions (at angle θ from the horizontal) followed by a persistent random walk (right panel—dashed lines). The angle of migration of step i, relative to the direction of the mitotic jump, is represented by Φi, and the distance travelled in each step is represented by dpers. (E) Plot showing the percentage (+/− SEM; n = 8) of simulated dividing and non-dividing cells entering a condensate in the model. Dashed blue and green lines indicate the proportion of dividing and non-dividing cells entering condensates, respectively, from experimental data. (F) Plot showing percentage of dividing and non-dividing cells entering a condensate against their initial position relative to the condensate centre from experimental data. Lineages with recent divisions can be recruited from further away. Time-lapse videos n = 8 from 4 independent embryos, mean number of dividing cells tracked per video = 42, non-dividing cells tracked per video = 50. (G) Initial locations of dividing (left) and non-dividing (right) agents that ultimately enter a condensate, from simulation. (H) For cells entering follicles, the probability density of entry from a given starting distance for dividing (blue) and non-dividing (green) cells–modelled (left) and experimental (right). The raw tracking data for A and E–H can be found in S1 Data. Scale bars in C = 100 μm; scale bar in G = 50 μm. SEM, standard error of the mean.</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

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

Overlaying of reaction-diffusion signalling and dermal condensation mechanisms.

<p>(A) A signalling network including bone morphogenetic protein–fibroblast growth factor–wingless-related integration site (BMP–FGF–WNT) interactions rapidly produces a periodic prepattern of hair placodes with high WNT/β-catenin activity. (B) FGF20 production by placodes leads to local dermal cell attraction and condensate formation, facilitated by widespread transforming growth factor (TGF) β activity. BMP production inhibits expansion of placode gene expression and further dermal cell attraction. (C) In the absence of epidermal patterning, no localised attractant signals from placodes are present, and dermal BMP signalling is uniform. TGFβ stimulates dermal cell–cell attraction, which is inhibited by BMPs. Suppression of BMP signalling permits TGFβ-driven mesenchymal patterning in the absence of a functioning epidermal reaction–diffusion network. Condensate expansion is restricted by mesenchymal cell depletion from the surrounding dermis.</p

Mitotic orientations are locally aligned in embryonic mesenchyme.

(A) Distribution of mitotic angles (n = 1,135) in a TCF/Lef::H2B-GFP skin explant culture (left panel) and representative frame showing coherence of mitotic orientations (right panel). Daughter nucleus pairs are connected by white lines, showing the angle of mitosis. The raw mitosis angle data for A can be found in S2 Data. (B) Schematic of whole embryo culture and multiphoton imaging with corresponding image of the trunk skin of an E13.5 TCF/Lef::H2B-GFP embryo. (C, D) Speed (C) and Euclidean distance travelled (D) by tracked dividing and non-dividing cells. Whole embryo time-lapse from 3 independent embryos, average number of divisions tracked/video = 65, non-dividing cells tracked/video = 50. In dividing cell plots, time 0 = mitosis. (E) Distribution of angles (n = 43) of division from TCF/Lef::H2B-GFP whole mouse embryo imaging; 0 and 180 degrees are parallel to the A-P axis. (F) Spatial distribution of mitotic angles in E (white overlaid lines), with heat map showing areas where angles are correlated. Right panel shows areas (overlain in black) containing no significant local correlation (i.e., mitotic angles are random). The coordinate system used is indicated in the top left-hand corner. The randomness ratio (proportion of black area to total area) calculated at a significance level of 0.01, ranged from 14% to 21% between the fields of view analysed. The raw tracking and mitosis position data for C–F can be found in S3 Data. Error bars represent SEM. Scale bar in A = 50 μm; scale bar in B = 100 μm. A-P, anterior-posterior; SEM, standard error of the mean.</p