Rapid mechanosensitive migration and dispersal of newly divided mesenchymal cells aid their recruitment into dermal condensates

Embryonic mesenchymal cells are dispersed within an extracellular matrix but can coalesce to form condensates with key developmental roles. Cells within condensates undergo fate and morphological changes and induce cell fate changes in nearby epithelia to produce structures including hair follicles, feathers, or intestinal villi. Here, by imaging mouse and chicken embryonic skin, we find that mesenchymal cells undergo much of their dispersal in early interphase, in a stereotyped process of displacement driven by 3 hours of rapid and persistent migration followed by a long period of low motility. The cell division plane and the elevated migration speed and persistence of newly born mesenchymal cells are mechanosensitive, aligning with tissue tension, and are reliant on active WNT secretion. This behaviour disperses mesenchymal cells and allows daughters of recent divisions to travel long distances to enter dermal condensates, demonstrating an unanticipated effect of cell cycle subphase on core mesenchymal behaviour

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

Time-lapse imaging of dividing mesenchymal cells in mTmG mouse skin.

Confocal time-lapse imaging of cultured E13.5 mTmG skin explant. Magenta and cyan dots highlight mesenchymal cells undergoing mitosis, magenta and cyan circles indicate the point at which cells divide. White arrowhead points to a cell process that persists through mitosis. (AVI)</p

Time-lapse imaging of dividing epithelial cell in mTmG mouse skin.

Confocal time-lapse imaging of cultured E13.5 mTmG skin explant, showing an example of a peridermal cell undergoing mitosis. The daughter cells remain in contact with one another. (AVI)</p

Data pertaining to Figs 1B–1G, 2A, 2F, 2H, S1B and S1C.

Data pertaining to Figs 1B–1G, 2A, 2F, 2H, S1B and S1C.</p

Data pertaining to Figs 2B, 2C, and 3A.

Embryonic mesenchymal cells are dispersed within an extracellular matrix but can coalesce to form condensates with key developmental roles. Cells within condensates undergo fate and morphological changes and induce cell fate changes in nearby epithelia to produce structures including hair follicles, feathers, or intestinal villi. Here, by imaging mouse and chicken embryonic skin, we find that mesenchymal cells undergo much of their dispersal in early interphase, in a stereotyped process of displacement driven by 3 h of rapid and persistent migration followed by a long period of low motility. The cell division plane and the elevated migration speed and persistence of newly born mesenchymal cells are mechanosensitive, aligning with tissue tension, and are reliant on active WNT secretion. This behaviour disperses mesenchymal cells and allows daughters of recent divisions to travel long distances to enter dermal condensates, demonstrating an unanticipated effect of cell cycle subphase on core mesenchymal behaviour.</div

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

Newly born cells in stretched skin show increased speed and displacement.

(A, B) Speed (A) and Euclidean distance travelled (B) of tracked dividing (top panels; time 0 = point of mitosis) and non-dividing (bottom panels) cells in skins that were relaxed (n = 3, average number of dividing cells tracked/video = 67, non-dividing cells tracked/video = 50), stretched laterally, (n = 4, average number of dividing cells tracked/video = 72, non-dividing cells tracked/video = 50), and stretched along the A-P axis (n = 3, average number of dividing cells tracked/video = 79, non-dividing cells tracked/video = 50). The raw tracking data for A and B can be found in S4 Data. Error bars represent SEM. (TIF)</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