Szabó A et al. (2016), In vivo confinement promotes collective migrati...

XB-ART-53724

J Cell Biol 2016 Jun 06;2135:543-55. doi: 10.1083/jcb.201602083.

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In vivo confinement promotes collective migration of neural crest cells.

Szabó A , Melchionda M , Nastasi G , Woods ML , Campo S , Perris R , Mayor R .

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Collective cell migration is fundamental throughout development and in many diseases. Spatial confinement using micropatterns has been shown to promote collective cell migration in vitro, but its effect in vivo remains unclear. Combining computational and experimental approaches, we show that the in vivo collective migration of neural crest cells (NCCs) depends on such confinement. We demonstrate that confinement may be imposed by the spatiotemporal distribution of a nonpermissive substrate provided by versican, an extracellular matrix molecule previously proposed to have contrasting roles: barrier or promoter of NCC migration. We resolve the controversy by demonstrating that versican works as an inhibitor of NCC migration and also acts as a guiding cue by forming exclusionary boundaries. Our model predicts an optimal number of cells in a given confinement width to allow for directional migration. This optimum coincides with the width of neural crest migratory streams analyzed across different species, proposing an explanation for the highly conserved nature of NCC streams during development.

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Species referenced: Xenopus
Genes referenced: dct egr2 eya1 fn1 foxi4 slc12a3 snai2 twist1 vcan
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	Figure 1. Versican V0â€“1 forms a delimiting boundary around migrating NCCs. (A) qPCR of versican isoforms. Bars, mean; error, SEM. (B) Western blot of V0â€“1 and V3 isoforms. (Câ€“G) Embryos showing expression of versican (C) and the NC marker Twist (D) using whole-mount in situ hybridization (ISH), and corresponding sections (E and F), with a scheme summarizing the sections (G). Sections on E and F have been enhanced by blurring and squaring the separated ISH signals and overlaid in pseudo-color on the section background. (Hâ€“L) NCC explants cultured on fibronectin (Fn) + BSA or Fn + versican barriers invade the barrier area only in absence of versican. (J and K) In vitro cluster trajectories with barrier boundary marked as solid line; n = 10; color: time. (L) Percentage of cells invading the barrier (n = 3 independent experiments with n = 251 and n = 336 explants in total for BSA and versican, respectively). Bars, mean; error, SEM. (Mâ€“Q) ISH for Twist of embryos grafted with PBS (M) or versican-soaked beads (N), with the corresponding sections (O and P), and inhibition of NC migration (Q; mean inhibition, PBS: n = 5/29, versican: n = 25/32 embryos). Arrowheads show migrating NCC and asterisks indicate grafted bead. **, P < 0.01.
	Figure 2. Versican is required for normal NC migration. (A and B) qPCR (A) and Western blot (B) analysis of CoMO- and VsMO-injected embryos. (Câ€“E) ISH of Twist in embryos injected with either CoMO (C) or VsMO (D), and inhibition of migration (E; C: n = 91, D: n = 135). (F and G) Graft experiments: VsMO + fluorescein-dextran (FDx)â€“injected NCC grafted into control host (F; 76% of migration, n = 17) or CoMO + FDx NCC grafted into VsMO-injected host (G; 13% of migration, n = 15). (H and I) ISH against Krox20, showing an uninjected versus VsMO-injected side of a stage 24 embryo (H, dorsal view; n = 53), and number of ectopic NCC (I; n = 51). Bars, mean; error, SEM. Arrowhead represents a migrating NC. **, P < 0.01.
	Figure 3. Computational model of NC migration. (A) Schematic embryo during NCC migration in the Xenopus head. (Bâ€“D) Cell interactions in the CPM: CIL (B), CoA (C), and dorsal and lateral confinements (D). (E) CPM configurations for versions of NC migration in constrained geometries with and without CIL and CoA. (F and J) Migration efficiencies. Error bars: min-max values, boxes: quartiles; central value: median; n = 50 simulations. Significance compared with relevant control, where significance bars (purple) compare data from the same conditions. (G) CPM configuration in unconstrained geometry. (H) Rules of the DEM model of NCC migration. (I) Configuration of the DEM model with and without confinement. ***, P < 0.001; ns, not significant.
	Figure 4. Confinement by versican enhances collective NC migration. (Aâ€“D) Lateral view of stage 27 embryos of wild-type NC (nuclear-GFP in cyan) transplanted into a CoMO-injected (A, n = 21) or VsMO-injected host (C, n = 24; Video 3), with cell trajectories (B and D). (Eâ€“H) In silico cell migration with and without confinement with cell trajectories (n = 50). (Iâ€“K) Comparison of cell speed, persistence, and direction of migration in vivo and in silico. (Lâ€“O) NC cluster migration in vitro. Frames of time-lapse movie of control NC cluster in versican confinement (L, green: versican, red: NC nuclei; n = 20) or without confinement (N; Video 2, n = 25) and cluster trajectories (M and O). (Pâ€“S) Simulations imitating the in vitro geometries (n = 50). (Tâ€“V) Comparison of cluster speed, persistence, and direction of migration in vitro and in silico. Error bars, minâ€“max, boxes: quartiles; bar, median. ***, P < 0.001; ns, not significant.
	Figure 5. Effect of confinement width and cluster size. (A) Schematic illustration and examples of maximal projection of a Z-confocal stack used to measure the number of cells in NC streams of different widths. White line illustrates how the stream width was measured. Width (W) and number of cells (N) are shown at the bottom, with SD in parentheses; ne, number of embryos analyzed. (B) Snapshots of simulations with increasing confinement widths (w, cell diameters) and cluster size (N, number of cells). (C and D) Cluster persistences and transport ratio (percentage of cells in the cluster migrating at least 150 Âµm away from the clusterâ€™s edge in 5 h). Heatmaps represent median values from n = 50 simulations. Dots show experimental values for NCs migrating in vivo in zebrafish and Xenopus embryos. Numbers on dots correspond to the same numbers shown in A.
	Figure S1. Versican is expressed in tissues that surround the migrating NC, related to Fig.Â 1.Â (A) Western blot analysis of extracts from control Xenopus embryos at stages 5, 18, 27, 38, 41, and 48. Î²-Actin was used as a loading control. Quantification by densitometry is shown in Fig.Â 1Â B.Â (B) Wholemount ISH of stage 28 Xenopus embryos, with a lateral view comparing the expression of Eya1/FoxI1C marking the tissues adjacent to the NC. Black line indicates the level of sections. (C) Section showing Eya1/FoxI1C expression. (D) Section showing Versican expression. The first, second, and third arch are labeled (1â€“3; migrating NC streams)
	Figure S2. Inhibition of versican leads to failure in NC migration, related to Fig.Â 2.Â (A) Western blot of extracts from wild-type (wt) embryos and embryos injected at the two-cell stage with 30 ng VsMO in each blastomere, harvested during NC migration. Lysates were probed with antibodies specific for the versican V0â€“1 and the V3 isoforms. Î²-Actin was used as a loading control. (Bâ€“E) Versican MO inhibits NC migration in vivo. Lateral view of stage 27 embryos, anterior to the left, showing that in comparison with control NC migration (B, arrows), versican depletion can result in mild (C) or severe (D) inhibition of NC migration (red arrowheads). (E) Quantification of NC migration inhibition in versican-depleted embryos (VsMO; dark brown represents mild phenotype [32%], light brown represents severe phenotype [49%, total n = 214]) compared with control (CoMO; dark brown represents mild phenotype 5%, n = 124). (F) Lateral view of control or VsMO-injected 32 Xenopus embryos at stage 32 revealing melanocytes (arrows) and NC derivative population. (G) The mean number of melanocytes is significantly reduced upon VsMO injection. Bars, SD. (H) Cartilage, another NC derivative, is affected in VsMOinjected embryos at stage 42 (ventral view). (I and J) Contact inhibition of locomotion is unaffected in NC explants in a confrontation assay. Clusters without CIL are expected to mix and form a larger overlap area (overlapping index [OI] âˆ¼0.5), and the lack of overlap is indicative of the presence of CIL (Becker et al., 2013). Bar, mean; error, SD; n = 23 pair of clusters per condition. (K and L) Coattraction of NC clusters explanted from two embryos onto fibronectin substrates is unaffected by VsMO in a CoA assay (Carmona-Fontaine et al., 2011). (M) Quantification of migration index as MI = d/D.Â Migration index in control host embryos with VsMO-injected NC grafts and control NC in VsMO-injected host embryos show that versican depletion in the surrounding tissues leads to significant reduction in NC migration. Bar, mean; error, SD. (N and O) Rescue of NC migration by versican protein. (N) NC expressing GFP were grafted into a VsMO-injected embryo, which impairs NC migration. In some embryos, microbeads soaked with versican protein were grafted at the border of the presumptive NC streams. (O) Migration index in embryos with beads soaked in PBS (n = 4) or versican (n = 5). Bar, mean; error, SD. Note that grafts of versican-soaked beads at the stream borders lead to a significant rescue of NC migration. (P) Placode localization is affected by VsMO injection, shown in ISH of a representative stage 25 embryo using Eya1/FoxI1C (88%, n = 24). *, P < 0.001; , P < 0.01; ns, not significant.
	Figure S3. Computational model of NC cluster migration, related to Fig.Â 3.Â (Aâ€“E) Analysis of the NC migration model with CIL and CoA in unconstrained simulations. (A) NCC persistence in isolation and in cluster matching experimentally measured values 0.1 and 0.5 (Theveneau et al., 2010). (B) Duration of cellâ€“cell contact in simulations of two-cell collisions. (C) Relative angle of velocities before and after two-cell collisions are largely uncorrelated, similar to previously reported experimental observations (Woods et al., 2014). (D) Cluster dispersion without confinement depends on cluster size. (E) Size dependence of dispersion is explained by lower levels of chemoattractant surrounding smaller clusters. Graph shows chemoattractant level profile along the cross section of 50 simulated clusters from simulations with n = 16, 50, and 100 cells each. (F) Net cluster displacements within 3Â h.Â (G) Cluster dispersion over 10Â h.Â Boxplots show median, quartiles as boxes, and minâ€“max values as error bars.

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