Collective chemotaxis requires contact-dependent cell polarity.
Directional collective migration is now a widely recognized mode of migration during embryogenesis and cancer. However, how a cluster of cells responds to chemoattractants is not fully understood. Neural crest cells are among the most motile cells in the embryo, and their behavior has been likened to malignant invasion. Here, we show that neural crest cells are collectively attracted toward the chemokine Sdf1. While not involved in initially polarizing cells, Sdf1 directionally stabilizes cell protrusions promoted by cell contact. At this cell contact, N-cadherin inhibits protrusion and Rac1 activity and in turn promotes protrusions and activation of Rac1 at the free edge. These results show a role for N-cadherin during contact inhibition of locomotion, and they reveal a mechanism of chemoattraction likely to function during both embryogenesis and cancer metastasis, whereby attractants such as Sdf1 amplify and stabilize contact-dependent cell polarity, resulting in directional collective migration.
PubMed ID: 20643349
PMC ID: PMC2913244
Article link: Dev Cell.
Grant support: Biotechnology and Biological Sciences Research Council , Medical Research Council , Wellcome Trust
Genes referenced: c3 cdh2 ctnnd1 cxcl12 cxcr4 psmd6 rac1 rhoa snai2 twist1
Antibodies referenced: Ctnnb1 Ab5
Morpholinos referenced: cdh2 MO1 cxcl12 MO1
Article Images: [+] show captions
|Figure 1. Sdf1-Cxcr4 Axis Is Required for NC Migration In Vivo(A–H) Premigratory ([A], Twist) and migratory ([B], Twist) NC cells (arrowheads) express Cxcr4 (C and D) and are surrounded by Sdf1-expressing ectoderm ([E and F], arrow, yellow dotted lines).(G) Summary of NC cells and Sdf1 distribution at premigratory and migratory stage.(H and I) Sections showing Cxcr4 expression in NC cells (H) and Sdf1 in the adjacent ectoderm (I); NC cells streams are delimited by dashed circles.(J–R) Embryos injected with Sdf1-Morpholino ([J and K], n = 132), treated with Cxcr4 inhibitor AMD3100 ([L and M], n = 128), injected with dominant-negative Cxcr4 (N and O, n = 77) or Cxcr4-Mo ([P and Q], n = 119) show clear inhibition of neural crest migration on the experimental side.(P′–Q′) Sections of Cxcr4-Mo-injected embryo. Arrowheads indicate border of the neuroepithelium and the front of NC cells migration.(R) Summary of phenotype after inhibition of Sdf1-Cxcr4 axis.(S–U) Rescue of Sdf1 inhibition by graft of Sdf1-expressing ectoderm ([T], n = 20) or coinjection of Sdf1-Mo and Sdf1 mRNA ([U], n = 68).(V and W) Rescue of Cxcr4-Mo by coinjection of Cxcr4-Mo and Cxcr4 mRNA (n = 14).(X–Z′) NC cells labeled with nRFP were grafted along side PBS ([X and X′], n = 4) or Sdf1 beads ([Y and Y′], n = 4). (X and Y) Frames of time-lapse movies showing in vivo NC migration. Green dot, grafted bead. Note that normal NC migration (X′) is partially affected by Sdf1 beads (Y′), with cell accumulating around the bead (arrowhead). (Z and Z′) Embryos analyzed by Twist in situ hybridization after graft of Sdf1 beads show ectopic NC cells located in between the streams (Z and Z′, arrowheads, n = 2).|
|Figure 3. Cell Cooperation Accounts for Efficient Collective Chemotaxis(A–D). Run and tumbling of NC cells. Single cell (A) and cells in a cluster (B) exposed to a source of Sdf1 (at the bottom) show alternation of run and tumbling. Asterisks indicate cell protrusions; white arrowhead marks a cell collapsing protrusion while moving forward. Time in minutes. Scale bars, 20 μm.(C and D) Migration speed during run and tumbling ([C], gray bars, single cells; black bars, groups) and tumbling duration (D).(E–M) Rescue of nonresponsive Sdf1 cells by wild-type cells.(E and F) Control NC cells (red nucleus, n = 6) or dnCxcr4 (green membarne; n = 6) were separately exposed to Sdf1.(G and H) Mix of control (red nucleus) and dnCxcr4 (green membrane) exposed to Sdf1 (n = 20). (I) Chemotaxis index of separated or mixed control (red bars) and dnCxcr4 (green bars) cells. (J) Tracks of NC cells shown in (E) –(H) as indicated. Time in minutes. Scale bars, 150 μm.(K–M) NC migration in vivo. Host NC cells were removed and replaced by control (K) or dnCxcr4 (L) NC cells, or both (M). Error bars show standard deviation. See also Figure S2 and Movies S5 and S6.|
|Figure 4. Sdf1 Stabilizes Cell Polarity Induced by Cell Interactions(A–D) Two-plane confocal image to show cell protrusions (red) and cell shape (green) in single cells (A and B) and groups (C and D), with (+) or without (−) Sdf, as indicated.(E–H) Orientation of cell protrusions analyzed from time-lapse movies in single cells (E and F) and groups (G and H), with (F and H) or without (E and G) Sdf1.(I–K) Size (I), duration (J), and numbers (K) of protrusions are shown for each condition (n = 50 per condition). Gray bar, single cells; black bar, group of cells. ∗p < 0.05; ∗∗∗p < 0.005.(L–U) FRET analysis of Rac1 activity in single, outer, and inner cells without (L–N) and with (O–Q) Sdf1 shows that Rac1 activity distribution is depending on cell contacts.(R) Levels of Rac1 activity in outer cells at the front (n = 26) or at the back (n = 25) of an explant with (+) or without (−, n = 20) Sdf1 and inner cells with (+, n = 6) or without (−, n = 6) Sdf1. ∗∗∗p < 0.005.(S–U) Summary of Rac1 activity distribution in single (S), outer (T), and inner (U) cells exposed to Sdf1. Circles under each bar represent different types of Rac1 polarities, which were quantified. Error bars show standard deviation. See also Figure S3.|
|Figure 5. N-Cadherin-Dependent Contacts Are Required for Collective Chemotaxis(A–F) N-cadherin expression in premigratory (A–C) and migratory (D–F) NC cells analyzed by whole mount in situ hybridization (A and D) and immunostaining (B, C, E, and F); NC cells streams are delimited by dotted lines.(G and H) N-cadherin loss-of-function using an antisense Morpholino (n = 87).(I and J) Full-length N-cadherin overexpression (n = 40).(K–S) NC cells labeled with rhodamine-dextran (RD) were grafted into unlabeled embryos and NC migration was monitored looking at the RD fluorescence. Immunostaining on sections for N-cadherin (N and O), β-catenin (P and Q), and p120-catenin (R and S) are shown in low and high magnification. Blue, DAPI staining. Scale bar, 20 μm.(T–V) Tracks of control cells ([J], n = 16) and NC pretreated with a control IgG ([K], n = 22) or with N-cadherin blocking antibody NCD2 ([L], n = 27) and exposed to Sdf1 showing that N-cadherin inhibition strongly blocks chemoattraction toward Sdf1. Chemotaxis index for each condition is shown in (W). b, branchial; h, hyoid. Error bars show standard deviation. See also Movies S7 and S8.|
|Figure 6. N-Cadherin-Dependent Cell Interactions Prevent Formation of Cell Protrusions through Local Inhibition of Rac1 at Cell Contacts(A–D) Embryos were injected with mbRFP (blue) at the 2 cell stage and with N-cadherin MO/mbGFP at the 32 cell stage to generate a mosaic expression of the MO. Two-plane confocal image to show cell protrusions (red) and cell shape (green) in control cells (A and B) and N-cadherin MO cells (C and D), with (+) or without (−) Sdf1, as indicated. N-cadherin loss-of-function induces formation of ectopic cell protrusions overlapping with neighboring cells ([C and D], arrowheads) regardless of Sdf1.(E) Size of inner cell protrusions (n = 10).(F–G′) In vivo, confocal images of migrating NC cells labeled with mbGFP and nRFP grafted into a control embryo. Not all the cells are labeled. Region shown equivalent to Figure 2O (middle of NC stream). Control cells (F and F′) have clear cell-cell boundaries while N-cadherin-Mo-injected cells (G and G′) have high membrane activity and show overlapping protrusions. Labeled cells surrounded by nonlabeled cells are presented in high magnification in F′ and G′. Scale bars, 15 μm.(H–J) FRET analysis of Rac1 activity distribution in control outer cells or outer cells treated with NCD2 antibody as indicated (n = 27). Scale bars, 10 μm.(K) Rac1 activity at the cell contacts region (n = 18).(L) Global Rac1 activity in outer cells (n = 29) ∗p < 0.05, ∗∗∗p < 0.005. Error bars show standard deviation. See also Figure S4 and Movie S9.|
|Figure 7. N-Cadherin Is Required for CIL(A–J) Collision assays between control (A–E) and NCD2-treated NC cells (F–J). Velocity (D–I) and acceleration (I–J) vectors for control (D and E) and NCD2-treated cells (I–J). Note the clear change in direction of migration upon collision in control cells (p < 0.005, n = 10) is lost in NCD2-treated cells (n = 10).(K–O) Invasion assays between control NC cells explants ([K and L], n = 36) and NCD2-treated explants (M and N, n = 47). Control explants do not invade each other (L and O), whereas N-cadherin inhibition allows NC cells to invade each other (O).(P–R) Model for Xenopus NC cells collective chemotaxis. The color gradient in the cytoplasm represents the levels and distribution of Rac1 (red) and RhoA (blue, after Carmona-Fontaine et al., 2008; Matthews et al., 2008) activities. The different thicknesses and directions of the arrows indicate the relative stabilities and orientation of protrusions, respectively. N-cadherin is represented as a green bar. Nuclei are shown as gray circles and the external gradient of Sdf1 as shades of green. (P) NC cells clusters exhibit radial symmetry where all outer cells are polarized with protrusions toward the free edge and inner cells are not polarized. When exposed to a gradient of Sdf1, protrusions at the front are further stabilized and the initial radial organization is broken leading to directional migration. (Q) If cell interactions are prevented (N-cadherin inhibition, cell dissociation), Rac1 distribution no longer matches cell-cell interactions and global levels are lowered thus inducing protrusions instability, loss of coordination among the cells, and the loss of directional migration. (R) Representation of the NC cells migration in vivo where NC cells are maintained on migratory routes by inhibitory cues (shades of purple) and attracted ventrally by chemotaxis to Sdf1. Protrusions can be seen at the border of the group and in between the cells only when gaps are generated. The NC cells population gets looser as migration proceeds ventrally and progressively breaks away as single cells. Error bars show standard deviation. See also Movie S10.|
|Figure 2. Cell Interactions Are Essential for NC Cell Chemotaxis toward Sdf1(A–H) In vitro attraction assay with control NC explant exposed to beads (green asterisk) soaked in PBS (A and B) or purified Sdf1 (C–H).(E and F) Control (ctl) and dominant-negative Cxcr4 (dn) explants compete over an Sdf1 bead (nctl = 25; ndnCxcr4 = 32).(G and H) Control (ctl) and Cxcr4 Morpholino-injected explants (Mo) compete over an Sdf1 bead (nctl = 12, nMo = 12). Tracks are shown at the right.(I–L) In vitro, cells were labeled with mbRFP (blue) and mosaic labeling of NC with mbGFP/nRFP. Optical sections of GFP mosaic labeled NC from the plane of the substrate (red, cell protrusions) and from 5 μm above (green, cell body) were overlaid with mbRFP image (blue, surrounding cells). Outer (I and K) and inner (J and L) cells showing no cryptic protrusions in between the cells and no influence of Sdf1 on the cluster organization.(M–P) In vivo, confocal images of migratory NC cells labeled with mbGFP and nRFP grafted in a control embryo. Not all cells are labeled. Early migrating cells located near the neuroepithelium show an epithelial-like organization with clear cell-cell boundaries (N). Cells in the middle of a stream show a more mesenchymal phenotype but have no clear protrusions in the cell contact region (O), while cells at the front of a migrating stream facing a free space have protrusions ([P], arrowheads).(Q and R) Tracks of dissociated and reaggregated cells (Q) and Chemotaxis index of cells dissociated, reaggregated, and small and large clusters (R).(S and T) Chemotaxis index of single cells (green, n = 25), single cells having transient contacts (purple, n = 21), single cells interacting with small clusters (red, n = 88), and large clusters (blue, n = 41). (T) Average chemotaxis index for each category analyzed in S (∗∗p < 0.01). Chemotaxis efficiency improves as cell density increases. Time in minutes. Scale bars in (A–H), 150 μm; (I–L), 10 μm. Error bars show standard deviation. See also Figure S1 and Movies S1–S4.|