Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo

Abstract

The induction and migration of neural crest cells (NCCs) are essential to the development of craniofacial structures and the peripheral nervous system. A critical step in the development of NCCs is the epithelial to mesenchymal transition (EMT) that they undergo in order to initiate migration. Several transcription factors are important for the NCC EMT. However, less is known about the effectors regulating changes in cell adhesion, the cytoskeleton, and cell motility associated with the EMT or about specific changes in the behavior of cells undergoing EMT in vivo. We used time-lapse imaging of NCCs in the zebrafish hindbrain to show that NCCs undergo a stereotypical series of behaviors during EMT. We find that loss of cell adhesion and membrane blebbing precede filopodial extension and the onset of migration. Live imaging of actin dynamics shows that actin localizes differently in blebs and filopodia. Moreover, we find that disruption of myosin II or Rho-kinase (ROCK) activity inhibits NCC blebbing and causes reduced NCC EMT. These data reveal roles for myosin II and ROCK in NCC EMT in vivo, and provide a detailed characterization of NCC behavior during EMT that will form a basis for further mechanistic studies.

Figure 1

NCCs arise from the neuroepithelium. A–C) In situ hybridization for myoD and snail2 (A), foxD3 (B), and sox10 (C) at the 8 somite stage, 13 hpf. Asterisk indicates gene expression in the otocyst in C. D–H) Epifluorescent images from a time-lapse sequence of a Tg(foxD3:gfp) embryo at the 15 somite stage, 16.5 hpf, showing half the neuroepithelium in the region of rhombomeres 5 and 6. Dividing cells and their daughters are pseudocolored. Arrowheads in H indicate daughters of two divisions that migrate to the edge of the neuroepithelium. Time is in minutes. I–J) Examples of embryos fixed at the 12 somite stage (I) and the 20 somite stage (J) after photo-uncaging of FITC-dextran 2 hours earlier in rhombomere 4. Embryo is labeled for uncaged FITC (purple) and krox20 (red). K) Plot of the number of migratory NCCs at various ages 2 hour post uncaging. All are dorsal views, anterior left. Scale bar = 100 μm for A–C; 20 μm for D–H; and 40 μm for I, J.

Figure 2

Imaging of cytokinesis in premigratory NCCs. A–M) Time-lapse sequences in wild-type embryos showing cytokinesis. A–E) Dorsal views of rhombomere 2 dorsal neuroepithelium. Parent cell is labeled with green asterisk before division and daughters are labeled with blue and yellow asterisks. A parent cell blebs extensively before division (arrowhead in A). Arrowhead in C indicates a filopodium extended from daughter cell. F–M) Lateral views of cytokinesis in a mesenchymal NCC adjacent to rhombomere 3. Parent cell is labeled with orange asterisk and daughters with red and yellow asterisks. Arrowhead in G indicates blebbing during cell rounding. Arrowhead in I indicates filopodial protrusion prior to the completion of cytokinesis. Arrowhead in J indicates blebbing after cytokinesis. Arrowhead in L indicates a narrow intercellular bridge connecting daughter cells. Time is in minutes. Scale bars = 20 μm.

Figure 3

Blebbing and filopodial protrusion occur during the NCC EMT. A) Time-lapse sequence showing loss of cell adhesion and onset of blebbing in cell pseudocolored orange. Asterisk indicates a neighboring cell. Time is in seconds. B–I) Time-lapse sequence showing dorsal view of NCCs undergoing EMT at the edge of the neuroepithelium. NCCs undergo blebbing (arrowheads in B, D) followed by migration out of the neuroepithelium accompanied by filopodial and lammelipodial extensions (arrowheads in F, G). Dorsal views of the basal edge of rhombomere 2/3, anterior is up, embryo is approximately 17 hpf. Time is in minutes.

Figure 4

Actin dynamics in protrusions of neuroepithelial cells undergoing EMT. A) Time-lapse sequence showing that F-actin localization coincides with membrane protrusion during filopodial extension (arrowhead). Cells are expressing GPI-GFP to label membranes (blue) and RFP-UtrCH to label F-actin (red). Dorsal views of basal edge of rhombomere 5/6, anterior is left, embryo is approximately 16 hpf. B–D) Cells labeled with GPI-GFP (green) and RFP-UtrCH (red) showing actin dynamics in blebs during EMT. B shows location of cells shown in time-lapse in C and D. Arrowheads in B delineate approximate location of edge of neuroepithelium. C) Cell within the neuroepithelium blebbing at the basal surface. D) Cell delaminating from the neuroepithelium. During extension, blebs do not contain actin (arrowheads in C, D). During bleb retraction, actin accumulates beneath the membrane (arrows in C, D). Dorsal view of basal edge of rhombomere 4, anterior is up, embryo is approximately 14 hpf. Time is in seconds. Scale bars = 5 μm for A and 10 μm for B–D.

Figure 5

Actin dynamics in blebs and lamellipodia in NCCs during EMT. Time-lapse sequence of NCCs at the edge of the neuroepithelium in embryos injected with sox10:CAAX-GFP DNA and RFP-UtrCh mRNA. A) Cell marked with asterisk extends a bleb (arrow). B) The bleb fills with actin signal upon retraction (arrowhead). C) The cell blebs again (arrow). D–F) The cell moves into the bleb while forming actin-filled lamellipodium and filopodia (arrowheads). Images are confocal projections. Dorsal views, anterior left. Scale bar=10 μm.

Figure 6

Inhibition of myosin II or ROCK disrupts blebbing and causes defects in NCC EMT. A–J) Time-lapse sequences of cells expression GPI-GFP (membranes, blue) and RFP-UtrCH (F-actin, red) in vehicle treated (DMSO) (A–E) or 50 μM blebbistatin treated (F–J) embryos. Arrowheads in A, C indicate normal blebs. Arrowhead in F indicates bleb that fails to retract after blebbistatin treatment. Dorsal view adjacent to rhombomere 2, anterior is right. Time is in seconds. K–N) In situ hybridization for dlx2 in embryos treated with DMSO (K), or 10 μM (L) or 50 μM (M) blebbistatin, or 50 μM ROCK inhibitor (N). Dorsal views, anterior left. O) Number of cells undergoing EMT per hour during 2 hour movies. Blebbistatin (BBS) and ROCKi were at 50 μM. Scale bar = 10 μm for A–J and 40 μm for K–N.

Figure 7

Myosin II activity is required for cytokinesis. A–F) Time-lapse sequences of cells expressing GPI-GFP (membrane, blue) and RFP-UtrCH (F-actin, red) in embryos treated with DMSO (A–C) or 50 μM blebbistatin (D–F). Arrowhead in D indicates site of cell fission. Time is in minutes. Scale bar = 10 μm.