Hayashi K et al. (2018), Intracellular calcium signal at the leading edg...

XB-ART-54542

Sci Rep 2018 Feb 05;81:2433. doi: 10.1038/s41598-018-20747-w.

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Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

Hayashi K , Yamamoto TS , Ueno N .

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During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca2+ signals play an essential role in the active cell migration during gastrulation.

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Species referenced: Xenopus laevis
Genes referenced: adm cer1 fn1 klf6 not rac1 tbxt
GO keywords: intracellular [+]

???displayArticle.omims??? NEURAL TUBE DEFECTS, SUSCEPTIBILITY TO; NTD

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	Figure 1. Ca2+ dynamics in a single cell. (a) Experimental design using cap-less explants. (1) The animal cap was removed at st12–12.5. (2) The cap-less explant was placed with the animal pole side down on a fibronectin-coated glass dish, and viewed from the bottom. (b) Snapshots from time-lapse calcium imaging of single cells. Upper panel: mRFP. Lower panel: FRET ratio of yellow cameleon-nano. The FRET ratio was converted to pseudocolours (bar at right). Scale bar: 50 μm. (c) Plot of the FRET ratio intensity over time for each of the areas shown in coloured circles in (b). Arrows indicate the points of maximum values. (d) Histogram of the calcium transient duration. n = 65 calcium transients.
	Figure 2. Ca2+ transients and their localization in LEM cells. (a) Snapshots of time-lapse imaging of the Ca2+ dynamics in the migrating LEM. FRET ratio images of yellow cameleon-nano were converted to pseudocolours (bar at right). White arrowheads indicate calcium transients. (b) Location of Ca2+ transients during mantle closing. n = 15 embryos. Error bars indicate s.e. ± Student’s t-test, **P < 0.005. (c) Illustration of the time course of mantle closure. (d) Frequency of Ca2+ transients during mantle closure. Red bars indicate average values. n = 15 embryos.
	Figure 3. Ca2+ transients and their localization in DMZ explants. (a) Preparation of DMZ explants including the LEM. (b) Snapshots of time-lapse imaging of the Ca2+ dynamics in DMZ explants. The FRET ratio of yellow cameleon-nano was converted to pseudocolours (bar at right). White arrowheads indicate calcium transients. Scale bar: 100 μm. (c) Frequency of calcium transients during LEM migration in DMZ explants. n = 22 embryos Error bars indicate s.e. ± Student’s t-test, **P < 0.005.
	Figure 4. Ca2+ transients are required for LEM migration. (a) Ca2+ transients in DMZ explants treated with BAPTA-AM for 3 hours. Red bars indicate average values. DMSO: n = 7 embryos and BAPTA-AM (50 μM): n = 8 embryos. Mann–Whitney U-test, P < 0.005. (b) Location of calcium transients during LEM migration in BAPTA-AM- or DMSO-treated DMZ explants. n = 8 embryos. Red bars indicate average values. Mann–Whitney U-test, P < 0.005, P < 0.05, n.s.: No significance. (c) Migration of DMZ explants treated with or [without] DMSO. Migration was suppressed by BAPTA-AM treatment. Scale bar: 100 μm. (d) Relative migration distance in DMSO- or BAPTA-AM-treated DMZ explants. Values were normalized to the migration distance of DMSO-treated explants. Error bars indicate s.e. ± Student’s t-test, P < 0.005. (e) Migration velocity of leader cells in DMSO- or BAPTA-AM-treated DMZ explants. Error bars indicate s.e. ± Student’s t-test, P < 0.005. (f) Migration velocity of follower cells (≥4th) in DMSO- or BAPTA-AM-treated DMZ explants. Error bars indicate s.e. ± Student’s t-test, n.s.: No significance. (g) Left: Snapshot from time-lapse imaging. Cytochalasin-D (1 μM) treatment suppressed the migration activity in DMZ explants. Right: Ca2+ imaging of DMZ explants treated with Cytochalasin-D (1 μM). White arrowheads indicate Ca2+ transients. (h) Location of Ca2+ transients during 3 hours of Cytochalasin-D (1 μM) treatment. Error bars indicate s.e. ± Student’s t-test, *P < 0.005.
	Figure 5. Intracellular Ca2+ signalling regulates migration activity in the LEM. (a) Snapshots from time-lapse imaging of the Ca2+ dynamics in DMZ explants treated with Ionomycin (2.5 μM). Upper panel: mRFP. Lower panel: FRET ratio of yellow cameleon-nano converted to pseudocolours (bar at right). (b) LEM migration into open space in Ionomycin- (2.5 μM) and DMSO-treated DMZ explants monitored by mRFP. (c) Relative migration distance in DMSO- and Ionomycin-treated DMZ explants. The migration distance was normalized to the migration distance of DMSO-treated DMZ. DMSO: n = 7 embryos. Ionomycin (Iono.): n = 8 embryos. Error bars indicate s.e. ± Student’s t-test, P < 0.05. (d) Mid-sagittal section of whole embryos at st12 and 12.5. Left side: dorsal. Right side: ventral. Yellow line indicates blastocoel roof not touching mesoderm. (e) Graph of the measured length of the yellow line of Fig. 5d at st12.5. Values were normalized to the length in DMSO-treated Embryos. DMSO: n = 71 embryos. Ionomycin: n = 74 embryos. Error bars indicate s.e. ± Student’s t-test, P < 0.05.
	Figure 6. Suppression of Ca2+ transients reduce the protrusive activity in LEM cells. (a) Snapshots of BAPTA-AM- and DMSO-treated DMZ explants. Scale bar: 50 μm. (b) Procedure for measuring the protrusion activity in LEM cells. (c) Protrusion size in DMSO- and BAPTA-AM-treated LEM cells. DMSO: n = 63 cells from 14 embryos, BAPTA-AM: n = 79 cells from 13 embryos. Error bars indicate s.e. ± Mann–Whitney U-test, P < 0.005. (d) Protrusion stability in DMSO- and BAPTA-AM-treated LEM cells. DMSO: n = 27 cells from 9 embryos, BAPTA-AM: n = 27 cells from 9 embryos. Red bars indicate average value. Student’s t-test, P < 0.005. (e) Protrusion dynamics in DMSO and BAPTA-AM-treated LEM cells. Left figure with yellow dotted line is entire view of leader cell. Right figure is kymograph along to dotted yellow line. Yellow arrowheads indicate retraction of cellular protrusion. Scale bar: 25 μm.
	Figure 7. Ca2+ signalling regulates Rac1 activity. (a) Rac1 activity after Ionomycin (Iono.) treatment for 3 minutes and 2 hours. DMZ explants were dissected and treated with Ionomycin. DMSO was used as a control. (b) Rac1 activity after Ionomycin treatment for 3 minutes. The active Rac1 intensity with Ionomycin was normalized to the intensity in DMSO-treated samples. Each western sample was prepared from 35–40 DMZ explants. Error bars indicate s.e. ± Student’s t-test, ns: No significance. (c) Rac1 activity after Ionomycin treatment for 2 hours. Student’s t-test, **P < 0.005.

References [+] :

Ataliotis, PDGF signalling is required for gastrulation of Xenopus laevis. 1995, Pubmed, Xenbase