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During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1alpha expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1alpha caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1alpha-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1alpha, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation.
Fig. 1.
Expression pattern of xCXCR4, and xSDF-1α and xSDF-1. (A) RT-PCR analysis of xCXCR4, and xSDF-1α and xSDF-1. xCXCR4 transcripts increased at the late blastula stage (St. 9). xSDF-1α was detected at the early gastrula stage (St.10), however xSDF-1 transcript levels increased at the tailbud stage (St.26). ODC was included as a loading control. (B–I) Spatial expression pattern of xCXCR4 (B–D,H) and xSDF-1α (E–G,I). Each panel demonstrates the lateral view of stage 10 (B,E), vegetal view of stage 11.5 (C,F), sagittal section of stage 10 (D,G–I), embryos. The panels of (H and I) indicate power magnified views of the dorsal side of (D and G), respectively. Arrowheads indicate the dorsal blastopore. White-dotted lines in (H and I) indicate the border of IMZ and BCR. Scale bar in (B–G), 0.5 mm, and 0.1 mm in (H and I).
Fig. 2.
Defects of gastrulation caused by xCXCR4 and xSDF-1α. (A–E) Phenotypes of the injected embryos. Control, uninjected (A), xCXCR4 over-expressed (B), xSDF-1α over-expressed (C), xCXCR4(ND) -expressed (D), xSDF-1α MO-injected (E) embryos at the late-gastrula stage (left images), neural stage (centre images) and tailbud stage (right images). (F,G) Expressions of mesodermal genes (Xbra and chordin) and endodermal gene (cerberus) in the xCXCR over-expressed and xCXCR4(ND)-expressed embryos at the late-gastrula stage (F); and epidermal genes (keratin) and neural genes (N-CAM and SoxD) at the neural stage (G). (H–L) Midsaggital sections of the injected embryos at the mid-gastrula stage. Control, non-injected (H), xCXCR4 over-expressing (I), xSDF-1α over-expressing (J), xCXCR4(ND)-expressing (K), xSDF-1α MO-injected (L) embryos. Arrows and arrowheads indicate the edge of mesendodermal cells and the blastopore region, respectively. Scale bars, 0.5 mm.
Fig. 3.
Migration of the LEM explants towards the BCR explants. (A) Experimental procedure. See Materials and methods. (B–H) Frames of time-lapse movies. The BCR explants are shown on the left, and the LEM explants on the right. The letters “l” and “h” indicate low-level over-expression and high-level over-expression, respectively. Control (BCR) and control (LEM) (B), xSDF-1α(l) and control (C), xSDF-1α(h) and control (D), xSDF-1α(l) and xCXCR4 (E), xSDF-1α(l) and xCXCR4(ND) (F), xSDF-1α(h) and xCXCR4(ND) (G), xSDF-1α(l) and control in 500 ng/ml hSDF (H). The time elapsed is indicated at the top right corner. Scale bar, 200 μm. See Supplemental movies of B–H. (B′–G′) Lines indicate the traces of the LEM movements over eight hours in panels of (B–G), respectively. The dot shows the initial position of the LEM. (I) The ratio of migration (N = 5). The ratio of the distance between the BCR explants and the LEM explants was measured every hour, and is shown by black for (B), red for (C), red-dotted for (D), blue for (E), green for (F), and green-dotted for (G) lines.
Supplemental Fig. 1
(A) Alignment of the predicted amino acid sequences of Xenopus laevis (xSDF-1α; BC073527
and xSDF-1; BC061945), human (hSDF-1α; ΝΜ_000609 and hSDF-1β; U16752), mouse
(mSDF-1α; L12029), rat (rSDF-1α; AF209976), chick (cSDF-1α; AF209976), and zebrafish
(zSDF-1α; AY147915) SDF-1. Shadow boxes represent shared amino acids. (B) Matrix plot of
xSDF-1 (BC061945; 2878 bp; X-axis) and xSDF-1α (BC073527; 6182 bp; Y-axis) cDNA. The
approximately 300 nucleotide sequences of 5’-region of each clone contain coding region, and
are homologous (red circle). However, the followed 3’-UTR has no significant homology
between them.
