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Int J Dev Biol
2013 Jan 01;571:95-100. doi: 10.1387/ijdb.120130af.
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Expression of xSDF-1α, xCXCR4, and xCXCR7 during gastrulation in Xenopus laevis.
Mishra SK
,
Nagata T
,
Furusawa K
,
Sasaki A
,
Fukui A
.
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Chemokines play a crucial role in developmental processes and recent studies have revealed that they also control gastrulation movements. In this paper, we report the expression patterns of xSDF-1α, xCXCR4 and xCXCR7 and regulation of the expression of xSDF-1α and xCXCR4 during gastrulation. We performed whole mount in situ hybridization (WISH) and quantitative real-time RT-PCR (qRT-PCR) analyses to examine the distribution of transcripts. The effect of activin/nodal signaling on the expression of xSDF-1α and its receptors was examined by animal cap assay and microinjection of cer-s mRNA. We have demonstrated that the xSDF-1αtranscript is increased in the blastocoel roof during gastrulation, but not in the involuted mesoderm. xCXCR4 was expressed in the mesendoderm at late blastula and was retained throughout gastrulation. xCXCR7 was found in the dorsal lip around the blastopore in the early gastrula stage and became localized in the presumptive notochord later. We also show that the expression of xCXCR4 and xSDF-1transcript is increased in the blastocoel roof during gastrulation, but not in the involuted mesoderm. xCXCR4 was expressed in the mesendoderm at late blastula and was retained throughout gastrulation. xCXCR7 was found in the dorsal lip around the blastopore in the early gastrula stage and became localized in the presumptive notochord later. We also show that the expression of xCXCR4 and xSDF-1α were reciprocally regulated by activin/nodal signaling. These results suggest that xSDF-1α and its receptors contribute to the cell arrangement of mesoderm cells and their expression patterns are partially regulated by activin/nodal signaling.
Fig. 1. Expression of xSDF-1a, xCXCR4 and xCXCR7. RT-PCR analysis of xSDF-1a, xCXCR4, and xCXCR7. Numbers show the developmental stage of each lane. UF is unfertilized egg. xCXCR4 and xCXCR7 transcripts increased at the late blastula stage (St. 9) and xSDF-1a was detected at the early gastrula stage (St.10). Xbra (Xenopus brachyury) was a stage marker and ODC (ornithine decarboxylase) was a loading control.
Fig. 2 (left). External view of the expression patterns of xSDF-1a, xCXCR4 and xCXCR7. xSDF-1a, xCXCR4 and xCXCR7 transcripts were examined by WISH in gastrula stage embryos. Panels show the results of probes using xSDF-1a (A-C), xCXCR4 (D-F), xCXCR7 (G-I) in stage 10 (A,D,G), stage 11 (B,E,H), and stage 12 (C,F,I) embryos. Embryos are vegetal view, dorsal side up. Dark blue staining shows signals of probes stained with BM purple. Numbers showing embryos with the same expression pattern to total number of embryos used for the experiment. Scale bar, 0.2 mm.
Fig. 3 (right). Sagittal view of the expression patterns of xSDF-1a, xCXCR4 and xCXCR7. WISH was performed in embryos bisected along the midline before probe hybridization. Panels show the results of hybridized probes using xSDF-1a (A-C), xCXCR4 (D-F), xCXCR7 (G-I) in stage 10 (A,D,G), stage 11 (B,E,H), and stage 12 (C,F,I) embryos. Sections are animal pole up and dorsal side to the right. Arrowheads indicate dorsal blastpores. Dark blue staining shows probes stained with BM purple. Numbers show embryos with the same expression pattern to total number of embryos used for the experiment. Scale bar, 0.2 mm.
Fig. 4. qRT-PCR analysis of separated explants. (A) Schematic diagram of the separation. Animal top (AT, blue), dorsoanimal (DA, light blue), dorsal marginal (D, red), mesendoderm (ME, yellow) explants were separated at stage 10 gastrula. (B) Explant separation was confirmed by RT-PCR using maker genes indicated in the panel as XIRG for BCR, Xbra for pan-mesoderm, and Cer for mesendoderm. ODC was used as a loading control. (C,D,E) qRT-PCR analysis of xSDF-1a, xCXCR4, and xCXCR7 expression, respectively. Total RNA was extracted from the explants 0h, 2h, and 4h after separation corresponding to early (st. 10), middle (st. 11), and late (st. 12) gastrula, respectively. Vertical axis shows the ratio of relative expression level normalized by xGAPDH expression to that for AT explant at 0h. Each column in graph is the same color as in (A). Experiment was performed twice and showed a similar tendency.
Fig. 5. Distribution of xCXCR4 and xSDF-1a transcripts in cer-s express- ing embryos Upper panels (A-C) show control LacZ-injected embryos and lower panels (D-F) are cer-s-injected embryos at stage 11. xSDF-1a (A,D), xCXCR4 (B,E), and Xbra (C,F) were used asWISH probes. Dark blue staining shows probes stained with BM purple. Arrowheads show mesendodermal regions. Numbers are embryos with the same expression pattern to total number of embryos used for the experiment. Scale bar, 0.2 mm.
Fig. 6. qRT-PCR analysis of activin-treated explants. Animal cap explants were treated with 5, 50, and 500 ng/ml activin A at stage 8.5. Expression levels of xSDF-1a (A), xCXCR4 (B), and xCXCR7 (C) were quantified by real-time RT-PCR. Vertical axis shows the ratio of relative expression level normalized by xGAPDH expression to that for the untreated explant. Results are the mean of three independent experiments, and error bars indicate the SE. Differences between means in no treatment (0 ng/ml) and each activin treatment were assessed with Student t-test. Asterisks (*) indicate p <0.01.
Fig. 7. Schematic diagram of the fate map of Xenopus gastrula and expression patterns of xSDF-1a, xCXCR4 and xCXCR7. Diagrams in top row show the fate map of sagittal section of gastrulaembryo stages from 10 to 12 and colors in diagrams indicate mesendoderm (orange), involut- ing mesoderm (red), presumptive neural tissue (light blue), and future epidermis (blue). Diagrams below are expression patterns of xSDF-1a (blue), xCXCR4 (red), and xCXCR7 (green) in stages corresponding with the fate map, respectively. Concentration of each color in the expression patterns corresponds to the predicted amount of expression of each mRNA examined by WISH and qRT-PCR analyses.
Supplementary Fig. S1. Sagittal view of the expression pattern of xCXCR4. WISH was performed in embryos bisected along the midline before probe hybridization. Panels show the results of hybridized probes using xCXCR4 in stage 10, stage 11, and stage 12 embryos. Sections are animal pole up and dorsal side to the right. White arrowheads indicate dorsal blastopores. Each panel in right column shows the magnified view of the boxed area in the panel of left column, respectively. White dotted lines represent the border between the inner surface of BCR and the involuting mesoderm. Black arrowheads indicate the area of probes stained with BM purple. The predominant expression of xCXCR4 was detected in mesendoderm during gastrulation. Scale bar, 0.5 mm.
