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Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.
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19770040
???displayArticle.link???Mech Dev
Fig. 1. Visualizing PGCs. NLD mRNA (mRNA coding for LacZ with its 3′-UTR fused with that of DEADSouth) was injected into single vegetal blastomeres of 32-cell stage embryos and fixed at stages (St.) 12 (A), 28 (B), 37 (C), 40 (D) and 46 (E), followed by x-gal staining. Bleached and cleared embryos were observed using a stereomicroscope. PGCs were identified by their blue nuclei (black arrows). (F) A higher magnification of (E). At stage 46, almost all the PGCs had reached the genital ridge. However, as shown by the white arrows, a few remained in the endoderm and were designated ‘ectopic PGCs’. Bar = 200 μm.
Fig. 2. CXCR4-knockdown experiments. (A) A schematic diagram of the microinjection procedure. Embryos at the 32-cell stage were rotated upside down using a hair loop in a well at the bottom of a hybridoma dish filled with 1.5% methylcellulose solution. NLD mRNA, mixed with or without morpholino oligos (MOs) including MO1, MO2, MO3 or a control MO, was injected into one of the vegetal blastomeres containing germ plasm. Embryos were fixed at stage 46 and stained. Bar = 500 μm. (B and C) Whole mount x-gal staining of the stage 46 tadpoles injected with NLD mRNA mixed with 10 ng of MO1 (B), 10 ng of control MO (C). The white ellipse indicates the position of PGCs in the genital ridge. Bar = 200 μm. (D–F) Effect of injected MOs on the numbers of successfully migrating PGCs. In each bar chart, the vertical axis shows the mean number of PGCs per embryo and the horizontal axis shows the quantity of MOs used: MO1 (D), MO2 (E) and MO3 (F). The bar indicates the standard error of the mean. Data are listed in Table 1. (G) NLD mRNA mixed with 10 or 20 ng of MO2 was injected into one of the blastomeres of 32-cell stage embryos and embryos were fixed at indicated stages. The percentages of injected embryos that were positive for PGC staining are shown.
Fig. 5. Expressing SDF-1 in PGCs reduced the numbers of PGCs reaching the genital ridge and increased the ratio of ectopic PGCs to total PGC numbers. SDF-1 mRNA fused with the DEADSouth 3′-UTR was co-injected with NLD mRNA into one of the vegetal blastomeres of 32-cell stage embryos. Embryos were fixed at stage 46, stained and scored. (A) Bleached and cleared embryos were observed using a stereomicroscope. PGCs were identified by their blue nuclei (white ellipse). Ectopic PGCs are indicated by arrows. Bar = 200 μm. (B) Effect of injected SDF-1 mRNA on the number of successfully migrating PGCs. The bar chart shows the results of two independent experiments, illustrated in different colors. In each bar chart, the vertical axis represents the mean number of PGCs per embryo and the horizontal axis shows the quantity of mRNA injected. The bars show the standard error. (C) Effect of injected SDF-1 mRNA on the proportion of ectopic PGC to total PGC numbers. Data are listed in Table 4.
