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Chemokines play important roles in early embryogenesis, including morphogenesis and cell differentiation, before the immune system is established. We characterized Xenopus laevis CC-type chemokine receptor 7 S (ccr7.S) to clarify its role during early development. ccr7 transcripts were detected ubiquitously in early embryos. Dorsal overexpression of ccr7.S inhibited gastrulation, and ccr7.S mRNA-injected embryos had short axes and widely opened neural folds. Because the Keller sandwich explants of the injected embryos elongated well, ccr7.S might affect cell migration, but not convergent extension movements. Ventral ccr7.S overexpression induced secondary axes and chrd.1 upregulation in gastrula-stage embryos. Animal cap assays showed increased expression of neural and cement gland marker genes at later stages. Ccr7.S knockdown reduced chrd.1 expression and inhibited gastrulation at the dorsal side. Our findings suggest that ccr7.S plays important roles in morphogenetic movement and cell differentiation.
Figure 1. Ccr7 alignments and ccr7-expression patterns. (a) Alignment of the Xenopus laevis S Ccr7, Xenopus laevis L Ccr7, Xenopus tropicalis Ccr7, human Ccr7, mouse Ccr7, and zebrafish Ccr7 amino acid sequences. Asterisks (*) indicates conserved amino acids. The seven transmembrane helices predicted using the TMSEG method are shaded yellow, and a protein-binding domain predicted using the ProNA2020 method is shaded red. (b and c) Expression patterns of ccr7. odc1 expression was used as an internal control. (b) Temporal expression patterns of ccr7. U indicates unfertilized eggs. The numbers indicate the developmental stages. (c) Spatial expression patterns of ccr7. Dissections were performed at the indicated developmental stages. Abbreviations: D, dorsal; V, ventral;Ec, ectoderm; Me, mesoderm; En, endoderm; H, head.
Figure 2. Phenotypes of embryos dorsally injected with ccr7.S mRNA and Keller sandwich explants. (a) Embryos dorsally injected with ccr7.S mRNA. The developmental stages are indicated in the upper panels. Vegetal views (st.12), dorsal views (st.16), and lateral views (st.30) are shown. The appearance rate (%) of indicated phenotype and the number of the examined embryos (n) are indicated. (b) Keller sandwich explants of embryos that were dorsally injected with ccr7.S mRNA. The upper panels indicate the stages of the time- control embryos. The right images represent fluorescent images of the explants
Figure 3. Effects of ccr7.S on differentiation. (a) Phenotypes of embryos ventrally injected with ccr7.S mRNA (st.28). The appearance rate of ventral gastrulation defects was 82.5% (n = 57), and the appearance rate of secondary axis formation was 17.5% (n = 57). Arrowheads indicate the secondary axis. (b) RT-PCR analysis of the dorsal or ventral sectors from embryos injected with ccr7.S mRNA (st.10). (c and d) RT- PCR analysis of the animal caps from embryos injected with ccr7.S mRNA. (c) (st.9) and (d) (st.25). (e) RT-PCR analysis of dorsal– ventral patterning like (b). (f) Phenotypes of embryos injected with human ccr7 mRNA (st.28). T Dorsal gastrulation defects, 94.9% (n = 59). Ventral gastrulation defects, 76.3% (n = 55). Secondary axis formation, 23.7% (n = 55). (g and h) RT-PCR analysis of the dorsal or ventral sectors (g) and the animal caps (h) from embryos injected with human ccr7 mRNA.
Figure 4. Effects of knocking down ccr7.S expression. (a) Western blotting analysis. The indicated mRNAs and MOs were injected, and hbg1-FLAG mRNA was co-injected as loading control.
(b) Phenotypes of embryos injected with ccr7.S-MO. Dorsal gastrulation defects, 73.7% (n = 57). Head defects, 26.3%
(n = 57). Ventral gastrulation defects, 89.6% (n = 58). Dorsal gastrulation defects, 85.7% (n = 49). Ventral gastrulation defects, 86.7% (n = 53). (c) RT-PCR analysis of the dorsal or ventral sectors of embryos injected with MO and the indicated mRNA(s) at the gastrula stage.
