ZFIN Image: Wu et al., 2012, Fig. 4

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Figure Caption

Fig. 4

Ccr7 functions as a GPCR and promotes calcium signaling.

(A) Misexpression of two dominant-negative Ccr7 mutant forms promotes organizer gene expression. Top panel, a schema of the mutant forms employed: DN1, C-terminal truncation, and DN2, DRY→DNY mutant. Bottom panel shows effects of injecting 100 pg DN2-ccr7 RNA on boz (a2, n = 14/18) and sqt (b2, n = 11/15), and of DN1-ccr7 RNA on chd (c2, n = 13/20) expression compared to uninjected control embryos (a–c) at sphere stage, 4 hpf. Animal views, dorsal to the right. (B) Expression of szl in control embryos (a, n = 8/8) was expanded in ccr7 RNA injected WT embryos (b, 150 pg, n = 14/14), and suppressed by co-injection of GDP-β-S (~300 nM) at shield stage (c, n = 12/15, two experiments). Animal views, dorsal to the right. (C) gsc expression in control (a) and thapsigargin-treated ich mutants (b; 4 µM; n = 20/20) at shield stage, 6 hpf. Animal views, dorsal to the right. (D) β-catenin immunostaining in control (a, a2), thapsigargin-treated ich mutants (b, b2 4 µM; n = 6/6), and thapsigargin-treated ich mutants injected with ccr7 RNA (c, c2 n = 5/6). (a2–c2) Higher magnification images of the boxed areas in a–c. (E) Thapsigargin (b, b2) and 2-APB-treated WT embryos (c, c2) showed increased level and nuclear ΔNβ-catenin-GFP signal, compared to control embryos (a, a2). H2B-RFP RNA was injected as nuclear background control (d, d3, e, e2, c, c2). (d) Western blot for total β-catenin and actin in control (DMSO-treated) and thapsigargin-treated (4 μM) WT embryos at 4 hpf, with relative quantification to β-actin from three independent experiments in the right panel. (F) Effects of ccr7 and thapsigargin on Ca²⁺ transients in superficial blastomeres. (a, b) Examples of Ca²⁺ transients revealed by in vivo ratiometric image analysis in a WT embryo at 512-cell stage. The arrows point out the Ca²⁺ transients at consecutive time points (still images). Note the rapid changes of Ca²⁺intensity (compare b to a, 8 s interval). (c) The number of Ca²⁺ transients normalized to 20 min in control embryos (WT1, n = 9, red; WT2, n = 7, blue), ccr7 morphants (n = 5), Ccr7 overexpressing blastulae (n = 10), ccr7/ccl19.1 double morphants (20 ng/2 ng; n = 8), and Ccr7/Ccl19.1 overexpressing (200 pg/200 pg RNA; n = 15), and thapsigargin-treated (n = 4) blastulae were quantified by in vivo time-lapse imaging with Calcium Green-1 at about 256-cell stage or 2.5 hpf (see Materials and Methods for detail). Red and blue data were collected over 10 and 20 min, respectively; * p<0.05, Student′s t test, unequal variance.

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Expression / Labeling details

Acknowledgments

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