Depletion of Ccr7 activity partially rescues axis formation in <i>ichabod</i> mutants.

<p>(A) Penetrance of strongly ventralized phenotypes displayed by maternal β-catenin-2, <i>ich</i>, mutant embryos (a, 92%, <i>n</i> = 110) was reduced by injection of MO1-<i>ccr7</i> (b–h, 10 ng; 40%, <i>n</i> = 98, five experiments). Arrows indicate partial axes and arrowheads indicate rudimentary head structures. (B) <i>myod1</i> expression in uninjected (a) and MO1-<i>ccr7-</i>injected <i>ich</i> embryos (b,b′) revealed somitic tissue. Arrows indicate partial double axes. (C) In contrast to uninjected <i>ich</i> mutants (a,b,c), in MO1-<i>ccr7</i>-injected <i>ich</i> embryos, the organizer genes <i>gsc</i> (a′, <i>n</i> = 11/12) and <i>chd</i> (b′, <i>n</i> = 8/8) were expressed; while expression of the ventral gene, <i>eve1,</i> was significantly reduced (c′, <i>n</i> = 30/40). Animal views. (D) Lack of β-catenin nuclear accumulation, detected by immunostaining at 256-cell stage, in <i>ich</i> mutants (a), was suppressed in MO1-<i>ccr7</i>-injected <i>ich</i> embryos (b,c; <i>n</i> = 9/10, two experiments). (a′–c′) Higher magnification of boxed areas in a–c. (E) Dorsal domain of nuclear accumulation of β-catenin, detected by immunostaining at 256-cell stage in WT embryos (a), was expanded in <i>ccr7</i> morphants (b; <i>n</i> = 6/13), while it was diminished in Ccr7 overexpressing embryos (c; <i>n</i> = 8/11). (a′–c′) Higher magnification of boxed areas in a–c. (F) Ectopic β-catenin nuclear accumulation, detected by immunostaining at 256–512-cell stage, in <i>ich</i> mutants injected with <i>β-catenin</i> RNA (a; 25 pg, <i>n</i> = 9/10), was suppressed by co-injecting <i>ccr7</i> RNA (b; 150 pg, <i>n</i> = 7/10). (a′–b′) Higher magnification of boxed areas in a–b. (G) Ccr7 gain-of-function decreased both levels of endogenous β-catenin and ectopic β-catenin-GFP. Western blotting of β-catenin and GFP protein from uninjected control, <i>β-catenin-GFP</i> RNA (10 pg) injected, or <i>β-catenin-GFP</i> RNA (10 pg)/<i>ccr7</i> RNA (200 pg) co-injected embryos (all at 3–3.3 hpf). Graphs below show the relative protein level (signal intensity) quantified from three separate immunoblots. * <i>p</i><0.05.