Extended Data Fig. 5: Characterization of Ackr3b ST/A and K/A cytoplasmic tail mutants.
From: A negative-feedback loop maintains optimal chemokine concentrations for directional cell migration
a, Amino acid sequence of the cytoplasmic tail of wild-type, ST/A and K/A mutant Ackr3b (in red). b, Design of ackr3b-sfGFP control and cytoplasmic tail mutant BAC transgenic lines. c, Quantification of migration distance of primordia in 48 hpf embryos of indicated genotypes. The mean difference for five comparisons against the shared ackr3b-/+ control embryos are shown as a Cumming estimation plot. The raw data is plotted on the left axis. On the right axis, the mean differences are plotted as bootstrap sampling distributions. Mean differences (dots) and 95% confidence interval (vertical bars) are indicated for ackr3b-/+ ( < x > = 1.00, n = 32), ackr3b-/- ( < x > = 0.35, n = 102, p = 1.7 × 10−17), ackr3b-/-; ackr3b:ackr3bwt tail-sfGFP ( < x > = 1.02, n = 15, p = 0.41), ackr3b-/-; ackr3b:ackr3bST/A tail-sfGFP ( < x > = 0.59, n = 19, p = 3.4 × 10−9), ackr3b-/-; ackr3b:ackr3bwt tail-sfGFP ( < x > = 1.03, n = 24, p = 0.03), and ackr3b-/-; ackr3b:ackr3bK/A tail-sfGFP ( < x > = 1.01, n = 34, p = 0.27), where < x > represents the mean, n represents the number of embryos, and p represents p-values (two-sided Mann-Whitney test). d,e, Left. Ackr3bwt tail-sfGFP and Ackr3bST/A tail-sfGFP (in d), and Ackr3bwt tail-sfGFP and Ackr3bK/A tail-sfGFP (in e) fluorescence intensities in the primordia are shown together with the primordium marker prim:lyn2mCherry (top panels) and separately as a heat map (bottom panels). The fluorescence intensities in all images are scaled identically. Right. Mean (dots) and SD (vertical bars) of Ackr3b-sfGFP fluorescence intensities along the front-back axis of primordia in wild-type and ST/A tail embryos (in d) and wild-type and K/A tail embryos (in e). n = 12 and 11 embryos for wt and ST/A tail, respectively and n = 5 embryos for wt and K/A tail. f, Response of Ackr3bwt tail-sfGFP and Ackr3bK/A tail-sfGFP fusion proteins in the primordium of embryos with increasing Cxcl12a levels. The control ackr3b:loxP-ackr3bwt tail-sfGFP-stop-loxP-ackr3bK/A tail-sfGFP; ackr3b-/-; prim:lyn2mCherry and the experimental ackr3b:ackr3bK/A tail-sfGFP; ackr3b-/-; prim:lyn2mCherry; hsp70:cxcl12a embryos were imaged at indicated times past a 30 min heat-shock that induced Cxcl12a expression from the heat shock promoter. The Ackr3b-sfGFP fluorescence intensities in the primordia are shown together with the primordium marker prim:lyn2mCherry (top panels) and separately as a heat-map (bottom panels). The fluorescence intensities in all images are scaled identically and quantified in g. g, Mean Ackr3bwt tail-sfGFP and Ackr3bK/A tail-sfGFP fluorescence intensities (dots) and SD (vertical bars) of heat-shocked control embryos (black, n = 4 and 3 embryos, respectively) and Cxcl12a-overexpressing embryos (blue, n = 3 and 5 embryos, respectively) along the front-back axis of primordia. In d–f, the scale bar corresponds to 20 μm. Anterior is to the left and the front of the primordium is to the right.
