Extended Data Fig. 4: Ackr3b BAC transgenes recapitulate Acrk3b function.
From: A negative-feedback loop maintains optimal chemokine concentrations for directional cell migration
a, Schematics of the ackr3b BAC transgenes. Exons 1 and 2 of ackr3b and upstream and downstream genomic regions are indicated. The unmodified genomic locus is shown for reference (top). For the ackr3b:ackr3b-GFP transgene, GFP is inserted before the stop codon in exon 2 (middle). For the ackr3b transcriptional reporter, the coding sequence of ackr3b in exon 2 was replaced with the coding sequence for sfGFP (bottom). b, Analysis of completed primordium migration in ackr3b-/- (n = 9 embryos) and ackr3b-/-; ackr3b:ackr3b-GFP embryos (n = 29 embryos) using in situ hybridization against the primordium marker epcam at 48 hpf. The arrowhead indicates the position of the primordium. Scale bar corresponds to 200 μm. c, Total Cxcr4b-GFP (n = 2 embryos) and Ackr3b-sfGFP (n = 5 embryos) fluorescence intensities within the primordia with increasing levels of Cxcl12a over time. Note that decreasing Cxcr4b-GFP intensity reflects increasing Cxcl12a levels outside the primordium. Fluorescence intensities in primordia of individual embryos (circles) and averaged total fluorescence intensities (horizontal lines) are indicated. The initial rate of Cxcr4b-GFP intensity decrease is 2.8 × 10−2 AU/s and initial rate of Ackr3b-GFP intensity increase is 1.8 × 10−3 AU/s. The experiment was independently repeated once with similar results. d, Increasing Ackr3b-sfGFP expression plotted against decreasing Cxcr4b-GFP expression from 50 min to 200 min after induction of increasing Cxcl12a expression as shown in c. The initial rate of Ackr3b-sfGFP intensity increase per Cxcr4b-GFP intensity decrease is 16. Mean (dot) and SD (grey bars) are indicated. n as indicated in c represents the number of embryos.
