Knockdown analysis of cxcr3.2 using splice donor and acceptor morpholinos revealed a significantly reduced accumulation of macrophages at sites of bacterial infection. (A) Schematic of the cxcr3.2 gene and MO targeting sites. Red boxes represent the exons in the cxcr3.2 gene. The intron (∼ 4 kb) is not drawn to scale. Red and blue bars represent the splice donor (D) and acceptor (A) MOs, which bind to the pre-mRNA on the intron/exon boundaries, inducing an insertion of the intron into the final transcripts. (B) RT-PCR detection of cxcr3.2 transcripts on MO knockdown. Altered splicing induced by D or A MOs resulted in an effective knockdown of the original cxcr3.2 mRNA at 24 hpf, whereas the combination of D + A MOs extended the knockdown effect until 48 hpf. The altered splice product with insertion of the approximately 4 kb intron is not amplified by RT-PCR. (C) Schematic of the experimental setup and representative images showing the attraction of macrophages and neutrophils to a local infection site. S typhimurium bacteria were injected into the embryo tail muscle at 28 hpf followed by a 3-hour incubation. Leukocytes were visualized at 3 hpi after double fluorescent in situ hybridization: (i) mfap4⁺ macrophages; (ii) mpx⁺ myeloid cells; (iii) merged; and (iv) bright-field image; bar represents 200 μm. (D) Migration of innate immune cells after MO knockdown. Accumulation of macrophages marked by mfap4 (i-iii) and accumulation of the nonoverlapping mpx⁺ myeloid cell population (iv) were quantified by determining the percentages of cells accumulated at the infection site with respect to the total number of cells in the tail. Data were collected from 3 independent experimental setups, in which the cxcr3.2 D MO (i), A MO (ii), or D + A MOs (iii-iv) were tested versus the Standard Control (SC) MO. Each data point represents an individual embryo, and each graph represents the combined results of 2 replicate experiments with the indicated MO combination. Lines indicate the mean, and P values indicate the level of statistical significance by Student t test. The knockdown of cxcr3.2 did not have a significant effect on migration of either macrophages or mpx⁺ myeloid cells in response to sterile injury (data not shown).