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Figure 5
(A) Schematic drawing of cell transplantation experiments. Wild-type donor embryos are labeled with Alexa-448-dextran and transplanted into slbp1 mutant recipient embryos at blastula stage. In slbp1 mutant recipient embryos, transplanted wild-type donor cells form retinal cell columns. The host slbp1 mutant line is combined with Tg[mfap4:tdTomato-CAAX], to investigate whether mfap4-positive microglial precursors (magenta) infiltrate the neural retina preferentially through Alexa-448-dextran-labeled, wild-type donor columns (green) in slbp1 mutant recipient embryos. (B) Live images of slbp1 mutant retinas with transplanted wild-type donor retinal cell columns at 48 hpf. Donor wild-type retinal cell columns are labeled with Alexa-488 dextran (green). Host microglial precursors are visualized with the transgene Tg[mfap4:tdTomato-CAAX] (magenta). Dotted circles indicate the outline of the optic cup. Many microglial precursors are associated with wild-type donor retinal columns in slbp1 mutant host retinas (right panel), compared with wild-type sibling host retinas (left panel). Scale bar: 30 μm (C) The fraction of mfap4-positive microglial precursors associated with donor transplanted retinal cell columns versus the total number of microglial precursors in the optic cup. The average fraction of mfap4-positive cells associated with donor retinal cell columns is significantly higher in slbp1 mutant host retinas than in wild-type host retinas. Bars and lines indicate means ± SD. *p < 0.05. (D) The trapping efficiency of mfap4-positive microglial precursors per donor column. The average trapping efficiency is significantly higher in slbp1 mutant host retinas than in wild-type host retinas, suggesting higher affinity of microglial precursors for neurogenic retinal cells. Bars and lines indicate means ± SD. **p < 0.01. (E) Schematic drawing of mosaic expression of NICD in retinas. A mixture of UAS:EGFP and UAS-myc-NICD plasmids was injected into fertilized eggs of the Tg[hsp:gal4; mfap4:tdTomato] transgenic line, which were treated by heat shock at 18 and 30 hpf. At 48 hpf, embryos were fixed to prepare serial retinal sections for imaging analysis. (F) Confocal scanning of retinal sections of Tg[hsp:gal4; mfap4:tdTomato] transgenic embryos injected with plasmids encoding UAS:EGFP or UAS:EGFP+ UAS:myc-NICD. Scale bar: 30 μm. (G) The fraction of mfap4-positive microglial precursors associated with EGFP-expressing retinal cell columns versus the total number of microglial precursors in the optic cup. The average fraction of mfap4-positive cells associated with EGFP-positive retinal columns is significantly lower in retinas injected with UAS:EGFP+ UAS:myc-NICD than with only UAS:EGFP control. Bars and lines indicate means ± SD. ***p < 0.005. (H) The trapping efficiency of mfap4-positive microglial precursors per EGFP-expressing retinal cell columns. The average trapping efficiency is significantly lower in retinas injected with UAS:EGFP+ UAS:myc-NICD than with only UAS:EGFP control, suggesting less affinity of microglial precursors for proliferative NICD-expressing retinal cells. Bars and lines indicate means ± SD. *p < 0.05.
Microglial precursors are preferentially associated with neurogenic retinal columns.