Fig. 3

il34 Deficiency Affects the Colonization of Microglial Precursors

(A) Schematic diagram showing the brain rudiment taken from the head of embryos and used for subsequent cDNA library construction.

(B) qRT-PCR of csf1a, csf1b, and il34 expression in the brain rudiments of 2-dpf, 3-dpf, and 4-dpf WT embryos. Pooled data from two or three separate experiments. n = 20 for each single experiment. Values represent means with SD.

(C and D) Neutral red (NR) staining showing microglia in 3-dpf WT, csf1a^−/−, csf1b^−/−, and il34^−/− embryos (C) and quantification (D) of NR⁺ microglia in the optic tectum of embryos with indicated genotype. n = 28, 47, and 24 for csf1a^+/+, csf1a^+/−, and csf1a^−/− embryos, respectively. n = 11, 36, and 12 for csf1b^+/+, csf1b^+/−, and csf1b^−/− embryos, respectively. n = 25, 35, and 16 for il34^+/+, il34^+/−, and il34^−/− embryos, respectively. Values represent means ± SD.

(E) Quantification of DsRedx⁺ microglia in different CNS regions of 3-dpf WT Tg(mpeg1:DsRedx) or il34^−/−;Tg(mpeg1:DsRedx) embryos. n = 6 for each group. Values represent means with SD. See also Figure S4C.

(F and G) Whole-mount in situ hybridization (WISH) showing mfap4 expression in 30-hpf WT or il34^−/− embryos (F) and quantification (G) of mfap4⁺ macrophages in the head region (upper panel) or the whole embryos (lower panel) with indicated genotype. n = 22, 37, and 19 for il34^+/+, il34^+/−, and il34^−/− embryos, respectively. Values represent means ± SD.

(H) Time-lapse imaging of the head region of WT Tg(mpeg1:DsRedx) and il34^−/−;Tg(mpeg1:DsRedx) embryos from 24 hpf to 32 hpf showing the migration of DsRedx⁺ macrophages to the anterior head regions. Left and middle panels show the beginning (24 hpf) and end (32 hpf) of time-lapse imaging, and right panels show the cell tracking of DsRedx⁺ macrophages entering the anterior head region. In WT embryos, DsRedx⁺ macrophages migrate to the anterior head regions from two preferential routes (arrows), while in il34^−/− mutants DsRedx⁺ macrophages rarely reached the anterior head region. The head is indicated by dotted lines. YS, yolk sac. See also Videos S1 and S2.

(I) Quantification of DsRedx⁺ macrophages migrate to the anterior head regions in the time-lapse imaging of WT Tg(mpeg1:DsRedx) and il34^−/−;Tg(mpeg1:DsRedx) embryos from 24 hpf to 32 hpf. n = 6 for each group. Values represent means ± SD. See also S2.

ns, p > 0.05; ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ^∗∗∗∗p ≤ 0.0001.