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Nkx3.1 function is required to regulate brain pericyte numbers. Lateral view of control nkx3.1+/− (A) and nkx3.1−/− MZ (B) mutants showing brain hemorrhage (arrowhead) at 52 hpf, and quantification (C; N = 3, proportions of hemorrhage). (D) nkx3.1−/− have decreased CtA vessel diameter in comparison to nkx3.1+/− hets. Dorsal images of nkx3.1+/− hets (E) and nkx3.1−/− (F) embryos expressing Tg(pdgfrβ:GFP) and Tg(kdrl:mCherry) showing fewer brain pericytes (arrows) in mutants at 75 hpf. Quantification of (G) decreased pericyte number and (H), decreased pericyte density (defined as the number of pericytes divided by the length of the vessel network) in mutants. In comparison to control wild-type embryos (I, Tg(hsp70l:tBFP), nkx3.1 gain-of-function (GOF) embryos expressing Tg(hsp70l:tBFP-2a-nkx3.1)) show more pericytes (J, arrowheads) (n = 20 control and 19 GOF)) as quantified (K). Pericyte density is also increased (L). Dorsal views of embryos labelled with TgBAC(nkx3.1:Gal4) and Tg(UAS:NTR-mCherry) under fluorescence (M, N) or under brightfield (O, P) that are untreated (M, O) or treated with metronidazole (N, P) to ablate nkx3.1-expressing cells. Ablated embryos show brain hemorrhage (P, arrowhead) at 48 hpf (P). (Q) Quantification of pericyte number in ablated embryos. Statistical significance was calculated using the Student t test (n = 5 wild type and 11 nkx3.1 mutants). Scale bars are 50 μm. The data underlying this figure can be found in S3 Table.
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