(a–b) Blood vessels are not detected in the OFT (outlined) of 21–28 dpf (fish size 5–7 mm) (a) Tg(flt1_9a_cFos:GFP); Tg(lyve1b:dsRed2) hearts (n = 6) or (b) Tg(fli1:EGFP);Tg(prox1a:KalTA4-UAS:uncTagRFP) (n = 4) fish, prior to OFT lymphatic sprouting (white arrows). (c) Diagram depicting a ventral view of a zebrafish larval head, indicating the approximate region imaged in (d-f). Facial lymphatics are colored in red (adapted from Okuda et al., 2012), yellow arrow points to the VFL. (d) OFT lymphatic sprouts (white arrows) arising from the VFL (outlined, yellow arrow) are detected at 24 dpf (Fish size 5–7 mm) in Tg(kdrl:EGFP);Tg(lyve1b:dsRed2);casper larvae (n = 5). (e) The VFL (arrow) connects to the LAA (inset). (f) No blood flow is detected in the VFL (arrows) of 22 dpf (fish size 5—7 mm) Tg(fli:EGFP);Tg(gata1a:dsRed2) larvae (n = 10). (g) Quantification of OFT lymphatics in 35 dpf (fish size 9–13 mm) prox1a transgenic zebrafish treated with 100 µM Atenolol (ncontrol = 43, nAtenolol = 43, *p<0.001). (h) mRNA levels of vegfc, cxcl12a and cxcl12b (nindependent experiments=5, *p<0.01) in the OFTs of 14–34 dpf larvae. (i–k) OFT of 19 wpf (22–25 mm) wt sibling (i) and flt4-/- (j) in the background of lyve1b demonstrating severe lymphatic defects in flt4 -/- hearts, quantified in (k) (nwt = 4, nflt4 -/-=5, *p<0.001). (l–n) OFT of 15 wpf (20–21 mm) wt sibling (l) and vegfc +/- (m) in the background of lyve1b showing malformed lymphatics in vegfc +/-, quantified in (n) (nwt = 8, nvegfc+/-=6, *p<0.01, **p<0.05). (o–q) OFT of 12 wpf (19–21 mm) age-matched wt control (o) and vegfd -/- (p) in the background of lyve1b showing normal OFT lymphatics in vegfd -/- hearts, quantified in (q) (nwt = 4, nvegfd-/-=5) (r–t) OFT lymphatics of 9.5 wpf (20–23 mm) wt sibling (r) and cxcr4a-/- (s) in the background of lyve1b showing mild defects in cxcr4a-/- OFT lymphatics, quantified in (t) (nwt = 4, ncxcr4a-/-=10, *p<0.01). (u–w) OFT lymphatics of 20 wpf (19–24 mm) wt sibling (u) and cxcl12b-/- (v) in the background of prox1a showing normal OFT lymphatics in cxcl12b-/- hearts, quantified in (w) (nwt = 4, ncxcl12b -/-=8). VFL, ventral facial lymphatics; LFL, lateral facial lymphatic; LAA, lymphatic branchial arches; MFL, medial facial lymphatic. Scale bars are 50 µm in a-f, 200 µm in i-v. Error bars, mean ± S.E.M.
|
