Differential response of cardiac lymphatics to injury. (a) Diagram depicting the cryoinjury procedure. Injured area is outlined in all images, insets show high-magnification of dashed boxes. (b) flt4;prox1a transgenic hearts at 40 hpci showing prox1a+ sprouts (yellow arrow) and isolated LECs (blue arrow) in the injured area (n = 5). (c) Tg(flt1_9a_cFos:GFP);Tg(prox1a:KalTA4-UAS:uncTagRFP) double labeled coronary sprouts (inset, yellow arrow), as well as prox1a+ isolated LECs (inset, blue arrow) are detected in the injured area at 40 hpci. (d–f) seven dpci injured hearts of (d) Tg(kdrl:nls-mCherry);Tg(flt4BAC:mCitrine) (n = 5), (e) Tg(kdrl:nls-mCherry);Tg(mrc1a:EGFP) (n = 5), and (f) Tg(prox1a:KalTA4-UAS:uncTagRFP);Tg(flt4BAC:mCitrine) (n = 5) fish, with white arrows pointing to OFT-connected ventricular lymphatics, and blue arrows pointing to isolated LECs in the injured area. (g–i) 14 dpci ventricles of (g) Tg(flt1_9a_cFos:GFP;Tg(lyve1b:dsRed2) (n = 8), (h) Tg(prox1a:KalTA4-UAS:uncTagRFP) (n = 5) and (i) Tg(prox1a:KalTA4-UAS:uncTagRFP);Tg(flt4BAC:mCitrine) (n = 5) showing isolated lymphatic sprouts in the injured area (blue arrows), which are not connected to ventricular lymphatics (white arrow) (j) Double labeled prox1a;flt4 ventricular lymphatic sprouts invade the injured area at 14 dpci (inset, white arrows) (n = 3). (k) 73 dpci Tg(kdrl:nls-mCherry);Tg(flt4BAC:mCitrine) heart showing increased lymphatic coverage in the injured vs. uninjured areas of the ventricle, quantified in (l). Error bars, mean ± s.e.m. *p<0.001. Scale bars are 200 µm. Fish size 25–30 mm.
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