The vascular anatomy of the larval swimbladder. (A - B) Erythrocyte-specific RFP expression (white arrowheads) and swimbladder EGFP expression in Et(krt4:EGFP)^sq33-2 xTg(gata1:dsRed)sd2 double transgenic larvae at 48 hpf, showing the active circulation in the vicinity of the developing swimbladder bud. (C - D) Blood circulation and swimbladder bud at 72 hpf in Et(krt4:EGFP)^sq33-2 × Tg(fli1:EGFP)^y1 (C) and Et(krt4:EGFP)^sq33-2 x Tg(gata1:dsRed)sd2 (D) double transgenic larvae. A pair of swimbladder artery (sba), as visualized by the GFP expression, could be seen to develop towards the swimbladder bud from both lateral sides in (C). (C′, D′) 2x magnification of the boxed regions in (C) and (D), respectively. Blood circulation was present in this vessel as observed in Et(krt4:EGFP)^sq33-2 x Tg(gata1:dsRed)sd2 double transgenic larvae (D). (E - F) Blood vessels (rete mirabile) in the swimbladder in Et(krt4:EGFP)^sq33-2 x Tg(fli1:EGFP)^y1 (E) and Et(krt4:EGFP)^sq33-2 x Tg(gata1:dsRed)sd2 (F) double transgenic larvae at 108 hpf. Each swimbladder artery contains two main countercurrent vessels formed through looping at the caudal end of the swimbladder (circular arrows in E). Blood circulation could be observed in the rete mirabile in Et(krt4:EGFP)^sq33-2 x Tg(gata1:dsRed)sd2 double transgenic larvae (white arrows in F). (G) A close-up view of the network of swimbladder vessels at 125 hpf. Direction of blood flow in these vessels is indicated by white arrows. Abbreviations: ama, anterior mesenteric artery; da, dorsal aorta; g, gut; sb, swimbladder; sba, swimbladder artery; siv, supra-intestinal vessel. Scale bars: 250 μm in (A-F), 100 μm in (G).

Role of endothelial cells during swimbladder budding and early growth phase. (A - C) Expression of swimbladder epithelial marker hb9 in control, clo^-/- mutant, and Tnnt2 morphant at the beginning of budding stage (40 hpf). Note the similar size of epithelial bud in all these embryos. (D - F) EGFP expression in control, clo^-/-, and Tnnt2 morphants on Et(krt4:EGFP)^sq33-2 background at 48 hpf. (D′ - F′) A 2.5x magnification of the swimbladder in (D - F). (G - I) Expression of fgf10a mesenchymal marker in swimbladders clo^-/-, mutant, and Tnnt2 morphant embryos was initiated at the same time as that in wild type, at 48 hpf. (J - L) EGFP expression in the same transgenic line during early growth stage at 54 hpf. Note the presence of swimbladder bud which has increased in size in all three larvae observed. (J′ - L′) A 2.5x magnification of the swimbladder in (J - L). Numbers represent anterior somites, Abbreviations: sb, swimbladder; g, gut; p, pancreas. Scale bars, 250 μm.

The role of ECs and blood circulation on swimbladder growth. (A - C) EGFP expression in control, clo^-/-, mutant, and Tnnt2 morphants on the background of Et(krt4:EGFP)^sq33-2 at 72 hpf. Swimbladder appeared smaller in larvae without endocardium and endothelium (clo^-/-, B) and without cardiac function (Tnnt2 MO, C) as compared to wild type (A). (A′ - C′) 2.5x magnification of the swimbladder in (A - C) respectively. The white arrow indicates the primordium of swimbladder anterior chamber. (D - F) Presence of swimbladder mesenchyme at 72 hpf as detected by fgf10a expression in wild type, clo^-/- mutant, and Tnnt2 morphant. (G - I) Cross section at the levels indicated by white dashed lines in panel (D - F) to show mesenchymal fgf10a expression in wild type, clo^-/- mutant, and Tnnt2 morphant larvae at 72 hpf. Red bar indicates swimbladder length, Gut (g) is demarcated by black dashed line. (J - L) Analysis of acta2 expression in swimbladder in wild type (J), clo^-/- mutant (K) and Tnnt2 morphant (L) at 72 hpf. (M - O) EGFP expression in swimbladder outer mesothelium in wild type (M), clo^-/- mutant (N), and Tnnt2 morphant (O) at 72 hpf. Note the severe reduction of outer mesothelium in clo^-/- mutant and its disorganization into two lateral stripes in Tnnt2 morphant. (M′ - O′) 2.5x magnification of the swimbladder in (M - O) respectively. Numbers indicate anterior somites. Abbreviations: pd, pneumatic duct; n, notochord; sb, swimbladder. Scale bars: 250 μm.

Swimbladder inflation is affected in the absence of blood circulation. (A - C) EGFP expression in swimbladder epithelium in control, clo^-/- mutant, and Tnnt2 morphants on the background of Et(krt4:EGFP)^sq33-2 at 5 dpf. (D - F) EGFP expression in swimbladder outer mesothelium in control, clo^-/- mutant, and Tnnt2 morphant on the background of Et(krt4:EGFP)^sqet3 at 5 dpf. All mutants and morphants failed to inflate their swimbladder by 5 dpf. Abbreviation: sb, swimbladder. Scale bars: 250 μm.