Quantitative TaqMan RT-PCR measurement of the relative expression of known lymphatic and blood vessel markers in HMVEC-dLy (Lymphatic Endothelial Cells, LEC) and HUVEC (Blood Endothelial Cells, BEC), normalized to expression levels in HMVEC-dLy (LEC). Four biological replicates were analyzed. All graphs are analyzed by t-test and the mean ± SD is shown.

Quantitative TaqMan RT-PCR measurement of the relative expression of known lymphatic and blood vessel markers in HMVEC-dLy (Lymphatic Endothelial Cells, LEC) and HUVEC (Blood Endothelial Cells, BEC), normalized to expression levels in HMVEC-dLy (LEC). Four biological replicates were analyzed. All graphs are analyzed by t-test and the mean ± SD is shown.

(a) Confocal images of the vasculature in the mid-trunk of a five dpf Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 double-transgenic animal with mCherry positive arterial blood vessels (red), EGFP positive lymphatic vessels (green), and mCherry and EGFP double positive venous blood vessels (yellow). The different vessels are labeled: DA, dorsal aorta; DLLV, dorsal longitudinal lymphatic vessel; DLAV, dorsal longitudinal anastomotic vessel; ISLV: intersegmental lymphatic vessel; aISV: arterial intersegmental vessel; vISV: venous intersegmental vessel; PCV, posterior cardinal vein; PL, parachordal line; TD, thoracic duct; CCL, collateral cardinal lymphatics; VTA, vertebral artery. (b) Confocal image from panel a with lymphatic vessels pseudocolored in green and other vessels in gray, for easier visualization of the lymphatic network. (c) Quantitative TaqMan RT-PCR measurement of the relative expression of lymphatic endothelial cell (LEC) genes lyve1b and prox1a in FACS-sorted zebrafish endothelial cell populations. Expression is normalized to the arterial (mCherry-positive) endothelial cell population. (d) Quantitative TaqMan RT-PCR measurement of the relative expression of blood endothelial cell (BEC) genes kdrl and cdh5 in FACS-sorted zebrafish endothelial cell populations. Expression is normalized to the lymphatic (EGFP-positive) endothelial cell population. Scale bars = 100 µm (a,b). Biological duplicates were analyzed. All graphs are analyzed by t-test and the mean ± SD is shown.

(a) Schematic diagram showing the location of miR-204 in intronic region of TRPM3 gene and the 100% conservation of its mature microRNA sequence amongst various vertebrate species. (b) Schematic of two additional zebrafish mir-204 loci located in intron 5 and 4 of trpm1a and trpm1b, respectively. (c) Bright field microscopic images of 5 dpf zebrafish larve that were injected with 0.5 ng of control MO (top) 0.5 ng of pan-204 MO (middle), or 0.75 ng of pan-204 MO (bottom).

(a) Schematic diagram showing the location of miR-204 in intronic region of TRPM3 gene and the 100% conservation of its mature microRNA sequence amongst various vertebrate species. (b) Schematic of two additional zebrafish mir-204 loci located in intron 5 and 4 of trpm1a and trpm1b, respectively. (c) Bright field microscopic images of 5 dpf zebrafish larve that were injected with 0.5 ng of control MO (top) 0.5 ng of pan-204 MO (middle), or 0.75 ng of pan-204 MO (bottom).

(a) Sequence alignment of MO1, 2, 3. (b-h) Representative confocal images of 5 dpf Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 double-transgenic animals injected with 0.5 ng of MO1 (b), MO2 (c), or MO3 (d); injected pairwise with 0.5 ng each of MO1+MO2 (e), MO1+MO3 (f), or MO2+MO3 (g), for a final combined MO dose of 1.0 ng in each case; injected with 0.5 ng each of MO1+MO2+MO3 (h), for a final combined MO dose of 1.5 ng. (i) Quantitation of the single MO injections in panels b-d. (j) Quantitation of the combined MO injections in panels e-h. All images are lateral views, rostral to the left. Scale bar: 100 μm (b). All graphs are analyzed by t-test and the mean ± SD is shown.

(a) Quantitative TaqMan RT-PCR measurement of the relative levels of mature mir-204 in one dpf mir-204–1^-/- mutants and wildtype siblings, normalized to wild type levels. (b,c) Confocal images of 2 dpf Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 mir-204–1^-/- mutant (c) or MO2-injected mir-204–1^-/- mutant (d) animals. (d) Quantitation of the number of PL sprouts in animals as in panels b and c. Scale bar: 100 μm (b). All graphs are analyzed by t-test and the mean ± SD is shown.

(a) Quantitative TaqMan RT-PCR measurement of the relative levels of mature mir-204 in one dpf mir-204–1^-/- mutants and wildtype siblings, normalized to wild type levels. (b,c) Confocal images of 2 dpf Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 mir-204–1^-/- mutant (c) or MO2-injected mir-204–1^-/- mutant (d) animals. (d) Quantitation of the number of PL sprouts in animals as in panels b and c. Scale bar: 100 μm (b). All graphs are analyzed by t-test and the mean ± SD is shown.

(a) Sequence alignment of mir-204–1, −2, and −3 wildtype and deletion mutant. The mature mir-204 sequence are noted with magenta lettering and the seed sequence is highlighted in blue. (b) Quantitative TaqMan RT-PCR measurement of the relative levels of mature mir-204 in five dpf mir-204 triple mutants and their mir-204–1^-/- siblings, normalized to mir-204–1^-/- mutants. Three biological replicates were analyzed. (c–e) Confocal image of 5 dpf Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 mir-204 triple mutant (c), mir-204–1 and −2 double mutant (d), and mir-204–1 and −3 double mutant (e) animals. All images are lateral views, rostral to the left. Scale bar: 100 μm (c). All graphs are analyzed by t-test and the mean ± SD is shown.

(a,b) Representative confocal images of mid-trunk of 3 dpf of (a) Tg(mrc1a:eGFP) or (b) Tg(mrc1a:mir204-eGFP). The thoracic duct is pseudocolored in green, with other vessels in gray. (c) Quantification of thoracic duct formation in animals as in panels a and b. The number of somitic segments with an intact thoracic duct was counted, with the same seven mid-trunk somites measured in each animal. (d) Quantification of the area between the dorsal aorta (DA) and posterior cardinal vein (PCV) in animals as in panels a and b, as an indicator of developmental timing. (e) Quantitation of mir-204 level measured by TaqMan qPCR from whole embryo at three dpf. All graphs are analyzed by t-test and the mean ± SD is shown.

(a,b) Representative confocal images of mid-trunk of 3 dpf of (a) Tg(mrc1a:eGFP) or (b) Tg(mrc1a:mir204-eGFP). The thoracic duct is pseudocolored in green, with other vessels in gray. (c) Quantification of thoracic duct formation in animals as in panels a and b. The number of somitic segments with an intact thoracic duct was counted, with the same seven mid-trunk somites measured in each animal. (d) Quantification of the area between the dorsal aorta (DA) and posterior cardinal vein (PCV) in animals as in panels a and b, as an indicator of developmental timing. (e) Quantitation of mir-204 level measured by TaqMan qPCR from whole embryo at three dpf. All graphs are analyzed by t-test and the mean ± SD is shown.

(a) Sequence alignment of mature miR-204 (middle line) and its target region in the human NFATC1 3’UTR (top line). A mutant form of the human NFATC1 3’UTR used for the luciferase assay in panel b is also shown (bottom line; four mismatches in the seed binding region are highlighted in red). (b) Quantitative luciferase reporter assay using wild type or mutant forms of the human NFATC1 3’UTR transfected into HEK293 cells together with either miR-204 or miR-126 (control). Four biological replicates were analyzed. (c) Quantitative TaqMan RT-PCR measurement of relative endogenous NFATC1 transcript levels in human LEC (HMVEC-dLy) transfected with miR-204-mimic or miR-204-antagomir, normalized to control mock transfected levels. (d) Sequence alignment of mature miR-204 (middle line) and its target region in the zebrafish nfatc1 3’UTR (top line). A mutant form of the zebrafish nfatc1 3’UTR used for the luciferase assay in panel e is also shown (bottom line; four mismatches in the seed binding region are highlighted in red). (e) Quantitative luciferase reporter assay using wildtype or mutant forms of the zebrafish nfatc1 3’UTR co-transfected into HEK293 cells together with either miR-204 or miR-126 (control). Four biological replicates were analyzed. (f) Quantitative TaqMan RT-PCR measurement of relative endogenous zebrafish nfatc1 transcript levels in five dpf animals that were injected with either control MO or pan-204 MO. Three biological replicates were analyzed. All graphs are analyzed by t-test and the mean ± SD is shown.

10.7554/eLife.46007.020Figure 5—source data 1.

(a,b) Confocal images of the mid-trunk of 5 dpf Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 control (a) and nfatc1 ATG MO-injected (b) animals. The dashed boxes in panels a and b show the areas magnified in panels c and d, respectively. (c,d) Magnified images from panels a and b, with the thoracic duct pseudocolored in green and other vessels in gray. (e) Quantitation of thoracic duct size measured as the area encompassed by the thoracic duct in confocal images of the same seven mid-trunk somitic segments in five dpf wildtype (n = 11) and nfatc1 MO-injected (n = 9) animals. (f,g) Confocal images of the mid-trunk of 5 dpf DMSO (f) and cyclosporine A (g) treated animals. The dashed boxes in panels f and g show the areas magnified in panels h and i, respectively. (h,i) Magnified images from panels f and g, with the thoracic duct pseudocolored in green and other vessels in gray. (j) Quantitation of thoracic duct size measured as the area encompassed by the thoracic duct in confocal images of 7 somitic segments from five dpf DMSO (n = 16) and cyclosporine A (n = 17) treated animals. All images are lateral views, rostral to the left. Scale bar: 100 μm (a,c). All graphs are analyzed by t-test and the mean ± SD is shown.

(a,b) Confocal images of the mid-trunk of 5 dpf Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 control (a) and nfatc1 ATG MO-injected (b) animals. The dashed boxes in panels a and b show the areas magnified in panels c and d, respectively. (c,d) Magnified images from panels a and b, with the thoracic duct pseudocolored in green and other vessels in gray. (e) Quantitation of thoracic duct size measured as the area encompassed by the thoracic duct in confocal images of the same seven mid-trunk somitic segments in five dpf wildtype (n = 11) and nfatc1 MO-injected (n = 9) animals. (f,g) Confocal images of the mid-trunk of 5 dpf DMSO (f) and cyclosporine A (g) treated animals. The dashed boxes in panels f and g show the areas magnified in panels h and i, respectively. (h,i) Magnified images from panels f and g, with the thoracic duct pseudocolored in green and other vessels in gray. (j) Quantitation of thoracic duct size measured as the area encompassed by the thoracic duct in confocal images of 7 somitic segments from five dpf DMSO (n = 16) and cyclosporine A (n = 17) treated animals. All images are lateral views, rostral to the left. Scale bar: 100 μm (a,c). All graphs are analyzed by t-test and the mean ± SD is shown.

(a–c) Confocal images of the mid-trunk of 5 dpf control (a) pan-204 MO-injected (b) or pan-204 MO and nfatc1 splice MO co-injected (c) animals. White dotted boxes in panels a-c show areas magnified in panels d-f, respectively, while white dashed boxes show areas magnified in panels g-i, respectively. (d–f) Magnified images from panels a-c with the thoracic duct (TD) pseudocolored in green and other vessels in gray. The TD is labeled, and the absence of the TD is noted with asterisks. (g–i) Magnified images from panels a-c with the dorsal longitudinal lymphatic vessel (DLLV) pseudocolored in green and other vessels in gray. The DLLV is labeled, and the absence of the DLLV is noted with asterisks. (j) Quantification of thoracic duct (TD) formation in five dpf control (n = 8), pan-204 MO-injected (n = 19), or pan-204 MO and nfatc1 splice MO co-injected animals (n = 21). A total of 7 mid-trunk somitic segments were scored in each animal for the presence or absence of an intact TD. (k) Quantification of dorsal longitudinal lymphatic vessel (DLLV) formation in five dpf control (n = 8), pan-204 MO-injected (n = 19), or pan-204 MO and nfatc1 splice MO co-injected animals (n = 21). A total of 7 mid-trunk somitic segments were scored in each animal for the presence or absence of an intact DLLV. (l) Schematic diagrams illustrating five dpf zebrafish trunk lymphatic vessels present in (i) normal control, (ii) mir-204 deficient, (iii) nfatc1-deficient, and (iv) mir-204- and nfatc1-deficient animals. Suppression of mir-204 leads to loss of lymphatic vessels (ii), while nfatc1 deficiency causes lymphatic (thoracic duct) hyperplasia (iii). The lymphatic defects in mir-204 deficient animals can be rescued by simultaneous suppression of nfatc1 (iv). All images are lateral views of Tg(mrc1a:eGFP)^y251, Tg(kdrl:mCherry)^y171 double-transgenic animals, rostral to the left. Scale bar = 100 μm (c,i). All graphs are analyzed by t-test and the mean ± SD is shown.

10.7554/eLife.46007.025Figure 7—source data 1.