Development and characterization of transgenic zebrafish lines expressing zFucci cell-cycle biosensors in endothelial cells (ECs). (A) Schematic representation of the plasmids used for delivering transgenes expressing mVenus-zGem (1/100) (mV-zGem) and mCherry-zCdt1(1/190) (mC-zCdt) under control of the flk1 promoter. (B) Upper, fluorescence image (mV-zGem [green] and mC-zCdt1 [red]) of an EC-zFucci, Tg(flk1:mV-zGem);(flk1:mC-Cdt) embryo, at 31 hpf; lower, bright field images. (C) 3D-rendered confocal stack fluorescence images of the caudal regions of the Tg(flk1:mV-zGem);(fli1:Myr-mC) embryo at 33 hpf. Upper, mVenus image; lower, the merged image of mVenus (green) and mCherry (red). All of the confocal fluorescence images are lateral views and displayed as anterior to the left, unless otherwise described, in the following images. (D) 3D-rendered confocal images of the caudal regions of the Tg(flk1:mC-Cdt);(fli1:Myr-GFP) embryo at 48 hpf. Upper, mCherry image; lower, the merged image of mVenus (green) and mCherry (red). (E) Images of the EC-zFucci embryo at 29.5 hpf treated with EdU for 1 h. mVenus images (green), mCherry images (red) and EdU images visualized by Alexa 647-azide (blue) of the trunk vessel are shown as indicated at the lower left corner of the image. Yellow, pink and white arrowheads indicate mV-zGem/EdU double-positive cells, mV-zGem-positive/EdU-negative cells and mC-zCdt-positive/EdU-negative cells, respectively. (F) Percentages of mV-zGem-positive, mC-zCdt-positive and mV-zGem/mC-zCdt double positive cells labeled without () or with (+) EdU as observed in E were quantified (n=55). Scale bars, 200 μm (B) and 100 μm (C–E).

Validation of the EC-zFucci Tg fish line. (A) Confocal 3D fluorescence image of the intersegmental vessel (ISV) of an EC-zFucci embryo at 36 hpf (first column) and the corresponding subsequent time-lapse images (from second to sixth columns) at the elapsed time (h:min) indicated at the bottom. Top, mVenus images; middle, mCherry images; bottom, the merged images. Note that an mV-zGem-positive cell (arrows at 0:00 and 0:20) divides into two daughter cells (arrows at 0:50), which subsequently lose mVenus fluorescence and start to emit mCherry fluorescence (arrows at 3:10, 3:50 and 8:10). (B) Confocal 3D fluorescence image of the ISV of an EC-zFucci embryo at 38 hpf (first column) and the corresponding subsequent time-lapse images (from second to seventh columns) at the elapsed time (h:min) indicated at the bottom are shown, essentially as in A. Note that the mC-zCdt-positive cell (arrows at 0:00 and 1:00) loses mCherry fluorescence (arrow at 1:10), starts to emit mVenus fluorescence (arrows at 2:30, 5:00 and 6:00), and subsequently divides into two daughter cells (arrows at 6:20 and 7:00). (C) 3D-rendered confocal images of the caudal regions of 48 hpf Tg(flk1:mV-zGem);(fli1:Myr-mC) embryos treated with either DMSO or both hydroxyurea and aphidicolin (HU/APH) from 30 to 48 hpf. Upper, mVenus images; lower, the merged images of mVenus and mCherry. Scale bars, 100 μm. (D) Duration: time that mVenus-positive cells in the EC-zFucci Tg embryos at 36 hpf continue to emit mVenus fluorescence. Tg(flk1:mV-zGem);(fli1:Myr-mC) embryos were treated from 30 hpf with either DMSO (closed circles) or HU/APH (open circles), and time-lapse imaged starting at 36 hpf for 9 h 20 min to track the mVenus-positive cells. The y-axis indicates the number of mVenus-positive cells, while the x-axis shows the time after starting the time-lapse imaging. Data are expressed as percentages relative to that observed at the beginning of the imaging (DMSO [n=35], HU/APH [n=41]).

Cell-cycle progression of ECs during ISV formation. (A, B) Time-lapse confocal imaging of ISV sprouting from the dorsal aorta (DA) in the trunk region of a Tg(flk1:mV-zGem);(flk1:NLS-mC) fish embryo. (A) ISVs used for imaging analyses. An ISV of the boxed region is presented schematically. (B) 3D-rendered confocal 3D image of the embryo at 24.5 hpf (left upper) and its subsequent time-lapse images at the elapsed time (hr:min) indicated at the bottom (mVenus [green] and mCherry [red]). Note that an EC in the S/G2/M phase sprouts from the DA (arrows in 0:00 and 1:50), divides during dorsal migration (arrows in 3:10, 3:50, 4:20), leaves the M-phase (arrows in 5:10 and 6:40), and further migrates dorsally (arrow in 9:00) to form an ISV. (C) Percentages of mV-zGem-negative () and mV-zGem-positive (+) ECs involved only in forming ISV by sprouting from the DA. Data are shown as means±s.d. (n=5). (D) Types of ECs that divide during ISV formation. Cell division was defined by the division of mV-zGem-positive ECs. The numbers of ISVs showing tip cell division (Tip), stalk cell division (Stalk) or division of both tip and stalk cells (Tip/Stalk) or showing no cell division (None) were divided by the total number of ISVs observed (n=34). (E) Quantification of the number of EC divisions during ISV formation. The numbers of ISVs showing no cell division, one, two, or three rounds of cell division divided by the total numbers of ISVs observed (n=34). (F) A model of cell-cycle regulation of ECs during ISV formation. (G) 3D-rendered confocal 3D images of the trunk regions of Tg(flk1:mV-zGem);(flk1:NLS-mC) embryos injected with control MO (upper panel) or dll4 MO (lower panel) at 48 hpf. Upper, merged images (mVenus [green] and mCherry [red]) of control morphants; lower, those of dll4 morphants. (H, I) The number of mV-zGem-positive cells (H) and the total number of ECs (I) within the five ISVs. Data are shown as means±s.d. (n=11). (J) 3D-rendered confocal images of the trunk regions of 32 hpf Tg(fli1:Myr-mC);( flk1:NLS-Eos) embryos treated from 20 hpf with DMSO (left column) or HU/APH (right column). Upper, Eos images; lower, the merged images of Eos and mCherry. Scale bars, 100 μm. (K) The numbers of ECs in the posterior cardinal vein (PCV), DA and ISV/dorsal longitudinal anastomotic vessel (DLAV) within the five ISVs as observed in J were counted, and shown as means±s.d. (DMSO [n=9], HU/APH [n=11]). ISV, intersegmental vessel; DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessels. ^NNp<0.01, ^NNNp<0.001 (C, H, I, K). n.s., no significance.

Cell-cycle progression of ECs during caudal vessel formation. (A) 3D-rendered confocal images of the caudal vessels of Tg(flk1:mV-zGem);(fli1:Myr-mC) embryos at 24, 30, 36 and 48 hpf as indicated in the first column from the left. Bright field images (left) and fluorescence images (as indicated at the top). The boxed areas of the second and the third columns are enlarged in the fourth and fifth columns, respectively. CA, caudal vessel; VC, vascular cord; ISV, intersegmental vessel; CVP, caudal vein plexus; DLAV, dorsal longitudinal anastomotic vessels. Scale bars, 100 μm. (B) Time-lapse confocal imaging of caudal vessel formation in the early stage (from 25 to 29 hpf) in the Tg(flk1:mV-zGem);(flk1:NLS-mC) embryos. The confocal 3D image of the embryo at 25 hpf (top) and subsequent time-lapse images (from second to fourth rows) as indicated by the elapsed time (hr:min) at the top right. Left, merged (mVenus and mCherry); center, enlarged mVenus images of the boxed region of the left panel; right, enlarged merged image of the boxed region of the left panel. The cell movement of the posterior-most cell at the beginning (crosses) and at the end (circles) during time-lapse imaging, as analyzed employing a cell tracking application. (C) Tg(flk1:mV-zGem);(flk1:NLS-mC) embryos in the late stage (from 33–37 hpf) were similarly time-lapse imaged and analyzed as in B. Note that NLS-mCherry-positive cells merge just posterior to the growing edges of caudal vessels in the early stage, but not in the late stage, of caudal vessel formation. (D) Time-lapse confocal imaging of caudal vessel formation at the early stage (from 23 to 27 hpf) in the Tg(flk1:NLS-Eos) embryos. Confocal 3D images (at 23 hpf) and subsequent time-lapse images of photoconverted NLS-Eos (red)-marked ECs (encircled) from original NLS-Eos (green)-marked cells by laser irradiation at 23 hpf. Elapsed time (hr:min). Upper, merged images of Eos (green) and photoconverted Eos (red); lower, photoconverted Eos (red) images. Arrowheads indicate the anteriormost and posteriormost of the photoconverted cells. (E) Tg(flk1:NLS-Eos) embryos in the late stage (from 33 to 40 hpf) were time-lapse imaged and analyzed as in D.

Effect of inhibiting EC proliferation on caudal vessel formation. (A) 3D-rendered confocal images of the caudal regions of 48 hpf Tg(fli1:Myr-mC);( flk1:NLS-Eos) embryos treated from 24 hpf with DMSO (upper) or HU/APH (lower). Left column, Eos images; from the second to fifth columns, the merged images of Eos (green) and mCherry (red). The boxed areas in the second column and the corresponding single scan confocal images are enlarged in the third and fourth columns, respectively. The cross-sectional single plane images of the areas indicated by dotted lines on the fourth column are also shown in the fifth column. ISV, intersegmental vessel; CA, caudal artery; CV, caudal vein; CVP, caudal vein plexus. Scale bars, 100 μm (first and fourth columns) and 50 μm (fifth column). (B) The numbers of ECs in the CV/ CVP, CA and ISV/DLAV within the five ISVs, as observed in A, were counted and shown as means±s.d. (DMSO [n=10], HU/APH [n=12]). ^NNp<0.01, ^NNNp<0.001. n.s., no significance.

Cell-cycle progression of ECs during formation of hindbrain vasculature. (A) 3D-rendered confocal images of the hindbrain vasculature in Tg(flk1:mV-zGem);(fli1:Myr-mC) embryos at 28 hpf. Left, mVenus image; right, the merged image of mVenus (green) and mCherry. All confocal images in this figure are dorsal views and displayed as anterior to the top. (B, C) 3D-rendered confocal fluorescence image of the hindbrain vasculature in Tg(flk1:mV-zGem);(fli1:Myr-mC) embryos at 28.5 hpf (B) or 34 hpf (C) and the corresponding subsequent time-lapse images at the elapsed time (h:min) indicated at the bottom of the upper panel. Upper, mVenus image; lower, the merged image of mVenus (green) and mCherry. Arrowheads indicate mVenus-positive ECs that sprout from the primordial hindbrain channels (PHBCs) (B) and the ECs that caudally migrate to form the basilar artery (BA) (C). (D) Confocal fluorescence image of the hindbrain vasculature in 36 hpf Tg(fli1:Myr-mC);( flk1:NLS-Eos) embryos treated from 27 hpf with DMSO (left) or HU/APH (right). mCherry and the merged images of mCherry (red) and Eos (green) are shown as indicated at the top. Arrowheads indicate defective formation of the BA. (E) Confocal fluorescence images of the hindbrain vasculature in Tg(flk1:mV-zGem);(fli1:Myr-mC) embryos at 39 hpf and the corresponding subsequent time-lapse images at the elapsed time (h:min) indicated at the bottom of the upper panel are shown, as in B. Arrowheads indicate mVenus-positive ECs that sprout from PHBCs to form the CtAs. (F) Confocal fluorescence images of the hindbrain vasculature in 48 hpf Tg(fli1:Myr-mC);( flk1:NLS-Eos) embryos treated from 34 hpf with DMSO (left) or HU/APH (right) are shown, as in D. Asterisks and arrowheads indicate CtAs and ECs failing to sprout from PHBCs to form the CtAs, respectively. PHBC, primordial hindbrain channels; LDA, lateral dorsal aorta; BA, basilar artery; CtA, central artery. Scale bars, 100 μm (A–F).

Cell-cycle progression of ECs during the late stage of vascular development. (A) 3D-rendered confocal images of the trunk (left) and caudal (right) vasculature in the Tg(flk1:mV-zGem);(fli1:Myr-mC) embryos at 2, 3 and 4 dpf as indicated at the left. Left, mVenus image; right, the merged image of mVenus (green) and mCherry (red). Scale bars, 100 μm. (B) The numbers of mVenus-positive ECs in the trunk and caudal vasculature at 2, 3 and 4 dpf, as observed in A, were counted and shown as means±s.d. (2 dpf [n=16], 3 dpf [n=14], 4 dpf [n=13]). (C) The numbers of mVenus-positive ECs in the DLAV, CA, arterial ISV (aISV), CV/CVP and venous ISV (vISV) of the caudal vasculature at 2 and 3 dpf were quantified, and then expressed as percentages of the total number (2 dpf [n=97], 3 dpf [n=28]).

Time-lapse confocal imaging of an EC-zFucci embryo. (A) 3D-rendered confocal images of the trunk regions of Tg(flk1:mV-zGem);(flk1:NLS-mC) embryos injected with control MO (upper panel) or cdkn1b MO (lower panel) at 48 hpf. Upper, merged images (mVenus [green] and mCherry [red]) of control morphants; lower, those of cdkn1b morphants. (B) The number of mV-zGem-positive cells within the five ISVs. Data are shown as mean ± s.d. (n=15). ***p<0.001.