1. Developmental Biology

Yap1 promotes sprouting and proliferation of lymphatic progenitors downstream of Vegfc in the zebrafish trunk

  1. Lin Grimm
  2. Hiroyuki Nakajima
  3. Smrita Chaudhury
  4. Neil I Bower
  5. Kazuhide S Okuda
  6. Andrew G Cox
  7. Natasha L Harvey
  8. Katarzyna Koltowska
  9. Naoki Mochizuki
  10. Benjamin M Hogan  Is a corresponding author
  1. The University of Queensland, Australia
  2. National Cerebral and Cardiovascular Centre Research Institute, Japan
  3. Peter MacCallum Cancer Centre, Australia
  4. University of Melbourne, Australia
  5. University of South Australia, SA Pathology, Australia
  6. Uppsala University, Sweden
https://doi.org/10.7554/eLife.42881
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Cite this article
  1. Lin Grimm
  2. Hiroyuki Nakajima
  3. Smrita Chaudhury
  4. Neil I Bower
  5. Kazuhide S Okuda
  6. Andrew G Cox
  7. Natasha L Harvey
  8. Katarzyna Koltowska
  9. Naoki Mochizuki
  10. Benjamin M Hogan
(2019)
Yap1 promotes sprouting and proliferation of lymphatic progenitors downstream of Vegfc in the zebrafish trunk
eLife 8:e42881.
https://doi.org/10.7554/eLife.42881
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5 figures, 6 videos, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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Nuclear EGFP-YAP changes dynamically in the developing trunk lymphatic vasculature.

(A–B) The TEAD reporter line [Tg(fli1:Gal4db-TEAD2ΔN-2A-mC);(UAS:GFP)] shows Yap1 activity in vasculature, parachordal LECs (PLs) and cardinal vein sprouts (arrowhead) of the 2 dpf trunk (A) as well as in the thoracic duct (TD) at 5 dpf (B). Scale bars: 40 μm. (C) Maximum projection of 8 PLs in a two dpf embryo showing EGFP-YAP in green [Tg(fli1:EGFP-YAP)] and the nucleus in red (C’), [Tg(flil:H2B-mCherry)] and merge (C”’). Scale bar: 25 μm. (D–E) High power single z-sections of selected PLs from C, showing nuclear YAP in PL2 and PL3, but low nuclear YAP in PL4 (D–D’) and PL7 (E–E’). Scale bars: 10 μm. (F) Quantification of nuclear EGFP/mCherry average pixel intensity across individual PLs from multiple embryos at 2 dpf. Each bar represents a single PL (n = 47), each grey shade a different embryo (n = 5). PLs in (C and D) highlighted in the green box. EGFP/mCherry Ratios have been calculated using mean fluorescent intensities in 3D. (G) Scatter Plot of the Nuclear EGFP/mCherry average pixel intensity for individual PLs (n = 5 embryos). Each colour indicates PLs from a different embryo. Values calculated in 3D measurements of the mean fluorescent intensity for EGFP and mCherry (0.66 ± 0.04, n = 47). (H) Pearson Correlation Plot of the Nuclear EGFP/mCherry Ratio values in F and the Nuclear/Cytoplasmic EGFP Ratio values in Figure 1—figure supplement 1B (r = 0.52, 95% confidence intervals: 0.27 to 0.70, R square = 0.27, p=0.0002(***)). The two distinct approaches produce correlative measurements. (I) Average nuclear pixel intensity graphs from cell tracks of single PLs time-lapse imaged from 2 to 3 dpf. EGFP-YAP intensity (green) is compared over time with H2B-mCherry (red) intensity in individual nuclei. Arrow points to a cell division during time-lapse.

https://doi.org/10.7554/eLife.42881.002
Figure 1—source data 1

Measurements of EGFP-YAP in PLs.

https://doi.org/10.7554/eLife.42881.005
Figure 1—figure supplement 1
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Measurements of EGFP-YAP intensity in lymphatic progenitor nuclei over time.

(A). Nuclear EGFP/mCherry average pixel intensity ratios measured manually on a single z-plane at the centre of the nucleus in 2D. Same PLs as in Figure 1F. (B). Nuclear/cytoplasmic EGFP average pixel intensity ratio measured manually on a single z-stack at the centre of the nucleus using the same approach A). Same PLs as in Figure 1F. (C) Scatter Plot of the Nuclear/Cytoplasmic EGFP average pixel intensity measured as a ratio across individual PLs (1.81 ± 0.11, n = 47) from multiple embryos (n = 5). Each colour indicates PLs from a different embryo. Values above one correspond to PLs with more EGFP-YAP in the nucleus while values below one correspond to PLs with more EGFP-YAP in the cytoplasm. (D) Scatter Plot of the fluorescent intensity values calculated manually in 2D on a single z-stack image for each PL selecting the centre of the nucleus (0.58 ± 0.04, n = 47). (E) Pearson Correlation comparing the similarity between the automated 3D- analysis and the manual 2D- analysis of the nuclear EGFP/mCherry mean intensity ratios. The values from both analyses show a strong correlation (r = 0.69, confidence intervals: 0.50 to 0.81, R squared = 0.47, p<0.0001 (****)). (F) Nuclear EGFP/mCherry ratios of single PLs (n = 6) tracked in 90 min high speed spinning disc time-lapse Videos (see Supplementary Video No 2). (F’–F”) Mean nuclear EGFP and mCherry intensities of single PLs from time-lapse Videos. Nuclear EGFP-YAP intensity remains relatively stable over the 90 min time interval (z-stack acquired every 1 min). The slow decrease in fluorescent intensity is due to signal bleaching. (G) Further examples of relative fluorescent intensity graphs generated from PL nuclei followed from 2 to 3 dpf in time-lapse Videos. High EGFP intensity corresponds to high Yap1 protein abundance in the nucleus (boxed area). H2B-mCherry fluorescence highlights the stable concentration of this control fluorophore in PL nuclei over time. Both fluorophores driven by the same promoter.

https://doi.org/10.7554/eLife.42881.003
Figure 1—figure supplement 1—source data 1

Methodological Analysis of EGFP-YAP Intensities in PLs.

https://doi.org/10.7554/eLife.42881.004
Figure 2 with 2 supplements
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Yap1 acts cell autonomously to control trunk lymphangiogenesis in zebrafish.

(A) Overall morphology of sibling (left) and MZyap1ncv101-/- mutant (right) at 5 dpf. Arrowheads indicate mild craniofacial defects and absent swim bladder. Scale bar 200 μm. (B–C) Trunk vasculature of sibling and MZyap1ncv101-/- mutant embryos at 5 dpf. Veins and lymphatics are displayed in white [Tg(dab2b:EGFP)], erythrocytes show normal blood flow in red [Tg(gata1:DsRed)]. Asterisks mark absent lymphatic vessels. (C) Trunk vasculature of sibling and MZyap1mw48-/- mutant at 6 dpf. Vascular nuclei are marked in green [Tg(fli1a:nEGFP)], venous and lymphatic vessels in white [Tg(−5.2lyve1b:DsRed)]. Asterisks indicate absent lymphatic vessels. Scale bars: 50 μm in B and C. (D) Percentage of TD fragments formed per somite, scored across six somites in total for siblings and MZyap1ncv101-/-mutants at 5 dpf (sibling: 100% ± 0, n = 23; MZyap1ncv101-/-: 49% ± 7.33, n = 22; p,0.0001). (E) Quantification of the total number of LECs across 6 somites at 6 dpf (sibling: 37 ± 1.91, n = 11; MZyap1mw48-/-: 8 ± 3.79; p<0.0001). (F) Schematic showing the cell transplantation technique. Blastomere cells are transplanted from donor (EGFP) into host (mCherry) embryos. This results in a chimeric host embryo (right) with randomly located, transplanted EC grafts. (G) Representative images of host chimeric trunk vessels at 5 dpf. Wildtype (wt) donor ECs contribute to all vascular EC types, while MZyap1ncv101-/- mutant ECs show reduced propensity to contribute to lymphatic structures. Asterisk marks missing TD. Scale bars: 50 μm. (H) Graft sizes analysed for vascular grafts. Numbers within bars represent the number of embryos scored for each graft size. (I) Percentage of embryos with EC grafts contributing to arterial (wt into wt: 96% ± 4; MZyap1ncv101-/- into wt: 100% ± 0; p=0.31, not significant (ns)), venous (wt into wt: 91% ± 6; MZyap1ncv101-/- into wt: 67% ± 10; p=0.04 (*)) and lymphatic vessels (wt into wt: 43% ± 10; MZyap1ncv101-/- into wt: 8% ± 6; p=0.005 (**)). Wildtype into wildtype total number of EC grafts: n = 23; MZyap1ncv101-/- mutant into wildtype total number of EC grafts: n = 24. Dorsal aorta (DA); Posterior Cardinal Vein (PCV); Thoracic Duct (TD); Intersegmental Lymphatic vessel (ISLV), Dorsal Longitudinal Lymphatic vessel (DLLV).

https://doi.org/10.7554/eLife.42881.009
Figure 2—source data 1

Measurements for the lymphatic phenotype of the MZyap1-/-mutant.

https://doi.org/10.7554/eLife.42881.014
Figure 2—figure supplement 1
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Zyap1-/- mutants only exhibit mild lymphatic defects in the trunk and MZyap1-/- mutants form facial lymphatics.

(A) Trunk lymphatics of sibling and Zyap1ncv101-/- mutant at 5 dpf. The vasculature is shown in red [Tg(kdrl:mCherry)], venous and lymphatics in green [Tg(dab2b:EGFP)]. Scale bar: 50 μm. (B) Heads of siblings and MZyap1ncv101-/- mutants showing facial lymphatics including the lateral facial lymphatic (LFL), medial facial lymphatic (MFL), otolithic lymphatic vessel (OLV), branchial arch lymphatics (LAA); and developing lymphatic loop (LL). Scale bar: 50 μm. (C) Percentage of thoracic duct (TD) segments formed across 6 somites at 5 dpf in siblings (98.17 ± 1.83, n = 18) and Zyap1ncv101-/- mutants (77.54 ± 10.14, n = 13; p=0.0269). (D) Score of facial lymphatics in siblings (LL: 0.75 ± 0.08, n = 10) and MZyap1ncv101-/- mutants (LL: 0.67 ± 0.11, n = 6; p=0.55 (ns)). Each point represents the vessel score for one embryo. Matrix scores for LFL, MFL and OFL between sibling and MZyap1ncv101-/- are identical, thus not significantly different.

https://doi.org/10.7554/eLife.42881.010
Figure 2—figure supplement 1—source data 1

Quantification of the lymphatic phenotype of the Zyap-/- mutant trunk and MZyap1-/-mutant craniofacial phenotype.

https://doi.org/10.7554/eLife.42881.011
Figure 2—figure supplement 2
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MZyap1-/- mutants do not show major defects in blood vessel formation.

(A) Blood vasculature (EC nuclei labelled in green) of sibling and MZyap1mw48-/- mutant at 24 hpf. S1-4 indicate the quantified sprouts of the sibling and M1-4 the equivalent for the MZyap1mw48-/- mutant in D–F). Dorsal aorta (DA). Scale bars in A–C): 50 μm. (B) Blood vasculature of [Tg(fli1a:nEGFP)] sibling and MZyap1mw48-/- mutant at 32 hpf. Posterior cardinal vein (PCV). (C) Trunk vasculature of sibling and MZyap1mw48-/- mutant at 50 hpf showing normal lumen formation in white ([Tg(kdrl:GFP)]). Missing parachordal LECs (PLs) are marked by asterisks. (D) Quantification of number of nuclei for all sprouts (S1-S4 for sibling, M1-4 for MZyap1mw48-/-) at 24 hpf (sibling: 2.73 ± 0.12, n = 56; MZyap1mw48-/-: 2.14 ± 0.13, n = 56; p=0.0013 (**)) and 32 hpf (sibling: 2.93 ± 0.09, n = 44; MZyap1mw48-/-: 2.91 ± 0.15, n = 44; p=0.90 (ns)). (E) Sprout length for all sprouts in μm (S1-S4 for sibling, M1-4 for MZyap1mw48-/-) at 24 hpf (sibling: 50.91 ± 1.70, n = 56; MZyap1mw48-/-: 44.74 ± 2.32, n = 56; p=0.034 (*)) and 32 hpf (sibling: 85.57 ± 2.98, n = 44; MZyap1mw48-/-: 74.57 ± 3, n = 44; p=0.01 (*)). (F) Quantification of nuclei number in single sprouts at 24 hpf (S1: 2.64 ± 0.20, n = 14, M1: 2.64 ± 0.23, n = 14, p>0.99 (ns); S2: 2.64 ± 0.19, n = 14, M2: 2.07 ± 0.25, n = 14, p=0.08 (ns); S3: 2.93 ± 0.37, n = 14, M3: 2.00 ± 0.28, n = 14, p=0.05 (ns); S4: 2.71 ± 0.16, n = 14, M4: 1.86 ± 0.27, n = 14, p=0.01 (*)) and 32 hpf (S1: 3.27 ± 0.14, n = 11, M1: 2.64 ± 0.24, n = 11, p=0.04 (*); S2: 3.09 ± 0.16, n = 11, M2: 2.91 ± 0.31, n = 11, p=0.61 (ns); S3: 2.63 ± 0.15, n = 11, M3: 3.18 ± 0.23, n = 11, p=0.06 (ns); S4: 2.73 ± 0.20, n = 11, M4: 2.91 ± 0.39, n = 11, p=0.68 (ns)).

https://doi.org/10.7554/eLife.42881.012
Figure 2—figure supplement 2—source data 1

Characterisation of angiogenic sprouting in the MZyap1-/-mutants.

https://doi.org/10.7554/eLife.42881.013
Figure 3
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MZyap1-/- mutants display defects in LEC numbers but not specification.

(A) Trunk vasculature (EC nuclei in green, veins and lymphatics in white) of sibling and MZyap1mw48-/- mutant at 2 dpf. Arrowheads indicate posterior cardinal vein (PCV) sprouts. Asterisks mark absent parachordal LECs (PLs). Dorsal aorta (DA). Scale bars: 50 μm. (B) Total number of lyve1-positive ECs departing the PCV across 6 somites at 2 dpf. (C) Number of endothelial cells in venous intersegmental vessels (vISV) across 6 somites at 2 dpf (sibling: 13 ± 0.94, n = 14; MZyap1-/-: 12 ± 1.33, n = 14; p=0.24 (ns)). (D) Number of PLs scored across 6 somites at 2 dpf (sibling: 3 ± 0.56, n = 14; MZyap1-/-: 0.36 ± 0.23, n = 14; p<0.0001 (****)). (E) Number of PLs scored across 6 somites at 3 dpf (sibling: 4.5 ± 0.20, n = 14; MZyap1-/-: 3.31 ± 0.36, n = 13; p=0.0075(**)). (F) Immunofluorescence staining for EC nuclei (green) and Prox1 (red) in sibling and MZyap1mw48-/- mutants at 36 hpf. Arrows point to Prox1+ LEC progenitors. Scale bars: 30 μm. (G) Quantification of Prox1 +cells in PCV and CV sprouts scored across 6 somites at 36 hpf (sibling: 3.47 ± 0.65, n = 19; MZyap1-/-: 3.31 ± 0.61, n = 16; p=0.86 (ns)). (H) Maximum projection stills from time-lapse Videos from 32 to 65 hpf. Sibling still images show normal lymphangiogenesis with PCV sprouts, PL formation, sprout detachment and PL proliferation (upper panels). MZyap1mw48-/- mutant one displays abnormal sprouting and looping of PCV sprouts that are retained until the end of the Video (central panels). MZyap1mw48-/- mutant two also exhibits abnormal sprouting and PCV loop formation but also forms PLs (lower panels). Scale bars: 25 μm. Timelapse imaging began at 32 hpf.

https://doi.org/10.7554/eLife.42881.015
Figure 3—source data 1

Cell counts for PCV-derived cells in the MZyap-/- mutants.

https://doi.org/10.7554/eLife.42881.016
Figure 4
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Yap1 mediates endothelial cell proliferation downstream of Vegfc.

(A) Trunk vasculature at 3 dpf displaying endothelial cells (ECs) in green from uninjected, p53 morpholino (MO) injected and p53 +yap1 MO injected embryos. Asterisks highlight missing parachordal LECs (PLs). Scale bars: 50 μm. (B) Trunk vasculature at 3 dpf from uninjected, p53 MO injected and p53 +yap1 MOs injected embryos of the Tg(prox1a:KalT4xUAS:uncTagRFP); Tg(10xUAS:vegfc) strain. yap1 MO injection rescues the EC proliferation phenotype. ECs in green. Asterisks mark missing PLs. Scale bars: 50 μm. (C) Quantification of total EC number across four somites in vegfc-unstimulated (uninjected: 343 ± 9, n = 18; p53 MO: 341 ± 9, n = 18; p53 +yap1 MOs: 248 ± 15, n = 18; p<0.0001(****)) and vegfc-stimulated embryos (uninjected: 811 ± 35, n = 18; p53 MO: 794 ± 38, n = 18; p53 +yap1 MOs: 212 ± 10, n = 18; p<0.0001(****)). (D) Schematic representation of vegfc-overexpressing cell transplantations for single muscle grafts in [Tg(fli1a:nEGFP);(−5.2lyve1b:DsRed2)] hosts of siblings and MZyap1mw48-/- mutants. (E) Schematic of transplanted host embryo at 3 dpf. Muscle grafts produce excessive Vegfc causing a hyperproliferation response in adjacent PLs. (F) Quantification of PL number within one somite responding to the vegfc-OE single muscle graft at 3 dpf (sibling: 23.00 ± 1.19, n = 18; MZyap1mw48-/-: 6.36 ± 0.71, n = 22; p<0.0001 (****)). (G) Examples of 3 different vegfc-OE muscle grafts (false coloured) in siblings (left panels) and MZyap1mw48-/- mutants (right panels). Merge images show ECs in green, lymphatic and venous ECs are red (yellow) [Tg(fli1a:nEGFP);(−5.2lyve1b:DsRed2)]. Scale bars: 25 μm.

https://doi.org/10.7554/eLife.42881.020
Figure 4—source data 1

Measurements of ECs in Vegfc-overexpression embryos.

https://doi.org/10.7554/eLife.42881.021
Figure 5 with 1 supplement
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Vegfc promotes nuclear Yap1 in developing lymphatic progenitors.

(A) Schematic showing the transplantation of vegfc-OE cells into EGFP-YAP reporter hosts. (B) Trunk vasculature of YAP reporter host embryos with neuron and muscle grafts expressing RFP (magenta) at 56 hpf. Control grafts (left) and vegfc-OE grafts (right). Posterior cardinal vein (PCV); Muscle (M); parachordal LECs (PLs); Neuron (N). Scale bars: 50 μm in B and C. (C) Heatmaps of maximum projections of EGFP-YAP from B) showing PLs and PCV. Red corresponds to high EGFP-YAP fluorescence. (C’) Lower panels show heatmaps of EGFP-YAP in PL nuclei. (D) EGFP intensity for each PL expressed as a ratio to the average of 6 dorsal aorta (DA) cells (unresponsive to vegfc). six embryos per group: untransplanted host (control) (PL n = 27), transplanted control without the UAS:vegfc construct (TP Control) (PL n = 40) and vegfc-OE transplanted embryos (TP vegfc-OE) (PL n = 251). Individual embryos indicated by an individual grey shade. (E) Heatmaps of EGFP-YAP depicting PL nuclei of embryos at 66 hpf that were treated with DMSO (left) and SL327 for 13 hr (hrs) (right). Max projections are from time-lapse Videos. Scale bars: 30 μM. (F) EGFP mean intensity for each PL/DA in panel D (Control: 1.61 ± 0.13, n = 27; TP Control: 1.93 ± 0.12, n = 40; p=0.08 (ns); TP vegfc-OE: 3.14 ± 0.07, n = 251; p<0.0001(****)). (G) Quantification of PL number across six somites for each group (Control: 5 ± 0.26, n = 6; TP Control: 8 ± 0.78, n = 16; p=0.0157 (*); TP vegfc-OE: 44 ± 8.04, n = 16; p=0.0001(***)). (H) Quantifications of the mean EGFP/mCherry fluorescent intensity ratio in PL nuclei from time-lapse Video stills. Embryos were mounted in DMSO as control (n = 5) or SL327 (15 μM) (n = 5) and continuously imaged for 12 hr starting at 54 hpf (1 hr drug treatment), finishing at 66 hpf (13 hr) (1 hr DMSO: 0.33 ± 0.02, n = 20; 1 hr SL327: 0.24 ± 0.02, n = 23; p=0.0036 (**)) (13 hr DMSO: 0.39 ± 0.02, n = 20; 13 hr SL327: 0.26 ± 0.03, n = 16; p=0.0006(***)). (I) Quantifications of mean EGFP/mCherry Ratio in PLs of embryos at 66 hpf treated with DMSO/SL327 for 12 hr from 54 hpf to 66 hpf without time-lapse imaging (12 hr DMSO: 0.47 ± 0.02, PLs n = 43; 13 hr SL327: 0.37 ± 0.03, PLs n = 22; p=0.02 (*)). (J) Quantification of PL number in embryos treated with DMSO/SL327 for 12 hr at 66 hpf without time-lapse imaging (12 hr DMSO: 8.6 ± 1.08, embryo n = 5; 13 hr SL327: 4.4 ± 0.81, embryo n = 5; p=0.014 (*)).

https://doi.org/10.7554/eLife.42881.022
Figure 5—source data 1

Measurements of LEC EGFP-YAP in response to Vegfc.

https://doi.org/10.7554/eLife.42881.025
Figure 5—figure supplement 1
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The autonomous EGFP-YAP response to Vegfc in transplanted ECs.

(A) Schematic displaying the transplantation of [Tg(fli1a:EGFP-YAP),(fli1a:H2B-mCherry)] endothelial cells (ECs) into control (no aberrant vegfc) and vegfc-overexpression (OE) host embryos at 3–4 hpf. (B) Average Nuclear EGFP Intensity as ratio of vegfc-responsive cells (parachordal LECs (PLs) and venous ECs (VECs))/vegfc unresponsive dorsal aorta (DA) cells. Grafted ECs in the TP vegfc-OE hosts show a higher amount of nuclear EGFP-YAP compared to the TP Control (TP Control: 1.52 ± 0.07, n = 52; TP vegfc-OE: 2.51 ± 0.14, n = 52; p=0.0001(****)). (C–D) Maximum projections of transplanted EGFP-YAP EC grafts in control hosts (C) and vegfc-OE hosts (D) at 2 dpf. Scale bars: 25 μm. (C’) shows the EGFP fluorescence as heatmap of the maximum projections for TP control and (D’) for TP vegfc-OE (red indicates high EGFP fluorescence) (central panels). (C”) and (D”) display nuclear EGFP-YAP in PCV and PL ECs as a heatmap (right panels). (E) Nuclear EGFP average pixel intensity as a ratio of vegfc- responsive cells (PLs and VECs) and vegfc-unresponsive cells (DA cells) for EC grafts in TP control (n = 7) and TP vegfc-OE host embryos (n = 6). Each grey shade represents a different embryo, each bar shows the EGFP-YAP fluorescent ratio of a vegfc-responsive cell nucleus. Statistics are shown in B.

https://doi.org/10.7554/eLife.42881.023
Figure 5—figure supplement 1—source data 1

Measurements of EGFP-YAP PLs in Vegfc-overexpression embryos.

https://doi.org/10.7554/eLife.42881.024

Videos

Video 1
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Time-lapse Video of the developing trunk vasculature of Tg(fli1:Gal4db-TEAD2ΔN-2A-mC);(UAS:GFP)] from 30 hpf – 2 dpf.

The EGFP signal can be seen to diminish in major trunk blood vessels but remains strong in the dorsal posterior cardinal vein (PCV) and parachordal LECs (PLs).

https://doi.org/10.7554/eLife.42881.006
Video 2
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High-speed time-lapse Video of the developing parachordal LECs (PLs) in [Tg(fli1a:EGFP-YAP),(fli1a:H2B-mCherry)] embryos from 2 dpf.

EGFP-YAP signal remains relatively stable over the imaged time period of 90 min. Z-stacks were acquired every minute. Fluorescence intensity of EGFP diminishes due to mild bleaching over time. The right panel shows the EGFP fluorescent intensity as a heatmap within the PL nuclei.

https://doi.org/10.7554/eLife.42881.007
Video 3
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Time-lapse Video of developing parachordal LECs (PLs) in [Tg(fli1a:EGFP-YAP),(fli1a:H2B-mCherry)] embryos from 2 to 3 dpf with images acquired in 20–22 min intervals.

The EGFP-YAP signal in individual PLs changes over time as shown in the lower panel displaying a heatmap of the nuclei. Dorsal aorta (DA), posterior cardinal vein (PCV).

https://doi.org/10.7554/eLife.42881.008
Video 4
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Time-lapse Video of the trunk vasculature of a sibling from 32 to 65 hpf in the [Tg(fli1a:nEGFP);(−5.2lyve1b:DsRed2)] background showing endothelial nuclei in green and lymphatic and venous endothelium in red.

Normal lymphangiogenesis involves sprouting from the posterior cardinal vein (PCV) to form PLs at the horizontal myoseptum. Dorsal aorta (DA). Scale bar: 25 μm.

https://doi.org/10.7554/eLife.42881.017
Video 5
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Time-lapse Video of MZyap1mw48-/- mutant 1 from 32 to 65 hpf with endothelial nuclei in green and lymphatic and venous endothelium in red.

Abnormal sprouts emerge from the posterior cardinal vein (PCV). Dorsal aorta (DA). Scale bar: 25 μm.

https://doi.org/10.7554/eLife.42881.018
Video 6
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Time-lapse Video of the trunk vasculature in MZyap1mw48-/- mutant 2 from 32 to 65 hpf, showing endothelial nuclei in green and lymphatic and venous endothelium in red.

Abnormal sprouts emerge from the posterior cardinal vein (PCV) anddisplaying loops as well as fusions with adjacent sprouts. Dorsal aorta (DA). Scale bar: 25 μm.

https://doi.org/10.7554/eLife.42881.019

Tables

Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information/
reagent source
Genetic reagent (D.rerio)yap1mw48-/-Miesfeld et al., 2015RRID:ZFIN_ZDB-ALT-160122-5Brian Link (Medical College of Wisconsin, Milwaukee, USA)
Genetic reagent (D.rerio)yap1ncv101-/-Nakajima et al., 2017RRID:ZFIN_ZDB-ALT-170522-16Naoki Mochizuki (National Cerebral and Cardiovascular
Centre Research Institute, Suita, Osaka)
Genetic reagent (D.rerio)Tg(fli1a:nEGFP)y7Lawson et al., 2002RRID:ZFIN_ZDB-ALT-060821-4Brant M Weinstein (National Institute of Child Health and Human Development, Bethesda, USA)
Genetic reagent (D.rerio)Tg(- 5.2lyve1b:DsRed)nz101Okuda et al., 2012RRID:ZFIN_ZDB-ALT-120723-3Phil and Kathy Crosier (Department
of Molecular Medicine
University of Auckland School of Medicine)
Genetic reagent (D.rerio)Tg(fli1:EGFP-YAP)ncv35Nakajima et al., 2017RRID:ZFIN_ZDB-ALT-170522-18Naoki Mochizuki (National Cerebral and Cardiovascular Centre Research Institute, Suita, Osaka)
Genetic reagent (D.rerio)Tg(fli1:Gal4db-TEAD2ΔN-2A-mC)ncv36Nakajima et al., 2017RRID:ZFIN_ZDB-ALT-170522-19Naoki Mochizuki (National Cerebral and Cardiovascular Centre Research Institute, Suita, Osaka)
Genetic reagent (D.rerio)Tg(UAS:GFP)y1Asakawa et al., 2008RRID:ZFIN_ZDB-ALT-011017-8Naoki Mochizuki (National Cerebral and Cardiovascular Centre Research Institute, Suita, Osaka).
Genetic reagent (D.rerio)Tg(fli1:Myr-mC)ncv1Kwon et al., 2013ZFIN ID: ZDB-FIG-150115–34Naoki Mochizuki (National Cerebral and Cardiovascular Centre Research Institute, Suita, Osaka)
Genetic reagent (D.rerio)Tg(fli1:H2B-mC)ncv31Yokota et al., 2015RRID:ZFIN_ZDB-ALT-160323–6Naoki Mochizuki (National Cerebral and Cardiovascular Centre Research Institute, Suita, Osaka)
Genetic
reagent (D.rerio)		Tg(gata1:DsRed)sd2Traver et al., 2003RRID:ZFIN_ZDB-ALT-051223-6Leonard I Zon (Howard Hughes Medical Institute, Boston, MA), European Zebrafish Resource Center (EZRC),Zebrafish International Resource Center (ZIRC).
Genetic
reagent (D.rerio)		TgBAC(prox1a: KalTA4-4xUAS-ADV.E1b:TagRFP)nim5Dunworth et al., 2014; van Impel et al., 2014RRID:ZFIN_ZDB-ALT-160323–6Elke Ober (The Danish Stem Cell Centre
(DanStem)
University of
Copenhagen)
Genetic reagent (D.rerio)Tg(10xUAS:vegfc)uq2bhKoltowska et al., 2015aRRID:ZFIN_ZDB-ALT-151208-1Ben M Hogan (Institute for Molecular Bioscience, The University of Queensland)
Genetic reagent (D.rerio)Tg(fli1a:H2B-mCherry)uq37bhthis paperBen M Hogan (Institute for Molecular Bioscience, The University of Queensland)
Genetic reagent (D.rerio)TgBAC(dab2b:EGFP)ncv67otherShigetomo Fukuhara and Shinya Yuge (Nippon Med. School)
Genetic reagent (D.rerio)Tg(kdrl:mCherry)ncv502otherNaoki Mochizuki (National Cerebral and Cardiovascular Centre Research Institute, Suita, Osaka).
Antibodychicken anti-GFPAbcamCat#ab13970Primary AB, Alexa Fluor-488 conjugated, 1:250
AntibodyC1 anti-GFPInvitrogenCat#A11039Secondary AB, 1:400
Antibodyrabbit anti-Prox1AngioBioCat#11–002Primary AB, 1:500
Antibodyanti-rabbit IgG-HRPCell SignalingCat#7074SSecondary AB, 1:1000
Sequence-based reagentMO4-yap1Loh et al., 2014ZFIN ID: ZDB-MRPHLNO-140915–5Genetools, LLC
Sequence-based reagentp53 MORobu et al., 2007SKU: PCO-ZebrafishP53-100Genetools, LLC
Commercial assay or kitTSA Plus Cyanine 3 SystemPerkin Elmer#NEL744001KTAmplification of signal detection of the Prox1-AB staining
Chemical compound, drugSL327 (MEK inhibitor)Sigma-AldrichS4069; CAS:305350-87-215 mM
Software, algorithmImageJImageJ (http://imagej.nih.gov/ij/)SCR:002285Image processing and analysis, Version 2.0.0-rc-49/1.51d
Software, algorithmImaris x64BitplaneSCR:007370Image processing and analysis, Version 9.0.2
Software, algorithmGraphPad PrismGraphPad
Prism (https://graphpad.com)		SCR:015807Statistics, Prism7: Version7.0 c and Prism8: Version 8.0.1

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https://doi.org/10.7554/eLife.42881.026

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  1. Lin Grimm
  2. Hiroyuki Nakajima
  3. Smrita Chaudhury
  4. Neil I Bower
  5. Kazuhide S Okuda
  6. Andrew G Cox
  7. Natasha L Harvey
  8. Katarzyna Koltowska
  9. Naoki Mochizuki
  10. Benjamin M Hogan
(2019)
Yap1 promotes sprouting and proliferation of lymphatic progenitors downstream of Vegfc in the zebrafish trunk
eLife 8:e42881.
https://doi.org/10.7554/eLife.42881