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The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function

Nature Cell Biology volume 23, pages 1136–1147 (2021)Cite this article

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Abstract

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc–Flt4 signalling. Ddx21 function is essential for Vegfc–Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc–Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

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Fig. 1: ddx21 is essential for lymphatic vascular development.
Fig. 2: ddx21 is enriched in secondary sprouts and cell-autonomously regulates lymphatic vessel development.
Fig. 3: ddx21 cell-autonomously regulates vegfc-induced EC proliferation.
Fig. 4: Loss of ddx21 leads to increased p53, p21 and EC cycle arrest.
Fig. 5: Increased p53 activity blocks embryonic lymphangiogenesis in ddx21 mutants.
Fig. 6: Ddx21 is required for ribosome biogenesis in ECs.
Fig. 7: Ddx21 regulates ribosomal biogenesis downstream of Vegfc–Flt4 signalling.

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Data availability

All RNA-seq data for this project are available at the Gene Expression Omnibus under accession number GSE180330. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

All code developed for this analysis is available as bitBucket scripts via ssh://git@atlassian.petermac.org.au:7999/~tyrone.chen/hogan_lab.git.

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Acknowledgements

This project was supported by NHMRC grants 1107755 and 1079670 and Ragnar Söderbergs Fellowship (M13/17). K.K. is supported by LE&RN and Wallenberg Fellowships (2017.0144), Vetenskapsådet (VR-MH-2016-01437) and Jeanssons Stiftelser. B.M.H. is supported by NHMRC/NHF (1083811) and NHMRC (1155221) Fellowships. We thank T. Chen, A. Klemm, A. Cox and A. Ong for technical assistance and N. Brajanovski for technical advice and staff at our imaging and genomics facilities for support.

Author information

Author notes

  1. Ignaty Leshchiner

    Present address: Broad Institute of MIT and Harvard, Cambridge, MA, USA

  2. These authors contributed equally: Katarzyna Koltowska, Kazuhide S. Okuda.

Authors and Affiliations

  1. Division of Genomics of Development and Disease, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland, Australia

    Katarzyna Koltowska, Kazuhide S. Okuda, Maria Rondon-Galeano, Huijun Chen, Neil I. Bower, Scott Paterson, Anne Karine Lagendijk, Gregory J. Baillie, Cas Simons, Kelly A. Smith & Benjamin M. Hogan

  2. Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

    Katarzyna Koltowska, Marleen Gloger, Hannah Arnold & Renae Skoczylas

  3. Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

    Kazuhide S. Okuda, Maria Rondon-Galeano, Elizabeth Mason, Jiachen Xuan, Stefanie Dudczig, Scott Paterson, Richard B. Pearson, Elaine Sanij & Benjamin M. Hogan

  4. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia

    Kazuhide S. Okuda, Maria Rondon-Galeano, Elizabeth Mason, Jiachen Xuan, Stefanie Dudczig, Scott Paterson, Richard B. Pearson, Elaine Sanij & Benjamin M. Hogan

  5. Massachusetts General Hospital, Harvard–MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA, USA

    Ignaty Leshchiner & Wolfram Goessling

  6. Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia

    Cas Simons

  7. Department of Anatomy and Physiology, University of Melbourne, Parkville, Victoria, Australia

    Kelly A. Smith & Benjamin M. Hogan

  8. Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Joan K. Heath

  9. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

    Joan K. Heath

  10. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia

    Richard B. Pearson

  11. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

    Richard B. Pearson

  12. Department of Clinical Pathology, University of Melbourne, Parkville, Victoria, Australia

    Elaine Sanij

  13. St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia

    Elaine Sanij

  14. Institute of Cardiovascular Organogenesis and Regeneration, Medical Faculty, WWU Münster, Münster, Germany

    Stefan Schulte-Merker

  15. Hubrecht Institute-KNAW and University Medical Centre, Utrecht, The Netherlands

    Stefan Schulte-Merker & Benjamin M. Hogan

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  1. Katarzyna Koltowska
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Contributions

K.K. and B.M.H. conceptualized the project, performed and analysed experiments and co-wrote the manuscript. K.S.O., M.G., M.R-G. and E.M. performed and analysed experiments and co-wrote the manuscript. S.D., H.C., H.A., R.S., N.I.B., S.P., A.K.L., G.J.B., I.L., E.S. and J.X. performed and analysed experiments. C.S., K.A.S., W.G., J.K.H., R.B.P. and S.S.-M. provided key unpublished reagents and data.

Corresponding authors

Correspondence to Katarzyna Koltowska or Benjamin M. Hogan.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Cell Biology thanks Leonard Zon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Genetic and additional phenotypic characterisation of the ddx21 mutant.

a) Positional cloning of ddx21uq20bh using whole genome sequencing and homozygosity mapping. Plot displays genomic homozygosity across 25 chromosomes (top), chromosome 13 (middle; 54 mega base region), the region of linkage (bottom; arrow points to ddx21 gene location within the region). (b) Sanger sequencing chromatograms confirming a missense mutation originally detected in the whole genome sequence data (arrow points to the position with a T/C nucleotide substitution). (c) Schematic representation of the DDX21 protein, displaying the position (422) of amino acid substitution and conservation of this region across vertebrates. (d) Left: Complementation test with a separate CRISPR allele for ddx21. Confocal projections of a sibling and a ddx21uq20bh;ddx21 uq21bh double mutant at 5 dpf. Scale bars= 30μm. Right: Quantification of LECs by scoring labelled nuclei present in lymphatics (right, n = 8 sibling and n = 9 double mutants). p < 0.0001. (e) Confocal projections of a sibling and a ddx21uq20bh mutant at 2 dpf. Scale bars= 30μm. (f) Left: Confocal projections of IF for endothelial nuclei (Tg(fli1a:nEGFP), grey) and Prox1 (red) in a sibling and a ddx21uq20bh mutant at 36 hpf. Scale bars= 30μm. Right: Quantification of the number of Prox1 and GFP double positive cells scored across 4 somites in 36 hpf siblings (n = 33) and ddx21uq20bh mutants (n = 12). p = 0.1731. Arrows indicate migrating Prox1-positive lymphatic progenitors. (g) Left: Confocal projections of IF for endothelial nuclei (Tg(fli1a:nEGFP), grey) and pErk (green) in a sibling and a ddx21uq20bh mutant at 32 hpf. Scale bars= 30μm. Right: Quantification of the number of pErk and GFP double positive cells in the posterior cardinal vein (PCV) scored across 4 somites in 32 hpf siblings (n = 16) and ddx21uq20bh mutants (n = 13). p = 0.35. Arrows indicate pErk-positive progenitors within the PCV. n=biologically independent samples (embryos) for d-g. (h) Bright field images showing overall phenotype of siblings (n = 35) and ddx21uq20bh mutants at 5 dpf (n = 19). Scale bars= 500μm. (i) Alcian blue staining in a 5 dpf sibling and ddx21uq20bh embryos labelling the craniofacial cartilage in the developing jaw. Ventral views (top), lateral views (bottom). Scale bars= 100μm. (j) Confocal projections of facial lymphatic vessels in a 5 dpf sibling (n = 11) and a ddx21uq20bh mutant (n = 11). Scale bars= 50μm. (k) Confocal projections of whole-larvae blood vasculature in a 6 dpf sibling (n = 4) and a ddx21uq20bh (n = 6) mutant larvae. Scale bars= 100μm. The number of animals analyzed in h-k is indicated in parentheses and a representative image is shown. aISV= arterial intersegmental vessel, DLLV = Dorsal longitudinal lymphatic vessel, ISLV = intersegmental lymphatic vessel, LEC = lymphatic endothelial cell, TD = thoracic duct, vISV= venous intersegmental vessel. Transgenic labels are indicated. Mean values + /-SEM shown. Two-tailed student’s t-test for (d), (f), and (g).

Source data

Extended Data Fig. 2 ddx21 is expressed in endothelium and genetically interacts with the Vegfc-Flt4 signalling pathway.

(a) in situ hybridisation for ddx21 showing expression at 4 cell-stage and 24 hpf (scale bars=100μm) and 48 hpf (bottom) lateral view (scale bar=100μm) and transverse section (zoom on right, n = 17, scale bars=20μm). Data shown represent 5 (4 cell), 6 (24 hpf), 16 (left, 48 hpf) and 17 (48 hpf) independent embryos analysed. (b,c) qPCR for ddx21 and endothelial genes on cDNA from sorted arterial endothelial cells (AECs, n = 4), lymphatic endothelial cells (LECs, n = 5 except for n = 4 for prox1a and ddx21) and venous endothelial cells (VECs, n = 4 for 60hpf and n = 5 for 3 dpf) at 60 hpf (b) and 3 dpf (c). Expression relative to kdrl for 60 hpf data and to rpl13 for 3 dpf data. n=biologically independent samples. (d) Confocal projections of TgBAC(ddx21:ddx21-Citrine) transgenics at 36 hpf and 5 dpf. Scale bars= 100μm. Data shown represent 15 independent embryos analysed. (e) Confocal projections showing nucleolar Ddx21-Citrine expression in haematopoietic stem cells (white arrows) in a 36 hpf TgBAC(ddx21:ddx21-Citrine);Tg(fli1a:H2BCherry) embryo (data shown represent 20 embryos analyzed). Image on the right shows TgBAC(ddx21:ddx21-Citrine) expression (grey). Scale bar= 25μm. (f) Confocal projections of an uninjected control sibling, ddx21uq20bh mutant, MO-dll4 injected sibling, and a ddx21uq20bhmutant at 7 dpf. Data shown represent n > 20 sibling control, n > 20 ddx21 mutant control, n = 70 MO-dll4 sibling and n = 67 MO-dll4 ddx21 mutant embryos analyzed in g). Arrows mark the ectopic branching and asterisk the absence. Scale bar= 50μm. (g) Quantification of the number of blood vessel branches per 10 somites in MO-dll4 injected siblings (n = 70) and ddx21uq20bh (n = 67) embryos. p = 0.0012. (h) Representative images for data shown in Fig. 3c. Confocal projections of siblings and ddx21uq20bh mutants in wild type or vegfr3+/- heterozygous backgrounds. Thoracic duct is false-coloured in magenta and asterisk indicate absent lymphatics. Scale bars= 50μm. DA = dorsal aorta, PCV = posterior cardinal vein. Transgenic labels are indicated. Mean values + /-SEM shown. Two-tailed Mann Whitney test for (g).

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Extended Data Fig. 3 Transcriptomic analysis of ddx21 mutant and siRNAi knockdown endothelial cells.

(a) Additional RNA Seq analysis for Fig. 4 zebrafish data. PCA and screeplots of all expressed genes (n = 18,549) before (top) and after (bottom) batch correction. Screeplot representing the percentage of variance explained by each principal component for all expressed genes prior to batch correction. X-axis reflects principal component (1 to 8 displayed) and Y-axis reflects the percentage of total variance explained by each component (top left). PCA of components 1 and 2 for all expressed genes and all samples prior to batch correction. Matched sibling and ddx21uq20bh mutant samples share the same shaped point, and sibling and ddx21uq20bh phenotype is depicted by light grey and dark grey colour respectively (top right). Screeplot representing the percentage of variance explained by each principal component for all expressed genes after batch correction. X-axis reflects principal component (1 to 8 displayed) and Y-axis reflects the percentage of total variance explained by each component (bottom left). PCA of components 1 and 2 for all expressed genes and all samples after batch correction. Matched sibling and ddx21uq20bh mutant samples share the same shaped point, and sibling and ddx21uq20bh phenotype is depicted by light grey and dark grey colour respectively (bottom right). (b) Additional RNA Seq analysis for Fig. 4 HUVEC data (as above). PCA and screeplots of all expressed genes (n = 14,588) before (top) and after (bottom) removing aberrant sample HUVEC_siRNA_01_DDX21_01. Samples of the same treatment group (Control, siRNA-construct-01, siRNA-construct-02) share the same shaped point and greyscale colour respectively, except the aberrant sample (HUVEC_siRNA_01_DDX21_01) which is coloured in red. Screeplot representing the percentage of variance explained by each principal component for all expressed genes after removing aberrant sample (HUVEC_siRNA_01_DDX21_01). PCA of components 1 and 2 for all expressed genes and all remaining samples after removing aberrant sample (HUVEC_siRNA_01_DDX21_01). Samples of the same treatment group (Control, siRNA-construct-01, siRNA-construct-02) share the same shaped point and greyscale colour. (c) Reactome pathways significantly enriched in genes upregulated in the ddx21 mutant zebrafish endothelial cells. Y-axis is pathway or GO term, X-axis is number of genes, bars are coloured according to BH (Benjamini-Hoschberg) adjusted p-value. (d) Upset plot indicating Reactome pathways significantly enriched in genes upregulated in ddx21 mutant zebrafish endothelial cells. Bar plot in upper panel: Y-axis represents the number of genes, X-axis the significantly enriched pathways. Points underneath a given bar indicate genes present in that pathway, a line between indicates the same genes present in more than one pathway. (e) Enrichment plot for p53 pathway (zebrafish data). The green curve corresponds to the calculated enrichment score, vertical black lines the position of individual genes in relation to the ranked gene list. Raw and adjusted p values in red. (f) Reactome pathway enrichment bar plot for upregulated genes upon DDX21-siRNA-knockdown. Presented as in (c). (g) Biological process GO term enrichment bar plot (top n = 15 significantly enriched terms) for in DDX21siRNA knockdown HUVECs. Y-axis is BP term, X-axis is number of genes, bars are coloured according to BH (Benjamini-Hoschberg) adjusted p-value. (h) Enrichment plot of p53 pathway (msigdb hallmark gene set) shows log fold change between DDX21-siRNA-knockdown and WT control samples. Presented as in (e). (i) Biological process GO term enrichment upset plot (top n = 15 significantly enriched terms) in DDX21siRNA knockdown HUVECs. Presented as in (d).

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Extended Data Fig. 4 Additional data related to cell death, senescence and Fig. 4.

(a) Confocal projections of Tg(-5.2lyve1b:DsRed2) (green) stained with TUNEL (magenta) in siblings and a ddx21uq20bh mutant at 48 hpf. Arrows indicate TUNEL-positive parachordal lymphatic endothelial cells (PLs). Scale bars= 50μm. (b) Confocal projections of IF for endothelial nuclei (Tg(fli1a:nEGFP), green) and cleaved Caspase-3 (magenta) in siblings and ddx21uq20bh mutants at 48 hpf. Arrows indicate cleaved Caspase-3-positive PLs or gut cells. Scale bars= 50μm. (c) Quantification of TUNEL-positive endothelial cells in siblings (n = 10) and ddx21uq20bh mutants (n = 10). p = 0.4737 for PCV. p = 0.5124 for LECs. p = 0.4737 for vISV. (d) Quantification of the number of cleaved Caspase-3 and GFP double positive cells in siblings (n = 11) and mutants (n = 25). p = 0.6239 for PCV. p = 0.2539 for LECs. p = 0.8707 for vISV. (e) Quantification of number of cell death in LECs between 36-56 hpf in siblings (n = 13) and ddx21uq20bh mutants (n = 13). p>0.9999. (f) Quantification of number of cell deaths in venous intersegmental vessel ECs between 36-56 hpf in siblings (n = 13) and ddx21uq20bh mutants (n = 13). p>0.9999. n=biologically independent samples (embryos) analysed. (g) Representative bright field images of senescence-associated ß-galactosidase (SAß-gal) stained 5 dpf sibling (18) and ddx21uq20bh mutant (14) larvae. Scale bars= 100μm. (h) Representative images of p21 in situ hybridisation on transverse sections of a sibling (7) and a mutant (7) at 2 dpf. 6 out of 7 mutants displayed darker stanning. Scale bars= 20μm. The number of animals (embryos) analyzed in g and h is indicated in parentheses. (i) Quantification of DDX21 (foci, left) intensity on control, siDDX21-1 and siDDX21-2 transfected HUVECs (n = 6 independent replicates for both siDDX21s). The variance in DDX21 foci intensity is larger in siDDX21-1 when compared to siDDX21-2. p<0.0001. (j) Quantifications of p53-positive aISV and PCV cells in 48 hpf siblings (n = 17) and ddx21uq20bh mutants (n = 17) from Fig. 4j. p = 0.2157 for aISV. p = 0.2265 for PCV. (k) Quantifications of total ECs and PCV cell numbers in siblings and ddx21uq20bh mutants across 3 somites. p = 0.016 for EC (siblings n = 40, ddx21uq20bhmutants n = 28). p = 0.8012 for PCV (siblings and ddx21uq20bhmutants n = 10). aISV= arterial intersegmental vessel, DA = dorsal aorta, LEC = lymphatic endothelial cell, PCV = posterior cardinal vein, vISV= venous intersegmental vessel. Mean values + /-SEM shown except for (i) which shows median, quartiles, and maximum/minimum values. Two-tailed student’s t-test for (j, PCV) and (k). Two-tailed Mann Whitney test for (c-f) and (j, aISV). Kruskal-Wallis test with Dunn’s multiple comparisons test for (i).

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Extended Data Fig. 5 Additional data related to DDX21 loss, cell cycle arrest and Fig. 4.

(a) Gating strategy for the cell cycle analysis on sorted LECs and VECs from sibling and ddx21uq20bh mutants. Top to bottom panels represent the cell population selection based on size and granularity (X-axis reflects FSC = Forward Scatter and Y-axis SSC = Side Scatter, A = Area), identification of double positive cells for fli1a:GFP and lyve1b:DsRed to select LECs and VECs (Y-axis reflects DsRed and X-axis GFP intensities), selection of single cells obtained by gating based on Hoechst staining (Y-axis reflects A = area and X-axis W = width), histogram of cell numbers with Hoechst staining (Y-axis reflects cell counts and X-axis W = width). Black line reflects Hoechst intensity distribution in cells, coloured lines reflect fitted model for cell cycle profiles based on analysis with Watson method (G1-phase=blue, S-phase=yellow and green=G-M-phase). (b) Gating strategy for the cell cycle analysis on control and siDDX21-treated HUVECs. Top to bottom panels represent the cell population selection based on size and granularity (Y-axis reflects FSC = Forward Scatter and X-axis SSC = Side Scatter, A = Area), selection of single cells obtained by gating based on Hoechst staining (Y-axis reflects A = area and X-axis W = width), histogram of cell numbers with Hoechst staining (Y-axis reflects cells counts and X-axis W = width. Black line reflects Hoechst intensity distribution in cells, coloured lines reflect fitted model for cell cycle profiles based on analysis with Dean-Jett-Fox method (G1-phase=blue, S-phase=yellow and green=G-M-phase). (c) The cell cycle profile obtained by FACS of LECs and VECs in siblings and ddx21uq20bh mutants (n = 4). G1-phase p = 0.0332, S-phase p = 0.737, G2-M-phase p = 0.0290. (d) The cell cycle profile obtained by FACS in control and siDDX21 transfected HUVECs (n = 9). G1-phase p = 0.004 (siDDX21-1) and p < 0.0001 (siDDX21-2), S-phase p = 0.0277 (siDDX21-1) and p = 0.0082 (siDDX21-2), G2-M-phase p = 0.3013 (siDDX21-1) and p = 0.0216 (siDDX21-2). (e) Left: Confocal projections showing endothelial nuclei (Tg(fli1a:nEGFP), green) and 5-Ethynyl-2′-deoxyuridine (EdU) staining (magenta) in a 36 hpf sibling and a ddx21uq20bh mutant. Arrows indicate EdU-positive secondary sprouts. Scale bars= 50μm. Right: Quantification of EdU-positive PCV or secondary sprout endothelial cells represented as a ratio of EdU-positive PCV or secondary sprout endothelial cells and total number of PCV or secondary sprout endothelial cells in siblings (n = 7) and ddx21uq20bh mutants (n = 7). p = 0.1200 for PCV. p = 0.5224 for secondary sprouts. (f) Confocal projections showing endothelial nuclei (green) and 5-Ethynyl-2′-deoxyuridine (EdU) staining (magenta) in a 48 hpf sibling and a ddx21uq20bh mutant. Arrows indicate EdU-positive parachordal lymphatic endothelial cells (PLs). Scale bars= 50μm. (g) Left: Confocal projections of IF for DAPI (blue), p53 (magenta) and phospho-histone H3 (PHH3, green) on control or siDDX21 transfected HUVECs. Scale bars= 25μm Right: Quantification of ratio of PHH3-positive cells in p53-low or p53-high cells on control or siDDX21 transfected HUVECs (n = 6 independent replicates). p < 0.0001 for control. p = 0.0143 for siDDX21. n = 6 biologically independent samples from 2 independent experiments. DA = dorsal aorta, PCV = posterior cardinal vein. Mean values + /-SEM shown. Paired one-tailed student’s t-test for (c). Two-tailed student’s t-test for (e, secondary sprout). Two-tailed Mann Whitney test for (e, PCV). Ordinary one-way ANOVA test with Dunnett’s or Tukey’s multiple comparisons test for (d) and (g), respectively.

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Extended Data Fig. 6 p53 function inhibits lymphangiogensis in the absence of Ddx21.

(a) Confocal projections of a wild type, and p53zdf1, ddx21uq20bh, ddx21uq20bh;p53zdf1, vegfchu5055, vegfchu5055;p53zdf1 mutants at 5 dpf. Scale bars= 100μm. (b) Quantification of lymphatic endothelial cells (LECs) by scoring labelled nuclei present in lymphatics. p = 0.9506 for siblings (n = 29, ddx21uq20bh) versus p53zdf1 (n = 17, ddx21uq20bh). p < 0.0001 for ddx21uq20bh (n = 19) versus ddx21uq20bh;p53zdf1 (n = 5). p = 0.8551 for siblings (n = 11, vegfchu5055) versus p53zdf1 (n = 8, vegfchu5055). p = 0.8453 for vegfchu5055 (n = 20) versus vegfchu5055;p53zdf1 (n = 8). (c) Confocal projections of IF for endothelial nuclei (Tg(fli1a:nEGFP), green) and p53 (magenta) in a control and p53 morpholino-injected 48 hpf sibling and ddx21uq20bh mutant embryos. Arrow indicates a p53-positive PL. Scale bar= 50μm. (d) Left: Quantification of the number of p53 and GFP double positive lymphatic endothelial cells (LECs) and venous endothelial cells (VECs) in control siblings (n = 10) and ddx21uq20bh mutants (n = 10), and MO-p53-injected siblings (n = 8) and ddx21uq20bh mutants (n = 8). p = 0.0006 for control siblings versus ddx21uq20bh. p = 0.9982 for MO-p53-injected siblings versus ddx21uq20bh. p = 0.9907 for control siblings versus MO-p53-injected siblings. p = 0.0009 for control ddx21uq20bh mutants versus MO-p53-injected ddx21uq20bh mutants. Right: Quantification of the number of p53 positive gut cells in control siblings (n = 16) and ddx21uq20bh mutants (n = 24), and MO-p53-injected siblings (n = 5) and ddx21uq20bh mutants (n = 5). p = 0.001 for control siblings versus ddx21uq20bh. p > 0.9999 for MO-p53-injected siblings versus ddx21uq20bh. p = 0.4499 for control siblings versus MO-p53-injected siblings. p = 0.0138 for control ddx21uq20bh versus MO-p53-injected ddx21uq20bh. n=biologically independent samples (embryos) analysed. DA = dorsal aorta, PCV = posterior cardinal vein. Mean values + /-SEM shown. Two-tailed Mann Whitney test for (b). Ordinary one-way ANOVA test with Tukey’s multiple comparisons test for (d, LECs and VECs). Kruskal-Wallis test with Dunn’s multiple comparisons test for (d, gut cells).

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Extended Data Fig. 7 Endothelial nucleolar Ddx21 is modulated by Vegfc-Flt4 signalling.

(a) Confocal projections of IF for endothelial nuclei (Tg(fli1a:nEGFP), green) and Fibrillarin (magenta) in a 36 hpf sibling and a ddx21uq20bh mutant. Scale bars = 10μm. White brackets were cropped for Fig. 6l. (b) Confocal projections of 3 dpf control and vegfc-induced embryos on TgBAC(ddx21:ddx21-Citrine);Tg(prox1a:Kalt4,4xUAS:TagRFP);(10xUAS:vegfc) background. Images on the right show Ddx21-Citrine expression (grey) of images on the left. Scale bars= 50μm. (c) Confocal projections of control and MO-ccbe1-injected 40 hpf TgBAC(ddx21:ddx21-Citrine);Tg(lyve1b:DsRed2) embryos. White arrows show nucleolar Ddx21 expression in PCV ECs. Quantification in Fig. 7d (right). Scale bars= 20 μm. Images represent 7 animals (embryos) analyzed for both control and MO-ccbe1. DA = dorsal aorta, PCV = posterior cardinal vein.

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Supplementary Video 1

Time-lapse imaging of venous and lymphatic sprouting in sibling (left) and ddx21uq20bh mutant (right) in Tg(-5.2lyve1b:DsRed2) background from 36 to 56 h.p.f. Images were taken every 20 min. Scale bar, 50 μm.

Supplementary Video 2

Time-lapse imaging of venous and lymphatic sprouting in sibling (left) and ddx21uq20bh mutant (right) in Tg(fli1a:nEGFP);(-5.2lyve1b:DsRed2) background from 36 to 56 h.p.f. Images were taken every 20 min. Scale bar, 50 μm.

Supplementary Video 3

Time-lapse imaging of TgBAC(ddx21:ddx21–Citrine);(fli1a:H2BCherry) from 36 to 72 h.p.f. (n = 5). Images were taken every 30 min. Arrows indicate proliferating endothelial (first and second video insets) and muscle (third video inset) cells. Scale bar, 15 μm.

Supplementary Video 4

Confocal z sections (2 μm) of a primary sprout in a 24 h.p.f. TgBAC(ddx21:ddx21–Citrine);(fli1a:H2BCherry) embryo. Scale bar, 15 μm.

Supplementary Video 5

Confocal z sections (2 μm) of a secondary sprout in a 40 h.p.f. TgBAC(ddx21:ddx21–Citrine);(lyve1b:DsRed2) embryo. Scale bar, 15 μm.

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Unprocessed western blots of Fig. 4f.

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Koltowska, K., Okuda, K.S., Gloger, M. et al. The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function. Nat Cell Biol 23, 1136–1147 (2021). https://doi.org/10.1038/s41556-021-00784-w

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