Fig. 1.

Mutations in the grb2b gene interfere with lymphatic development. a–d Confocal projections of the trunk region in siblings (a, c) and tabula rasa mutants (b, d) at 48hpf (a, b) and at 5dpf (c, d) with flt4:mCitrine highlighting venous and lymphatic structures in green and the flt1:tdTomato transgene showing arterial endothelial cells in red. Arrows indicate the presence of PLs at the HM (a) and of a fully developed TD (c) in siblings, whereas asterisks indicate the lack of PLs (b) and of TD fragments (d) in mutants. e Quantification of TD-containing segments scored over the length of 10 consecutive somites at 5dpf. Most tabula rasa mutants lack the whole TD or they form only few fragments. wt: n = 24; het: n = 45; mut: n = 27. f Quantification of the number of PLs per embryo at 48hpf shows a significant decrease in the number of PLs in tabula rasa mutants compared to both wild types and heterozygotes. wt: n = 4; het: n = 12; mut: n = 6. ** Between wt and mut: P value 0.0095 (Mann–Whitney). ***Between het and mut: P value 0.003 (Mann–Whitney). g Quantification of the number of aISVs and vISVs in 20 consecutive segments (bilateral) at 48hpf in the zebrafish trunk. tabula rasa mutants display significantly higher numbers of arteries than veins. wt n = 4; het: n = 12; mut: n = 6. **Between vISVs wt and vISVs mut: P value = 0.0048 (Mann–Whitney). ***Between vISVs het and vISVs mut: P value  = 0.0001 (Mann Whitney). ** Between aISVs mut and vISVs mut: P value 0.0022 (Mann–Whitney). h, i Schematic representations of the Grb2b protein containing two SH3 domains and one SH2 domain. h I₄₈ > N indicates the point mutation in the tabula rasa mutant: the isoleucine at position 48 is replaced by an asparagine. i A 2 bp deletion in the grb2b^mu404 allele is predicted to cause a frame-shift after amino acid 19, within the first SH3 domain of the protein. j, k Confocal projections showing the lack of complementation in tabula rasa and grb2b^mu404 trans-heterozygous embryos. j tabula rasa heterozygous embryos have a full TD, as indicated by arrows. k tabula rasa^+/−; grb2b^mu404+/− trans-heterozygous embryos lack the TD (indicated by asterisks). flt4:mCitrine is shown in green and flt1:tdTomato in red. SH2 Src homology domain 2, SH3 Src homology domain 3, aISVs arterial intersegmental vessels, vISVs venous intersegmental vessels, PL parachordal lymphangioblast, HM horizontal myoseptum, TD thoracic duct, ns not significant. Scale bars: 50 µm. Data in f, g are mean ± sd