Fig. 3.

Loss of grb2a does not cause lymphatic defects in the trunk, but Grb2a can functionally compensate for the loss of grb2b upon over-expression. a In situ hybridization of grb2a shows wide expression in the zebrafish embryo at 32hpf. b Schematic representation of the Grb2a protein, with two SH3 domains and one SH2 domain, highlighting the − 5 bp mutation in the SH2 domain of the grb2a^mu405 allele. c Quantification of TD fragments shows that grb2a is not essential for trunk lymphatic development. The presence of TD was quantified over 10 segments. wt: n = 19; het: n = 35; mut: n = 27. d Quantification of the number of PLs at 48hpf in the different genotypes of a grb2a^mu405; grb2b^mu404 double heterozygous in-cross. Lymphatic precursor cells do not form when embryos are mutant for grb2b, independent of the number of grb2a wild-type copies. * Between wt and grb2a wt; grb2b^−/−: P value 0.0119 (Mann–Whitney); **Between wt and grb2a^+/−; grb2b^−/−: P value = 0.0045 (Mann–Whitney); *Between wt and grb2a^−/−; grb2b^−/−: P value = 0.0179 (Mann–Whitney). Note that the comparison between wt and each one of the other genotypes resulted in a non-significant difference. e Quantification of TD⁺ segments in embryos from grb2a^mu405; grb2b^mu404 double heterozygous parents at 5dpf. The development of the main lymphatic vessel depends on grb2b, and not on grb2a. d, e wt: n = 5; grb2a^+/−; grb2b wt: n = 14; grb2a^−/−; grb2b wt: n = 7; grb2a wt; grb2b^+/−: n = 14; grb2a^+/−; grb2b^+/−: n = 25; grb2a^−/−; grb2b^+/−: n = 11; grb2a wt; grb2b^−/−: n = 6; grb2a^+/−; grb2b^−/−: n = 13. SH2 Src homology domain 2, SH3 Src homology domain 3, PL parachordal lymphangioblast, TD thoracic duct. Data in d represent the mean ± sd