Fig. 2

The orthologous human MET mutations, I284N and E217K, impair HGF signaling.

A The spatiotemporal expression of hgfa and met in zebrafish embryos. hgfa is expressed around the thyroid primordium at 48 hpf and 54 hpf; met is colocalized in a portion of thyrocytes with tg in the embryos at 48–60 hpf, as detected by WISH. hgfa mRNA: blue, arrowhead on panel A; met mRNA: blue, red arrow on panel A; T: thyroid; tg:GFP caax: brown, white arrow on panel A. hgfa and met mRNA were detected by WISH, and thyrocytes were marked by immunohistochemical staining for GFP. a’, b’, a”, b”: left lateral position; a”’, b”’: right lateral position; c’–e’ and c”–e”: dorsal position. Scale bars: 100 μm. e:eye; hgfa mRNA was detected by fluorescence in situ hybridization, and thyrocytes were marked by immunofluorescence staining for GFP in Aa”’ and Ab”’. Three independent experiments were carried for A. B–D A single-cell atlas of the zebrafish thyroid gland showing that hgfa was expressed in some stromal cells but met was restrictedly expressed in thyrocytes (https://sumeet.shinyapps.io/zfthyroid). E Mutation sites in the secondary structure of the MET protein. Diagram of MET showing that the E217K and I284N substitutions are localized to the semaphorin-like domain (Sema), which contains the furin cleavage site (residues 302–307). F Immunofluorescence staining of HEK293T cells transfected with the zmet^WT, zmet^I217K and zmet^I284N plasmids, respectively. WGA staining (red) marks the cell membrane, and DAPI staining (blue) marks the nuclei. Three independent experiments were repeated with similar results. Scale bars: 100 μm. G The E217K and I284N substitutions in the mutants represent a significant shift in amino acids that are relatively conserved in vertebrates. H Western blotting was performed to analyze the expression and phosphorylation of MET in TOV112D cells transfected with plasmids containing human MET^WT, MET^E221K or MET^L288N after treatment with or without the human HGFα protein. Three independent experiments were repeated with similar results. I The rescue effects of human HGF factors or zebrafish wild-type met injection (pTol2tg:met-mCherry) on thyroid development are displayed. DMSO con, embryos injected with dimethyl sulfoxide (DMSO). Vehicle con, embryos injected with pTol2tg:mCherry. Scale bars: 100 μm. Three independent experiments were carried for I.