The hinge motif F88 in LMO2 is required for functional interaction with SCL/TAL1. (A) Details of the LMO2 structure model around hinge residue F88 (region in green inset) shown in a balls-and-stick representation and the corresponding electron density map (contoured at a σ level of 1). The structurally proximal residues K74 and R77 belonging to the LIM1 domain are also shown. (B) Mammalian 2-hybrid assays in HEK293 cells were carried out to detail the effect of mutating the hinge motif F88. SCL/TAL1 is expressed as a VP16 fusion protein and LMO2 (wild-type and mutants) as Gal4DBD fusion proteins (diagram bottom right). (Top) Interaction between SCL/TAL1 and wild-type LMO2 activates luciferase gene expression. Mutation-dependent effects are observed when the LMO2 mutants F88A and F88D are coexpressed with SCL/TAL1. Error bars represent SD. (Bottom) Western blot analysis of expression of SCL-VP16 (top panel) and LMO2-GAL4DBD (wild-type and mutated forms, bottom panel) in transfected HEK293 cells. Antibodies are indicated on the right. Untr indicates untransfected cells. *Nonspecific band; arrow, the LMO2-Gal4DBD fusion protein. (Ci) Overexpression assay in zebrafish embryos. The hemangioblast marker Fli1 is expressed in the anterior and posterior lateral plate mesoderm in uninjected wild-type embryos. Note that Fli1 expression is absent from the cardiac mesoderm (square brackets). (Cii) Co-overexpression of wild-type Scl/Tal1 and Lmo2 mRNA causes an expansion of Fli1 expression in the head mesoderm (red arrowheads) and cardiac mesoderm (square brackets). This is abolished when Scl/Tal1 is coexpressed with the Lmo2 mutants LMO2-F88A (Ciii) or LMO2-F88D (Civ). Embryos were staged at 10 somites and flat mounted. Anterior to the top. The number of embryos displaying the phenotype is shown in each panel.