Elucidating the mechanism of action of Lmo4 in vertebrate eye development

We have identified the lmo4 locus as a regulator of the size and organization of the eye. Using the yeast 2 hybrid system, we have identified and characterized Lmo4-Ldb interactions. We show that Lmo4 interacts with zebrafish Ldb1, Ldb2, and Ldb3 (LIM-domain binding) proteins, albeit more strongly with Ldb1. This interaction can be mediated by both LIM domains of Lmo4, though the interaction is much stronger with LIM B rather than LIM A. Mutating both LIM domains through strategic point mutations of key zinc coordinating residues abolishes their ability to interact with Ldbs. Mutant overexpression studies showed that overexpressing LIM B results in a mild small eye phenotype whereas overexpressing LIM A does not. Furthermore, Ldb1, Ldb2, and Ldb3 can heterodimerize and all can interact with zebrafish Islet3, which belongs to the LIM-homeodomain class of proteins and is suggested by others to play an essential role in eye development. We have tested the model of islet3 antagonism by lmo4 through morpholino knock down of islet3 and attempted rescue of the lmo4 gain of function phenotype by islet3 overexpression. Knock down of islet3 does not result in an eye phenotype, and rescue experiments show that the lmo4 overexpression phenotype can not be rescued by ectopic isl3 overexpression. We conclude that islet3 is not necessary for eye development in zebrafish, and the role of lmo4 with respect to eye development must be mediated through an antagonism of other lhx genes or through an lhx-independent mechanism