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In the experiment group, injection of as little as 20?pg of synthetic RNA per embryo restored RBC formation and rescued coloboma, inflated head and vascular abnormalities (see below) in the mutants (mutant phenotype: 3/174, 1.7%; p[http://www.selleckchem.com/products/GDC-0941.html Selleckchem GDC 941] by genotyping. In contrast, injection of synthetic RNA encoding Lmo2C94X mutant protein at similar or much higher doses (250?pg per embryo), failed to rescue any of the described phenotypes (mutant phenotype: 96/343, 28%; p=0.1; Fig. 1G). It has been shown that forced expression of a truncated Lmo2 protein missing its second LIM domain in transgenic mice resulted in decreased hematopoiesis, suggesting that in the absence of the second LIM domain, Lmo2 functioned as a dominant negative (Terano et al., 2005). We therefore injected very high doses of lmo2C94X synthetic RNA into wild-type embryos (750?pg per embryo), however, we could not detect any abnormal phenotypes in the injected embryos (Suppl. Fig. S2C and D). Altogether, the results indicate that eye, head, vasculature and blood phenotypes result from lmo2vu270 mutation and suggest that the mutant protein lost its activity. The lack of RBCs does not explain the enlarged head and coloboma phenotypes observed in lmo2 mutants, suggesting that loss of Lmo2 function results in additional abnormalities, which underlie these defects. [http://en.wikipedia.org/wiki/Diglyceride diglyceride] To understand why loss of Lmo2 function resulted in an inflated-looking head and ocular coloboma, we first sectioned and compared heads of 2 dpf mutant and normal embryos. Transverse sections through the forebrain of mutants revealed abnormal spaces around the eyes and lenses and within head tissues ( Fig. 2A and B). Additionally, brain ventricles were grossly inflated ( Fig. 2A and B). These findings suggest that mutant embryos suffer from severe [http://www.selleckchem.com/products/AZD0530.html Selleckchem AZD0530] head edema. Edema could be a consequence of vascular abnormalities, for example, increased vascular permeability. We therefore examined ocular blood vessels in live Tg(kdrl:EGFP) embryos whose endothelial cells express EGFP ( Jin et al., 2005). Early embryonic ocular vasculature comprises two systems, the intraocular hyaloid system and a superficial system that gives rise to choroidal vasculature ( Alvarez et al., 2007, Kitambi et al., 2009?and?Saint-Geniez and D'Amore, 2004). In normal embryos at 2 dpf, the hyaloid artery enters the eye through the medially located optic disk and supplies a network of capillaries that partially envelope the lens ( Fig. 2C and E). The superficial system at this stage comprises the nasal radial vessel (nrv) through which blood enters, and the dorsal radial vessel (drv) and ventral radial vessel (vrv) through which blood drains from the system ( Fig. 2E) (following nomenclature by ( Kitambi et al., 2009)).