Multiple functions for Eya3 in mammalian organism

The important function of Eya (eyes absent) in eye development of D.melanogaster is well known, the function of its mammalian orthologs Eya1-4 is only partially understood. Yet, no phenotype for Eya2 and Eya3 in humans or mouse have been described. In our study we analysed new Eya-deficient mouse model generated by insertional mutagenesis. Expression analysis of Eya3 by in-situ hybridizations and beta-Gal-staining revealed a broad expression of the gene throughout development, e.g. in brain, eyes, branchial arches, somites and limbs. This diverse expression pattern suggests pleiotropic effects of Eya3 in mammalian organisms. In contrast to D. melanogaster, a close examination of the eye by various methods did not show any obvious ocular defects in mutant animals. A broad phenotypical characterization of homozygous mutants displayed decreased body weight and body length. Additionally, behavioral changes as a educed forward locomotor activity and altered exploratory behaviour were identified. A significant reduction of forelimb grip strength and an impairment of cardiac processes in Eya3-/- mice suggest a role for Eya3 in muscle development and heart function. Our results implicate that Eya3 plays various roles outside the eye with effects particularly on heart and muscle function in the mouse. Keywords: Eya3-deficient mice, cDNA microarrays, brain, heart, muscle analysis of 4 Eya3 mutant mice versus a pool of 5 wild type animals; brain, heart, and muscle; 8 hybridisations for each tissue: two technical replicates for each mutant animal including one dye-swap experiments小眼缺失（eyes absent，简称Eya）在黑腹果蝇（Drosophila melanogaster，D.melanogaster）眼睛发育中的关键功能已广为人知，但其哺乳动物同源基因Eya1-4的功能仍仅部分阐明。目前尚未见人类与小鼠中Eya2及Eya3相关表型的报道。本研究针对通过插入诱变构建的新型Eya缺陷型小鼠模型展开分析。通过原位杂交（in-situ hybridizations）与β-半乳糖苷酶染色（beta-Gal-staining）对Eya3进行表达分析，结果显示该基因在发育全程广泛表达，例如在脑、眼、鳃弓、体节与四肢中均有分布。这种广泛的表达模式提示Eya3在哺乳动物体内存在多效性作用。与黑腹果蝇的情况不同，通过多种方法对突变小鼠的眼部进行细致检查，未发现任何明显的眼部缺陷。对纯合突变体的全面表型分析显示，其体重与体长均有所降低。此外，还检测到其行为发生改变：如自主活动能力下降、探索行为异常。Eya3基因敲除（Eya3-/-）小鼠的前肢握力显著下降，且心脏功能出现异常，这提示Eya3在肌肉发育与心脏功能中发挥作用。本研究结果表明，在小鼠体内，Eya3在眼部之外发挥多种功能，尤其对心脏与肌肉功能具有重要影响。关键词：Eya3缺陷型小鼠、cDNA微阵列（cDNA microarrays）、脑、心脏、肌肉；实验设置：以4只Eya3突变小鼠与5只野生型动物的混合样本开展对照实验；检测组织为脑、心脏与肌肉；每个组织进行8次杂交实验，每只突变小鼠设置2次技术重复（technical replicates），其中包含1次染料互换实验（dye-swap experiments）。