Eyeless: Pitx3 mutant mouse line, Expression profiling of liver

A new mouse mutant arose spontaneously at the 5th inbred generation in a congenic C3H/He line. Since the mice suffered from anophthalmia or microphthalmia, we referred to this new mutant as eyeless (provisional gene symbol: eyl). The mutation follows an autosomal recessive pattern of inheritance and was mapped to chromosome 19 close to Pitx3; complementation tests confirmed that eyl is an allelic variant of aphakia (gene symbol: ak). Sequence analysis of the Pitx3 gene identified an insertion of a G in exon 4 (after cDNA position 416: 416insG). The altered open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but 121 new amino acids instead of the OAR domain at its C-terminal part. The new Pitx3eyl allele shares the ophthalmological and brain defects of the aphakia mouse including the loss of dopamine transporter 1 transcripts in the midbrain. Moreover, we observed in the mutants an increased extramedullary hematopoiesis in the spleen, and frequently also hepatosteatosis, combined with a reduced body weight. Fat mass, fat content and subcutaneous fat are significantly decreased in females, but not in males. Additionally, we observed a broad spectrum of behavioural changes in the homozygous mutants including reduced forelimb grip strength and increased nociception. In addition to these alterations being apparent in both sexes, we observed in female Pitx3eyl-/- mice increased anxiety, reduced locomotor activity, reduced object exploration and increased social contacts, but decreased anxiety and increased arousal in males. Most of these defects identified in the new Pitx3 mutation can also be observed in Parkinson patients. Therefore, the Pitx3eyl mutant is a valuable new model, because it is the first mutant mouse line carrying a point mutation within the coding region of the Pitx3 gene. 5 male animals, 5 experiments including 2 dye swap hybridisations本研究在同源导入C3H/He品系（congenic C3H/He line）的第5代近交代数中，自发获得一株新型小鼠突变体。该突变小鼠表现为无眼（anophthalmia）或小眼（microphthalmia）表型，故将此新突变命名为无眼（eyeless，临时基因符号：eyl）。 该突变遵循常染色体隐性（autosomal recessive）遗传模式，定位于19号染色体靠近Pitx3的区域；互补试验（complementation tests）结果证实，eyl为无晶状体眼（aphakia，基因符号：ak）的等位变异体。 对Pitx3基因的序列分析显示，其第4外显子（exon 4）的互补DNA（cDNA）位置416后存在一个G碱基插入（416insG）。该插入突变会改变基因的开放阅读框（open reading frame），预测可编码一种嵌合蛋白：该蛋白仍保留Pitx3同源结构域（homeobox），但在C端区域，原本的OAR结构域被替换为121个全新的氨基酸残基。 新型Pitx3^eyl等位基因与无晶状体眼小鼠具有一致的眼科与脑部缺陷，包括中脑多巴胺转运体1（dopamine transporter 1）转录本的缺失。此外，我们在突变体中观察到脾脏髓外造血（extramedullary hematopoiesis）增强，常伴随肝脂肪变性（hepatosteatosis），且突变小鼠体重显著降低。 雌性突变小鼠的脂肪总量、脂肪含量及皮下脂肪水平均显著下降，而雄性小鼠未出现该表型。我们还在纯合突变体中发现广泛的行为学改变，包括前肢抓握力减弱、痛觉敏感性升高。 除上述两性共有的表型外，雌性Pitx3^eyl^-/-小鼠还表现出焦虑水平升高、自主活动减少、物体探索能力下降及社交接触增多；而雄性突变小鼠则呈现焦虑水平降低、觉醒水平升高的表型。 本新发现的Pitx3突变所导致的多数缺陷，在帕金森病（Parkinson disease）患者中均有体现。因此，Pitx3^eyl突变小鼠是极具价值的新型动物模型——它是首个在Pitx3基因编码区携带点突变的小鼠品系。 本研究共使用5只雄性实验动物，开展5组实验，其中包含2次染料交换杂交（dye swap hybridisations）。