Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia

During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358A->C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71T->C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development. Time course analysis of mouse embryonic tissues from Aey069 mice and controls在一次ENU（N-乙基-N-亚硝基脲，N-ethyl-N-nitrosourea）诱变筛选实验中，我们观测到一种显性小眼突变小鼠，其纯合突变个体可正常存活。研究团队随后建立了对应的突变品系，命名为Aey69（眼部异常#69，abnormality of the eye #69）。在德国小鼠诊所（German Mouse Clinic）对纯合Aey69突变小鼠开展的全面表型分析显示，野生型个体与纯合突变体之间仅存在少量具有统计学显著性的表型差异。该突变会引发无晶状体小眼畸形，并伴随视网膜过度增殖。连锁分析证实，该突变位点定位于小鼠3号染色体D3Mit188与D3Mit11标记区域之间。测序结果显示，Hist2h3c1基因存在一处358A→C突变（对应氨基酸改变为Ile120Leu），Gja8基因存在一处71T→C突变（对应氨基酸改变为Val24Ala）。对纯合突变小鼠眼部发育的详细分析表明，晶状体发育紊乱始于晶状体泡阶段，伴随晶状体蛋白以及晶状体特异性转录因子（如PITX3、FOXE3）的表达水平显著下调。与之相反，我们观测到以多种标志物为特征的视网膜祖细胞早期异常表达，其中包括BRN3（视网膜神经节细胞标志物）与OTX2（视锥感光细胞标志物）。在胚胎发育阶段E11.5至E15.5，视网膜出现异常改变的同时，晶状体在对应阶段发生凋亡过程。过度的视网膜增殖以Ki67表达水平升高为特征。尽管过度增殖的视网膜最终会覆盖整个眼球，但该异常增殖并未影响出生后阶段主要视网膜细胞类型的分化与形态构建。通过吗啉代寡核苷酸介导的斑马鱼（zebrafish）hist2h3ca1基因敲低实验，证实该基因会特异性扰乱晶状体发育。将吗啉代寡核苷酸注射入斑马鱼受精卵后，仅突变型小鼠的mRNA会引发严重发育畸形，表型范围从独眼畸形到重度小眼畸形。野生型Hist2h3c1 mRNA可挽救吗啉代寡核苷酸诱导的发育缺陷，进一步证实了其在晶状体发育中的特异性功能。基于上述实验数据，我们得出结论：Hist2h3c1基因（编码经典H3.2组蛋白变体）的眼部功能在进化过程中具有高度保守性。此外，本研究结果还凸显了Hist2h3c1在眼部发育过程中晶状体与视网膜协同形成过程中的关键作用。对Aey69小鼠及对照小鼠的胚胎组织开展的时间进程分析