Manuel Martine, Pratt Thomas, Liu Min, Jeffery Glen, Price David J
Genes and Development Group, Centres for Integrative Physiology and Neuroscience Research, Hugh Robson Building, George Square, University of Edinburgh, Edinburgh EH8 9XD, UK.
BMC Dev Biol. 2008 May 28;8:59. doi: 10.1186/1471-213X-8-59.
The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey) and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections.
Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the optic chiasm vary differently with gene dosage. Increasing dosage increases the proportion projecting ipsilaterally regardless of the size of the total projection.
Pax6 overexpression does not obviously impair the initial formation of the eye and its major cell-types but prevents normal development of the retina from about E14.5, leading eventually to severe retinal degeneration in postnatal life. This sequence is different to that underlying microphthalmia in Pax6+/- heterozygotes, which is due primarily to defects in the initial stages of lens formation. Before the onset of severe retinal dysplasia, Pax6 overexpression causes defects of retinal axons, preventing their normal growth and navigation through the optic chiasm.
转录因子Pax6在发育中的眼睛的多种细胞类型中表达。在纯合功能丧失型小鼠突变体（Pax6Sey/Sey）中眼睛无法形成，而在Pax6Sey/+突变体中眼睛异常小。在除了内源性Pax6之外还表达多个拷贝的人类PAX6的PAX77小鼠中，眼睛也异常小；这两个物种的蛋白质序列相同。导致PAX77小鼠发生小眼症的发育事件尚未得到充分表征，因此尚不清楚Pax6/PAX6的过表达和低表达是否通过相似机制导致小眼症。在此，我们研究了过表达对眼睛及其轴突连接的影响。
PAX77+/+胚胎中眼睛形成，但随后退化。在胚胎发育12.5天时，我们未发现眼部形态、视网膜细胞周期参数及视网膜细胞死亡发生率有异常。从胚胎发育14.5天起，我们观察到视盘畸形。从胚胎发育16.5天到出生后，视网膜发育异常和小眼症逐渐加重。基因表达模式分析表明，PAX77+/+视网膜产生正常范围的细胞类型，包括视网膜神经节细胞（RGCs）。在胚胎发育14.5天和16.5天时，用一系列与视网膜发育相关分子的探针进行定量逆转录聚合酶链反应（RT-PCR），仅显示出一个显著变化：在胚胎发育16.5天时，编码分泌型形态发生素Shh的mRNA水平略有降低。在胚胎发育16.5天时，用羰花青染料对PAX77+/+胚胎进行束路追踪，发现RGC轴突在视网膜内导航存在错误，到达丘脑的RGC轴突数量减少，且在那些确实到达丘脑的RGC轴突中，同侧投射的比例增加。对具有不同Pax6/PAX6基因剂量（Pax6Sey/+、Pax6+/+、PAX77+和PAX77+/+）的胚胎进行的一项调查显示：（1）视网膜投射的RGC轴突总数；（2）在视交叉处被分类到同侧和对侧视束的比例随基因剂量的变化方式不同。无论总投射大小如何，增加剂量都会增加同侧投射的比例。
Pax6过表达不会明显损害眼睛及其主要细胞类型的初始形成，但从胚胎发育约14.5天起会阻止视网膜的正常发育，最终导致出生后严重的视网膜退化。这个过程与Pax6+/-杂合子中小眼症的潜在过程不同，后者主要是由于晶状体形成初始阶段的缺陷所致。在严重视网膜发育异常开始之前，Pax6过表达会导致视网膜轴突缺陷，阻止它们通过视交叉正常生长和导航。
