Mouse models of forebrain defects caused by Pax6 haploinsufficiency

在人类中，约有1/50，000的活产婴儿患有一种称为先天性无虹膜的疾病，这种疾病是由破坏PAX 6基因功能的突变引起的。这种基因编码一种蛋白质，这种蛋白质是其他基因活性的高水平调节剂，在发育中的眼睛和大脑中表达，对它们的正常形成至关重要。在这里，我们有兴趣了解PAX 6的一个拷贝的功能丧失对大脑最高级部分-前脑发育的影响。如果PAX 6基因的两个拷贝都发生突变，以至于根本没有功能性PAX 6，结果是在出生前或出生后不久就致命，并且大脑严重异常。具有导致PAX 6的一个拷贝丧失功能的突变的患者具有正常的预期寿命，但显示出大脑结构和功能的异常。这些异常包括连接大脑两个半球的主要结构胼胝体的体积减少，以及一系列神经和精神缺陷。众所周知，缺失Pax 6基因功能的突变小鼠为先天性无虹膜中发现的至少一些缺陷提供了极好的模型。杂合突变小鼠（即Pax 6的两个拷贝中只有一个是异常的突变体，称为Pax 6 +/-）是先天性无虹膜的最佳小鼠模型，因为患有该病症的人是PAX 6功能丧失（PAX 6 +/-）的杂合小鼠。虽然之前在Pax 6 +/-小鼠中发现了眼睛，嗅球和特定后脑结构的缺陷，但直到现在还没有确定模拟人类患者的前脑缺陷。我们现在有新的试点数据显示，小鼠中的杂合Pax 6突变导致特定的前脑缺陷。这些发现对于理解人类先天性无虹膜可能非常相关。我们发现：（i）成年Pax 6 +/-小鼠中胼胝体的显著变窄，该结果与患有先天性无虹膜的杂合PAX 6 +/-人中胼胝体异常的观察结果非常吻合。(ii)一组特定类型的Pax 6 +/-细胞（激活或过去激活称为Zic 4的基因的细胞：即所谓的Zic 4谱系细胞）在前脑的一个区域中密度增加，称为膝状体核，与大脑皮层连接;尚未报道PAX 6 +/-人类中相应缺陷的研究。我们的目的是为了更好地了解这些前脑缺陷及其原因，以期提高我们对与人类先天性无虹膜相关的前脑异常的认识。在第一部分中，我们将研究Pax 6 +/-小鼠的胼胝体缺陷，其发病年龄，它们的细胞原因以及Pax 6 +/-小鼠是否表现出先前作者认为与胼胝体异常有关的行为异常。在第二部分中，我们将研究突变Pax 6的一个拷贝对膝状体核中Zic 4系细胞发育的影响。我们将首先研究Zic 4谱系细胞中特异性突变Pax 6对这些细胞的出生后发育及其与皮质的连接的影响。然后，我们将研究在所有细胞中携带一个Pax 6突变拷贝的小鼠中Zic 4谱系细胞发生了什么，这种情况最接近人类先天性无虹膜模型。总之，我们的目标是利用我们最近发现的缺陷，这些缺陷可能与理解先天性无虹膜的前脑异常高度相关。一旦我们知道了这里提出的问题的答案（例如，缺陷是早期还是晚期发作，它们是细胞自主的还是细胞非自主的？）我们将能够设计未来的实验，为我们的发现提供分子解释，并测试拯救可能适用于人类的异常的方法。

In the human population, ~1/50,000 live births suffer from a condition called congenital aniridia, which results from mutations that disrupt the function of a gene called PAX6. This gene, which codes for a protein that is a high-level regulator of the activity of other genes, is expressed in developing eye and brain and is critical for their normal formation. Here, we are interested in understanding the consequences of the loss of function of one copy of PAX6 for development of the most advanced part of the brain, the forebrain. If both copies of the PAX6 gene are mutated so that there is no functional PAX6 at all, the result is fatal before or shortly after birth and there are severe abnormalities of the brain. Patients with mutations causing one copy of PAX6 to lose function have normal life expectancy but show abnormalities of brain structure and function. These abnormalities include a reduced volume of the major structure that links the two hemispheres of the brain, the corpus callosum, and a range of neurological and psychiatric defects. It is well-recognized that mutant mice that have lost Pax6 gene function provide excellent models of at least some of the defects found in congenital aniridia. Heterozygous mutant mice (i.e. mutants in which just one of the two copies of Pax6 is abnormal, designated Pax6+/-) are the best mouse models for congenital aniridia, since humans surviving with the condition are heterozygous for PAX6 loss-of-function (PAX6+/-). Whereas defects of the eye, olfactory bulbs and specific hindbrain structures have been found before in Pax6+/- mice, until now forebrain defects that model those in human patients have not been identified. We now have new pilot data showing that heterozygous Pax6 mutations in mice cause specific forebrain defects. These findings are potentially very relevant for understanding human congenital aniridia. We have found: (i) A significant narrowing of the corpus callosum in adult Pax6+/- mice, a result that fits well with observations of corpus callosum abnormalities in heterozygous PAX6+/- humans with congenital aniridia. (ii) An increased density of a set of Pax6+/- cells of a specific type (cells that activate, or in their past activated, a gene called Zic4: i.e. so-called Zic4-lineage cells) in a region of the forebrain, known as the geniculate nuclei, that connects with the cerebral cortex; studies of corresponding defects in PAX6+/- humans have not yet been reported. Our objective in this proposal is to gain a better understanding of these forebrain defects and their causes in mice, with a view to improving our knowledge of the forebrain abnormalities associated with human congenital aniridia.In the first part, we shall study callosal defects in Pax6+/- mice, their age of onset, their cellular causes and whether Pax6+/- mice show behavioural abnormalities that have been linked by previous authors with callosal abnormalities. In the second part, we shall study the effects of mutating one copy of Pax6 on the development of Zic4-lineage cells in the geniculate nuclei. We shall first study the effects of mutating Pax6 specifically in Zic4-lineage cells on the postnatal development of these cells and their connections to the cortex. We shall then study what happens to Zic4-lineage cells in mice carrying one mutant copy of Pax6 in all cells, a condition that most closely models human congenital aniridia. In summary, our objective is to capitalize on our recent identification of defects that are likely to be highly relevant for understanding forebrain abnormalities in congenital aniridia. Once we know the answers to the questions posed here (e.g. are defects early or late onset, are they cell autonomous or cell non-autonomous?) we shall be able to design future experiments to provide molecular explanations for our discoveries and to test ways of rescuing abnormalities that might be applicable to humans.