The Queensland Brain Institute, The University of Queensland, Brisbane 4072, Australia.
Dev Biol. 2012 May 1;365(1):36-49. doi: 10.1016/j.ydbio.2012.02.004. Epub 2012 Feb 11.
The Slit molecules are chemorepulsive ligands that regulate axon guidance at the midline of both vertebrates and invertebrates. In mammals, there are three Slit genes, but only Slit2 has been studied in any detail with regard to mammalian brain commissure formation. Here, we sought to understand the relative contributions that Slit proteins make to the formation of the largest brain commissure, the corpus callosum. Slit ligands bind Robo receptors, and previous studies have shown that Robo1(-/-) mice have defects in corpus callosum development. However, whether the Slit genes signal exclusively through Robo1 during callosal formation is unclear. To investigate this, we compared the development of the corpus callosum in both Slit2(-/-) and Robo1(-/-) mice using diffusion magnetic resonance imaging. This analysis demonstrated similarities in the phenotypes of these mice, but crucially also highlighted subtle differences, particularly with regard to the guidance of post-crossing axons. Analysis of single mutations in Slit family members revealed corpus callosum defects (but not complete agenesis) in 100% of Slit2(-/-) mice and 30% of Slit3(-/-) mice, whereas 100% of Slit1(-/-); Slit2(-/-) mice displayed complete agenesis of the corpus callosum. These results revealed a role for Slit1 in corpus callosum development, and demonstrated that Slit2 was necessary but not sufficient for midline crossing in vivo. However, co-culture experiments utilising Robo1(-/-) tissue versus Slit2 expressing cell blocks demonstrated that Slit2 was sufficient for the guidance activity mediated by Robo1 in pre-crossing neocortical axons. This suggested that Slit1 and Slit3 might also be involved in regulating other mechanisms that allow the corpus callosum to form, such as the establishment of midline glial populations. Investigation of this revealed defects in the development and dorso-ventral positioning of the indusium griseum glia in multiple Slit mutants. These findings indicate that Slits regulate callosal development via both classical chemorepulsive mechanisms, and via a novel role in mediating the correct positioning of midline glial populations. Finally, our data also indicate that some of the roles of Slit proteins at the midline may be independent of Robo signalling, suggestive of additional receptors regulating Slit signalling during development.
缝隙分子是脊椎动物和无脊椎动物中线轴导向的化学排斥配体。在哺乳动物中，有三个 Slit 基因，但只有 Slit2 与哺乳动物脑连合形成的关系进行了详细研究。在这里，我们试图了解缝隙蛋白对最大脑连合胼胝体形成的相对贡献。缝隙配体与 Robo 受体结合，先前的研究表明 Robo1(-/-) 小鼠的胼胝体发育缺陷。然而，在胼胝体形成过程中，Slit 基因是否仅通过 Robo1 信号传导尚不清楚。为了研究这一点，我们使用扩散磁共振成像比较了 Slit2(-/-) 和 Robo1(-/-) 小鼠胼胝体的发育情况。该分析表明这些小鼠的表型存在相似之处，但关键是还突出了细微的差异，特别是在交叉后轴突的导向方面。对 Slit 家族成员的单一突变分析显示，100%的 Slit2(-/-) 小鼠和 30%的 Slit3(-/-) 小鼠出现胼胝体缺陷（但不是完全缺失），而 100%的 Slit1(-/-); Slit2(-/-) 小鼠的胼胝体完全缺失。这些结果表明 Slit1 在胼胝体发育中起作用，并表明 Slit2 是体内中线交叉所必需的，但不是充分的。然而，利用 Robo1(-/-) 组织与表达 Slit2 的细胞块进行共培养实验表明，Slit2 足以介导 Robo1 在预交叉新皮层轴突中的导向活性。这表明 Slit1 和 Slit3 也可能参与调节胼胝体形成的其他机制，例如中线胶质细胞群体的建立。对这方面的研究揭示了多个 Slit 突变体中灰结节胶质细胞发育和背腹定位的缺陷。这些发现表明缝隙通过经典的化学排斥机制以及通过调节中线胶质细胞群体正确定位的新作用来调节胼胝体的发育。最后，我们的数据还表明，缝隙蛋白在中线的一些作用可能独立于 Robo 信号，表明在发育过程中还有其他受体调节缝隙信号。
