Loss of <i>Usp9x</i> Disrupts Cortical Architecture, Hippocampal Development and TGFβ-Mediated Axonogenesis

The deubiquitylating enzyme Usp9x is highly expressed in the developing mouse brain, and increased Usp9x expression enhances the self-renewal of neural progenitors in vitro. USP9X is a candidate gene for human neurodevelopmental disorders, including lissencephaly, epilepsy and X-linked intellectual disability. To determine if Usp9x is critical to mammalian brain development we conditionally deleted the gene from neural progenitors, and their subsequent progeny. Mating Usp9xloxP/loxP mice with mice expressing Cre recombinase from the Nestin promoter deleted Usp9x throughout the entire brain, and resulted in early postnatal lethality. Although the overall brain architecture was intact, loss of Usp9x disrupted the cellular organization of the ventricular and sub-ventricular zones, and cortical plate. Usp9x absence also led to dramatic reductions in axonal length, in vivo and in vitro, which could in part be explained by a failure in Tgf-β signaling. Deletion of Usp9x from the dorsal telencephalon only, by mating with Emx1-cre mice, was compatible with survival to adulthood but resulted in reduction or loss of the corpus callosum, a dramatic decrease in hippocampal size, and disorganization of the hippocampal CA3 region. This latter phenotypic aspect resembled that observed in Doublecortin knock-out mice, which is an Usp9x interacting protein. This study establishes that Usp9x is critical for several aspects of CNS development, and suggests that its regulation of Tgf-β signaling extends to neurons.去泛素化酶（deubiquitylating enzyme）Usp9x在发育中的小鼠脑中呈高表达状态，且Usp9x表达上调可在体外增强神经祖细胞的自我更新能力。USP9X是人类神经发育障碍的候选致病基因，相关病症包括无脑回畸形（lissencephaly）、癫痫以及X连锁智力障碍（X-linked intellectual disability）。 为明确Usp9x对哺乳动物脑发育是否具有关键作用，我们通过条件性敲除技术敲除了神经祖细胞及其后续子代细胞中的该基因。将Usp9xloxP/loxP小鼠与巢蛋白（Nestin）启动子驱动Cre重组酶（Cre recombinase）表达的小鼠交配，可在全脑范围内敲除Usp9x，该操作导致小鼠出现出生后早期致死表型。 尽管整体脑结构保持完整，但Usp9x的缺失会扰乱脑室区（ventricular zone）、室下区（sub-ventricular zone）以及皮质板（cortical plate）的细胞组织形态。Usp9x的缺失还会在体内与体外环境中引发轴突长度的显著缩短，这一表型可部分通过转化生长因子-β（TGF-β）信号通路功能异常得到解释。 仅通过与Emx1-Cre小鼠交配，在背侧端脑（dorsal telencephalon）特异性敲除Usp9x，可使小鼠存活至成年，但会引发胼胝体（corpus callosum）缩小或缺失、海马体积显著下降，以及海马CA3区结构紊乱。后一种表型特征与双皮质素（Doublecortin）敲除小鼠的表型相似，而双皮质素是Usp9x的互作蛋白。 本研究证实Usp9x对中枢神经系统（CNS）发育的多个环节至关重要，并表明其对TGF-β信号通路的调控作用可延伸至神经元。