The role of Arl13b in transcription-independent Sonic hedgehog regulation of axon guidance

项目总结： Sonic hedgehog(Shh)信号是已知的调节细胞命运的关键发育途径 规格和细胞增殖。这一过程需要初级纤毛，并涉及Shh激活 下游效应因子(Smo)和Gli转录因子(依赖于转录)。另外， Shh信号调节神经系统发育中的轴突引导和成纤维细胞迁移 通过一条截然不同的、不依赖转录的途径。Arl13b编码富含纤毛的调节性GTP酶 通过Smo调节转录依赖的Shh信号。Arl13b基因的隐性突变导致 纤毛病Joubert综合征(JS)，一种由智力障碍、小脑发育不良和 身体畸形。JS被诊断为后脑畸形的MRI证据，这种畸形被称为臼齿 体征，部分是由组成小脑上脚的白质束(SCP)引起的 没有穿过大脑的中线。JS患者还表现出视交叉中线交叉缺陷和 皮质脊髓束。总体而言，这些表型表明JS患者存在轴突引导缺陷； 然而，纤毛基因如arl13b与轴突引导的调节机制仍然存在。 不清楚。因为(1)Arl13b调节转录依赖的Shh信号，(2)转录不依赖 Shh信号调节轴突引导；(3)Arl13b突变导致JS轴突引导缺陷。 我们假设Arl13b调节转录非依赖性Shh信号以指导轴突引导。 发育中的大脑。为了测试这一点，我检查了缺少Smo或Smo的小鼠SCP的方向和一致性 Arl13b通过进行扩散张量成像(DTI)。我展示了缺乏Smo的SCP显示出显著的轴突 后脑的引导缺陷，表明这些投射神经元中的轴突引导是Smo依赖的 暗示Shh可能是一个指导信号。此外，SCP中存在轴突引导缺陷。 Arl13b，提示Arl13b调控这一转录非依赖性Shh信号通路。另外， 缺乏Arl13b的成纤维细胞向Shh迁移减少。然而，成纤维细胞能够在 纤毛的缺失，提示Arl13b在转录调控中可能具有非纤毛功能。 独立的嘘信号。因此，我们假设Arl13b在纤毛外调节 转录非依赖的Sonic Hedgehog信号对成纤维细胞体外和轴突迁移的直接作用 神经发育过程中的体内指导。本项目的目标是1)使用非纤毛Arl13b和Smo 通过改良Boyden测试Arl13b在成纤维细胞迁移中的功能是否发生在纤毛外的变体 小室分析和2)通过检测SCP轴突的能力来确定Arl13b在轴突引导中的作用 通过DTI和逆行追踪跨越Arl13b和Smo突变小鼠的大脑中线。 通过这些方法，我的目标是确定Arl13b在转录非依赖Shh信号和 在纤毛相关基因和轴突引导之间建立一种新的联系。

Project Summary: Sonic hedgehog (Shh) signaling is a critical developmental pathway best known to regulate cell fate specification and cell proliferation. This process requires primary cilia and involves Shh activation of downstream effector Smoothened (Smo) and Gli transcription factors (transcription-dependent). Additionally, Shh signaling regulates axon guidance in the developing nervous system as well as fibroblast migration through a distinct, transcription-independent pathway. Arl13b encodes a regulatory GTPase enriched in cilia that regulates transcription-dependent Shh signaling through Smo. Recessive mutations in ARL13B cause the ciliopathy Joubert syndrome (JS), a disorder defined by intellectual disability, cerebellar hypoplasia, and physical deformities. JS is diagnosed by MRI evidence of a hindbrain malformation known as the molar tooth sign, which is caused in part by the white matter tracts comprising the superior cerebellar peduncles (SCPs) failing to cross the midline of the brain. JS patients also exhibit midline crossing defects in the optic chiasm and the corticospinal tract. Collectively, these phenotypes indicate JS patients display axon guidance defects; however, the mechanism connecting cilia genes such as ARL13B to the regulation of axon guidance remains unclear. Because (1) Arl13b regulates transcription-dependent Shh signaling, (2) transcription-independent Shh signaling can regulate axon guidance, and (3) ARL13B mutations result in axon guidance defects in JS, we hypothesize that Arl13b regulates transcription-independent Shh signaling to direct axon guidance in the developing brain. To test this, I examined the direction and uniformity of mouse SCPs lacking either Smo or Arl13b by performing diffusion tensor imaging (DTI). I showed that SCPs lacking Smo display significant axon guidance defects in the hindbrain, indicating axon guidance in these projection neurons is Smo-dependent and suggesting Shh as a possible guidance cue. Furthermore, there is an axon guidance defect in SCPs lacking Arl13b, suggesting Arl13b regulates this transcription-independent Shh signaling pathway. Additionally, fibroblasts lacking Arl13b display decreased migration toward Shh. However, fibroblasts are able to migrate in the absence of cilia, suggesting that Arl13b may have a non-ciliary function in the regulation of transcription- independent Shh signaling. Therefore, we hypothesize that Arl13b works outside of the cilium to regulate transcription-independent Sonic hedgehog signaling to direct fibroblast migration in vitro as well as axon guidance in vivo during neural development. The aims of this project are 1) to use non-ciliary Arl13b and Smo variants to test whether Arl13b function in fibroblast migration occurs outside the cilium via modified Boyden chamber assays and 2) to define the role of Arl13b in axon guidance by examining the ability of SCP axons to cross the midline of the brain in Arl13b and Smo mouse mutants through DTI and retrograde tract tracing. Through these methods, I aim to define the role of Arl13b in transcription-independent Shh signaling and establish a novel connection between cilia-associated genes and axon guidance.