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

项目摘要： Sonic hedgehog（Shh）信号传导是调节细胞命运的重要发育途径 特异化和细胞增殖。这个过程需要初级纤毛，并涉及Shh激活 下游效应物Smoothened（Smo）和Gli转录因子（转录依赖性）。此外，本发明还 Shh信号调节神经系统发育中的轴突导向以及成纤维细胞迁移 通过一个独特的不依赖转录的途径。Arl 13 b编码在纤毛中富集的调节性GT3 通过Smo调节转录依赖的Shh信号。ARL 13 B的隐性突变导致 睫状体病Joubert综合征（JS），一种由智力残疾、小脑发育不全和 身体畸形JS是通过MRI证据诊断的后脑畸形称为臼齿 部分由包含上级小脑脚（SCP）的白色物质束引起的体征 无法穿过大脑中线JS患者还表现出视交叉中线交叉缺陷， 皮质脊髓束总的来说，这些表型表明JS患者显示轴突导向缺陷; 然而，连接纤毛基因如ARL 13 B与轴突导向调节的机制仍然存在， 不清楚因为（1）Arl 13 b调节转录依赖性Shh信号，（2）转录非依赖性Shh信号， Shh信号可以调节轴突导向，（3）ARL 13 B突变导致JS中轴突导向缺陷， 我们假设Arl 13 b调节转录非依赖性Shh信号传导，以指导轴突导向。 大脑发育为了测试这一点，我检查了缺乏Smo或Smo的小鼠SCP的方向和均匀性。 Arl 13 b通过执行扩散张量成像（DTI）。我发现缺乏Smo的SCP显示出显著的轴突 引导缺陷，表明这些投射神经元中的轴突引导是Smo依赖性的， 暗示“嘘”可能是一种引导信号此外，在缺乏轴突导向的SCP中， Arl 13 b，表明Arl 13 b调节这种转录非依赖性Shh信号通路。此外，本发明还 缺乏Ar 113 b的成纤维细胞显示向Shh的迁移减少。然而，成纤维细胞能够迁移到 纤毛的缺乏，表明Arl 13 b可能在转录调节中具有非纤毛功能- 独立的Shh信号。因此，我们假设Arl 13 b在纤毛外起作用，以调节 转录非依赖性Sonic hedgehog信号转导在体外指导成纤维细胞迁移以及轴突 在神经发育过程中的体内指导。本项目的目的是：1）使用非纤毛Arl 13 b和Smo 变体以测试Arl 13 b在成纤维细胞迁移中的功能是否通过修饰的Boyden在纤毛外发生 2）通过检查SCP轴突的能力来确定Ar 113 b在轴突引导中的作用， 通过DTI和逆行追踪，在Ar 113 b和Smo突变小鼠中穿过脑中线。 通过这些方法，我的目标是确定Arl 13 b在转录非依赖性Shh信号传导中的作用， 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.