Bolatto Carmen, Nieves Sofía, Reyes Agustina, Olivera-Bravo Silvia, Cambiazo Verónica
Developmental Biology Laboratory, Histology and Embryology Department, Faculty of Medicine, Universidad de la República (UdelaR), Montevideo, Uruguay.
Cell and Molecular Neurobiology Laboratory, Computational and Integrative Neuroscience (NCIC) Department, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay.
Front Neurosci. 2022 Aug 23;16:920670. doi: 10.3389/fnins.2022.920670. eCollection 2022.
(), classified primarily as a neuroectodermal gene, encodes a protein with predicted topology and domain organization closely related to those of Patched (Ptc), the canonical receptor of the Hedgehog (Hh) pathway. To investigate the physiological function of Ptr in the developing nervous system, null mutant embryos were immunolabeled and imaged under confocal microscopy. These embryos displayed severe alterations in the morphology of the primary axonal tracts, reduced number, and altered distribution of the Repo-positive glia as well as peripheral nervous system defects. Most of these alterations were recapitulated by downregulating expression, specifically in embryonic nerve cells. Because similar nervous system phenotypes have been observed in and mutant embryos, we evaluated the Ptr participation in the Hh pathway by performing cell-based reporter assays. Clone-8 cells were transfected with -specific dsRNA or a DNA construct and assayed for changes in Hh-mediated induction of a luciferase reporter. The results obtained suggest that Ptr could act as a negative regulator of Hh signaling. Furthermore, co-immunoprecipitation assays from cell culture extracts premixed with a conditioned medium revealed a direct interaction between Ptr and Hh. Moreover, overexpression in the domain of the imaginal wing disc where Engrailed and Ptc coexist produced wing phenotypes at the A/P border. Thus, these results strongly suggest that Ptr plays a crucial role in nervous system development and appears to be a negative regulator of the Hh pathway.
（）主要被归类为一种神经外胚层基因，编码一种蛋白质，其预测的拓扑结构和结构域组织与刺猬信号通路（Hh）的经典受体帕奇（Ptc）密切相关。为了研究Ptr在发育中的神经系统中的生理功能，对null突变胚胎进行免疫标记并在共聚焦显微镜下成像。这些胚胎在初级轴突束的形态、Repo阳性神经胶质细胞的数量减少和分布改变以及外周神经系统缺陷方面表现出严重变化。通过下调表达，特别是在胚胎神经细胞中，这些变化大多得以重现。因为在和突变胚胎中观察到了类似的神经系统表型,我们通过进行基于细胞的报告基因分析来评估Ptr在Hh信号通路中的参与情况。用特异性dsRNA或DNA构建体转染Clone-8细胞，并检测Hh介导的荧光素酶报告基因诱导的变化。所得结果表明Ptr可能作为Hh信号的负调节因子。此外，从与条件培养基预混合的细胞培养提取物中进行的免疫共沉淀分析揭示了Ptr和Hh之间的直接相互作用。此外，在翅成虫盘的Engrailed和Ptc共存区域过表达会在A/P边界产生翅表型。因此，这些结果强烈表明Ptr在神经系统发育中起关键作用，并且似乎是Hh信号通路的负调节因子。
