Zhang Tingting, Cui Shiquan, Xiong Xinrui, Liu Ying, Cao Qilin, Xia Xu-Gang, Zhou Hongxia
Department of Environmental Health Sciences, Robert Stempel College of Public Health and Social Work, Florida International University, Miami, FL, United States.
The Center for Translational Sciences, Port St Lucie, FL, United States.
Front Cell Dev Biol. 2023 Oct 12;11:1282787. doi: 10.3389/fcell.2023.1282787. eCollection 2023.
Recessive mutation of the X-linked gene, (), causes familial ciliopathy. PIH1D3 deficiency is associated with the defects of dynein arms in cilia, but how PIH1D3 specifically affects the structure and function of dynein arms is not understood yet. To gain insights into the underlying mechanisms of the disease, it is crucial to create a reliable animal model. In humans, rats, and mice, one copy of the gene is located on the X chromosome. Interestingly, mice have an additional, intronless copy of the gene on chromosome 1. To develop an accurate disease model, it is best to manipulate the X-linked gene, which contains essential regulatory sequences within the introns for precise gene expression. This study aimed to develop a tailored rat model for PIH1D3-associated ciliopathy with the ultimate goal of uncovering the intricate molecular mechanisms responsible for ciliary defects in the disease. Novel Pih1d3-knockout (KO) rats were created by using TALEN-mediated non-homologous DNA recombination within fertilized rat eggs and, subsequently, underwent a comprehensive characterization through a battery of behavioral and pathological assays. A series of biochemical and histological analyses were conducted to elucidate the identity of protein partners that interact with PIH1D3, thus shedding light on the intricate molecular mechanisms involved in this context. PIH1D3-KO rats reproduced the cardinal features of ciliopathy including situs inversus, defects in spermatocyte survival and mucociliary clearance, and perinatal hydrocephalus. We revealed the novel function of PIH1D3 in cerebrospinal fluid circulation and elucidated the mechanism by which PIH1D3 deficiency caused communicating hydrocephalus. PIH1D3 interacted with the proteins required for the pre-assembly and uploading of outer (ODA) and inner dynein arms (IDA), regulating the integrity of dynein arm structure and function in cilia. PIH1D3-KO rats faithfully reproduced the cardinal features of ciliopathy associated with PIH1D3 deficiency. PIH1D3 interacted with the proteins responsible for the pre-assembly and uploading of dynein arms in cilia, and its deficiency led to dysfunctional cilia and, thus, to ciliopathy by affecting the pre-assembly and uploading of dynein arms. The resultant rat model is a valuable tool for the mechanistic study of PIH1D3-caused diseases.
X连锁基因（）的隐性突变会导致家族性纤毛病。PIH1D3缺乏与纤毛中动力蛋白臂的缺陷有关，但PIH1D3如何具体影响动力蛋白臂的结构和功能尚不清楚。为了深入了解该疾病的潜在机制，建立一个可靠的动物模型至关重要。在人类、大鼠和小鼠中，该基因的一个拷贝位于X染色体上。有趣的是，小鼠在1号染色体上还有一个无内含子的该基因拷贝。为了开发一个准确的疾病模型，最好操纵X连锁的该基因，其内含子中含有精确基因表达所需的关键调控序列。本研究旨在开发一种针对PIH1D3相关纤毛病的定制大鼠模型，最终目标是揭示导致该疾病纤毛缺陷的复杂分子机制。通过在受精大鼠卵内使用TALEN介导的非同源DNA重组创建了新型Pih1d3基因敲除（KO）大鼠，随后通过一系列行为和病理检测进行了全面表征。进行了一系列生化和组织学分析，以阐明与PIH1D3相互作用的蛋白质伙伴的身份，从而揭示其中涉及的复杂分子机制。PIH1D3-KO大鼠重现了纤毛病的主要特征，包括内脏反位、精母细胞存活和黏液纤毛清除缺陷以及围产期脑积水。我们揭示了PIH1D3在脑脊液循环中的新功能，并阐明了PIH1D3缺乏导致交通性脑积水的机制。PIH1D3与外动力蛋白臂（ODA）和内动力蛋白臂（IDA）预组装和加载所需的蛋白质相互作用，调节纤毛中动力蛋白臂结构和功能的完整性。PIH1D3-KO大鼠忠实地重现了与PIH1D3缺乏相关的纤毛病的主要特征。PIH1D3与负责纤毛中动力蛋白臂预组装和加载的蛋白质相互作用，其缺乏导致纤毛功能失调，从而通过影响动力蛋白臂的预组装和加载导致纤毛病。所得的大鼠模型是研究PIH1D3所致疾病机制的宝贵工具。
