目的:脯氨酸羟化酶结构域2(PHD2),由Egln1基因编码,作为缺氧诱导因子(HIF)途径的关键调节剂,并充当细胞氧传感器。小鼠体内PHD2的体细胞失活导致红细胞增多症和充血性心力衰竭。然而,由于PHD2缺乏症的胚胎致死性,它在发展中的作用仍然难以捉摸。这里,我们研究了两个egln1同源基因的功能,egln1a和egln1b,斑马鱼。
方法:使用CRISPR/Cas9系统产生egln1无效斑马鱼。采用实时定量PCR和Westernblot分析检测egln1缺乏对缺氧信号通路的影响。通过分析心率评估egln1突变斑马鱼的缺氧反应,g搅动频率,和血流速度。随后,采用邻茴香胺染色和原位杂交技术研究egln1在斑马鱼造血功能中的作用。
结果:我们的数据表明,单独损失egln1a或egln1b对生长速率没有明显影响。然而,egln1a;egln1b双突变体在约2.5个月大时表现出明显的生长迟缓和死亡率升高。egln1a-null和egln1b-null斑马鱼胚胎对缺氧的耐受性均增强,全身缺氧反应,包括Hif通路激活,心脏活动增加,和红细胞增多症。
结论:我们的研究将斑马鱼egln1突变体作为PHD2的第一个先天性胚胎有活力的系统性脊椎动物动物模型,为缺氧信号和PHD2相关疾病的进展提供了新的见解。
OBJECTIVE: Prolyl hydroxylase domain 2 (PHD2), encoded by the Egln1 gene, serves as a pivotal regulator of the hypoxia-inducible factor (HIF) pathway and acts as a cellular oxygen sensor. Somatic inactivation of Phd2 in mice results in polycythemia and congestive heart failure. However, due to the embryonic lethality of Phd2 deficiency, its role in development remains elusive. Here, we investigated the function of two egln1 paralogous genes, egln1a and egln1b, in zebrafish.
METHODS: The egln1 null zebrafish were generated using the CRISPR/Cas9 system. Quantitative real-time PCR assays and Western blot analysis were employed to detect the effect of egln1 deficiency on the hypoxia signaling pathway. The hypoxia response of egln1 mutant zebrafish were assessed by analyzing heart rate, gill agitation frequency, and blood flow velocity. Subsequently, o-dianisidine staining and in situ hybridization were used to investigate the role of egln1 in zebrafish hematopoietic function.
RESULTS: Our data show that the loss of egln1a or egln1b individually has no visible effects on growth rate. However, the egln1a; egln1b double mutant displayed significant growth retardation and elevated mortality at around 2.5 months old. Both egln1a-null and egln1b-null zebrafish embryo exhibited enhanced tolerance to hypoxia, systemic hypoxic response that include hif pathway activation, increased cardiac activity, and polycythemia.
CONCLUSIONS: Our research introduces zebrafish egln1 mutants as the first congenital embryonic viable systemic vertebrate animal model for PHD2, providing novel insights into hypoxic signaling and the progression of PHD2- associated disease.