Tmod3-/- mouse fetal liver compared with wild type

Tropomodulins (Tmods) cap the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse Tmod3 leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. BFU-E and CFU-E colony numbers are greatly reduced, indicating defects in progenitor populations. Flow-cytometry of fetal liver erythroblasts shows late stage populations are also decreased, including reduced percentages of enucleated cells. AnnexinV staining indicates increased apoptosis of Tmod3-/- erythroblasts, and cell cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3-/- embryos. Notably, enucleating Tmod3-/- erythroblasts are still in the process of proliferation, suggesting impaired cell cycle exit during terminal differentiation. Tmod3-/- late erythroblasts often exhibit multi-lobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3-/- fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis, due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation. Total RNAs from Tmod3+/+ and Tmod3-/- fetal livers at E14.5 were extracted and prepared for microarray analysis using the MoGene-1_0-st-v1 Affymetrix chip in the Scripps Research Microarray Core Facility. Each experiment was repeated with three independent embryos.肌动蛋白丝尖端封端蛋白（Tropomodulins, Tmods）可在红系与非红系细胞中包裹肌动蛋白丝的尖状末端。对小鼠Tmod3进行定向敲除会导致胚胎在E14.5-E18.5阶段出现致死，同时因胎肝内定型红细胞生成缺陷引发贫血。爆式红系集落形成单位（BFU-E）与红细胞系集落形成单位（CFU-E）的集落数量显著减少，提示祖细胞群体存在功能缺陷。对胎肝成红细胞的流式细胞术检测显示，晚期成红细胞群体数量同样下降，包括脱核细胞占比降低。膜联蛋白V染色结果表明，Tmod3敲除（Tmod3-/-）的成红细胞凋亡率升高；细胞周期分析则显示，Tmod3-/-胚胎中处于S期的Ter119高表达细胞比例更高。值得注意的是，正在进行脱核的Tmod3-/-成红细胞仍处于增殖状态，提示其在终末分化过程中细胞周期退出机制受损。Tmod3-/-晚期成红细胞常呈现多叶核形态，且在脱核过程中出现F-肌动蛋白组装异常。此外，Tmod3-/-胎肝中成红细胞岛的形成过程受损，且Tmod3在成红细胞与巨噬细胞中均发挥必要作用。综上，Tmod3功能缺失会导致定型红细胞生成受损，其机制包括祖细胞数量减少、成红细胞岛形成障碍，以及成红细胞的细胞周期进程与脱核过程缺陷。Tmod3介导的肌动蛋白重塑可能对于成红细胞-巨噬细胞黏附、细胞周期与分化的协同调控，以及成红细胞脱核过程中的F-肌动蛋白组装与重塑均不可或缺。研究人员提取了E14.5阶段Tmod3野生型（Tmod3+/+）与Tmod3敲除（Tmod3-/-）胎肝的总RNA，使用MoGene-1_0-st-v1 芯片（Affymetrix品牌）在斯克里普斯研究所芯片核心实验室进行芯片分析，每组实验均采用3个独立胚胎样本重复。