Deletion of Stk40 impairs definitive erythropoiesis in the mouse fetal liver. Mus musculus

The serine threonine kinase Stk40 has been shown to involve in mouse embryonic stem cell differentiation, pulmonary maturation and adipocyte differentiation. Here we report that targeted deletion of Stk40 leads to fetal liver hypoplasia and anemia in the mouse embryos. The reduction of erythrocytes in the fetal liver is accompanied by increased apoptosis and compromised erythroid maturation. Stk40-/- fetal liver cells have significantly reduced colony forming units (CFUs) capable of erythroid differentiation, including burst forming unit-erythroid (BFU-E), colony forming unit-erythroid (CFU-E), and CFU-granulocyte, erythrocyte, megakaryocyte and macrophage (CFU-GEMM), but not CFU-granulocyte/macrophages (CFU-GM). Purified Stk40-/- megakaryocyte-erythrocyte progenitors (MEPs) produced substantially fewer CFU-E colonies compared to control cells. Moreover, Stk40-/- fetal liver erythroblasts failed to form normal erythroblastic islands in association with wild type or Stk40-/- macrophages, indicating an intrinsic defect of Stk40-/- erythroblasts. Furthermore, the hematopoietic stem and progenitor cell pool is reduced in Stk40-/- fetal livers but still retains the multi-lineage reconstitution capacity. Finally, analysis of microarray data of E14.5 fetal liver cells suggests a potential role of aberrantly activated TNF-α signaling in Stk40 depletion induced dyserythropoiesis with a concomitant increase in cleaved Caspase-3 and decrease in Gata1 proteins. Altogether, the identification of Stk40 as a regulator for fetal erythroid differentiation, maturation and survival provides new clues to the molecular regulation of erythropoiesis and related diseases. Overall design: Six E14.5 mouse fetal liver cells of same genotype were pooled together for each sample. There are two replicates for each genetype. mRNA extraction and hybridization on Affymetrix microarrays were performed by Shanghai Biochip Company.

丝氨酸苏氨酸激酶（serine threonine kinase）Stk40已被证实参与小鼠胚胎干细胞分化、肺脏成熟及脂肪细胞分化过程。本研究报道，靶向敲除Stk40会导致小鼠胚胎出现胎儿肝脏发育不全与贫血症状。胎儿肝脏内红细胞数量减少的同时，伴随细胞凋亡增加及红细胞成熟障碍。Stk40基因敲除（Stk40-/-）的胎儿肝脏细胞中，具备红细胞分化能力的集落形成单位（CFUs）显著减少，包括爆式红系集落形成单位（BFU-E）、红系集落形成单位（CFU-E）以及粒-红-巨核-巨噬细胞集落形成单位（CFU-GEMM），但粒-巨噬细胞集落形成单位（CFU-GM）未受明显影响。与对照细胞相比，纯化获得的Stk40-/-巨核细胞-红细胞祖细胞（MEPs）所形成的CFU-E集落数量大幅降低。此外，Stk40-/-胎儿肝脏成红细胞无法与野生型或Stk40-/-巨噬细胞构建正常的红细胞岛，提示Stk40-/-成红细胞存在内在功能缺陷。进一步研究发现，Stk40-/-胎儿肝脏中的造血干祖细胞池规模缩减，但仍保留多系造血重建能力。最后，对E14.5天小鼠胎儿肝脏细胞的芯片数据分析表明，异常激活的肿瘤坏死因子-α（TNF-α）信号通路可能参与了Stk40缺失诱导的红细胞生成障碍，同时伴随剪切型半胱天冬酶-3（cleaved Caspase-3）表达上调及Gata1蛋白水平下调。综上，本研究证实Stk40作为胎儿红细胞分化、成熟及存活的调控因子，为红细胞生成的分子调控机制及相关疾病研究提供了新的线索。实验设计：每份样本均将6只同基因型E14.5天小鼠的胎儿肝脏细胞混合制备。每种基因型设置2次生物学重复。RNA提取及Affymetrix芯片杂交实验由上海生物芯片有限公司（Shanghai Biochip Company）完成。