Expression data from B220+ Hspa9+/+ and Hspa9+/- CFU-PreB colonies isolated on Day 7 of culture

CFU-PreB colonies are reduced in number and size in Hspa9+/- mice compared to wildtype littermates. We compared the expression profiles of these colonies to gain insight into the mechanism driving this difference. HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndromes (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. While homozygous knockout of Hspa9 is embryonic lethal, mice with heterozygous deletion of Hspa9 (Hspa9+/-) are viable and have a 50% reduction in Hspa9 expression. Hspa9+/- mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9+/- mice have a cell-intrinsic reduction in bone marrow CFU-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9+/- B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNAi and observe reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B-lymphopoiesis in vivo. Knockdown of Hspa9 in an IL-7 dependent mouse B-cell line reduced Stat5 phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B-cells. Collectively, these data implicate a role for Hspa9 in B-lymphopoiesis and Stat5 activation downstream of IL-7 signaling. Bone marrow cells from 5 Hspa9+/+ and 5 Hspa9+/- mice were independently cultured in CFU-PreB methylcellulose medium for 7 days. PreB colonies were isolated and B220+ cells were flow sorted for RNA isolation.

与野生型同窝对照小鼠相比，Hspa9+/-小鼠的CFU-PreB集落数量与体积均显著减少。本研究通过比较两类集落的基因表达谱，以解析介导该差异的分子机制。人类HSPA9基因定位于5号染色体q31.2区域，该区域在髓系恶性血液病[del(5q)]患者中常发生缺失，包括骨髓增生异常综合征（myelodysplastic syndromes, MDS）。伴del(5q)的MDS患者体内HSPA9基因表达水平降低50%，符合单倍体剂量不足的表达特征。斑马鱼突变体模型以及人源和小鼠细胞的基因敲低实验均证实，HSPA9在造血过程中发挥关键作用。为全面评估Hspa9单倍体剂量不足对造血功能的影响，本研究构建了Hspa9基因敲除小鼠模型。尽管Hspa9纯合敲除会导致胚胎致死，但Hspa9杂合缺失（Hspa9+/-）小鼠可存活，且其Hspa9基因表达水平降低50%。Hspa9+/-小鼠的基础造血功能正常，且不会自发发生MDS。然而，Hspa9+/-小鼠的骨髓CFU-PreB集落形成能力呈现细胞自主性降低，但其体内B细胞祖细胞的数量并无明显变化，这与Hspa9+/-小鼠B细胞祖细胞存在功能缺陷的结论相符。本研究进一步通过RNA干扰（RNAi）技术将Hspa9基因表达水平降至50%以下，结果观察到小鼠体内B细胞祖细胞数量减少，这表明正常的B淋巴细胞生成需要Hspa9基因维持≥50%的适宜表达水平。在依赖白细胞介素7（IL-7）的小鼠B细胞系中敲低Hspa9基因，会降低IL-7受体刺激后的信号转导与转录激活因子5（Stat5）磷酸化水平，证实Hspa9在B细胞的Stat5信号通路中发挥重要作用。综上，上述实验数据表明Hspa9在B淋巴细胞生成以及IL-7信号通路下游的Stat5激活过程中具有关键作用。本研究分别将5只Hspa9野生型（Hspa9+/+）和5只Hspa9+/-小鼠的骨髓细胞接种于CFU-PreB甲基纤维素培养基中培养7天。随后分离PreB集落，并通过流式细胞术分选B220+细胞以提取RNA。