Extensive Crosstalk between BMP and Notch Signaling Pathways in Activated Adult Muscle Stem Cells [array]. Extensive Crosstalk between BMP and Notch Signaling Pathways in Activated Adult Muscle Stem Cells [array]

Muscle stem cells (MuSC) are the cellular source for generation and regeneration of skeletal muscle. To ensure correct muscle growth, MuSC self-renewal and differentiation need to be tightly regulated. Several signaling systems have been implicated in the control of MuSCs, among them Bone Morphogenetic Proteins (BMPs) and Notch, both of which promote MuSC proliferation and suppress differentiation. To better understand the mechanisms of function and the target genes regulated by BMP signaling in myogenesis, we investigated the transcriptional responses of adult mouse MuSCs to BMP6/4 using RNA-sequencing. BMP6/4-stimulation of freshly isolated MuSCs for one hour rapidly increased the expression of classical BMP target genes like Id1 and strongly induced expression of genes of the Notch pathway (Hes1, Hey1, Lfng, Snai1). In parallel, using Cleavage Under Targets and Tagmentation (CUT&Tag), we generated whole-genome binding profiles for the BMP pathway effectors pSMAD1/5/9 and SMAD4 and detected binding in promoters and potential regulatory elements of BMP targets and Notch pathway genes (Hes1, Hey1, Lfng, Snai1) indicating that BMP signaling directly influences Notch and that crosstalk between the two pathways regulates myogenesis. Overall design: Microarray of BMP6-stimulated and control adult mouse muscle stem cells, three biological replicates UPDATE: [Mar-06-2026] The Sample titles were updated. The old titles are in the Sample description field.

肌肉干细胞（Muscle stem cell, MuSC）的激活、增殖、自我更新与分化是受严格调控的生物学过程，可在不耗竭干细胞库的前提下支持肌肉发育与再生。骨形态发生蛋白（Bone morphogenetic protein, BMP）信号通路可维持MuSC处于增殖状态，并阻止其分化。本研究借助RNA测序（RNA-sequencing）、基因表达芯片（RNA expression arrays）、通路特异性定量PCR芯片（pathway-specific qPCR arrays），以及可实现高空间分辨率识别转录因子结合位点与特定组蛋白修饰的靶向切割与标签化（Cleavage Under Targets and Tagmentation, CUT&Tag）技术，探究了骨形态发生蛋白4与6（BMP4和BMP6）对成年小鼠MuSC的转录调控及细胞内信号通路的影响。新鲜分离的MuSC培养42至72小时后，于无血清条件下培养6小时，随后用BMP4/6进行刺激。添加BMP4/6后，典型BMP靶基因DNA结合抑制因子1（Inhibitor of DNA binding 1, Id1）的表达迅速升高，并于1小时后达到峰值。正如预期，BMP4/6刺激可显著上调BMP靶基因（如Smad7、Nog、Id1、Klf10及Lif）的转录水平，同时也可强烈激活Notch通路基因（如Hes1、Lfng及Hey1）的转录。本研究证实，关键BMP通路效应因子SMAD4与磷酸化SMAD1/5/9（pSMAD1/5/9）可结合于上述Notch靶基因的启动子区域。研究结果表明，Notch信号通路相关编码基因的快速表达源于BMP信号通路的直接调控。总体实验设计：从6至8周龄雌性小鼠的肌肉组织中分离MuSC，于基质胶（Matrigel®）上扩增培养2天。为消除培养基（如血清补充成分）中内源性BMP的干扰，细胞在BMP刺激前需于含200 ng/mL BMP可溶性受体（重组小鼠BMPR-IA/ALK3-Fc嵌合体，R&D Systems，货号437-MR）的无血清培养基中培养6小时。随后，用100 ng/mL的BMP4或BMP6对细胞进行1小时的脉冲刺激，对照组则仅使用用于溶解BMP的溶媒处理。之后收集细胞并提取总RNA，对经血清饥饿后分别用BMP4或溶媒处理的成年小鼠肌肉干细胞的3个生物学重复样本进行芯片分析。