AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMPK, a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK-/- EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs over-expressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. 2 WT and 2 AMPK DKO ESC lines were differentiated into embryoid bodies (EBs) for various lengths of time (2, 4, 8, and 12 days) in high and low glucose conditions. Both ESC and EB samples were profiled by mRNA-seq to examine how global gene expression changes associated with ESC differentiation are affected by AMPK deletion.

发育程序的忠实执行依赖于多能祖细胞获取独特的细胞身份，这一过程受众多信号级联反应的组合输入调控，最终决定谱系特异性的转录输出。尽管越来越多的证据表明代谢与诸多分子网络存在整合，但控制能量稳态的通路如何影响细胞命运决策，在很大程度上仍未明确。本研究显示，腺苷酸活化蛋白激酶（AMPK）作为核心代谢调控因子，在谱系特化中发挥关键作用。尽管AMPK缺陷型胚胎干细胞（ESCs）在多能状态下表现正常，但这些细胞在分化过程中出现显著缺陷：无法形成嵌合胚胎，且在拟胚体（EB）形成过程中优先向外胚层分化，以内胚层为代价。AMPK敲除的拟胚体中，溶酶体核心转录调控因子转录因子EB（Tfeb）的表达水平降低，导致内体溶酶体功能受损。值得注意的是，Tfeb的遗传缺失同样会引发内胚层发育缺陷，而在AMPK缺失的胚胎干细胞中过表达该转录因子可使其分化潜能恢复正常，这揭示了Tfeb/溶酶体通路与胚层特化之间的紧密联系。AMPK或Tfeb失活导致的内体溶酶体系统功能受损会削弱Wnt信号通路，而上调该通路则可恢复内胚层标志物的表达。综上，本研究发现AMPK通路是分化过程中细胞命运决定的新型调控因子。本研究将2株野生型（WT）和2株AMPK双敲除（DKO）胚胎干细胞系分别在高糖和低糖条件下诱导分化为拟胚体，分化时长分别为2、4、8和12天。通过mRNA测序（mRNA-seq）对胚胎干细胞及拟胚体样本进行转录组分析，以探究AMPK缺失如何影响与胚胎干细胞分化相关的全局基因表达变化。