Mesenchymal SLMAP coordinates with MST3 to govern gut elongation during development (scRNA-Seq)

Developing gut in mice undergoes rapid elongation during late embryogenesis, yet significantly slows down after birth. Precise regulatory mechanism of this dynamic morphogenetic process remains unknown. Utilizing single-cell RNA-sequencing analysis, we show that YAP activity in Cxcl13high intestinal fibroblasts is the major molecular contributor to gut elongation. To determine how mesenchymal YAP activity is controlled, we identified canonical Sarcolemma membrane-associated protein (SLMAP) as its critical regulator during embryonic gut morphogenesis. Deleting Slmap in gut mesenchyme impairs YAP activity, leading to short gut and a significant decrease in intestinal epithelial cell proliferation. Mechanistically, SLMAP activates YAP by directly regulating MST3 kinase. Physiologically, MST3 levels prominently increase over the developmental time, reaching their peak on postnatal day P14, when gut elongation in mice slows down. Depleting Mst3 in mesenchyme results in increased gut length at P14 accompanied by enhanced YAP activity. Importantly, short gut phenotype in mesenchyme-specific Slmap mutant mice is partially compensated by concomitant deletion of mesenchymal Mst3. Taken together, our findings demonstrate that SLMAP interacts with MST3 kinase to dynamically regulate mesenchymal YAP activity that governs dynamic gut elongation across its embryonic and postnatal development. To elucidate the cellular mechanism governing gut elongation, we performed scRNA-seq analysis on cells from mouse small intestine at E15.5, E17.5 and postnatal day 7 (P7).小鼠肠道在胚胎发育后期会经历快速伸长，但出生后伸长速度显著放缓。这一动态形态发生过程的精确调控机制至今仍未明确。本研究借助单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）分析，证实Cxcl13高表达肠成纤维细胞中的YAP活性是驱动肠道伸长的核心分子贡献因素。为明确间充质YAP活性的调控方式，我们鉴定出经典肌膜相关蛋白（Sarcolemma membrane-associated protein, SLMAP）是胚胎肠道形态发生过程中的关键调控因子。在肠道间充质中敲除Slmap会削弱YAP活性，进而引发短肠表型，并使肠道上皮细胞增殖能力显著下降。机制层面研究显示，SLMAP通过直接调控MST3激酶来激活YAP。生理条件下，MST3的表达水平随发育进程显著升高，并在出生后第14天（P14）达到峰值，而此时小鼠肠道的伸长过程恰好放缓。在间充质中敲除Mst3会使P14天时的肠道长度增加，并伴随YAP活性增强。值得注意的是，间充质特异性Slmap突变小鼠出现的短肠表型，可通过同时敲除间充质Mst3得到部分代偿。综上，本研究结果表明，SLMAP与MST3激酶相互作用，动态调控间充质YAP活性，进而主宰胚胎及出生后阶段的肠道动态伸长过程。为阐明调控肠道伸长的细胞机制，我们对小鼠胚胎第15.5天（E15.5）、第17.5天（E17.5）以及出生后第7天（P7）的小肠细胞开展了scRNA-seq分析。