Mesenchymal SLMAP coordinates with MST3 to govern gut elongation during development [RNAseq_TL]

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 scRNA-seq 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 SlmapcKO 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 gut elongation across its embryonic and postnatal development. To test whether spliceosome-mediated alternative splicing (AS) partakes in gut elongation, we analyzed full-length bulk RNA-seq on small intestine mesenchyme between E17.5 and P14, We conducted gene expression profiling analysis using data obtained from RNA-seq of the small intestinal mesenchyme from four wild-type E17.5 and four P14 mice小鼠胚胎发育后期的肠道会经历快速伸长，但出生后该过程显著放缓。目前，这一动态形态发生过程的精确调控机制仍未阐明。本研究借助单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析发现，Cxcl13高表达肠道成纤维细胞中的YAP（Yes-associated protein）活性是驱动肠道伸长的核心分子诱因。为明确间充质YAP活性的调控路径，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，引发短肠表型，并显著降低肠上皮细胞的增殖能力。从机制层面而言，Slmap通过直接调控Mst3激酶来激活YAP。生理层面，Mst3的表达水平随发育进程持续升高，在小鼠出生后第14天（P14）达到峰值，而此时小鼠的肠道伸长恰好进入放缓阶段。敲除间充质中的Mst3则会使P14天小鼠的肠道长度增加，并伴随YAP活性升高。值得注意的是，同时敲除间充质中的Mst3可部分挽救Slmap条件性敲除（SlmapcKO）小鼠的短肠表型。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，进而精准调控小鼠胚胎期及出生后发育阶段的肠道伸长过程。为探究剪接体介导的可变剪接（alternative splicing, AS）是否参与肠道伸长过程，我们对E17.5天至P14天小鼠小肠间充质的全长批量RNA测序（bulk RNA-seq）数据进行了分析；本研究利用4只野生型E17.5天小鼠和4只野生型P14天小鼠的小肠间充质RNA测序数据开展了基因表达谱分析。