在胚胎发育过程中,通过一系列时空紧密协调的细胞行为,组织和器官逐渐形成其功能形态。跨后生动物的高度保守的器官形状是上皮管。管形态发生是一个复杂的多步骤过程,包括精心编排的细胞行为,如会聚延伸,细胞伸长,和管腔形成。协调这些复杂的形态发生步骤的信号分子的身份仍然难以捉摸。脊索是存在于脊索门所有成员的胚胎中线区域的重要管状器官。这里,使用基因组编辑,药理学和定量成像在早期脊索酸盐Ciona肠我们表明Ano10/Tmem16k,进化上古老的跨膜蛋白家族的成员称为Anoctamin/TMEM16是会聚延伸所必需的,管腔扩张,和脊索形态发生过程中的连接。我们发现Ano10/Tmem16k与位于SERCA的质膜(PM)局部Na/Ca2交换剂(NCX)和内质网(ER)协同工作,RyR,和IP3R蛋白建立发育阶段特异性Ca2+信号分子模块,调节脊索形态发生和Ca2+动力学。此外,我们发现高度保守的Ca2传感器钙调蛋白(CaM)和Ca2/钙调蛋白依赖性蛋白激酶(CaMK)显示出Ano10/Tmem16k依赖性亚细胞定位。它们的药理抑制作用导致收敛延伸,肾小管发育缺陷,和疯狂的Ca2+动力学,这表明Ano10/Tmem16k参与了发育Ca2+信号的“编码”和“解码”。此外,Ano10/Tmem16k在脊索形态发生过程中介导细胞骨架重组,可能是通过改变两种重要的细胞骨架调节剂的定位,小GTP酶Ras同源物家族成员A(RhoA)和肌动蛋白结合蛋白Cofilin。最后,我们在组织培养中使用电生理记录和杂乱酶测定法来证明Ano10/Tmem16k可能充当离子通道,而不是磷脂杂乱酶。我们的结果将Ano10/Tmem16k确立为脊椎动物前分子工具包中的新颖参与者,该工具包可跨尺度控制器官形态发生。
During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The
notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during
notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca2+ signaling molecular modules that regulate
notochord morphogenesis and Ca2+ dynamics. In addition, we find that the highly conserved Ca2+ sensors calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the \"encoding\" and \"decoding\" of developmental Ca2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during
notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales.