Center for Regenerative Therapies TU Dresden (CRTD), Technische Universität Dresden, Fetscherstr 105, 01307, Dresden, Germany.
Max Perutz Labs, University of Vienna, A-1030, Vienna, Austria.
Cell Tissue Res. 2021 Mar;383(3):987-1002. doi: 10.1007/s00441-020-03318-2. Epub 2020 Dec 28.
Extracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.
细胞外信号在胚胎模式形成中起着至关重要的作用，通过以浓度依赖的方式提供位置信息，许多这样的信号，如 Wnt、成纤维细胞生长因子 (FGF)、Hedgehog (Hh) 和视黄酸，通过分泌到细胞外空间来发挥作用，从而在其他细胞中引发受体介导的反应。Isthmin1 (ism1) 是一种分泌蛋白，其基因表达模式与早期背侧决定因素、 nodal 配体基因（如 sqt 和 cyc）以及斑马鱼发育的各个阶段的 fgf8 一致。Ism1 在早期胚胎模式形成和发育中的功能知之甚少；然而，最近有研究表明它与 nodal 通路基因相互作用，以控制鸡的器官不对称性。在这里，我们表明 ism1 缺失构建体的异位表达会破坏斑马鱼的胚胎模式形成，并与 Fgf 和 nodal 信号通路表现出遗传相互作用。与 Fgf 和 nodal 通路突变体不同，CRISPR/Cas9 工程化的 ism1 突变体没有表现出明显的发育缺陷。此外，体内单分子荧光相关光谱 (FCCS) 表明 Ism1 在细胞外空间中自由扩散，扩散系数与 Fgf8a 相似；然而，我们的测量结果不支持 Ism1 与 nodal 配体或 Fgf8a 之间在发育中的斑马鱼胚胎中直接的分子相互作用。总之，功能获得和功能丧失实验的数据表明，斑马鱼 Ism1 在调节早期胚胎发育中外源信号中发挥着复杂的作用。
