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.

中文翻译：

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Isthmin1 是一种分泌性信号蛋白，作用于发育中的不同胚胎模式中心的下游

细胞外信号通过以浓度依赖性方式提供位置信息，在胚胎模式形成过程中发挥重要作用，许多此类信号，如 Wnt、成纤维细胞生长因子 (FGF)、刺猬 (Hh) 和视黄酸，通过分泌到细胞外而发挥作用。空间，从而触发其他细胞中受体介导的反应。Isthmin1 (ism1) 是一种分泌蛋白，其基因表达模式与早期背部决定簇、节点配体基因（如 sqt 和 cyc）以及在斑马鱼发育的各个阶段中的 fgf8 一致。Ism1 在早期胚胎模式和发育中的功能知之甚少；然而，最近显示它与节点通路基因相互作用以控制鸡的器官不对称。这里，我们表明 ism1 缺失结构的错误表达会破坏斑马鱼的胚胎模式，并表现出与 Fgf 和节点信号传导的遗传相互作用。与 Fgf 和节点通路突变体不同，CRISPR/Cas9 改造的 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）显示Ism1在细胞外空间自由扩散，扩散系数与Fgf8a相似；然而，我们的测量不支持 Ism1 与发育中的斑马鱼胚胎中的节点配体或 Fgf8a 之间的直接分子相互作用。总之，来自功能获得和功能丧失实验的数据表明，斑马鱼 Ism1 在早期胚胎发育过程中在调节细胞外信号方面发挥着复杂的作用。