Diverse Fgfr1 signaling pathways and endocytic trafficking regulate early mesoderm development II

The Fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1 null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth, but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM-interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identify processes regulating early mesoderm development by mechanisms involving both canonical and non-canonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation. To investigate changes in transcription during mesoderm development with or without canonical FGF signaling, we created mouse lines containing point mutations in key effector binding residues. We then collected tailbud tissue from E9.5 mouse embryos to use for gene expression profiling. Comparative gene expression profiling between wild-type and mutant FGFR1 tissue was used to determine changes in GO and KEGG term gene sets.成纤维细胞生长因子（Fibroblast growth factor, FGF）信号通路是一类保守的信号通路，对胚胎发育至关重要。激活的成纤维细胞生长因子受体1（Fibroblast growth factor receptor 1, FGFR1）可驱动多条胞内信号级联反应通路，包括ERK/MAPK与PI3K/AKT通路，上述通路共同被称为经典信号通路（canonical signaling）。然而，与Fgfr1基因敲除胚胎不同，携带Fgfr1功能减退（hypomorphic）突变且无法激活经典下游信号的胚胎仍可发育至出生，但在所有中胚层来源组织中均表现出严重缺陷。引入额外的信号突变会进一步降低FGFR1的活性，导致胚胎更早致死、体节发生减弱以及转录产物谱出现更为显著的变化。参与细胞迁移、细胞外基质（extracellular matrix, ECM）相互作用以及磷脂酰肌醇信号通路的基因显著下调；蛋白质组学分析发现其与内吞通路组分的互作发生改变；表达突变受体的细胞其胞内吞运输过程亦出现异常。综上，本研究明确了通过经典与非经典FGFR1通路调控早期中胚层发育的相关过程，其中包括与细胞黏附组分的直接相互作用以及内吞调控机制。为探究存在或缺失经典FGF信号通路时中胚层发育过程中的转录变化，我们构建了携带关键效应蛋白结合残基点突变的小鼠品系。随后我们收集了胚胎发育第9.5天（embryonic day 9.5, E9.5）小鼠胚胎的尾芽组织用于基因表达谱分析。通过对比野生型与FGFR1突变型组织的基因表达谱，我们确定了基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）基因集的变化情况。