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

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 E8.5 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. We compared wild type to Fgfr1 FCPG, wild type to Fgfr1 FCSPG, and Fgfr1 FCPG to Fgfr1 FCSPG.成纤维细胞生长因子（Fibroblast growth factor, FGF）通路是一类高度保守的信号通路，对胚胎发育至关重要。活化的成纤维细胞生长因子受体1（Fibroblast growth factor receptor 1, FGFR1）可激活多条胞内信号级联通路，包括ERK/MAPK与PI3K/AKT通路，此类通路统称为经典信号通路（canonical signaling）。然而，与Fgfr1纯合缺失胚胎不同，携带Fgfr1功能减退型突变且无法激活经典下游信号的胚胎仍可发育至足月分娩，但所有中胚层来源的组织均会出现严重发育缺陷。引入额外的信号通路突变会进一步降低FGFR1的活性，进而导致胚胎更早死亡、体节发生（somitogenesis）受阻以及转录产物的改变更为显著。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表达显著下调；蛋白质组学分析显示，其与内吞通路组分的互作模式发生改变；表达突变型受体的细胞其内吞运输（endocytic trafficking）过程亦出现异常。综上，本研究明确了通过经典与非经典（non-canonical）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互作以及内吞调控机制。为探究存在/不存在经典FGF信号通路时中胚层发育过程中的转录变化，本研究构建了在关键效应蛋白结合残基处携带点突变的小鼠品系。随后，我们收集了胚胎发育第8.5天（E8.5）小鼠胚胎的尾芽组织，用于基因表达谱（gene expression profiling）分析。通过对野生型与FGFR1突变型组织开展比较基因表达谱分析，本研究确定了基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）基因集的表达变化。我们将野生型样本与Fgfr1 FCPG样本、野生型样本与Fgfr1 FCSPG样本，以及Fgfr1 FCPG样本与Fgfr1 FCSPG样本进行了对比分析。