A Non-Canonical Raf Function Is Required for Dorsal-Ventral Patterning in Drosophila Embryogenesis. A Non-Canonical Raf Function Is Required for Dorsal-Ventral Patterning in Drosophila Embryogenesis

Embryonic development requires the establishment of directional axes. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGF-beta signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NF-kB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGF-beta. Gene expression analysis showed cells adopting ectodermal fates similar to loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning. Overall design: Embryo RNA profiles of wild type (WT) and Raf926-/- Drosophila

胚胎发育依赖于方向性体轴的建立。在果蝇（Drosophila）中，转录因子的空间离散表达决定了早期胚胎的前后体轴构型，而Toll与转化生长因子-β（TGF-beta）信号通路则调控早期背腹模式的形成。胚胎中的丝裂原活化蛋白激酶/细胞外调节蛋白激酶（MAPK/ERK）信号通路，既参与胚胎末端区域的前后模式构建，也在卵子发生与胚胎阶段调控背腹模式的形成。我们在此报道了Raf激酶基因的一种全新功能缺失突变体，该突变可导致腹侧细胞命运丧失，具体表现为：腹沟消失、Dorsal/核因子-κB（NF-kB）核定位缺失、中胚层决定因子Twist与Snail表达缺失，以及转化生长因子-β（TGF-beta）的表达扩张。基因表达分析显示，该突变体中的细胞呈现出类似Toll信号通路缺失时的外胚层细胞命运。本研究结合新型突变体、活细胞成像、光遗传学（optogenetics）与转录组学（transcriptomics）技术，证实了Raf在胚胎模式构建中存在一种全新的、不依赖于MAPK级联反应的功能。整体实验设计：野生型（WT）与Raf926基因纯合缺失（Raf926-/-）果蝇的胚胎转录组RNA谱分析