Dorsoventral axis formation in the cricket Gryllus bimaculatus as example of convergent evolution of Toll’s morphogen function

Toll和BMP信号传导对于迄今为止研究的所有昆虫的背腹（DV）轴形成至关重要。此外，果蝇中的Toll信号梯度代表了以浓度依赖性方式决定细胞命运的最佳理解的形态发生系统之一。虽然BMP对于大多数双侧动物中DV轴的建立是至关重要的，但Toll的DV功能似乎是昆虫进化上的一种新奇。在过去，我们已经研究了DV轴的形成在主要的昆虫谱系的代表，并发现一个越来越少的昆虫Toll更远的相关果蝇的需求。同时，BMP信号传导的重要性增加，接近BMP主导的图案化网络已知从其他双侧动脉。令人惊讶的是，蟋蟀，Gryllus bimaculatus，一个直翅目物种，代表了我们迄今为止研究的最基部的昆虫谱系，并不符合这一趋势。蟋蟀的DV系统与果蝇的DV系统有着惊人的相似之处。在这个提议中，我们希望解开蟋蟀DV模式化的分子机制，从而探索Toll形态发生功能趋同进化的可能性。我们的工作计划是基于蟋蟀活体成像，转基因，CRISPR/Cas9介导的基因组编辑和基因组测序的最新进展，它包括四个具体目标。我们计划（1）通过可视化转录因子Dorsal/NF-κB的核积累来研究Toll信号传导的时空分布，（2）通过差异转录组分析来鉴定Toll和BMP靶基因，（3）通过ATAC-seq、计算基序搜索和基于ATAC-seq的足迹法来表征靶基因的顺式调控元件，（4）标记靶基因用于活体成像并进行报告基因测定以确认预测的顺式调节元件的功能性。总之，这些数据将提供分子见解苍蝇和板球DV图案之间的意想不到的相似之处，并补充我们的图片DV轴形成昆虫的复杂进化历史。

Toll and BMP signaling are essential for dorsoventral (DV) axis formation in all insects studied so far. The Toll signaling gradient in Drosophila represents moreover, one of the best-understood morphogen systems determining cell fates in a concentration-dependent manner. While BMP is crucial for the establishment of the DV axis in most bilaterian animals, Toll’s DV function appears to be an evolutionary novelty of insects. In the past we have studied DV axis formation in representatives of major insect lineages and found a diminishing requirement for Toll in insects more distantly related to Drosophila. Concomitantly, the importance of BMP signaling increases approaching the BMP dominated patterning networks known from other bilateria. Surprisingly, the cricket, Gryllus bimaculatus, an orthopteran species representing the basal-most insect lineage we have studied so far, did not conform to this trend. The Gryllus DV system rather has striking similarities to that of Drosophila. In this proposal, we want to unravel molecular mechanisms of Gryllus DV pattering and thereby explore the possibility of convergent evolution of Toll’s morphogen function. Our work programme is based on recent advances in Gryllus live-imaging, transgenesis, CRISPR/Cas9 mediated genome editing and genome sequencing, and it encompasses four specific objectives. We plan (1) to study the spatiotemporal profile of Toll signaling by visualizing the nuclear accumulation of the transcription factor Dorsal/NF-κB, (2) to identify Toll and BMP target genes by differential transcriptome analysis, (3) to characterize cis-regulatory elements of target genes by ATAC-seq, computational motif searches and ATAC-seq based footprinting, (4) to tag target genes for live imaging and perform reporter assays to confirm the functionality of predicted cis-regulatory elements. Together, these data will provide molecular insights into the unexpected parallels between fly and cricket DV patterning and complement our picture of the complex evolutionary history of DV axis formation in insects.