Patterning and morphogenesis of the insect body via genetic analysis and cell tracking at single-cell resolution

黑腹果蝇是主要的昆虫模式生物，其细胞和发育生物学机制已经使用过多的工具和资源来解决。然而，重要的发育机制在果蝇中是不容易处理的，因为它们在这个物种中被二次丢失或修改。例如，大多数昆虫通过在后端连续添加新的节来产生它们的躯干，类似于脊椎动物的体节发生，但在果蝇中不存在。此外，果蝇的幼虫头部高度缩小并向胸部内卷，这是昆虫的非典型情况。新的实验工具和基因组资源（RNA干扰（RNAi），转基因，CRISPR，实时成像）现在允许在更典型的昆虫模型中研究这些发育过程，红粉甲虫Tribolium castaneum。在这个项目中，我们将联合收割机结合在赤拟谷盗中的四种强大的实验方法：在小细胞群内操纵基因功能（例如通过克隆分析），CRISPR介导的基因编辑，细胞的克隆标记，通过光片显微镜（SPIM）的高分辨率实时成像和通过RNAi的无偏见大规模筛选。在过去的几年里，我们的实验室一直致力于在赤拟谷盗中建立这些技术，我们现在想利用它们来研究头部发育和躯干分割，这在新兴模式生物中是前所未有的。我们将以单细胞分辨率对这些形态发生过程进行成像，并跟踪标记的细胞组，以生成精确的命运图，并识别有助于头部和躯干形态发生的细胞行为。为了探索基因调控的相互作用，我们将通过产生遗传镶嵌和通过局部RNAi来操纵候选基因的功能和有限细胞群内的信号通路。克隆分析将使我们能够区分发育基因的细胞自主和非细胞自主作用，并揭示它们的功能。我们将利用目前在赤拟谷盗（iBeetle）中进行的大规模RNAi筛选来鉴定在这些过程中具有重要作用的先前未知的基因，我们将详细分析它们的功能。我们的研究结果将揭示在一个典型的昆虫胚胎的头部和躯干形态发生的机制，基于对潜在的基因功能和单细胞分辨率的细胞行为的理解。在赤拟谷盗的头部和躯干中解决类似的问题将使我们能够探索新的发育机制，并开拓新的实验策略，在新兴的昆虫模型中探索这些机制。这里生成的工具将大大拓宽Tribolium可能的机械研究范围，这将影响远远超出本项目范围的发展研究。

Drosophila melanogaster is the major insect model organism where mechanisms of cell and developmental biology have been addressed using a plethora of tools and resources. However, important developmental mechanisms are not tractable in Drosophila because they are secondarily lost or modified in this species. For example, most insects generate their trunk by successive addition of new segments at the posterior end similar to vertebrate somitogenesis, but it is absent in Drosophila. Further, the larval head becomes highly reduced and involuted into the thorax in Drosophila, which is an atypical situation for insects. New experimental tools and genomic resources (RNA interference (RNAi), transgenesis, CRISPR, live imaging) now allow to study these developmental processes in a more typical insect model, the red flour beetle Tribolium castaneum. In this project, we join forces to combine four powerful experimental approaches in Tribolium: the manipulation of gene function within small groups of cells (e.g. by clonal analysis), CRISPR-mediated gene editing, clonal marking of cells, high resolution live imaging via light-sheet microscopy (SPIM) and unbiased large-scale screening via RNAi. In the last years our labs have worked to establish these techniques in Tribolium, and we now want to exploit them to study head development and trunk segmentation on a level unprecedented in emerging model organisms. We will image these morphogenetic processes at single-cell resolution and follow marked groups of cells to generate precise fate maps and to identify cell behaviours contributing to morphogenesis of the head and trunk. To probe the gene regulatory interactions, we will manipulate the function of candidate genes and signalling pathways within restricted populations of cells, by generating genetic mosaics and through localized RNAi. Clonal analysis will allow us to discriminate the cell-autonomous and non-cell-autonomous roles of developmental genes and to uncover their function. We will exploit the large-scale RNAi screen currently performed in Tribolium (iBeetle) to identify previously unknown genes with essential roles in these processes, and we will analyse their functions in detail. Our results will uncover mechanisms of head and trunk morphogenesis in a typical insect embryo, based on an understanding of underlying gene function and cell behaviour at single-cell resolution. Addressing similar questions in the head and trunk of Tribolium will allow us to explore new developmental mechanisms and to pioneer new experimental strategies to probe these mechanisms in an emerging insect model. The tools generated here will significantly broaden the scope of mechanistic research possible in Tribolium, which will impact developmental research far beyond the scope of this project.