Functional genomics of the enigmatic stellate cell

昆虫是地球上最多的物种，几乎占据了从北极、沙漠到水生的所有环境生态位。有害昆虫造成巨大的健康和经济损失——作物受损、昆虫传播的动植物疾病，导致全球GDP损失20%。鉴于昆虫对所有杀虫剂的抗性和日益增加的环境问题，昆虫控制的压力越来越大，因此了解昆虫生存和环境耐受性的机制可能是昆虫控制新途径的关键。对于所有昆虫来说，生存依赖于渗透调节和流体平衡（体内平衡），这也使它们能够承受低湿度环境中的干燥，包括英国。昆虫的“肾脏”——马氏小管——是维持体液平衡的关键组织。多年来，我们已经研究并建立了遗传易感性的马氏小管的细胞信号传导、离子转运、功能基因组学和整合生理学，它是昆虫小管的一个很好的模型，特别是双翅目物种。与英国经济相关的双翅目害虫的例子是小麦球茎蝇和卷心菜根蝇（作物害虫）；蠓（蓝舌，动物健康）；越来越多的蚊子（人类健康）。在这里，我们计划了解小管液分泌和反应的机制和神经内分泌控制，特别是通过小管星状细胞；并由氯离子和水分在正常和干燥条件下通过星状细胞运输。拟议的工作计划将包括在正常和干燥条件下从特定的小管细胞类型（主细胞和星状细胞）中分离活性转录基因（‘翻译组’），使用一种新的细胞特异性翻译组分离方法以及基因阵列（微阵列）的组合。这将使我们能够将基因分配给主细胞或星状细胞，从而了解每种细胞类型的基因特征；鉴定在干燥胁迫下积极转录的细胞特异性基因；并通过评估鉴定出的基因在全蝇干燥耐受性试验中的作用来确定与干燥耐受性有关的基因。我们还计划确定在正常和干燥条件下，通过星状细胞的氯和水通量的流体分泌和流体稳态的机制；同时也是对神经激素控制的反应。为了做到这一点，我们将在分子和生理水平上定义氯离子通道（CLCs）和星状细胞特异性水通道蛋白的作用，利用转基因果蝇敲除星状细胞特异性CLC和水通道蛋白基因；小管液分泌测定；分子方法;利用新型转基因报告基因进行生物成像细胞生物学;生理测量（与美国离子通道电生理学小组专家合作）；并对全蝇进行干燥耐受性测定。总之，这将提供对流体稳态中氯化物和水运输的第一次全面了解，以及对干燥耐受性的见解，这可能有助于及时识别仅针对一小部分昆虫的新的，更环保的杀虫剂。

Insects comprise the most species on earth, occupying almost all environmental niches, from arctic, desert to aquatic. Harmful insects incur enormous health and economic costs - crop damage, insect-borne plant and animal diseases, cause the loss of 20% of GDP worldwide. There is increasing pressure for insect control, given insect resistance to all insecticides and increasing environmental concerns, so understanding mechanisms of insect survival and environmental tolerance may be key to novel routes for insect control. For all insects, survival depends on osmoregulation and fluid balance (homeostasis), which also allows them to withstand desiccation in low humidity environments, including that of the UK. The insect 'kidneys' - Malpighian tubules - are key tissues for fluid homeostasis. Over some years, we have investigated and established cell signalling, ion transport, functional genomics and integrative physiology in the genetically tractable D. melanogaster Malpighian tubule, which is an excellent model for insect tubules, especially Dipteran species. Examples of Dipteran insect pests relevant to the UK economy are the wheat bulb fly and the cabbage root fly (crop pests); the midge (blue-tongue, animal health); and increasingly, mosquitoes (human health). Here, we plan to understand the mechanisms and neuroendocrine control of tubule fluid secretion and responses, specifically by the tubule stellate cell; and by chloride and water transport through the stellate cell under normal and desiccation conditions. The proposed programme of work will include isolation of actively transcribed genes (the 'translatome') from specific tubule cell-types (principal and stellate cells) under normal and desiccation conditions, using a combination of a novel cell-specific translatome isolation method, as well as gene arrays (microarrays). This will allow us to assign genes to either principal or stellate cells and so understand the gene signature of each cell type; to identify cell-specific genes which are actively transcribed in response to desiccation stress; and to define those genes implicated in desiccation tolerance by also assessing the role of identified genes in whole fly desiccation tolerance assays. We also plan to define the mechanism of chloride and water flux through the stellate cells for fluid secretion and fluid homeostasis under normal and desiccation conditions; and also in response to neurohormone control. In order to do this, we will define the role of chloride channels (CLCs) and aquaporins specific to the stellate cell at the molecular and physiological level using transgenic flies to knock-down specific CLC and aquaporin genes in only stellate cells; tubule fluid secretion assays; molecular methods; bioimaging using novel transgenic reporters; cell biology; physiological measurements (this in collaboration with a US group expert in ion channel electrophysiology); and whole fly desiccation tolerance assays. Together, this will provide the first comprehensive understanding of chloride and water transport in fluid homeostasis, as well as insights into desiccation tolerance, which may in time, help identify, new, greener insecticides that target only a subset of insects.