The genetic dissection of seamless tube shape control in the Drosophila trachea

 描述(申请人提供)：为了建立一个有功能的血管网络，内皮管必须采用定型的拓扑结构。在脊椎动物的血管中可以观察到多细胞和单细胞的管状拓扑结构。无缝管是单细胞的，不受连接的约束。在脊椎动物的血管网络中，可以在多种情况下发现无缝管。无缝管在这些环境中的功能作用还不是很清楚，因为对无缝管形态发生的细胞机制了解很少。然而，无缝管在整个门中是保守的，包括在果蝇幼虫的呼吸(气管)系统中，在那里可以利用强大的正向遗传学方法来研究无缝管的形态发生。为了建立一个有功能的血管网络，内皮管还必须采用刻板的形状。血管内皮细胞分泌一种称为糖萼的管腔(顶端)细胞外基质(AECM)，由分泌和膜结合的糖蛋白和蛋白多糖组成。糖萼结构缺陷会导致血管内皮细胞扩张缺陷。事实上，AECM在调节气管生长方面具有保守的作用，包括在果蝇的气管中。果蝇气管的无缝管分泌一种基于甲壳素的AECM，称为角质层，其在无缝管形态发生中的作用尚不清楚。我们对单个分泌的基质因子被组织成有序的AECM的分子途径知之甚少。此外，AECM塑造顶膜以调节管形态发生的分子途径还完全不清楚。我将以果蝇幼虫气管系统的无缝管为模型，阐明通过调节AECM的结构/功能来控制无缝管形状的新的遗传途径。我已经确定了无缝管AECM在调节管子形状和完整性方面的作用。甲壳素合成酶缺乏的终末细胞表现为顶膜包囊和不连续。我还鉴定了一组新的突变体(HICOR和哮喘)，这些突变体的表型与“甲壳素生物发生”突变体相似。我推测这些突变可能影响调控无缝管AECM结构或功能的新途径(S)。哮喘和哮喘分别编码锌指转录因子(CG11966和ZIF)，表明它们调节AECM成分的表达或靶向到管腔。在具体目标1中，我将确定无缝管中AECM组织的遗传要求，包括测试已知位于ZIF下游的保守极性因子在将AECM成分靶向管腔中的作用。在具体目标2中，我将确定无缝管AECM调节管形状的细胞和分子途径。我将使用遗传和创新的活细胞成像技术来测试无缝管AECM在调节顶端肌动球蛋白组织中的作用。

 DESCRIPTION (provided by applicant): To build a functional vascular network, endothelial tubes must adopt stereotyped topologies. Both multicellular and unicellular tube topologies are observed in vertebrate vasculatures. Seamless tubes are unicellular and unbound by junctions. Seamless tubes are found in multiple contexts within vertebrate vascular networks. The functional roles of seamless tubes in these contexts are poorly understood because the cellular mechanisms of seamless tube morphogenesis are poorly understood. Nevertheless, seamless tubes are conserved across phyla, including in the respiratory (tracheal) system of Drosophila melanogaster larvae, where powerful forward genetic approaches can be utilized to study seamless tube morphogenesis. To build a functional vascular network, endothelial tubes must also adopt a stereotyped shape. Endothelial cells secrete a luminal (apical) extracellular matrix (aECM) called the glycocalyx, comprised of secreted and membrane-bound glycoproteins and proteoglycans. Defects in glycocalyx structure lead to defects in endothelial tube expansion. Indeed, aECMs have a conserved role in regulating tube growth, including in Drosophila tracheal tubes. The seamless tubes of the Drosophila trachea secrete a chitin-based aECM called the cuticle, whose role in seamless tube morphogenesis not known. We have a poor understanding of molecular pathways through which individual secreted matrix factors are organized into an ordered aECM. Moreover, the molecular pathways through which aECMs shape the apical membrane to regulate tube morphogenesis are entirely unknown. I will use the seamless tubes of the Drosophila larval tracheal system as a model to elucidate novel genetic pathways controlling seamless tube shape by regulating aECM structure/function. I have identified a role for the seamless tube aECM in regulating tube shape and integrity. Chitin synthase-deficient terminal cells exhibit apical membrane cysts and discontinuities. I have also identified a set of novel mutants (ichor and asthmatic) that phenocopy "chitin biogenesis" mutants. I hypothesize that these mutants may affect novel pathway(s) regulating the structure or function of a seamless tube aECM. ichor and asthmatic encode zinc- finger transcription factors (CG11966 and zif, respectively), suggesting they regulate the expression or targeting of aECM components to the lumen. In Specific Aim 1, I will identify the genetic requirements for aECM organization in seamless tubes, including testing the role of a conserved polarity factor known to be downstream of Zif, in targeting aECM components to the lumen. In Specific Aim 2, I will identify cellular and molecular pathways through which the seamless tube aECM regulates tube shape. I will test a role for the seamless tube aECM in regulating apical actomyosin organization using both genetic and innovative live cell imaging techniques.