Functional analysis of novel molecular components in head organiser establishment and maintenance in the lower metazoan Hydra

组织者在脊椎动物胚胎发育的体轴的建立中起着至关重要的作用。由Spemann-Mangold组织者组织产生和分泌的多种形态因子通过形成离散的激活或抑制因子梯度来协调这一过程。在九头蛇中，在稳定状态的息肉和萌芽或再生过程中，下气孔头部组织者作为信号中心维持和启动主体轴。Wnt蛋白家族的分泌因子作为控制这一过程的主要信号信号。一种普遍接受的假说认为，源自Hydras头部组织者的β -连环蛋白活性梯度通过向体柱上皮细胞提供位置信息来负责模式的形成。根据Gierer-Meinhardt理论，一种可扩散的远程抑制剂通过与自催化活化中心相互作用来维持这种梯度是至关重要的。在组织体形成过程中，Hydra Wnt3作为最早的Wnt信号活动级联中的Wnt配体，已经清楚地证明了基于其启动子区域TCF结合位点的自催化反馈回路。虽然像Hydra Dkk1/2/4这样的几种候选拮抗剂被讨论为可能的拮抗剂，但对于Hydra中所提出的激活剂-抑制剂系统中所涉及的分子参与者还没有完整的描述。在本应用中，提出了一种可以在此过程中起关键作用的新因素。一种astacin家族的金属蛋白酶，被命名为Hydra nas -15样，已经在Hydra Wnt3加工因子的筛选中被鉴定出来。nas -15样表达被证明是β - catenin依赖的，并且在稳态息肉中形成一个梯度，反映了Hydras体内β - catenin活性的梯度。我们假设Hydra nas -15样蛋白在蛋白水平上作为Wnt活性的负反馈调节器作用于β -连环蛋白的下游，从而限制了Wnt活性对下口组织者的高规范。该应用的主要目的包括阐明nas -15样的底物特异性及其对Wnt活性和模式的影响。此外，通过cDNA筛选，我们希望确定增加水螅体柱中Wnt稳定性的因素，从而促进远程信号传导。

The organizer has a crucial role in setting up the body axes of the developing vertebrate embryo. Diverse morphogens produced and secreted from the Spemann-Mangold organizer tissue orchestrate this process by forming discrete gradients of activatory or repressive factors. In Hydra, the hypostomal head organizer acts as a signalling center that maintains and initiates the primary body axis in steady state polyps and during budding or regeneration. Secreted factors of the Wnt protein family act as primary signalling cues controlling this process. A commonly accepted hypothesis states that a gradient of beta-Catenin activity emanating from Hydras head organizer is responsible for pattern formation by providing positional information to the epithelial cells of the body column. According to the Gierer-Meinhardt theory of de novo pattern formation a diffusible long-range inhibitor is crucial in maintaining this gradient by interacting with the autocatalytic activation center. An autocatalytic feedback-loop based on TCF binding sites in its promotor region has been clearly demonstrated for Hydra Wnt3 that acts as the earliest Wnt ligand in a cascade of Wnt signalling activity during organizer formation. Although several candidates like Hydra Dkk1/2/4 are discussed as possible antagonists, there is no complete picture of the molecular players involved in the proposed activator-inhibitor system in Hydra. In the present application a novel factor is presented that could play a crucial role in this process. A metalloproteinase of the astacin family, designated as Hydra NAS-15-like, has been identified in a screen for Hydra Wnt3 processing factors. NAS-15-like expression is shown to be beta-Catenin-dependent and, in steady-state polyps, to form a gradient reflecting the proposed gradient of beta-Catenin activity in Hydras body. We hypothesize that Hydra NAS-15-like acts downstream of beta-catenin as a negative feedback regulator of Wnt activity on the protein level providing a limitation of high canonical Wnt activity to the hypostomal organiser. Major aims of the application include the elucidation of the substrate specificity of NAS-15-like and its influence on Wnt activity and patterning. In addition, by using a cDNA screen, we want to identify factors that increase Wnt stability in the body column of Hydra and thus facilitate long-range signalling.