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

组织者在建立发育中的脊椎动物胚胎的体轴方面起着至关重要的作用。从Spemann-Mangold组织者组织产生和分泌的各种形态原通过形成激活或抑制因子的离散梯度来协调这一过程。在九头蛇中，下头组织者作为一个信号中心，在稳定状态的息肉和萌芽或再生期间维持和启动主体轴。Wnt蛋白家族的分泌因子作为控制这一过程的主要信号线索。一种普遍接受的假说认为，从Hydras头部组织者发出的β-连环素活性梯度通过向体柱上皮细胞提供位置信息来负责图案的形成。根据Gierer-Meinhardt的从头模式形成理论，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。Hydra WNT3已经清楚地证明了一个基于其启动子区域TCF结合位点的自动催化反馈环，它在组织者形成过程中作为Wnt信号活动的级联活动中最早的Wnt配体。尽管像Hydra Dkk1/2/4这样的几个候选者被讨论为可能的拮抗剂，但还没有关于Hydra中拟议的激活剂-抑制剂系统所涉及的分子参与者的完整图景。在本申请中，提出了一种可能在该过程中起关键作用的新因素。在Hydra WNT3加工因子的筛选中，已经发现了一种ASTIN家族的金属蛋白酶，命名为Hydra NAS-15-like。NAS-15样蛋白的表达是β-连环蛋白依赖性的，在稳态息肉中，NAS-15的表达形成了一个梯度，反映了水龙体中β-连环蛋白活性的梯度。我们推测，Hydra NAS-15类似物在β-连环蛋白下游作为Wnt活性的负反馈调节因子，在蛋白质水平上为下位组织者提供了对高规范Wnt活性的限制。应用的主要目的包括阐明NAS-15-like的底物专一性及其对Wnt活性和构型的影响。此外，通过使用cDNA筛选，我们想要确定增加九头蛇体体柱中Wnt稳定性的因素，从而促进远程信号传递。

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.