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过程中的体轴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不同形态发生素通过形成激活或抑制因子的离散梯度来协调这一过程。在水螅中，口下的头组织者作为一个信号中心，在稳定状态的息肉和出芽或再生期间维持和启动初级体轴。Wnt蛋白家族的分泌因子作为控制该过程的主要信号线索。一个普遍接受的假说指出，从水螅头组织者发出的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图案形成。根据Gierer-Meinhardt从头模式形成理论，可扩散的长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。基于其启动子区域中的TCF结合位点的自催化反馈环已被清楚地证明为水螅Wnt 3，其在组织者形成期间的Wnt信号传导活性级联中充当最早的Wnt配体。虽然几个候选人，如水螅Dkk 1/2/4作为可能的拮抗剂进行了讨论，有没有完整的图片的分子参与水螅中提出的激活剂-抑制剂系统。在本申请中，提出了一种新的因素，其可以在该过程中发挥关键作用。一种被命名为Hydra NAS-15样的astacin家族的金属蛋白酶已经在Hydra Wnt 3加工因子的筛选中被鉴定。NAS-15样表达被证明是β-连环蛋白依赖性的，并且在稳态息肉中形成反映水螅体中β-连环蛋白活性的拟议梯度的梯度。我们假设，水螅NAS-15样行为的β-连环蛋白下游的Wnt活性的负反馈调节蛋白质水平上提供了一个限制的高规范Wnt活性的口下组织者。本申请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，通过使用cDNA筛选，我们希望确定增加Wnt在水螅体柱中稳定性的因素，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.