Molecular and cellular characterization of prominin-like molecules in urodele amphibian - A unique model to study central nervous system regeneration

本研究的目的是研究尾目两栖动物Axolotl中的显著蛋白分子的特征，并评估它们是否可以作为再生中枢神经系统(CNS)中正常和/或刺激的神经发生的标志。与哺乳动物不同，水生脊椎动物具有巨大的再生能力，能够再生许多复杂的解剖结构，使它们成为研究表型再生机制的诱人系统。然而，由于缺乏合适的标记，在低等脊椎动物中干细胞和祖细胞的细胞和分子特征是特别困难的。在哺乳动物中，显著蛋白-1定义了广泛的胚胎和躯体干细胞和祖细胞，包括中枢神经系统的干细胞和祖细胞。最近，我们在Axolotl中发现了三个不同的显著分子。值得注意的是，它们中的一个(即显著蛋白-1)在尾部截肢后上调，表明突出蛋白阳性细胞可能参与了脊髓再生。在这里，我们建议剖析突起蛋白在Axolotl中的生理作用，并重要的是确定它们是否可以作为细胞表面标记用于分离稀有的胚胎后干细胞。为此，我们将首先对这些分子在正常发育和成年动物中的普遍表达进行详细的分析，特别是胚胎后轴突的神经源性基质区域。其次，它们的表达将在再生中枢神经系统中进一步记录。第三，我们将解决它们的功能方面，例如在脊髓再生中，通过反义抑制下调它们的表达。最后，我们将调查再生脊髓中的神经前体细胞是否可以根据重要蛋白-1的表达进行前瞻性分离。综上所述，建议的方法将为未来鉴定、表征和最终分离低等脊椎动物的干细胞和祖细胞提供一个独特的机会。

The purpose of this research program is to characterize prominin molecules in urodele amphibian axolotl and to assess whether they can be established as markers of normal and/or provoked neurogenesis in the regenerating central nervous system (CNS). As opposed to mammals, aquatic vertebrate animals have an enormous regenerative ability being able to regenerate a number of complex anatomical structures making them attractive systems for studying mechanisms underlying epimorphic regeneration. However, the cellular and molecular characterization of stem and progenitor cells in lower vertebrates is particularly difficult due to the lack of suitable markers. In mammals, prominin-1 defines a broad range of embryonic and somatic stem and progenitor cells including those of the CNS. Recently, we have identified three distinct prominin molecules in axolotl. Remarkably, one of them (i.e. prominin-1) is upregulated upon tail amputation indicating a potential involvement of prominin–positive cells in spinal cord regeneration. Here we propose to dissect the physiological role of prominins in axolotl, and importantly to determine whether they can be used as cell surface markers for the isolation of rare postembryonic stem cells. To that end, we will perform first a detailed analysis of the general expression of these molecules both in the normal developing and adult animals with special emphasis on neurogenic matrix zones of the postembryonic axolotl. Second, their expression will be further documented in regenerating CNS. Third, we will address their functional aspect, for instance in spinal cord regeneration, by down regulating their expression by antisense-inhibition. Finally, we will investigate whether the neural progenitors in the regenerating spinal cord could be prospectively isolated based on the expression of prominin-1. Taken together, the proposed approach will offer a unique opportunity for prospective identification, characterization and eventual isolation of stem and progenitor cells in lower vertebrates.