BACKGROUND Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts) can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system. RESULTS We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes--macvasa and macpiwi--and studied effects of hydroxyurea (HU) treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories. CONCLUSION We show that Macrostomum lignano is a suitable organism to perform high throughput large scale whole mount in situ hybridization. Genes identified in such screens--together with BrdU/H3 labeling--can be used to obtain information on flatworm neoblasts.

中文翻译：

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木质大果的特殊干细胞系统：筛选基因表达并通过羟基脲处理和辐射研究细胞增殖。

背景技术扁虫的特征在于出色的干细胞系统。这些干细胞（成胚细胞）可以产生包括生殖细胞在内的所有细胞类型，并赋予许多扁虫物种非凡的再生能力。木质大头孢菌是研究扁虫干细胞生物学的新兴模型系统。它因其体积小，透明度高，简单的培养维护，基础分类学地位以及与螺旋虫更紧密相关的较少的胚胎发生而与经过精心研究的平面虫互补。细胞，组织和器官特异性标记的发展对于进一步表征扁虫干细胞的分化潜力是必要的。大规模原位杂交是鉴定可能标记的合适工具。在大规模筛选中鉴定出的独特基因与通过羟基脲或辐射处理成胚细胞相结合，将使我们对扁虫干细胞系统的分化和调控有更深入的了解。结果我们为扁虫Macrostomum lignano建立了高通量大规模整装原位杂交的协议。在先导筛选中，鉴定了许多细胞，组织或器官特异性表达模式。我们选择了两个与干细胞和生殖细胞相关的基因-巨噬细胞和macpiwi-并研究了羟基脲（HU）处理或辐射对基因表达的影响。此外，我们观察了在不同时期的HU暴露或不同照射水平后，使用有丝分裂标记物和S期细胞的溴脱氧尿苷标记进行的细胞增殖。HU介导的细胞增殖耗竭和HU诱导的基因表达降低被用于通过抑制性消减杂交生成cDNA文库。对147个差异表达的基因进行了测序，并将其分配给不同的类别。结论我们证明了Macrostomum lignano是一种适合进行高通量大规模大规模原位杂交的有机体。在这种筛选中鉴定出的基因与BrdU / H3标记一起可用于获得有关扁虫成神经细胞的信息。结论我们证明了Macrostomum lignano是一种适合进行高通量大规模大规模原位杂交的有机体。在这种筛选中鉴定出的基因与BrdU / H3标记一起可用于获得有关扁虫成神经细胞的信息。结论我们证明了Macrostomum lignano是一种适合进行高通量大规模大规模原位杂交的有机体。在这种筛选中鉴定的基因与BrdU / H3标记一起可用于获得有关扁虫成神经细胞的信息。