Almazan Eugene Matthew P, Lesko Sydney L, Markey Michael P, Rouhana Labib
Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, United States.
Department of Biochemistry and Molecular Biology, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, United States.
Dev Biol. 2018 Jan 15;433(2):433-447. doi: 10.1016/j.ydbio.2017.07.022. Epub 2017 Aug 1.
Planarian flatworms are popular models for the study of regeneration and stem cell biology in vivo. Technical advances and increased availability of genetic information have fueled the discovery of molecules responsible for stem cell pluripotency and regeneration in flatworms. Unfortunately, most of the planarian research performed worldwide utilizes species that are not natural habitants of North America, which limits their availability to newcomer laboratories and impedes their distribution for educational activities. In order to circumvent these limitations and increase the genetic information available for comparative studies, we sequenced the transcriptome of Girardia dorotocephala, a planarian species pandemic and commercially available in North America. A total of 254,802,670 paired sequence reads were obtained from RNA extracted from intact individuals, regenerating fragments, as well as freshly excised auricles of a clonal line of G. dorotocephala (MA-C2), and used for de novo assembly of its transcriptome. The resulting transcriptome draft was validated through functional analysis of genetic markers of stem cells and their progeny in G. dorotocephala. Akin to orthologs in other planarian species, G. dorotocephala Piwi1 (GdPiwi1) was found to be a robust marker of the planarian stem cell population and GdPiwi2 an essential component for stem cell-driven regeneration. Identification of G. dorotocephala homologs of the early stem cell descendent marker PROG-1 revealed a family of lysine-rich proteins expressed during epithelial cell differentiation. Sequences from the MA-C2 transcriptome were found to be 98-99% identical to nucleotide sequences from G. dorotocephala populations with different chromosomal number, demonstrating strong conservation regardless of karyotype evolution. Altogether, this work establishes G. dorotocephala as a viable and accessible option for analysis of gene function in North America.
涡虫纲扁形虫是体内再生和干细胞生物学研究的热门模型。技术进步和遗传信息可用性的提高推动了对涡虫中负责干细胞多能性和再生的分子的发现。不幸的是,全球范围内进行的大多数涡虫研究都使用了非北美本土的物种,这限制了新实验室获取这些物种的机会,并阻碍了它们用于教育活动的传播。为了规避这些限制并增加可用于比较研究的遗传信息,我们对北美广泛分布且可商业获取的涡虫物种多头吉拉德涡虫(Girardia dorotocephala)的转录组进行了测序。从完整个体、再生片段以及多头吉拉德涡虫(MA - C2)克隆系的新鲜切除的耳状物中提取的RNA获得了总共254,802,670对序列读数,并用于其转录组的从头组装。通过对多头吉拉德涡虫中干细胞及其后代的遗传标记进行功能分析,验证了所得的转录组草图。与其他涡虫物种的直系同源物类似,发现多头吉拉德涡虫Piwi1(GdPiwi1)是涡虫干细胞群体的一个可靠标记,而GdPiwi2是干细胞驱动再生的一个重要组成部分。早期干细胞后代标记PROG - 1的多头吉拉德涡虫同源物的鉴定揭示了在上皮细胞分化过程中表达的一个富含赖氨酸的蛋白质家族。发现MA - C2转录组的序列与具有不同染色体数目的多头吉拉德涡虫群体的核苷酸序列有98 - 99%的同一性,表明无论核型进化如何都有很强的保守性。总之,这项工作确立了多头吉拉德涡虫作为北美基因功能分析的一个可行且可获取的选择。
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