SummaryWhile ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anteriorposterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 up-regulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubulebased movement and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many up-regulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes such as dual-specific tyrosine-(Y)-phosphorylation regulated kinases and MORN domain containing proteins that seemingly act as key-regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies, our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.Keywords: cell damage repair / cell regeneration / single cell transcriptomics / Stentor / ciliate / RNAseq / microbial eukaryotes / protist . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/232363 doi: bioRxiv preprint first posted online Dec. 16, 2017; 3
Results and discussionThe regenerative capabilities of the heterotrichous ciliate Stentor have been first documented over a century ago [1]. Since then, Stentor has become a model organism for studying cellular regeneration and repair, and, eventually, for developing new therapeutic approaches for curing human disease [2]. Yet, the molecular basis for Stentor's regenerative capability is largely unknown. Some pioneering studies employing microsurgery have revealed that regeneration and cell division share many similarities on nuclear level [3,4]: Weisz observed macronuclear changes in posterior regenerating fragments, which he compared to dividing cells, while Guttes and Guttes found that the micronuclei in regenerating posterior fragments underwent mitosis. In this study, we employed RNA sequencing (RNAseq) of single Stentor polymorphus cells and cell fragments to gain insight into the molecular basis of cellular regeneration and to identify key determinants underlying this...