Why do you think that stem cell progeny expressed nearly twice as many transcription factors than stem cells or differentiated tissues (p 1742)?
How does GSEA work?
Also, I don't fully understand why they irradiated the worms.
The 'Not your father's planarian' article discusses the ability of a planarian to "de-grow" in times of resource depletion, capable of shrinking to 1/20th its original size. How do you think planarian are able to signal mass apoptosis of cells without losing their bodily form or the viability of their organs?
In the primary article, why did they exclude ORFs of <100 amino acids? Couldn't there still be some viable genes of interest there?
On p. 1737 of the Labbe et al. article they mentioned that there is no GO database for planarian genes so they just used the mouse and human orthologs to search for gene ontology information. Since genes can sometimes have different localizations and functions in different species, especially those as distantly related as planarians and mammals, do you this this methodology could have skewed their data?
The primary article by Labbe et.al. states on page 1743 that all the genes tested on planarians were "...100% penetrant and lethal". The genes tested were to show the effects on the adult stem cells and there regenerative properties into pluripotent cells. These genes tested were thought to be available in mice and humans, though there is not a GO database for planarians to compare homologs. Thus, because these genes killed the planarian in time and had great developmental effects on regeneration, would you expect there to be similar results when tested on mice and human tissue? Would the mice or human tissue die as a result and not lead to the benefits of regenerative medicine of adult stem cells?
In the discussion of the primary article, they mention that "no stem cell purification was applied, and thus having all tissues present can also complicate analyses and diminish the dynamic range of the sequencing." What differences would be seen in the results if they had purified the sample vs not?
Do you think that the planarians being fed RNAi bacteria only after starved for 7 days could have affected the planarian's ability to fully process the dsRNA and could have skewed the results?
In the article it talks about the worms being exposed to lethal amounts of irradiation. This rids the planaria of there stem cells. I don't understand how this allows for the rest of the organism to remain intact - how is it specific to stem cells.
Would you please explain how the stem cells were purified? The Labbe et al. paper mentioned radiation but said that it killed the cells, and then it mentioned flow cytometry.
Maybe I missed this somewhere, but isn't it relevant how the transcription factors relate to other genes? For instance, I think looking at the transcription factors and the genes they regulate independently may mask some important findings -- maybe what is really important for stem cell identity are the transcription factors, which are sufficient to influence gene expression. I think this is one of the limits to their approach -- they've got such huge sets of data that it seems easy to miss important patterns.
How can they be sure that irradiation doesn't affect the transcriptome of differentiated tissues in planaria?
Also, how do you think researchers could circumvent or make up for the fact that these functional studies conducted in planaria used only RNAi? I'm not sure how feasible this is, but what if they were to look at stem cells in Drosophila gut that are being studied in the Ohlstein lab at Columbia and Micchelli lab at Washington University?
In the primary article, what is the advantage of analyzing the transcriptome over the genome or proteome? What is to be gained from transcriptomics that genomics and proteomics cannot provide in this study?
1) Why is clonal lineage tracing cannot be carried out on planarians?
2) How high is the purity of stem cell isolated? As far as I know the stem cell count is extremely low, take human as example, actual hematopoietic stem cells consist of >0.01% of blood sample, but traditional flow cytometry can only purify it down to ~0.1%, of which ~0.09% of the "stem cells" isolated are actually progenitor cells. I am doubtful of the purity of stem cell samples obtained in this paper.
In the review paper “RNA-Seq: a revolutionary tool for transcriptomics”, the authors say generally, as the genome is larger, the transcriptome is more complex and more sequencing depth is required. Moreover, they say it is tricky to calculate the transcriptome coverage since the true number and different transcript isoforms' level is not well known. Here, i don’t really get what the true number and level of different transcript isoforms are. And i wonder whether there is any way to overcome the challenge or not.
Gamma radiation seems like a very harsh way to isolate stem cells from the rest of the organism. The authors even acknowledge that it has effects on transcription in differentiated tissues. Isn't it possible that this radiation is having a similar effect on the stem cells and progeny that they've separated?
The paper mentions, that for gene expression quantification, only the human and mouse canonical transcripts were used. What exactly does this mean? How does Ensembl filter the transcripts?
The paper by Labbe et al. talks about three categories of genes that are differentially expressed in planarian stem cells. “(a) genes with no 1:1:1 ortholog but greater than 5-fold differential expression in planarian stem cells (b) genes with a 1:1:1 ortholog and greater than 5-fold differential expression in planarian stem cells but no differential expression in mammalian ESCs, and (c) genes with a 1:1:1 ortholog and greater than 5-fold differential expression in mouse, human, and planarian stem cells.” I understand how knowledge about category c could be very useful to medicine but what about categories a and b? What are the practical applications of research on stem cell genes in planarians if they either don’t have a mammalian ortholog or are not differentially expressed in mouse and human stem cells (suggesting they don’t play a role in mammalian stem cells)?
What is the reasoning behind irradiation of the worms? I feel that there are more precise ways of causing mutations within the worms that would yield more definitive results.