In this study we introduce the use of Smed-H2B
RNAi as a means for the genetic ablation of NBs. We demonstrate that it eliminates the expression of NB-expressed genes in a short period of time and use this in order to elucidate the transcriptional profiles of NBs. Smed-H2B
RNAi induces a quick NB loss that happens in a period of 5 days, faster than most RNAi knockdowns of genes involved in NB biology. For instance, during Smedwi-2
RNAi, NBs and their mitotic activity are detected for more than 10 to 15 days [16
], and similar periods were described for the gene knockdowns of Smed-bruno-like
] and Smed-CHD4
]. The speed of the Smed-H2B
RNAi phenotype makes this knockdown an excellent tool for the genetic ablation of NBs.
is a NB-specific histone isoform that is likely needed for the progression of NBs through the S-phase of the cell cycle. Its RNAi-driven knockdown probably causes a cycling failure. It is still unknown if NBs die at this point or simply differentiate. The consistent peak in NB progeny markers [24
] suggests that at least part of the NB population commits to differentiation, and this is what creates the peaking dynamics of category 2 and 3 markers. It is likely that this also happens after irradiation, since some progeny markers also display a prominent peak after irradiation. Some transcripts with a typical NB expression pattern were found to peak during Smed-H2B
RNAi as well. This suggests that some of the expression of these transcripts is also localized to early NB progeny, and they are only downregulated later in the differentiation process, although further work is still needed in order to elucidate this question.
RNAi approach for the discovery of transcripts expressed in NBs is very specific in comparison to irradiation. Irradiation is known to affect not only the proliferative cells, but to introduce DNA damage in all cells and therefore induce a very potent stress response. As a consequence of this, nearly half of our transcriptomic dataset is consistently downregulated upon irradiation, with a very significant portion of these transcripts not being NB specific. This fact was already observed by Eisenhoffer and co-workers [24
], and solved by carefully checking the WMISH patterns of expression of the transcripts downregulated by irradiation. Our approach solves this question in a very specific way, since all categories described by Eisenhoffer and co-workers behave differently after Smed-H2B
RNAi. Therefore, Smed-H2B
RNAi is informative because of its specificity and discriminative power compared to irradiation.
Our RNA-seq quantification of transcripts expressed in NBs by irradiation and Smed-H2B RNAi reveals that a significant amount of the downregulated transcripts do not disappear after NB ablation, and therefore are expressed at significant levels outside of the NB cellular compartment. However, we show that the quantification of the amount of transcript insensitive to NB ablation differs significantly by both methods. Our analyses of this question indicates that these differences are likely an artifact of irradiation, since the values for irradiation tend to be lower but random when compared to the values determined with Smed-H2B(RNAi). More precisely, for several of the functional gene sets that we describe through annotation of our data, the expression left after NB ablation is consistent for Smed-H2B RNAi but random for irradiation. This fact highly supports that our Smed-H2B RNAi approach is more successful in estimating the amount of transcript left after NB ablation and therefore the amount of expression outside of NB stem cells. Therefore, irradiation not only downregulates nearly half of the planarian transcriptome, but also downregulates the portion of expression left after NB ablation for a very large number of transcripts. The extent of this artifact can also vary according to the detection method used (qRT-PCR, WMISH or RNA-seq), therefore introducing confusion when elucidating the expression patterns of a very significant number of genes. Our Smed-H2B RNAi approach solves this problem and will therefore be a valuable tool for the functional studies of these transcripts and for NB ablation in the future.
The transcripts described as expressed in NBs by our combined approach recapitulate the known morphological features of NBs. A strong enrichment of nuclear components is found, reflecting the high nucleus versus cytoplasm ratio of planarian stem cells. A strong enrichment for chromatin remodeling factors and putative CB components is also found, paralleling the morphological observations of NB chromatin and CBs. Furthermore, a very prominent enrichment of the cell proliferation machinery is also found in our NB-expressed transcripts, agreeing with the fact that NBs are the only proliferative cell type of asexual planarians.
For most of the genes studied here and in the literature, transcripts that do not localize only in NBs are also localized in the planarian CNS [15
]. Interestingly, neurons in other organisms also contain RNA granules, often called neuronal granules [62
], which are similar at the morphologic and biochemical levels to other kinds of RNA granules [50
], and are believed to have similar RNA processing roles. Neuronal RNA granules function in the translational repression and transport of nuclear mRNAs to their different destinations in order to be translated locally. Neuronal granules are found in a variety of organisms, and have been described as well in freshwater planarians [27
]. Interestingly, the S. polychroa
homolog of Smedtud-1
and the D. japonica
have both been localized to perinuclear granules in NBs and neurons, further evidence of the amount of overlap that exists between the molecular machinery of CBs and neuronal granules in planarians. Our data reveal that this overlap is very substantial. Most of the NB-expressed transcripts detected by our approach have a significant portion of their expression that is insensitive to NB ablation. This fact suggests that they are likely present in the planarian CNS, and is consistent with the presence of neuronal granules in the planarian brain as well.
The most enriched process found in our dataset is RNA splicing, revealing that splicing must be of fundamental importance for the regulation of planarian stem cells. Significantly, the regulation of alternative splicing has recently been linked to stem cell biology and embryonic stem cells [66
]. Our data uncover that splicing must also be of fundamental importance for the regulation of planarian stem cells and therefore suggest that it is a key conserved process in the regulation of stem cells.
Our data offer an integrated view, in which the PTGR control is fundamental for the regulation of both NBs and neurons, and in concert with chromatin remodeling and maintenance of the undifferentiated state [73
]. RNA granules and the CBs of NBs are probably a fundamental hub for this control, integrating the processes of splicing and RNA transport, and other RNA related cell functions. CBs have been classically associated with nuclear pores from the morphological point of view. Our data are consistent with this observation, identifying several nuclear pore components as expressed in NBs, suggesting that CBs are the repository of nuclear transcribed mRNAs, where PTGR occurs. This regulation is probably affected by, and ultimately affects, the processes of chromatin remodeling [73
]. Splicing and mRNA quality processes are also probably integrated in CBs. Overall, our NB ablation transcriptome approach reveals that regulation of planarian stem cells relies heavily on PTGR mechanisms.