brucine Determining whether the specification of most or all

Determining whether the specification of most, or all, cell lineages occurs within neoblasts is essential for understanding the cellular basis for planarian regeneration. Specifically, at what cellular step in regeneration is the identity of new brucine specified? On the basis of prior results demonstrating the specialization of smedwi-1 cells for case study tissues, such as the eye, we sought to test the breadth of the specialized neoblast model. Because it is possible that the smedwi-1cell population contains both dividing cells (neoblasts) and immediate nondividing neoblast progeny cells, we purified S and G2/M phase neoblasts (X1 neoblasts) using fluorescence-activated cell sorting (FACS) and used RNA sequencing (RNA-seq) to identify transcription factors upregulated in X1 neoblasts following wounding. We combined this approach with broad gene expression screening to identify transcription factors expressed in many different tissues and in neoblasts of wounded planarians. Several conserved transcription factors expressed in neoblasts following wounding were required for regeneration of specific cell types, as predicted by the specialized neoblast model. Our results identify a large collection of transcription factors expressed in small subsets of neoblasts at wounds. Together with previous data, these results indicate that the neoblasts that produce regenerative lineages are a highly heterogeneous population consisting of pluripotent stem cells and lineage-restricted progenitors.
Results
Discussion Planarian neoblasts have long attracted interest as adult dividing cells that are required for regeneration and cell turnover. A largely unaddressed question has been whether neoblasts are a homogeneous population or are constituted of multiple different cell types, such as stem cells and lineage-committed progenitors. Two models for regeneration have been considered (Reddien, 2013): the naive neoblast model unites all neoblasts as pluripotent stem cells, with fate specification occurring in nondividing neoblast progeny. The specialized neoblast model posits that fate of regenerating cells is specified in neoblasts themselves. A variety of recent reports have demonstrated instances of smedwi-1 cell specialization. Transcription factors required for the regeneration of the eye (Lapan and Reddien, 2011, 2012), nephridia (Scimone et al., 2011), the anterior pole (Scimone et al., 2014), and several neuron classes (Cowles et al., 2013; Currie and Pearson, 2013; Wenemoser et al., 2012) are expressed in smedwi-1 cells during regeneration. These data provide support for the specialized neoblast hypothesis. If this hypothesis is correct, it predicts that numerous additional transcription factors - perhaps for the specification of every missing cell type - would be expressed in distinct subsets of neoblasts during regeneration. We tested this prediction here by seeking transcription factors expressed in small numbers of X1 neoblasts and/or smedwi-1 cells following wounding. We coupled an RNA-seq approach with expression screening approaches using transcription factors expressed in specific differentiated planarian tissues. In total, 41 transcription factors (8 known from prior reports and 33 reported here) were expressed in X1 neoblasts and/or smedwi-1 cells (Figure S1E). A total of 36 out of these 41 transcription factors were detectably expressed in sorted X1 neoblasts from wounded planarians, indicating expression in cells that will complete at least one round of division. These identified transcription factors displayed expression in specific differentiated cells and in small numbers of neoblasts at wound sites, consistent with the specialized neoblast model. Many specific lineages could be investigated to understand the formation of specific cell types during regeneration, and the transcription factors described here provide a resource for such inquiry. In vivo lineage-tracing methods are presently limited in planarians. Therefore, in principle, a transcription factor induced in neoblasts at wounds might have some role in neoblast physiology other than lineage specification. However, two lines of evidence suggest that many or all of these transcription factors will be involved in specifying progenitors for the regeneration of specific differentiated cell types. First, RNAi of a number of identified transcription factors, reported here and in published work, ablate the regeneration of their specific tissues. Here, we demonstrated a requirement for FoxA for the pharynx; pax3/7 for DBH ventral midline neurons, and klf for cintillo+ sensory neurons. In prior work, sp6-9, eya, and ovo were required for the eye (Lapan and Reddien, 2011, 2012); six1/2-2, POU2/3, and sall were required for the nephridia (Scimone et al., 2011); FoxD was required for the anterior pole (Scimone et al., 2014); and ap-2, lhx1/5-1, pitx, coe, hesl, and sim were required for different subsets of neurons (Cowles et al., 2013; Currie and Pearson, 2013; Wenemoser et al., 2012). Second, we found that transcription factors that were expressed in different differentiated tissues were expressed in distinct subsets of neoblasts. By contrast, in multiple cases, transcription factors expressed together in the same differentiated tissues were expressed together in the same neoblasts. Some described transcription factors might prove to have roles in the specification of more than one lineage. For instance, klf is required for the specification of both photoreceptor neurons as well as for the sensory cintillo+ neurons (this study and Lapan and Reddien, 2012). Similarly, eya has been shown to be required for the specification of eye progenitors (Lapan and Reddien, 2011) as well as nephridia cells (Scimone et al., 2011).