The planarian Schmidtea mediterranea maintains and regenerates all its adult tissues through the proliferation and differentiation of a single population of pluripotent adult stem cells (ASCs) called neoblasts. Despite recent advances, the mechanisms regulating ASC differentiation into mature cell types are poorly understood. Here, we show that silencing of the planarian EGF receptor egfr-1 by RNA interference (RNAi) impairs gut progenitor differentiation into mature cells, compromising gut regeneration and maintenance. We identify a new putative EGF ligand, nrg-1, the silencing of which phenocopies the defects observed in egfr-1(RNAi) animals. These findings indicate that egfr-1 and nrg-1 promote gut progenitor differentiation, and are thus essential for normal cell turnover and regeneration in the planarian gut. Our study demonstrates that the EGFR signaling pathway is an important regulator of ASC differentiation in planarians.
Adult stem cells (ASCs) usually mediate the normal turnover of adult tissues and their repair after injury. The balance between ASC proliferation and differentiation needs to be tightly regulated because any alteration can lead to depletion or overgrowth of the ASC population, resulting in premature aging or cancer (Oh et al., 2014; Visvader, 2011). The decision to either self-renew or differentiate is controlled by intrinsic determinants present in ASCs and/or extrinsic signals from neighboring differentiated tissues (Morrison and Spradling, 2008). However, a comprehensive understanding of the exact molecular mechanisms controlling ASC maintenance and differentiation is lacking.
Freshwater planarians constitute an attractive model for the in vivo study of stem cell biology in homeostatic and regenerating conditions (Gentile et al., 2011; Newmark and Sánchez Alvarado, 2002; Rink, 2013). These organisms have an impressive capacity to regenerate missing body parts (Morgan, 1898; Reddien and Sánchez Alvarado, 2004), and to grow or degrow depending on environmental conditions (Baguña, 2012; Oviedo et al., 2003; Pellettieri et al., 2010; Romero and Baguña, 1991). These unique abilities arise from a population of pluripotent ASCs, the so-called neoblasts (Baguña et al., 1989; Reddien and Sánchez Alvarado, 2004; Rink, 2013; Wagner et al., 2011). Neoblasts are the only dividing cells of planarians (Morita and Best, 1984; Newmark and Sánchez Alvarado, 2000) and thus can differentiate into any cell type. Accordingly, the neoblast population consists of a compartment of pluripotent ASCs (Wagner et al., 2011) and a heterogeneous pool of lineage-restricted progenitors (Hayashi et al., 2010; Moritz et al., 2012; Reddien, 2013; Scimone et al., 2014; van Wolfswinkel et al., 2014), which can be distinguished by the expression of tissue-specific transcription factors (Adler et al., 2014; Cowles et al., 2013; Lapan and Reddien, 2012; Scimone et al., 2014, 2011; Wagner et al., 2011). Despite recent advances, the genes and molecular pathways that regulate the differentiation of known progenitors into distinct mature cell types remain poorly understood.
The epidermal growth factor receptor (EGFR) pathway is implicated in numerous biological processes (Cela and Llimargas, 2006; Jiang and Wu, 2014; Lyons et al., 2005) and is commonly involved in stem cell proliferation, maintenance and differentiation (Aguirre et al., 2010; Aroian et al., 1990; Biteau and Jasper, 2011; Li et al., 2015). In the planarian Schmidtea mediterranea, one putative EGF ligand (epiregulin-1) (Wenemoser et al., 2012) and four EGF receptors (egfr-1, egfr-2, egfr-3 and egfr-5) (Fraguas et al., 2011; Rink et al., 2011) have been identified to date. Although no function has yet been attributed to epiregulin-1 or egfr-2, the receptors egfr-1, egfr-3 and egfr-5 are required for regeneration and maintenance of various planarian tissues. However, the exact role of the EGFR pathway in neoblast biology is still unclear.
Here, we demonstrate that EGFR signaling controls the differentiation of gut progenitors, and hence the regeneration and homeostasis of the planarian intestine. We show that egfr-1(RNAi) animals are unable to regenerate and maintain the normal morphology of the gut. The loss of gastrodermis is not due to increased apoptosis within this tissue, but to impaired differentiation of gut progenitors into mature gastrodermal cells. Moreover, we identify a new putative EGF ligand, nrg-1, which is expressed in the mesenchyme and pharynx. Silencing of nrg-1 phenocopies the defects caused by egfr-1 RNAi, suggesting that nrg-1-mediated signaling might induce the differentiation of gut progenitors via egfr-1. Our findings support a conserved role of EGFR signaling in controlling the differentiation and overall stability of neoblasts.
egfr-1 is required for gut regeneration
Consistent with previous studies (Fraguas et al., 2011), fluorescent in situ hybridization of egfr-1 (Fig. 1A) revealed expression in the eye pigment cells (Fig. 1Aa), pharynx and mouth opening (Fig. 1Ab), and gut (Fig. 1Ac), and in discrete cells within the mesenchyme (Fig. 1Ac). The colocalization of egfr-1 and SMEDWI-1, a specific neoblast marker (Guo et al., 2006; Marz et al., 2013), indicated that egfr-1 is expressed in neoblasts and/or immediate neoblast descendants (Fig. 1B).
The planarian intestine is composed of one anterior and two posterior main gut branches, which ramify into secondary, tertiary and quaternary branches (Fig. 2A) (Forsthoefel et al., 2011). We used the specific marker panthotenate kinase 1 (pk-1) (Fraguas et al., 2014) to examine gut morphology during regeneration after egfr-1 RNAi (Fig. 2B-S). During posterior regeneration in head fragments (Fig. 2B-G), control animals developed new intestinal branches, which projected around the newly formed pharynx after 1 week (Fig. 2B). In egfr-1(RNAi) animals, these regenerating branches were shorter and failed to completely enclose the pharynx (Fig. 2C). After 2 weeks, the new posterior primary branches extended towards the posterior end and began to produce secondary branches in controls (Fig. 2D). By contrast, the new posterior primary branches in egfr-1(RNAi) animals did not elongate and failed to produce secondary branches (Fig. 2E). After 3 weeks, new secondary and tertiary gut branches were observed in controls (Fig. 2F), but not in egfr-1(RNAi) animals (Fig. 2G).
In regenerating trunk pieces (Fig. 2H-M), elongation of the pre-existing anterior and posterior primary branches within the blastema was observed after 1 week of regeneration in controls (Fig. 2H,H′). After 2 weeks, new primary branches ramified to form secondary and tertiary gut branches (Fig. 2J,J′), restoring the original pattern 3 weeks after amputation (Fig. 2L,L′). Although elongation of the pre-existing primary branches was also observed in egfr-1(RNAi) animals (Fig. 2I,I′), no new secondary or tertiary branches were formed (Fig. 2K,K′), and these animals ultimately failed to regenerate a normal gut (Fig. 2M,M′).
In regenerating tail fragments (Fig. 2N-S), the pre-existing posterior branches converged and fused in the midline to form the new anterior branch 1 week after amputation in controls (Fig. 2N). While this event was also observed in egfr-1(RNAi) animals, the new anterior branch was shorter (Fig. 2O). After 2 weeks, secondary branches began to develop in controls (Fig. 2P), but were almost absent in most egfr-1(RNAi) animals (Fig. 2Q). Finally, after 3 weeks, the normal gut pattern had been restored in controls (Fig. 2R), whereas egfr-1(RNAi) animals had failed to regenerate nearly any secondary or tertiary branches (Fig. 2S).
Remarkably, all egfr-1(RNAi) animals analyzed lost almost all secondary and tertiary branches of the pre-existing gut (Fig. 2G,M,S). Similarly, in line with previous results (Fraguas et al., 2011), all egfr-1(RNAi) animals showed defects in pharynx regeneration (Fig. 2G,S), exhibiting a reduced pharynx cavity compared with control animals (Fig. 2F,R). Taken together, these results indicate that egfr-1 is necessary for proper regeneration of the planarian digestive system, and suggest a possible role of this gene in the maintenance thereof.
egfr-1 is required for the maintenance of gut morphology
We next examined the morphology of the gut in intact control and egfr-1(RNAi) animals (Fig. 3). Two weeks after the final egfr-1 dsRNA injection, both control and egfr-1(RNAi) animals showed the typical gut morphology (Fig. 3A,E). However, after 4 weeks, egfr-1(RNAi) animals differed from controls in that they exhibited a less ramified gut, with fewer tertiary and quaternary branches (Fig. 3B,F). Moreover, the gut branches of egfr-1(RNAi) animals were thinner than those of controls (Fig. 3Bb,Fb). By 6 weeks, egfr-1(RNAi) animals showed more severe defects: few tertiary and almost no quaternary branches remained in these animals, in contrast to controls, in which both branches were maintained (Fig. 3C,G). The most extreme phenotype was observed 8 weeks after RNAi: egfr-1(RNAi) animals had completely lost all tertiary and quaternary branches, with only some secondary branches remaining (Fig. 3H). At this time point, the anterior primary branch, and to a lesser extent the posterior primary branches, were shorter in egfr-1(RNAi) animals compared with controls (Fig. 3D,H). As seen during regeneration, egfr-1(RNAi) animals already exhibited a wider and thicker pharyngeal cavity 4 weeks after treatment (Fig. 3B,F).
To better understand the progression of the egfr-1 RNAi phenotype during homeostasis, we quantified the length of the primary gut branches and the number of secondary, tertiary and quaternary branches (Fig. 3I). Although the effect was less severe in the posterior region, from 4 weeks onwards both the anterior and posterior primary gut branches were significantly shorter in egfr-1(RNAi) animals compared with controls (Fig. 3J). By 2 weeks, a significant reduction in the number of quaternary branches in the anterior region was also observed, followed by a decrease in the numbers of tertiary and secondary branches by 4 and 8 weeks, respectively (Fig. 3K). In the posterior region the dynamics of the phenotype were similar to those of the anterior region, albeit with a slight delay (Fig. 3L). Overall, quantitative analysis of the number and length of gut branches in egfr-1(RNAi) animals indicated that the disappearance of the branches occurred in a stereotypical, organized fashion (Fig. 3J-L), sequentially affecting the quaternary, tertiary and finally secondary branches, accompanied by progressive shortening of the primary branch. Our data thus indicate that while control animals were capable of maintaining their gut morphology over time, egfr-1(RNAi) animals were unable to preserve the integrity of the gut, as evidenced by the gradual shortening of this structure and the loss of gut branches.
egfr-1 silencing affects gastrodermis integrity
The planarian gastrodermis is a monostratified epithelium composed of two cell types, absorptive phagocytes and secretory goblet cells, surrounded by an enteric muscular plexus (Bowen et al., 1974; Bueno et al., 1997; Kobayashi et al., 1998). To better characterize the loss of the gut after egfr-1 RNAi, we performed a detailed histological analysis of the gastrodermis. Specifically, we studied intact egfr-1(RNAi) animals that were fixed 4 weeks after the last RNAi injection, the earliest time point at which all the features of the egfr-1 phenotype were evident (Fig. 3). As observed in representative transverse sections corresponding to four different positions along the antero-posterior axis of the planarian body, egfr-1(RNAi) animals had fewer and smaller gut diverticula per section than controls (Fig. 4A). Importantly, the defects in the gastrodermis in egfr-1(RNAi) animals were not correlated with loss of the enteric musculature surrounding the diverticula (anti-MHC) (Cebrià et al., 1997), which appeared to be unaffected (Fig. 4A). Moreover, Mallory's staining in sagittal sections revealed a reduced and aberrant gastrodermis in egfr-1(RNAi) animals, without the characteristic columnar disposition of cells evident in control animals (Fig. 4B).
To support our qualitative observations, we performed quantitative analyses of the area of the gastrodermis in anterior and posterior sections of control and egfr-1(RNAi) animals (Fig. 4C). Consistently, egfr-1 RNAi resulted in a significant decrease in the gastrodermal area in both regions (Fig. 4D). Quantification of the number of nuclei per diverticulum showed that the gastrodermis of egfr-1(RNAi) animals contained fewer nuclei than that of controls. However the number of nuclei relative to the area of the diverticulum remained constant, as this area was also significantly reduced in egfr-1(RNAi) animals (Fig. 4E). To determine whether cell loss affected one or both gastrodermal cell types, we used an anti-RPZ-1 antibody to specifically label goblet cells (Reuter et al., 2015) (Fig. 4F). Immunostaining of anterior and posterior sections revealed a significant reduction in the number of goblet cells in egfr-1(RNAi) animals compared with controls (Fig. 4F,G). As observed with Mallory's staining, the goblet cells of egfr-1(RNAi) animals showed abnormal morphologies (Fig. 4F). We estimated the number of phagocytes by subtracting the number of goblet cells from the total number of gastrodermal cells as identified using the nuclear marker DAPI. The gastrodermis of egfr-1(RNAi) animals contained fewer phagocytes than that of controls, although the number of phagocytes relative to the area of the diverticulum remained constant in both control and egfr-1(RNAi) animals (Fig. 4H). Together, these results indicate that the morphological defects observed in egfr-1(RNAi) animals involve a reduction in gastrodermis area and depletion of both gastrodermal cell types.
Impaired gut progenitor cell differentiation accounts for the loss of gastrodermis in egfr-1(RNAi) animals
To determine whether the decrease in the number of cells in the gastrodermis of egfr-1(RNAi) animals was due to increased apoptosis, we performed TUNEL assays to quantify apoptotic cells (Pellettieri et al., 2010) in transverse sections of intact animals after egfr-1 RNAi (Fig. 5A). egfr-1 RNAi resulted in a significant increase in the number of apoptotic mesenchymal cells (Fig. 5B). However, there were no significant differences in the number of apoptotic gastrodermal cells between control and egfr-1(RNAi) animals (Fig. 5C), suggesting that the decrease in cell number seen in RNAi animals was not caused by an abnormal increase in apoptosis within this tissue.
We next investigated whether impaired differentiation of new cells could explain the defects seen after egfr-1 RNAi. To test this hypothesis we labeled S-phase neoblasts using the thymidine analog F-ara-EdU in control and egfr-1(RNAi) animals (Zhu et al., 2015). After a 6-day chase, the presence of EdU-labeled cells was assessed in histological sections (Fig. 5D). While control animals showed EdU-labeled cells within the gastrodermis, almost no EdU incorporation was observed in the gut tissue of egfr-1(RNAi) animals (Fig. 5D,E). By contrast, there were no significant differences in the number of mesenchymal EdU-labeled cells between control and egfr-1(RNAi) animals (Fig. 5F). These findings suggest that the reduced number of gastrodermal cells in egfr-1(RNAi) animals is likely to be due to impaired differentiation of gut progenitor cells.
egfr-1 silencing increases the number of gut progenitor cells
Because neoblasts are the only source of new gastrodermal cells (Forsthoefel et al., 2011), we hypothesized that abnormal neoblast proliferation and/or commitment dynamics could account for the impaired differentiation observed in egfr-1(RNAi) animals. In agreement with previous studies (Fraguas et al., 2011), we found that neoblast proliferation was significantly increased in egfr-1(RNAi) animals (Fig. S1A,B). Moreover, we observed a clear increase in the total number of neoblasts, as determined using the specific marker histone-2B (Solana et al., 2012) (Fig. S1C), the anti-SMEDWI-1 antibody (Fig. S1D) and smedwi-1 (Fig. S1E,F).
The expanded neoblast population observed in egfr-1(RNAi) animals allowed us to rule out defective stem cell maintenance as a possible source of the egfr-1 RNAi phenotype and to consider the possibility of impaired commitment of neoblasts to intestinal progenitors. The transcription factors hnf4 and gata4/5/6 are expressed by differentiated gut cells and by a sub-class of neoblasts (γ-neoblasts) (van Wolfswinkel et al., 2014) proposed to be progenitors of this cell lineage (Scimone et al., 2014; Wagner et al., 2011). Therefore, we quantified the number of hnf4+/SMEDWI-1+ and gata4/5/6+/SMEDWI-1+ cells in control and egfr-1(RNAi) animals (Fig. 6). Silencing of egfr-1 resulted in a significant increase in the number of both hnf4+/SMEDWI-1+ (Fig. 6A,B,E) and gata4/5/6+/SMEDWI-1+ (Fig. 6C,D,F) cells. Moreover, we observed co-expression of egfr-1 and gata4/5/6 (Fig. 6G), as well as egfr-1 and hnf4 (Fig. 6H) in mesenchymal cells. Altogether, these results suggest that the gastrodermis defects observed in egfr-1(RNAi) animals are due to impaired differentiation of gut progenitors into mature gastrodermal cells rather than in their commitment to this cell lineage.
Silencing of the putative ligand nrg-1 phenocopies the defects observed in egfr-1(RNAi) animals
To date, only one putative EGF ligand, epiregulin-1, has been identified in planarians. Its expression is limited to the digestive system (Wenemoser et al., 2012) (Fig. S2A) and its silencing failed to affect gut regeneration or homeostasis (Fig. S2B). We thus performed new in silico searches and identified a new putative EGF ligand, which we named nrg-1 (Fig. S3). In intact animals, nrg-1 was mainly expressed in the mesenchyme and pharynx (Fig. 7A). During regeneration, nrg-1 was expressed within blastemas from 3 days onwards, as well as in the mesenchyme and the newly regenerating pharynx (Fig. 7A). nrg-1 RNAi affected both regeneration (Fig. 7B,C) and homeostasis (Fig. 7D-G), phenocopying the defects observed after egfr-1 knockdown (Fraguas et al., 2011). After nrg-1 RNAi, regenerating head pieces formed smaller pharynxes than controls, although the pre-existing pharynx of trunk pieces appeared unaffected, as evidenced by the expression profile of the specific marker laminin (Cebrià and Newmark, 2007) (Fig. 7B-C). Regenerating nrg-1(RNAi) trunks formed smaller eye pigment cups than those seen in controls (Fig. 7B). Consistent with our observations in egfr-1(RNAi) animals, nrg-1(RNAi) animals were unable to regenerate new gut branches and gradually lost the pre-existing branches (Fig. 7C). Furthermore, an increase in the neoblast population was observed in regenerating nrg-1(RNAi) animals (Fig. 7C).
During homeostasis, nrg-1 RNAi led to a reduction in the number of eye pigment cells (Fig. 7D), although the pharynx appeared unaffected (Fig. 7E). We also observed fewer gut branches in nrg-1(RNAi) animals compared with controls (Fig. 7E), although these defects were milder than those seen in the pre-existing tissue of regenerating animals (Fig. 7C). Finally, as in regenerating animals, the neoblast population was increased (Fig. 7E-G). Two rounds of dsRNA injection in both regenerating (Fig. S4A) and homeostatic animals (Fig. S4B) led to more severe defects in the pharynx, eyes and gut, and ultimately to death of the animal after a few days. Our functional analyses revealed that nrg-1(RNAi) animals exhibit a phenotype strikingly similar to that of egfr-1(RNAi) animals, supporting the view that nrg-1 might act via egfr-1.
If nrg-1 does indeed activate egfr-1, a reduction in gut cell differentiation and an increase of the pool of gut progenitors (γ-neoblasts) would be expected after nrg-1 silencing. To investigate this hypothesis, we labeled S-phase neoblasts in control and nrg-1(RNAi) animals 20 days after a single round of dsRNA injection. After a 6-day chase, we observed a significant reduction of EdU incorporation in the gut tissue of nrg-1(RNAi) animals compared with controls (Fig. 8A,B). By contrast, no significant differences in the number of mesenchymal EdU-labeled cells were observed (Fig. 8C). Next, we assessed the γ-neoblast population by quantifying the number of hnf4+/SMEDWI-1+ and gata4/5/6+/SMEDWI-1+ cells in controls and in nrg-1(RNAi) animals (Fig. 8D-I). As predicted, nrg-1 silencing resulted in a significant increase in the number of hnf4+/SMEDWI-1+ cells (Fig. 8D,E,H). However, no significant differences in the number of gata4/5/6+/SMEDWI-1+ cells (Fig. 8F,G,I) were observed, probably because the gut defects were less severe after only one round of nrg-1 inhibition. Overall, these results suggest that the gut defects observed in nrg-1(RNAi) animals are due to impaired differentiation of neoblasts into gastrodermal cells, further supporting our hypothesis that the putative EGF ligand nrg-1 controls differentiation of gut progenitors via egfr-1.
Silencing of egfr-1 and nrg-1 induces an increase in the expression of σ- and ζ-neoblast markers, but exclusively affects gastrodermal cell differentiation
To elucidate how the general increase in the neoblast population in egfr-1(RNAi) and nrg-1(RNAi) animals affected the primary neoblast subclasses σ and ζ, we performed whole-mount in situ hybridizations with the σ-neoblast marker soxP-2 and the ζ-neoblast marker zfp-1 (van Wolfswinkel et al., 2014). We observed an apparent increase in both neoblast subclasses in egfr-1(RNAi) and nrg-1(RNAi) animals (Fig. S5A,D). qPCR analysis of soxP-2 and zfp-1 expression levels revealed a significant increase in the expression of both σ- and ζ-neoblasts in egfr-1 (RNAi) animals (Fig. S5B,C), but only in ζ-neoblasts in nrg-1(RNAi) animals (Fig. S5E,F). These results demonstrate that the expansion of the neoblast population observed after silencing of egfr-1 and nrg-1 affects all described primary neoblast subclasses.
To determine whether this expansion of the neoblast population affected other cell lineages, we analyzed a set of known markers of differentiated cells and progenitor cells from diverse planarian tissues. We observed no significant differences in the populations of dopaminergic (th) (Nishimura et al., 2007) or octopaminergic (tbh) (Nishimura et al., 2008) neurons between control and egfr-1(RNAi) animals (Fig. S6A-C), and no change in the expression of the neural progenitor marker coe (Cowles et al., 2013) (Fig. S6A). Analysis of the expression of the protonephridial cell marker CAVII-1 (Sánchez Alvarado et al., 2002) and the excretory system progenitor marker pou2/3 (Scimone et al., 2011) revealed no differences between control and egfr-1(RNAi) animals (Fig. S6D,E). Similarly, expression of the epithelial lineage-specific marker AGAT-1 (Eisenhoffer et al., 2008; Tu et al., 2015) did not differ significantly between control and egfr-1(RNAi) animals (Fig. S6F,G). Similar results were obtained after nrg-1 silencing (Fig. S7). Altogether, these data suggest that the observed expansion of the neoblast population seen after both egfr-1 and nrg-1 RNAi does not affect the commitment/differentiation of neural, epidermal or excretory cell lineages.
Finally, because pharynx regeneration and maintenance is also impaired in egfr-1 and nrg-1 RNAi animals (Fraguas et al., 2011), we sought to determine whether those alterations were attributable to an impaired differentiation of new pharyngeal cells. Both egfr-1(RNAi) and nrg-1(RNAi) animals showed statistically similar numbers of EdU+ cells within this organ (Fig. S8A-D). Analysis of the expression of the pharynx-progenitor marker foxA (Adler et al., 2014) showed no significant differences between treated animals and controls (Fig. S8E,F). Taken together, these results suggest that egfr-1 and nrg-1 are implicated in pharynx regeneration and homeostasis via a mechanism other than neoblast differentiation.
egfr-1 and nrg-1 regulate gut regeneration and homeostasis
Gut regeneration relies on both remodeling of the pre-existing gastrodermis and the supply of newly differentiated gut cells (Forsthoefel et al., 2011). This de novo differentiation occurs within the blastema, as well as in other regions of the pre-existing intestine that need to remodel the normal proportions and symmetry of this organ (Forsthoefel et al., 2011). Our results uncover a new function of egfr-1 and nrg-1 in controlling regeneration of the planarian gut and maintaining the morphology of the pre-existing tissue. In egfr-1(RNAi) animals, pre-existing primary branches are partially capable of elongation during the first week after amputation, suggesting that the contribution of the differentiated tissue to gut regeneration is not impaired. Accordingly, the incomplete elongation of the primary branches and the failure to form new secondary and tertiary branches, together with the marked defects in remodeling of the pre-existing gastrodermis, are consistent with compromised differentiation of new gastrodermal cells in these animals.
During planarian homeostasis, adult tissues preserve their shape and integrity through cell turnover, which involves the constant death of differentiated cells, the division of ASCs, and the differentiation and integration of ASC progeny into the pre-existing tissue to replace the lost cells (Pellettieri and Sánchez Alvarado, 2007). In the planarian gut, the uniform integration of new gastrodermal cells throughout the intestine maintains this tissue and contributes to the formation of new gut branches, which are always in proportion to the animal's body size (Forsthoefel et al., 2011). Silencing of egfr-1 and nrg-1 impairs the differentiation of new gut cells. This, together with a normal ratio of cell loss by apoptosis, disrupts the normal turnover of the gut in egfr-1(RNAi) animals, compromising maintenance of its normal morphology. Accordingly, the defects in gut tissue are observed gradually, from the most terminal branches (i.e. quaternary and tertiary), which consist of fewer cells, to the most principal branches, which contain a larger number of gastrodermal cells. Notably, this phenotype differs from those described after silencing other genes required for intestinal integrity (Forsthoefel et al., 2012). The impairment of intestinal adhesion, polarity and phagocyte maturation completely compromises gut regeneration and homeostasis, eventually leading to the death of the animal (Forsthoefel et al., 2012).
The EGFR pathway controls the differentiation of gut progenitor cells
Planarian neoblasts are heterogeneous, consisting of pluripotent ASCs and lineage-committed progenitors (Scimone et al., 2014; van Wolfswinkel et al., 2014). Gene expression profiling shows the existence of three subclasses of neoblasts: ζ-neoblasts, regarded as a epidermal progenitors (Tu et al., 2015; van Wolfswinkel et al., 2014); γ-neoblasts, identified by the expression of the endodermal transcription factors hnf4 and gata4/5/6 (Ang et al., 1993; Azzaria et al., 1996; Murakami et al., 2005), and thus hypothesized to include gut progenitors (van Wolfswinkel et al., 2014); and σ-neoblasts, which are regarded as truly pluripotent neoblasts with the ability to generate all the other subclasses and cell lineages (van Wolfswinkel et al., 2014). In addition to these advances, recent studies have begun to elucidate the process by which neoblast dynamics are regulated. The RNA-binding protein mex3-1 (Zhu et al., 2015) and the transcription factor egr-5 (Tu et al., 2015) are key regulators of the specification of all major known lineage progenitors and the differentiation of the epidermal lineage, respectively.
Our results indicate that egfr-1 and nrg-1 are essential regulators of the differentiation of gut progenitors into mature gastrodermal cells. Remarkably, the specification of gut progenitors (γ-neoblasts) is not affected after silencing of egfr-1 and nrg-1, and the expansion of this population is probably due to their incapacity to differentiate. This phenomenon is reminiscent of the phenotype observed after egr-5 silencing, whereby epidermal differentiation is impaired whereas numbers of epidermal progenitors increase (Tu et al., 2015). Importantly, the analysis of other differentiated cell/progenitor populations, including epidermal, excretory, neural and pharyngeal cells, suggests that the silencing of egfr-1 and nrg-1 specifically affects differentiation of the gut lineage. We thus propose that in normal conditions, pluripotent neoblasts commit to gut progenitors and start expressing hnf4 and/or gata4/5/6 with egfr-1. Upon nrg-1 binding, these cells proceed to differentiate into mature gut cells (Fig. 9A). Silencing of either egfr-1 or its putative ligand nrg-1 disrupts EGFR signaling, and hence the differentiation of gut progenitors, leading to their accumulation in the mesenchyme (Fig. 9B). However, several aspects of this scenario require further study, such as the cellular origin of nrg-1, the dynamics of nrg-1/egfr-1 binding, and the mechanisms underlying nrg-1 proteolysis and diffusion. Finally, the EGF receptors egfr-3 and egfr-5 control the regeneration and homeostasis of particular planarian cell types, such as neuronal and excretory cells (Fraguas et al., 2011; Rink et al., 2011). Further studies will thus help us to elucidate whether the model proposed here can be extrapolated to these other receptors, eventually uncovering a general role of the planarian EGFR pathway in the regulation of progenitor cell differentiation.
Strikingly, the silencing of egfr-1 and nrg-1 also results in the hyperproliferation and expansion of the ζ- and σ-neoblasts. However, this neoblast expansion does not appear to correlate with an increase in the number of differentiated cells of any of the cell lineages examined. One possibility is that the loss of gut tissue stimulates neoblasts to proliferate as part of a stress response (Tu et al., 2015). Alternatively, gut loss and disorganization could affect some signaling processes, normally originating in the gastrodermis, that regulate neoblast dynamics (Adler and Sánchez Alvarado, 2015). The latter possibility is supported by a study of transcription factor nkx-2.2. Silencing of this gene, which is expressed in the gut, disrupts gut integrity and blocks neoblast proliferation (Forsthoefel et al., 2012). Accordingly, future studies will need to elucidate the precise causes of this hyperproliferation and determine whether the observed increased in apoptosis in the mesenchyme reflects a compensatory response to this increase.
In summary, our study uncovers a role of the EGFR signaling pathway in controlling the differentiation of gut progenitors in the planarian S. mediterranea. This role in stem cell differentiation appears to be widespread among bilaterian animals (Freeman et al., 1992; Galvez-Contreras et al., 2015; Yoo et al., 2004). Moreover, studies performed in Drosophila and mouse have demonstrated the role of the EGFR pathway in the maintenance of the intestine (Jiang et al., 2011; Wong et al., 2012), as demonstrated here in planarians. However, contrary to our observations, the EGFR pathway appears to regulate the proliferation of intestinal stem cells in the aforementioned organisms. Therefore, our results support a conserved role of this pathway in controlling the dynamics of ASCs in bilaterian animals and underscore the utility of the unique planarian ASC system as a model in which to study the role of the EGFR pathway in stem cell biology.
MATERIALS AND METHODS
Asexual S. mediterranea from the BCN-10 clonal line were maintained in artificial water (Cebrià and Newmark, 2005). Animals were fed with veal liver and starved for at least 1 week before all experiments.
Gene identification and cloning
All genes were amplified by gene-specific PCR and cloned into either pCRII (Life Technologies) or pGEM-T Easy (Promega) vectors. The putative EGF ligand nrg-1 was identified by in silico searches querying Prostheceraeus vittatus putative EGF ligands (BioProject accession ID: PRJNA277637).
Silencing by RNAi was performed as previously described (Sánchez Alvarado and Newmark, 1999) and validated by qPCR (Fig. S9). Control animals were injected with double-stranded RNA (dsRNA) for green fluorescent protein (GFP). In egfr-1(RNAi) analyses, all animals received two rounds of injections separated by 3-4 days, whereas only one round of injections was performed for nrg-1(RNAi) analyses.
Quantitative real-time PCR
Total RNA was extracted from a pool of five control, egfr-1(RNAi), and nrg-1(RNAi) animals. Quantitative real-time PCR was performed as described previously (Solana et al., 2012) and data were normalized based on the expression of the internal control URA-4. All experiments were performed using three biological and three technical replicates for each condition. The following primers (5′ to 3′) were used: egfr-1F, CTGATTGGAAAGGATGTACTCAATGT; egfr-1R, ACCATTACACTGCGGATGACAC; nrg-1F, TGACGAATCTAGAAGGAAATGTAGC; nrg-1R, CGACGTCCGTAGAAACCATTT; soxP-2F, CCAGCAATTTTCCCAAAG; soxP-2R, CCCCTTCTGAATCATCCAT; zfp-1F, AAATTTTCCCGTGCCTG; zfp-1R, TGATCTTTGAGTGAAGCTGGT.
Whole-mount in situ hybridization and immunohistochemistry
Single and double fluorescent in situ hybridizations (FISH) were performed as previously described (King and Newmark, 2013) using the Tyramide Signal Amplification Kit (TSA; Perkin Elmer). After probe development, neoblasts were visualized with rabbit anti-SMEDWI-1 antibody (kindly provided by Kerstin Bartscherer, Max Planck Institute for Molecular Biomedicine, Münster, Germany; 1:1100) (Guo et al., 2006; Marz et al., 2013).
In situ hybridization, immunohistochemistry and histological staining of paraffin sections
For in situ hybridization and immunohistochemistry, animals were killed in 2% HCl in Milli-Q water, fixed with 4% paraformaldehyde in PBS for 4 h at 4°C, and stored in 70% ethanol in Milli-Q water. Paraffin embedding, sectioning, and subsequent in situ hybridization and immunostaining were performed as previously described (Adell et al., in press; Cardona et al., 2005a). The antibodies used were rabbit anti-SMEDWI-1 (1:1100), mouse anti-MHC (kindly provided by Rafael Romero, University of Barcelona, Spain; 1:20) (Cebrià et al., 1997), anti-phospho-histone-H3-Ser10 (anti-PH3) (Cell Signaling, #3377S; 1:300) and rabbit anti-RPZ-1 (kindly provided by Kerstin Bartscherer; 1:200) (Reuter et al., 2015). Mallory's staining was performed as previously described (Cardona et al., 2005b; Sluys, 1989).
TUNEL and EdU staining of paraffin sections
Animals were fixed and sectioned as described above. Staining of apoptotic cells was performed in accordance with the manufacturer's recommendations (ApopTag Red In Situ Apoptosis Detection Kit, Merck-Millipore, S7165), followed by immunostaining with anti-MHC (as described above). Samples were counterstained with DAPI (1:5000 in PBS) and mounted with 70% glycerol in PBS. For EdU experiments, a single pulse of F-ara-Edu (Sigma) was injected into control, egfr-1(RNAi) and nrg-1(RNAi) animals at a concentration of 60 μg/ml (diluted in 10% DMSO/planarian artificial water). After a 6-day chase, staining of EdU-labeled cells was performed using the EdU Click-555 kit (Baseclick, BCK-EdU555), following the manufacturer's recommendations, with the addition of a prior step consisting of incubation in proteinase K (20 μg/ml) for 10 min. Samples were counterstained with anti-MHC and DAPI, and mounted in 70% glycerol in PBS.
FISH samples were imaged under either a MZ16F stereomicroscope (Leica) equipped with a ProgRes C3 camera (Jenoptik) or an SPE confocal laser-scanning microscope (Leica). Histological sections were observed under an Axiophot microscope (Zeiss) and imaged with a DFC300FX camera (Leica) or an SP2 confocal laser-scanning microscope (Leica). Images were processed using Fiji and Photoshop CS6 (Adobe) software. Brightness/contrast and color balance adjustments were always applied to the entire image, rather than specific regions thereof.
We thank José M. Martín-Durán and all the members of Emili Saló's lab for support and discussion. We thank Kerstin Bartscherer for the anti-SMEDWI-1 and the anti-RPZ-1 antibodies, and Rafael Romero for the anti-MHC antibody. We thank Owen Howard for advice on English style.
S.B. and F.C. conceived and designed the study. S.B. and S.F. conducted the experiments and S.B. drafted the manuscript. All authors discussed and commented on the data. F.C. edited the paper.
This study was funded by the Ministerio de Economía y Competitividad (MICINN, Spain) [BFU2011-22510 and BFU2012-31701]; and Generalitat de Catalunya [2009-SGR-1018 to F.C.]. S.B. was supported by a fellowship from the Generalitat de Catalunya and a collaboration fellowship from the Institute of Biomedicine of the University of Barcelona (IBUB). S.F. was supported by a fellowship from MICINN.
The sequence of smed-nrg-1 has been deposited in GenBank with accession number KU850495.
The authors declare no competing or financial interests.