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. Author manuscript; available in PMC: 2022 Sep 23.
Published in final edited form as: Dev Biol. 2021 Sep 3;480:105–113. doi: 10.1016/j.ydbio.2021.08.011

Wnt/β-catenin signaling pathway regulates cell proliferation but not muscle dedifferentiation nor apoptosis during sea cucumber intestinal regeneration

Miosotis Alicea-Delgado 1, José E García-Arrarás 1
PMCID: PMC9503151  NIHMSID: NIHMS1835810  PMID: 34481794

Abstract

Regeneration is a key developmental process by which organisms recover vital tissue and organ components following injury or disease. A growing interest is focused on the elucidation and characterization of the molecular mechanisms involved in these regenerative processes. We have now analyzed the possible role of the Wnt/β-catenin pathway on the regeneration of the intestine in the sea cucumber Holothuria glaberrima. For this we have studied the expression in vivo of Wnt-associated genes and have implemented the use of Dicer-substrate interference RNA (DsiRNA) to knockdown the expression of β-catenin transcript on gut rudiment explants. Neither cell dedifferentiation nor apoptosis were affected by the reduction of β-catenin transcripts in the gut rudiment explants. Yet, the number of proliferating cells decreased significantly following the interference, suggesting that the Wnt/β-catenin signaling pathway plays a significant role in cell proliferation, but not in cell dedifferentiation nor apoptosis during the regeneration of the intestine. The development of the in vitro RNAi protocol is a significant step in analyzing specific gene functions involved in echinoderm regeneration.

Keywords: RNAi, Echinoderm, Explants, Electroporation, Regeneration

Introduction

Regeneration is an astonishing process characterized by the restitution of lost body parts. Many organisms with regenerative capabilities have the ability to develop a functional organ after losing it. This regenerative capacity has intrigued the scientific community for centuries, and scientists have been studying how and why this process seems to be restricted in some species while widely displayed by others. For example, the coelenterate Hydra and the flatworm Planaria have the ability to regenerate a whole organism after suffering an amputation (Petersen et al., 2015; Witchley et al., 2013). Other species, such as the zebra fish and the salamander, can regenerate lost body parts, including the lens, heart, fins, limbs, among others (Jopling et al., 2010; Meyers et al., 2012; Morrison et al., 2006). In contrast, regenerative capacities are highly limited in other groups, such as adult insects and mammals (Bely & Nyberg, 2010; Maden, 2018).

The understanding of this phenomenon begins with the identification of the cells that are involved and the mechanisms that trigger and achieve these regenerative responses. It has been documented that regenerative models either have stem cells or cells that undergo dedifferentiation (Ferrario et al., 2020; Saló et al., 2009; Sánchez Alvarado, 2006; Sánchez Alvarado & Kang, 2005). For instance, regeneration in flatworms depends on the presence of neoblasts, proliferating stem cells that give rise to every cell in the organisms (Saló et al., 2009; Sánchez Alvarado, 2006; Sánchez Alvarado & Kang, 2005). In contrast, limb regeneration in salamanders depends on the dedifferentiation and proliferation of the cells adjacent to the injury site (Morrison et al., 2006; Tsonis, 2000a, 2000b). Whether regenerating cells originate from dedifferentiating cells or from stem cells depend on various factors, such as the species and/or the organ involved. Perhaps the most striking demonstration of the duality of cell origins was demonstrated in newts, where the cellular mechanism for limb regeneration switches from a stem/progenitor-based mechanism at the larval stage to a dedifferentiation-based mechanism in the adult (Tanaka et al., 2016).

Experimental evidence from other phyla, provides additional examples of cellular dedifferentiation involvement in organ regeneration (Garcia-Arraras, 2017; San Miguel-Ruiz & García-Arrarás, 2007). In sea cucumbers (Holothuroidea, Echinodermata), muscle cells and coelomic epithelial cells (peritoneocytes) regress to a less-differentiated state giving rise to the cell population that will restore the organ. Our lab has actively studied the regeneration of the digestive system using the sea cucumber Holothuria glaberrima. This species has the ability to naturally expel out its digestive tract through the cloaca after being exposed to hostile conditions (García-Arrarás et al., 1998). Organ regeneration is based on the mesentery where the expulsed organs were previously attached (García-Arrarás et al., 2019; García-Arrarás et al., 1998; García-Arrarás & Greenberg, 2001). The mesentery undertakes a series of cellular and molecular changes that signal the formation of a rudiment and eventually the formation of the new intestine (García-Arrarás et al., 1998; Mashanov and García-Arrarás, 2011). Previous studies have elucidated that cell division, cell dedifferentiation and apoptosis are some of the mechanisms that contribute to intestinal regeneration in H. glaberrima (Candelaria et al., 2006; Dolmatov, 2021; Dolmatov & Ginanova, 2001; Ferrario et al., 2020; García-Arrarás & Dolmatov, 2010; Mashanov et al., 2010; San Miguel-Ruiz & García-Arrarás, 2007).

Several lines of evidence suggest that the Wnt signaling pathway plays an important role in these early regenerative events. The sea cucumber Apostichopus japonicus presented a high peak of expression of Wnt6 during the first week of regeneration (Sun et al., 2013). Also, in this organism, a genome-wide analysis on the signaling pathways that might participate in the regeneration process showed that Wnt7, Fz7 and Dvl were significantly upregulated at early stages of intestinal regeneration (Yuan et al., 2019). Likewise, several genes of the Wnt and Frizzled families were characterized in another regenerating holothurian, Eupentacta fraudatrix (Girich et al., 2017).

Concomitantly, in the sea cucumber H. glaberrima, gene expression profiling demonstrated that Wnt9, a Wnt homolog, was also upregulated during early intestinal regenerative stages (Mashanov et al., 2012; Ortiz-Pineda et al., 2009). In an attempt to elucidate the role of the canonical Wnt signaling pathway in H. glaberrima intestinal regeneration, our laboratory implemented pharmacological treatments to modulate the Wnt signaling activity (Bello et al. 2020). The results suggested that cell division, but not cell dedifferentiation was controlled by the canonical Wnt/β-catenin pathway. Moreover, it was proposed that a GSK3 Wnt-independent pathway was responsible for the cellular dedifferentiation observed.

Although intriguing, the results obtained in these pharmacological experiments remained inconclusive, mainly due to the lack of an experimental protocol to modulate a specific genetic product. To tackle this gap-in-knowledge, we implemented the use of Dicer-substrate interference RNA (DsiRNA) to knockdown the expression of β-catenin transcripts (Alicea-Delgado et al., 2021). Our results strongly support the contention that the Wnt/β-catenin signaling pathway plays an important role in cell proliferation but is not the primary signaling pathway for muscle dedifferentiation or apoptosis.

Results

The results presented here address the possible role of the canonical Wnt signaling pathway activation in the process of intestinal regeneration in the sea cucumber H. glaberrima. The data are presented in 2 interrelated sections: (1) the determination of the sequential genetic expression of β-catenin during intestinal regeneration in vivo, (2) the results obtained using RNA interference (RNAi) to evaluate the functional role of β-catenin during regeneration in intestinal explants.

β-catenin and canonical Wnt downstream elements temporal expression in vivo.

The expression patterns of β-catenin, Myc and G-cadherin might help to clarify their role in different cellular events during the regenerative process. β-catenin is a co-transcription factor involved in the activation of Wnt downstream elements, such as Myc and G-cadherin. The latter two genes are implicated in regenerative processes occurring in echinoderms as well as in other regenerative models. The expression of mRNAs in the regenerating tissues were compared with two different controls, the normal mesentery and the normal intestine. The reason for the dual comparison is that the early intestinal regenerates (3 – 7 days post-evisceration or dpe) mainly consist of mesenterial tissue with a thickening at one end, sharing most of the tissue components and their distribution with the normal mesentery (Quispe-Parra et al., 2021). Thus, the results obtained from 3 – 7 dpe regenerates were compared to those of the normal mesentery. On the other hand, later regenerative stages (14 – 21 dpe) share tissue components and distribution with normal intestines (i.e., luminal epithelium). Thus, stages 14 to 21 dpe were compared to normal, non-eviscerated intestines. A mid-stage of 10 dpe regenerating intestines can share anatomical characteristics with both (dashed lines on figure), representing a milestone in the intestinal regeneration process of this organism.

Surprisingly, β-catenin expression showed no changes when compared with either control mesenteries in the early stages, or with control intestines in the later stages of regeneration (Fig. 1). G-cadherin and Myc, on the other hand, were differentially expressed in almost all regenerative stages compared to normal mesentery and intestine (Figure 1). At 3 and 5 dpe, G-cadherin presented ~ 6-fold change to the control then decreased slightly at 7dpe but still maintained a high expression (~ 4-fold change) compared to normal mesentery. At 10 dpe, G-cadherin genetic expression was comparable to control mesentery but increased in comparison to control intestine. This increased expression (in comparison to control intestine) persisted at 14 and 21 dpe. In the case of Myc transcript, stages 3-, 5- and 7- dpe presented the highest expression compared to normal mesentery. The expression returned to levels comparable to those of control mesentery, or control intestine at 10 dpe but then increased again to ~ 3-fold at 21 dpe, when compared to expression in control intestines.

Figure 1.

Figure 1

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Relative gene expression patterns of β-catenin, G-cadherin and Myc transcripts in the regenerating digestive tube as determined by qRT-PCR. NADH gene expression was used as reference. Transcripts abundance of early regenerative stages (left side of the dashed lines) are expressed as X-fold relative to the mesentery and late regenerative stages (right side of the dashed lines) are expressed as X-fold relative to the normal intestine. The 10 dpe stage represents the mid-stage of the intestinal regeneration, sharing anatomical characteristics with early and late stages. Results are presented as mean ± S.E. of 3 to 6 animals. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

β-catenin downregulation hinders cell proliferation in regenerating intestine explants.

To determine the functional role of β-catenin in early regenerative stages, 4 dpe explants were electroporated with 1, 10 and 100 μM of DsiRNA-targeting β-catenin transcript and kept in vitro for 2 days. The levels of β-catenin mRNA were measured by qRT-PCR and showed that the electroporation with 100 μM DsiRNA-targeting β-catenin transcript produced a significant reduction of ~ 3-fold, when compared to explants electroporated with the control GFP-targeting DsiRNA (Alicea-Delgado et al., 2021).

To determine the role of β-catenin in the cell proliferation that takes place during intestinal regeneration, we studied the incorporation of BrdU in gut rudiment explants electroporated with β-catenin RNAi. After 2 days in culture, a smaller number of BrdU-labeled cells were observed in the gut rudiment explants electroporated with β-catenin RNAi when compared to the gut rudiment explants electroporated with GFP RNAi (Fig. 2). Quantification of the number of BrdU labeled cells per total number of cells showed a reduction of almost 50% in β-catenin RNAi treated animals (Fig. 2).

Figure 2.

Figure 2

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β-catenin downregulation reduces cell proliferation. Sections were labeled with anti-BrdU antibody (red) and DAPI (blue) to determine cell proliferation of gut rudiment explants electroporated with GFP (control; left) or β-catenin RNAi (right). Percentage of dividing cells was determined in three different regions of the gut rudiment explants. Each point represents the mean ± S.E. of 7 gut rudiment explants. *P<0.05.

β-catenin downregulation in regenerating intestine explants has no effect on cell dedifferentiation.

The process of cell dedifferentiation is another important event that takes place during early regenerative stages. In holothurians, this process is characterized by the condensation of filaments into spindle-like structures (SLSs) that are expelled into the extracellular space or degraded by neighboring cells (Candelaria et al., 2006; Dolmatov & Ginanova, 2001; García-Arrarás & Dolmatov, 2010). To visualize the presence of SLSs in gut rudiment explants, tissue sections were stained with rhodamine-labeled phallodin (Fig. 3). Equivalent levels of dedifferentiation were quantified in experimental and control groups, as indicated by the number of SLSs per nuclei in explants electroporated with DsiRNA-targeting β-catenin or the control DsiRNA-targeting GFP (Fig. 3).

Figure 3.

Figure 3

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β-catenin downregulation has no effect on cell dedifferentiation in rudiment explants. Gut rudiment explants electroporated with GFP RNAi (left) and β-catenin RNAi (right) were stained with rhodamine-labeled phalloidin (red) to identify and quantify spindle-like structures (SLSs; white arrows) and nuclei DAPI stain (blue). The ratio between SLSs and nuclei was measured in 3 different areas of the explants. Each point represents the mean ± S.E. of 5 gut rudiment explants.

Programmed cell death in regenerating intestine explants is independent of β-catenin downregulation.

Apoptosis, as well as cell proliferation and dedifferentiation, also takes place during the intestinal regeneration of H. glaberrima (García-Arrarás et al., 2011; Mashanov et al., 2010). We performed TUNEL assays to determine the level of programmed cell death in gut rudiment explants electroporated with GFP RNAi and β-catenin RNAi. The percentage of apoptotic cells in regenerating intestine explants electroporated with DsiRNA-targeting β-catenin remained similar to those of control DsiRNA-targeting GFP (Fig. 4).

Figure 4.

Figure 4

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Programmed-cell death is not altered by the downregulation of β-catenin in rudiment explants. Apoptotic cells (green) and nuclei (blue) of gut rudiment explants electroporated with GFP RNAi (left) and β-catenin RNAi (right) were stained with TUNEL and DAPI, respectively. The ratios between Tunel+ cells and nuclei were measured in three different areas of the explants. Each point represents the mean ± S.E. of 3 to 7 gut rudiment explants.

Discussion

The experiments described here are part of our continuing search to determine the signaling pathways that regulate regenerative processes. Specifically, they address the role of Wnt/β-catenin pathway in the process of intestinal regeneration in our model system, the sea cucumber H. glaberrima. Here we explore two additional lines of evidence to determine a role for Wnt signaling. First, the in vivo expression patterns, during early stages of regeneration, of genes known to be associated with the canonical Wnt signaling pathway. Second, the consequences of RNAi experiments where the expression of β-catenin, one of the key molecules in the Wnt signaling cascade, is downregulated.

A. In vivo expression of Wnt signaling associated genes

Gene expression patterns can provide insights into the molecular pathways involved in the intestinal regenerative process. In our particular case, the finding that β-catenin mRNA expression is not altered during different regenerative stages suggests that overexpression of its coding protein is not required for intestinal regeneration. This coincides with a recent RNAseq expression analysis showing that β-catenin genetic expression in the mesentery and in the 3 dpe rudiment do not differ (Quispe-Parra et al., 2021). However, it contrasts with regeneration studies in other model systems where an increase in β-catenin expression was documented. For example, during Hydra head regeneration Iachetta and colleagues (2018) observed a peak of β-catenin expression and Petersen and colleagues (2015) found a 5- to 6-fold upregulation of β-catenin transcripts. Yet, increasing evidence has suggested that post-translational modifications of the Wnt/β-catenin pathway components, and not necessarily increases in mRNA expression, may play a pivotal role in the activation of the pathway (Gao et al., 2014).

The expression of two Wnt downstream elements was analyzed, Myc and G-cadherin. Both genes showed an increase expression during early regenerative stages that slowly decreased in the first two weeks of regeneration. The increase in Myc expression had been previously described for the 3 dpe stage of intestinal regeneration (Mashanov et al., 2015a). Additionally, previous studies in our laboratory also indicated a significant upregulation of Myc transcript in regenerating radial nerve cord of H. glaberrima. Myc expression upregulation is in line with what has been described in other species. In the newt Notophthalmus viridescens, c-myc is upregulated at day 8 during lens regeneration and at day 7 during limb regeneration (Maki et al., 2009). It has been proposed that c-myc has a putative role in the regulation of cell proliferation, dedifferentiation and/or programmed-cell death (Géraudie et al., 1990; Meyer & Penn, 2008). Similarly, G-cadherin overexpression at different regenerative stages suggest that it might play a significant role during the intestinal regeneration of H. glaberrima. Studies of different cadherins have implied this protein family to be associated with regenerative processes. For example, in mouse embryos M-cadherin in situ hybridization signals increase at 3 days after inducing muscle regeneration by transient ischemia (Moore & Walsh, 1993). Likewise, an upregulation of cadherin-2 (cdh-2) and cadherin-4 (cdh-4) was detected in the retinal ganglion cells and their axons, following lesions to the eye and the optic nerve in adult zebra fish (Liu et al., 2002).

The overexpression of Myc and G-cadherin during the intestinal regeneration of H. glaberrima might be a subsequent effect of the activation of Wnt/β-catenin signaling pathway. These genes are considered to be Wnt-target genes, being c-myc one of the most-studied β-catenin responsive genes (Sancho et al., 2004). Consonant with this, a previous study demonstrated that regenerating intestinal crypts of rodents depend on high levels of β-catenin and c-myc, thus suggesting, that c-myc transcription depends on the activation of the canonical Wnt signaling pathway (Ireland et al., 2004).

B. RNAi results

Wnt/β-catenin pathway plays an important role in cell division

While measurements of mRNA and protein products of specific genes provide some insight into regenerative processes, these values remain purely correlational. Thus, experiments are required, where the levels of the gene products are modulated, and the effects of such modulation determined. Herein lies the importance of the RNAi experiments. Quantitative RT-PCR results confirmed that the RNAi protocol successfully decreased β-catenin expression in the gut rudiment explants, providing us with the tool to evaluate the role of β-catenin in the regenerative process (Alicea-Delgado et al., 2021).

Wnt action on cell proliferation during regenerative events has been well documented (Li et al., 2017; Yang et al., 2017; Zhu et al., 2014). In Hydra, interference of β-catenin and of its target gene CTNNB1, reduced cell proliferation during head regeneration (Chera et al., 2009). In contrast, in another animal system, β-catenin upregulation prevented retinal pigment cells from entering the cell cycle (Zhu et al., 2014). In mammals, maintenance and proliferation of stem cells involved in luminal epithelium regeneration, is controlled by the activation of the canonical Wnt signaling pathway (Ashton et al., 2010; Cordero & Sansom, 2012; Pinto et al., 2003). In our system, control of cellular proliferation was characterized in the mesothelium. We cannot rule out a similar effect on H. glaberrima luminal epithelium, however, this tissue layer is absent at the regenerative stage studied here.

Wnt/β-catenin is not involved in cellular dedifferentiation nor in apoptosis

Not all cellular events respond to changes in β-catenin mRNA as the RNAi does not appear to have an effect on cell dedifferentiation nor on programmed-cell death. This result correlates with previous findings from our laboratory, where cell dedifferentiation was not altered by pharmaceutical inhibition of the canonical Wnt signaling pathway (Bello et al., 2020). In fact, our lab has hypothesized that dedifferentiation is under the control of a GSK-3 Wnt-independent signaling pathway, that could be analogous to the dedifferentiation of the vascular smooth muscle via PI3K/Akt pathway activation (Bello et al., 2020; Frismantiene et al., 2016).

In other organisms the inhibition of β-catenin showed different results. In the chick retina, β-catenin inhibition promoted the dedifferentiation of the pigmented cells (Zhu et al., 2014). In a study made using human osteoarthritic cartilage, β-catenin upregulation stimulated chondrocytes dedifferentiation, which is a hallmark of cartilage destruction (Hwang et al., 2004). Similarly, in human aged epidermal cells treated with GSK-3β, accumulation and translocation of β-catenin regulated the dedifferentiation of these cells into stem cell-like cells (Lim et al., 2008). Therefore, these results suggest that β-catenin’s role in cell dedifferentiation depends on the tissue and model system.

We also studied the effect of lowering β-catenin expression on the number of apoptotic cells. Previous cellular studies from our laboratory suggested that programmed-cell death is an early event during intestinal regeneration (García-Arrarás et al., 2011; Mashanov et al., 2010). However, the decrease in β-catenin mRNA levels resulting from the RNAi, did not show any significant effect on this process. Once again, our results support those of Bello et al. (2019), where neither Wnt activators nor inhibitors altered programmed-cell death during intestinal regeneration in H. glaberrima. However, in other systems, upregulation of β-catenin can induce apoptosis as was shown in normal fibroblasts and tumor cells, (Kim et al., 2000).

For both dedifferentiation and apoptosis an alternate hypothesis is that, in our system, the residual β-catenin levels that remained following RNAi, are sufficient to allow these processes to continue. In this scenario, decreasing β-catenin expression even lower might have an effect on cellular dedifferentiation and apoptosis. Future experiments should address this possibility.

Wnt involvement in H. glaberrima intestinal regeneration

These results together with previous studies from the laboratory strongly suggest a role for Wnt in the regenerative response of H glaberrima. A summary of the available data includes: First, microarray experiments showed that a Wnt9/14 homolog was differentially expressed during the intestinal regeneration (Ortiz-Pineda et al., 2009). Second, in situ hybridization identified the spatiotemporal expression of Wnt9 in regenerating and non-regenerating intestines. The Wnt9 transcripts were mostly undetectable in non-regenerating intestines compared to those that were regenerating. These results were confirmed using RT-PCR (Mashanov et al., 2012). Third, and most important is the recent study using pharmacological treatments to modulate the activity of the canonical Wnt signaling pathway performed in our laboratory (Bello et al. 2020). These experiments provided direct evidence that the Wnt/β-catenin pathway modulates cell proliferation during intestinal regeneration, as shown by the decrease in cell proliferation produced by Wnt pathway inhibitors. This result closely mirrors our present experiment showing that decreasing β-catenin mRNA levels greatly reduces cell division. Moreover, pharmacological inhibition of the Wnt pathway, similar to our RNAi downregulation of β-catenin, showed no effect on apoptosis. Finally, the effect on muscle dedifferentiation caused by the inhibition of the canonical Wnt signaling pathway using pharmacological agents was somewhat complex. Only drugs that targeted directly the GSK-3 activity showed an effect on cell dedifferentiation while drugs that targeted upstream molecules in the signaling pathways did not. Thus, we postulated that dedifferentiation was mediated by a Wnt-independent, GSK-3 pathway. Our present experiments strengthen our hypothesis that the Wnt pathway is not involved in the dedifferentiation response, and therefore no effect is observed when β-catenin levels were reduce. However, it remains unclear as to what is the signaling pathway that mediates dedifferentiation. In summary, the side-by-side comparison of two different experimental approaches, pharmacological and RNAi, is an important step in determining the sequence of actions that take place during intestinal regeneration. It also strengthens our proposition of separate signaling systems mediating cell proliferation, cell dedifferentiation and apoptosis.

Researchers working with other holothurian models have also identified Wnt genes as possible signaling molecules for intestinal regeneration. A genetic expression profile performed in the sea cucumber Apostichopus japonicus established an upregulation of Wnt6 and WntA transcript at 3 dpe. (Ferrario et al., 2020; Li et al., 2017; Sun et al., 2013). Also, in this organism, Yuan, et al. (2019), performed a genome-wide analysis on the signaling pathways that participate in the regeneration process and determined that the Wnt signaling pathway was under positive selection in echinoderms. They observed that Wnt7, Fz7 and Dvl were significantly upregulated at early stages of intestinal regeneration. Thus, they interfered with the mRNA levels of Wnt7 and Dvl resulting in the inhibition of the intestinal extension. Likewise, several genes of the Wnt and Frizzled families were characterized in another holothurian, Eupentacta fraudatrix. The gene expression of wnt16, and of the Wnt receptors frizzled 1/2/7, and frizzled4 increased significantly at 3 dpe. Within 5 and 7 dpe, expression is increased in frizzled1/2/7, frizzled4, wntA, wnt4, and wnt6. At 10 dpe, frizzled5/8 is the only gene significantly expressed, and at the latest regeneration stages (14 and 20 dpe), only frizzled1/2/7 and frizzled4 retain their expression above normal (Girich et al., 2017). A recent publication addresses the possible role of various signaling pathways (including Wnt) in holothurian regeneration (Dolmatov, 2021).

C. Set up of an RNAi for echinoderm tissues in vitro

It is important to highlight an additional achievement reported here: the establishment of an RNAi protocol for echinoderm tissues in vitro. Setting up a protocol for modulating gene expression in echinoderms in vitro represents a major advance in the field. A detailed description of the method has been published elsewhere (Alicea-Delgado et al., 2021). We chose electroporation as a reliable transfection method to introduce DsiRNAs into 4 dpe gut rudiment explants based on previous studies performed by our group for radial nerve tissue in vivo (Mashanov et al., 2015b). Working with gut rudiment explants provide a better control of the environmental factors and helps reduce the cost of the experiments and the number of animals needed. The implementation of this silencing mechanism for echinoderms in vitro has given us the opportunity to characterize the role of the canonical Wnt signaling pathway during intestinal regeneration in H. glaberrima. Moreover, it has opened a world of possibilities to understand and characterize other mechanisms involved in regeneration not only in H. glaberrima, but in other organisms from the phylum Echinodermata.

Conclusion

In summary, our data provide information regarding the utility of DsiRNAs to characterize the role of the canonical Wnt signaling pathway and its relationship with the cellular events occurring during intestinal regeneration in H. glaberrima. The genetic dynamic changes of β-catenin, Myc and G-cadherin establish that these genes are expressed during intestinal regeneration in vivo, providing us another hint about the mechanisms involved. These results led to the improvement of the electroporation of gut rudiment explants with DsiRNAs-targeting β-catenin to elucidate its role during intestinal regeneration. The interference of β-catenin reduced the proliferative rate of the cells during regeneration, but not cell dedifferentiation nor programmed-cell death. This protocol is a reliable tool to overcome the limitation of performing genetic modulations in adult echinoderms and gives us the opportunity to elucidate the role of other genes and/or pathways that might be regulating the regenerative process.

Methods

Animal and tissue collection.

Adult individuals of the sea cucumber H. glaberrima were collected from the Northeast coast of Puerto Rico and transported to the laboratory. They were kept at room temperature (RT; 22 – 24 °C) with constant aeration overnight. Evisceration of the sea cucumbers was induced by injections of 0.35 M KCl (2 – 4 mL) into the coelomic cavity as previously described (García-Arrarás et al., 2011). Regenerating (3, 5, 7, 10, 14 and 21 days-post evisceration or dpe) and non-regenerating animals were kept in aerated indoor seawater aquaria. Sea cucumbers were anesthetized by immersion with 0.2% 1,1,1-trichloro-2-methyl-2-propanol hemihydrate (chlorobutanol, Sigma) in sea water for 45 min on ice. The anterior region of the sea cucumbers was cut near the calcareous ring and along the dorsal interambulacral region under RNAse-free conditions. Regenerating intestines, non-eviscerated intestines and mesentery were dissected and placed in different solutions depending on the experimental procedure that ensued. For analysis of mRNA expression tissues were placed directly in RNAlater (1 mL) at − 80 °C and for immunohistological experiments, tissues were placed in 4% paraformaldehyde (PFA) at 4 °C.

RNA extraction and qRT-PCR for temporal expression and RNAi validation.

Total RNA from regenerating (3, 5, 7, 10, 14 and 21 days-post evisceration or dpe) and non-regenerating guts and mesentery were obtained using TRI reagent (Sigma) and treated with DNase I (Qiagen) to reduce the possibility of genomic DNA contamination. The concentration and integrity of the RNA was determined using the NanoDrop-1000 Spectrophotometer (Nano Drop Technologies). cDNA was synthesized from ~ 500 ng of total RNA isolated from using ImPromt-II Reverse Transcriptase (Promega). Sense and antisense PCR primers for β-catenin (5′ – AGA GTG CCA AGT TAC GGA TG – 3′ and 5′ – AAT TCA GTA GCG GCT GGC TTA T – 3′), G-cadherin (5′ – CAA CCA TCT CCC ATT ATT GCT AGA – 3′ and 5′ – CCG TAG AAC CCT CTC CTT CAT A – 3′), Myc (5′ – CAA GAC TGT ATG TGG AGT GCT TTT – 3′ and AGT CCG AGG GTT GAC TAC AAT CAC – 3′) and NADH (5′ – CAA TGG TTG TTG CTG GAG TCT TT – 3′ and 5′ – CGC AGA AGT AGC CGC GAA TAT – 3′) were designed using the guidelines of Integrated DNA Technologies, Inc (IDT). qPCR reactions (20 μL) were performed using iQ™ SYBR Green Supermix (Bio-Rad) and ran in a Mastercycler® ep realplex with the following parameters: 95 °C for 3 min (denaturation step) followed by 35 cycles of 95 °C for 15 s, (55 °C, 54 °C, and 57. °C) for 30 s and 68 °C for 40 s (amplification step). Fluorescence data were collected during the amplification step. Melting curve analysis was performed for each PCR product to ensure primer specificity. Real time PCR reactions were performed in three independent biological replicates and three technical replicates from each of the regenerating stages, non-regenerating guts and mesentery. The expression of β-catenin was normalized relative to the expression of NADH dehydrogenase subunit 5 using the Livak method (ΔΔCt method). For RNAi validation, total RNA was collected from one half of the explants treated with DsiRNA-targeting β-catenin and GFP transcripts after 2 days in culture.

DsiRNA Design.

Dicer-substrate small interfering RNA (DsiRNA) were designed using siDirect (v2.0) software and edited using the guidelines of IDT, Inc. Longer DsiRNAs were used based on its efficiency regulating transcripts compared with the classical 21-mers RNAis (Kim et al., 2005). β-cat Dsi2 was designed to target a region of the coding sequence of H. glaberrima β-catenin (sense strand 5′ – GAU GUU GAC GAA AAU UGC UAU GGT T – 3′ and antisense strand 5′ – AAC CAT AGC AAU UUU CGU CAA CAU CUC – 3′) and the control was designed to target green fluorescent protein (GFP) transcript (sense strand 5′ - AGC UGA CCC UGA AGU UCA UCU GCA C - 3′ and antisense strand 5′ - GUG CAG AUG AAC UUC AGG GUC AGC UUG – 3′). The DsiRNA reagents were resuspended in the RNase-free Duplex Buffer (IDT) to 100 μM stock solution, which was heated to 94 °C for 2 min and then slowly cool down at RT. Dilutions of the DsiRNA solution were performed to 1 and 10 μM from the stock solution.

Explants culture.

Explant culture has been previously described (Bello et al., 2015). In brief, 4-day regenerating sea cucumbers were anesthetized by immersion with 0.2% 1,1,1-trichloro-2-methyl-2-propanol hemihydrate (chlorobutanol, Sigma) in sea water for 45 min on ice. Animals were placed in 10% sodium hypochlorite for 1 min, 70% ethanol for 5 min and rinsed with distilled water. Gut rudiments were dissected and cleaned.

RNAi procedure.

The details and validation of this technique has been detailed in a book chapter (Alicea-Delgado et al., 2021). Abridgedly, we experimented with four electroporation parameters (15 V, time pulse: 45 ms, interval pulse: 955 ms; 20 V, time pulse: 40 ms, interval pulse: 960 ms; 27 V, time pulse: 30 ms, interval pulse: 970 ms; 35 V, time pulse: 25 ms, 975 ms) in the BTX/Harvard Apparatus ECM 830 Square Wave Pulse Electroporation System and determined the efficiency of electroporation by transfecting the gut rudiments explants with tetramethylrhodamine-conjugated anionic dextran 3,000 MW, lysine fixable. We selected the last electroporation parameter (35 V, time pulse: 25 ms, 975 ms) to perform the transfection of the gut rudiment explants with 1, 10 and 100 μM of β-cat Dsi2 or GFP Dsi. Explants treated with 100 μM of DsiRNAs were then used for molecular and histological purposes and placed, one half per well, in 24-well plates containing L-15 media conditioned for marine species. Explants were incubated at RT in an incubator chamber (Billups-Rothenberg, Inc.) for 2 days.

Histological Analyses.

After 2 days in culture, explants were fixed in 4% PFA for 24 hrs, washed 4 times with 0.1 M Phosphate Buffered Solution (PBS) pH 7.4 for 5 min and cryoprotected with 30% sucrose/PBS at 4 °C. Cryosections (20 μm) were performed using a Leica CM1850 cryostat and placed on poly-L-lysine covered slides. Sections were used to analyze electroporation efficiency, cellular proliferation, dedifferentiation and apoptosis.

Cell proliferation –

To determine the level of proliferation, we evaluated BrdU incorporation during the regenerative process. For this, following electroporation, explants were placed in wells containing bromodeoxyuridine (BrdU; 50 μM) and incubated at RT in an incubator chamber for 2 days. Explants were then fixed and sectioned as explained above. Cryosections were washed with 0.2% Triton X-100 for 15 min, washed twice with 0.1 M PBS for 15 min and treated with 0.05 M HCl for 1 hr. After another wash with 0.1 M PBS (15 min), sections were blocked with goat serum for 1 hr. Slides were incubated with mouse monoclonal anti-BrdU (1:5, GE Healthcare RPN202) in a humid chamber overnight. The following day, slides were washed with 0.1 M PBS for 15 min three times and incubated with the secondary antibody Cy3 goat anti-mouse antibody (1:1000, Jackson Immuno Research Laboratories) for 1 h in a humid chamber. Following the three 0.1 M PBS washes, slides were mounted in buffered glycerol containing DAPI (1 μg/mL, Sigma).

Cell dedifferentiation -

The intestinal regeneration of H. glaberrima is characterized by the condensation of contractile filaments of the mesentery muscle cells into spindle-like structures (SLSs), a cellular process known as dedifferentiation. During this process, muscle cells eject SLSs and turn into what has been defined as a less differentiated state. To evaluate cell dedifferentiation in the explants, cryosections were incubated with Phalloidin-TRITC (1:2,500, Sigma), a toxin that interacts with muscle fibers and SLSs, for 1 h in a humid chamber at RT. After 1 h, slides were washed 3 times with 0.1 M PBS for 15 min, mounted in buffered glycerol containing DAPI (1 μg/mL, Sigma).

Programmed cell death –

Cells undergoing programmed cell death were identified by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using the FragEL DNA Fragmentation Kit, Fluorescent – TdT Enzyme (Calbiochem). Different to what is suggested by the manufacturer, cryosections were treated with Proteinase K for 1 min instead of 5 min.

Analyses -

Cryosections were visualized and analyzed using a Nikon Eclipse Ni fluorescent microscope. BrdU+, SLSs, TUNEL+ and DAPI counts were taken with 100X objective in three different regions of at least 3 different explants. Regions with 10 cells or more were chosen for statistical purposes. An average of the BrdU+/DAPI ratio was obtained from each region of the explants and for all the explants to calculate the percentage of BrdU-positive cells. The same was applied to obtain SLSs/DAPI ratio and Tunel+/DAPI ratio.

Statistical Analyses.

To evaluate statistical differences between groups, control and experimental, we applied unpaired t-test or two-way ANOVA followed by Tukey’s multiple comparisons test using the software GraphPad Prism 5. Values are reported as mean ± standard error (S.E.).

Highlights.

  • Wnt target genes are differentially expressed during intestinal regeneration

  • DsiRNA-targeting β-catenin was tested in gut rudiment explants

  • Dedifferentiation and apoptosis are unaffected by β-catenin DsiRNA

  • β-catenin DsiRNA affects cell proliferation during intestinal regeneration

Acknowledgements

We thank Ms. Griselle Valentín-Tirado and Dr. Samir A. Bello-Melo, and the Molecular Science Research Center for technical support.

Funding

We acknowledge support from the NIH (Grant R15GM124595), NSF (Grant IOS-1252679) and the University of Puerto Rico.

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