2. Regeneration after Fission

2.1. Morphological Aspects of Fission and Regeneration

Echinoderms exhibit different ways of asexual reproduction, of which the most common one is fission [63]. To date, 16 species of fissiparous holothurians are known that can be model objects for studying the mechanisms of fission with subsequent regeneration [59]. Most holothurian species have a thick body wall composed of connective tissue [56]. Accordingly, ECM remodeling is an important component of the transverse fission mechanism [63,64,65]. It has been shown that connective tissue in holothurians possesses a unique ability to alter its mechanical properties under effects of various factors [66,67]. Thus it is called mutable collagenous tissue (MCT) [68] or catch connective tissue [69]. The body-wall connective tissue in holothurians can occur in three states: Stiff, standard, and soft [65]. The mechanical stretching results in the decreased stiffness of the connective tissue and promotes its transition into the soft state [65].

Furthermore, stiffness of MCT depends on the interaction of three protein groups: Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and cross-link complexes connecting collagen fibrils to one another [70]. As the activity of TIMPs increases, MMPs are blocked. As a result, cross-links are formed between collagen fibrils, and MCT strengthens. Conversely, an increase in the MMPs concentration or activity in connective tissue leads to the destruction of cross-link complexes. This destruction enables collagen fibrils to slide along one another, which brings MCT into a compliant state. Thus, a local change in properties of the body-wall connective tissue allows holothurian to divide into two parts.

After division the anterior fragment of holothurian contains the AB, the gonad, and the anterior half of the intestinal tube (Figure 1a,b). The posterior part of the gut, the cloaca, and the respiratory organs (respiratory trees) remain in the posterior fragment (Figure 1a,e).

The first morphological signs of regeneration in both fragments are detected after 5 days post-fission [45,60]. In the anterior fragment of holothurians, the remaining part of the gut becomes slightly shortened, probably due to the destruction of a portion of cells, and its end region is narrowed. A connective-tissue thickening grows out from the gut along the edge of the gut mesentery and gradually extends posteriorly [45]. Simultaneously with this process, enterocytes in the wound zone begin to dedifferentiate and divide mitotically. Intercellular junctions between them are not broken. The luminal epithelium of the gut grows into the connective-tissue thickening (Figure 1c). Subsequently, the anlage gradually extends backwards and reaches the posterior wall of the body, where the cloaca and respiratory trees have already formed by this time (Figure 1d) [45].

In the posterior fragment, regeneration begins with the formation of AB, which develops at the anterior end of the animal [60]. The gut regeneration in the posterior fragment occurs through the transformation of the anterior part of the digestive tube. The anterior end of the gut remnant becomes thinner. It gives rise to a connective-tissue thickening, which grows anteriorly along the mesentery edge and connects to AB (Figure 1f). Enterocytes are dedifferentiated and divide mitotically, but intercellular junctions between them are not destroyed. As a result, the luminal epithelium of the gut grows into the connective-tissue thickening and then into AB. Subsequently, tentacles and organs of the water–vascular system are formed (Figure 1g). Thus, the main regeneration processes in holothurians after fission are ECM remodeling, cell dedifferentiation, cell proliferation, and epithelial morphogenesis.

In regeneration, the significance of ECM remodeling is associated with the fact that most organs, both in holothurians and in all echinoderms, are epithelial formations (mostly tubular), containing a large amount of connective tissue. Accordingly, when organs regenerate after fission, the connective-tissue base (connective-tissue thickening) is formed first, and then certain cells or epithelia migrate into it [45,60]. This pattern of organ formation is typical of regeneration not only after fission, but also after evisceration (see below). For this reason, major attention was paid to the structure of connective tissue and the mechanisms of its modification during asexual reproduction in holothurians [71].

2.2. Structural Components of Connective Tissue

Transcripts of genes of many ECM components that are characteristic of most multicellular animals—collagens, proteoglycans, and glycoproteins—have been found in holothurians [71,72,73,74,75]. On the other hand, the differences in connective tissue of echinoderms and vertebrates have also been identified. In particular, one of the major ECM components in vertebrates is elastin, whose fibers are formed through the polymerization of tropoelastin [76]. Echinoderms lack the tropoelastin gene [71].

Another distinguishing feature of holothurian ECM is the absence of tenascins and fibronectins [77]. These proteins play an important role in the structural integrity of connective tissues in vertebrates [78,79]. In some of holothurians, transcripts have been found that are blasted as tenascin-like proteins [71,72,80]. These contigs encode the domains characteristic of tenascins, EGF, FBG, and TILa. Nevertheless, according to Hynes [81], all these domains are ancient in origin and are found in many animals; however, in the combination characteristic of tenascins, they occur only in chordates. No transcripts of the fibronectin gene were found in C. schmeltzii [71]. Thus, the lack of such proteins as elastin, fibronectin, and tenascin indicates significant differences in the connective tissue organization between echinoderms and chordates.

Due to the lack of elastin, its function in echinoderms is probably performed by fibrillin. In ECM, it forms a network consisting of microfibrils 10–14 nm in diameter that surrounds and penetrates bundles of collagen fibrils [82]. It is suggested that fibrillin microfibrils may be involved in ligament contraction in sea urchins [83]. As has been shown recently, fibrillin microfibrils play an important role in ECM functioning [84]. They are involved in the distribution, accumulation, and modulation of signals from transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) that regulate various aspects of cell activity, including ECM formation and remodeling [85]. In addition, fibrillins can bind to integrin receptors and a number of other molecules and, as a result, signals about changes in the extracellular microenvironment are transmitted to cell. In fact, fibrillin microfibrils form niches accumulating various factors [86].

Holothurians have laminins, nidogens, fibulins, agrin, dystroglycan, perlecan, and thrombospondins. The proteins encoded by these genes, along with collagens and fibrillins, are included in the basic set of “basement membrane ECM toolkit”, typical of all Bilateria [87]. The genes are involved in the construction and functioning of connective tissue. For example, fibulins can bind to many ECM components such as fibrillin, and play an important role in stabilizing supramolecular complexes of connective-tissue [88]. Fibulin 1 has been shown to accelerate the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-mediated aggrecan proteolysis and, thus, participate in tissue renewal [89].

Thrombospondins (TSPs) play certain role in the organization of ECM, since they can serve as molecular bridges between various components of connective tissue [90]. They have been shown to interact with MMPs, fibrillar collagen, and TGF-β. TSP-1 and TSP-2 can inhibit the activity of MMP2 [91] and regulate its level in extracellular matrix [92,93]. Thus, TSPs interacts with the molecules that in echinoderms may be involved in mechanisms responsible for modifying ECM during fission and regeneration.

2.3. Proteins Modifying ECM

In addition to the genes of connective-tissue structural components, holothurians have a wide variety of genes encoding ECM-modifying proteins. Many different serine, cysteine, aspartyl, and metal peptidases and their inhibitors have been identified [71,94,95,96,97,98]. In the study of regeneration in holothurians, much attention is paid to MMPs that can degrade collagen [96,97,99,100,101]. In the transcriptomes of holothurians, products of MMPs genes and genes of their inhibitors (TIMPs) have been found [71,72,75,102]. In some of holothurian species, the number of TIMPs can reach 45 [103]. The increase in the number of TIMPs occurred, apparently, due to the increased role of connective tissue in the vital activities of echinoderms such as asexual reproduction, autotomy, and regeneration [71,103].

One of the factors that have an effect on the connective tissue strength in echinoderms is assumed to be tensilin [67,104]. Tensilins are TIMP-like proteins [67,71]. It is worth mentioning that the tensilin gene is found only in species belonging to members of relatively young groups of holothurians and, consequently, have formed within the class Holothuroidea [71]. No similar proteins are found in Apodida (the most ancient order of holothurians [105]), as well as in other echinoderms. This may indicate evolutionary changes in the mechanisms of functioning of ECM that occurred in the phylogeny of echinoderms. Depending on needs, the properties and functions of TIMP-like proteins varied in different classes of Echinodermata. Holothurians differ from other echinoderms by their more developed body wall and the almost complete reduction of skeleton [56]. The increased role of connective tissue was probably accompanied by the modification of ECM remodeling mechanisms and the emergence of a separate group of TIMP-like genes, tensilins.

3. Regeneration after Anterior Evisceration

3.1. Morphological Aspects of Regeneration

The morphological aspects of gut regeneration after anterior evisceration have been described in sufficient detail only from one holothurian species, E. fraudatrix [24,42,43,46,61]. The early post-evisceration stage is approximately the same as in A. japonicus and H. glaberrima (Figure 4a–f). Within the first 3 dpe, the wound at the anterior end of the animal is healed [42], immunity is activated, the cell composition of the coelomic fluid is restored [26,27], and connective-tissue thickenings are formed along the mesentery edge (Figure 4d–f) [42,61]. The main difference from A. japonicus and H. glaberrima is that E. fraudatrix ejects not only the gut, but also the aquapharyngeal bulb (AB) during evisceration. As a result, regeneration begins with the formation of the AB anlage, and then, after 3 dpe, the anterior connective-tissue thickening begins to grow from it along the mesentery edge [42].

Figure 4. Scheme of regeneration of anterior part of digestive system after anterior evisceration. (a) Anterior part of holothurian just after evisceration; (b) Longitudinal section of the region of the mesentery, which is marked by the rectangle in (a). (c) Transverse section of the region of mesentery which is marked by solid line in (b). (d) Anterior part of holothurian on third day post-evisceration; (e) Longitudinal section of the region of the tissue thickening, which is marked by the rectangle in (d). (f) Transverse section of the region of connective tissue thickening which is marked by solid line in (e). (g) Anterior part of holothurian on seventh day post-evisceration; (h) Longitudinal section of the region of gut anlage, which is marked by the rectangle in (g). (i) Transverse section of the site of embedding of coelomic epithelial cells into connective tissue thickening which is marked by solid line in (h). ab, aquapharyngeal bulb; bw, body wall; ce, coelomic epithelium; ct, connective tissue; ctt, connective tissue thickening; dc, de-differentiated coelomic epithelial cell; dm, de-differentiating myoepithelial cell; dp, de-differentiating peritoneal cell; ga, anterior gut anlage; m, mesentery; my, myoepithelial cell; nc, nerve cell; pc, peritoneal cell; sls, myofilaments grouped into spindle-like structures, tc, coelomic epithelial cell during transdifferentiation.

After 5–7 dpe, the coelomic epithelial cells are embedded into the connective-tissue thickening and begin transdifferentiation (Figure 4g–i) [46]. The embedding occurs in a quite limited area on the ventral side of mesentery, near the AB anlage. The embedded cells divide and gradually migrate anteriorly and posteriorly inside the thickening, forming the luminal epithelium of the anterior gut anlage. The cells do not lose relationships with each other, and intercellular junctions are maintained. Transdifferentiation proceeds quite quickly, and the signs of specialization of enterocytes in luminal epithelium appear after 10 dpe [46]. The posterior anlage forms in the same way as in A. japonicus and H. glaberrima. Subsequently, both anlagen of the gut grow along the mesentery edge towards each other and merge into a single digestive tube within 15–20 dpe.

 

3.2. Transcriptomic Data

A total of 11 genes of TFs were identified and characterized in E. fraudatrix that showed higher transcriptional activity during transdifferentiation (5–7 dpe) than in the previous and subsequent stages (3 dpe and 10 dpe, respectively) [102]. They belong to 6 TF classes: Tryptophan cluster (Ef-elf), C2H2 zinc finger (Ef-prdm9, Ef-egr1, Ef-klf1/2/4 (klf2), Ef-snai2), bHLH (Ef-tcf24, Ef-msc, Ef-id2), C4 zinc finger (Ef-gata3), polycomb group ring finger (Ef-pcgf2), and T-box (Ef-tbx20). During this period, in addition to the embedding and transdifferentiation of a part of coelomic epithelial cells, the dedifferentiation and proliferation of coelomic epithelial cells on the surface of mesentery and anlage, and also the ECM synthesis and modification continue in the anterior gut anlage of E. fraudatrix. It can be assumed that genes whose expression increases during this period are involved in the regulation of one or more of the above mentioned processes.

The dedifferentiation, proliferation, and maintenance of the undifferentiated state of coelomic epithelial cells can involve Ef-elf, Ef-prdm9, Ef-klf1/2/4, and Ef-egr1. Ef-ELF belongs to the Ets family, whose members are powerful regulators of cell proliferation, angiogenesis, hematopoiesis, tumor transformation, and differentiation [179,180]. Proteins of the PRDM family are important epigenetic regulators of development, cell differentiation, and pluripotency in mice [181]. The KLF proteins play diverse roles in cell proliferation, differentiation, and development [182]. EGR1 is involved in the cell cycle progression of various tumor types, and also in hepatic regeneration in mammals [183,184].

The Ef-gata3, Ef-egr1, and Ef-klf1/2/4 genes may be involved in the regulation of transdifferentiation. The Ef-gata3 gene is of particular interest as its homolog in Caenorhabditis elegans is involved in transdifferentiation [185]. Moreover, it regulates the mesoderm and endoderm specification in echinoderms and mammals [186,187]. In planarians, EGR1 is a “putative pioneer factor to directly activate wound-induced genes” in whole-body regeneration [188]. KLF2 and KLF4 are key TFs that maintain a stem cell-like state and somatic cell reprogramming [189]. It is worth noting that in the holothurians H. glaberrima and A. japonicus the expression of klf1/2/4 does not change during gut regeneration [28,75,109]. The different dynamics of klf1/2/4 expression between these two species and E. fraudatrix confirms presence of different gut regeneration mechanisms in holothurians [58].

Ef-elf and Ef-id2 can be involved in processes associated with the digestive system development and enterocyte differentiation. The expression of sea urchin elf is detected everywhere in late gastrula, being more concentrated in the gut [190]. The id2 gene encodes a protein that, strictly speaking, is not a TF, since it lacks the basic DNA-binding domain. Nevertheless, it plays an important role in the digestive system development in vertebrates by preventing the precocious differentiation of the embryonic intestinal epithelium [191].

As is mentioned above, in the period when transdifferentiation occurs, the dedifferentiation of myoepithelial cells continues on the surface of the gut anlage. Among the identified TFs, Ef-msc and Ef-tbx20 may probably be involved in the regulation of this process. In mammals, MSC (musculin) is a lineage-restricted repressor of embryonic skeletal muscle development [192]. TBX20 is a crucial cardiogenic TF in mammals [193]. The tbx20 gene is also expressed in the nervous system and embryonic lateral mesoderm in mice [193,194].

Homologs of three TFs, Ef-pcgf, Ef-snai2, and Ef-id2, in vertebrates are involved in the mechanisms of the epithelial–mesenchymal transition (EMT). PCGF2 is a chromatin-modifying protein that inhibits EMT and negatively regulates stem cell-like properties [195,196]. SNAI proteins play a crucial role in EMT and in repressing the mesenchymal-epithelial transition, being important in embryonic and tumor development [197,198,199]. On the other hand, a complex of SNAI and ID2 inhibits EMT [200,201]. Co-expression of Ef-snai2 and Ef-id2 may indicate a partial involvement of the EMT mechanisms during the gut regeneration in E. fraudatrix. It is probable that, while embedding in the connective-tissue thickening, coelomic epithelial cells acquire some mesenchymal features [202,203,204].

Gene tcf24 was first described in 2002 from humans as a paralog of the tcf23 [205]. Since then, no information about its functions has been obtained. Activation of Ef-tcf24 during regeneration in holothurians is the first description of its involvement in regeneration. The increase in the Ef-tcf24 expression after 5-7 dpe in E. fraudatrix suggests participation of this gene in gut regeneration in holothurians.

Moreover, genes of Cyclins, Cdcs, Retinoic acid receptors and some other proteins participating in retinoic acid anabolism, are up-regulated during regeneration of anterior gut anlage in E. fraudatrix [102].

 

3.3. Wnt Signaling Pathway

The Wnt signaling pathway is activated in the regeneration of the organs of the anterior part of the body in E. fraudatrix (AB and the anterior part of the gut). Gene transcripts of four ligands (wntA, wnt4, wnt6, and wnt16) and three receptors (frizzled1/2/7, frizzled4, and frizzled5/8) have been identified in the AB and gut anlagen [206]. All of them exhibit different expression dynamics. According to preliminary data, transcripts of the studied genes are localized in coelomic epithelium and organs of the water–vascular system of forming AB [207].

The increase in the wnt16 expression occurs earliest of all, after 3 dpe. In vertebrates, the wnt16 expression is often associated with the development or regeneration of connective-tissue structures, the transformation of the extracellular matrix, and the activation of metalloproteinases [208,209,210]. In E. fraudatrix, an increase in the activity of MMPs and ECM remodeling is observed in this period [97].

After 5-7 dpe, the expression of wntA, wnt4, wnt6, frizzled1/2/7, and frizzled4 increases abruptly and reaches its maximum. At this time, the main structures of AB and luminal epithelium are formed [42,46]. It is probable that Wnt signaling, performed through these ligands and receptors, influences the regeneration of the canals of the AB water–vascular system (water–vascular ring and radial water–vascular canals), and is also involved in the regulation of transdifferentiation.

The following regeneration period (10 dpe) is characterized by either reduced or insignificantly changed expression of most of the studied genes. The exception is frizzled5/8, whose number of transcripts reaches its maximum. Subsequently, after 14 and 20 dpe, changes in the expression of all Wnt and frizzled genes are minimal. It is worth mentioning that the dynamics of frizzled1/2/7 differ between E. fraudatrix and A. japonicus [167,206]. This may be explained by different functions of pathways which activated through the receptor Frizzled1/2/7.

Furthermore, the Wnt5 expression is revealed during the regeneration of the anterior structures in E. fraudatrix [211]. Unlike other ligands, Wnt5-positive cells were localized mainly in the connective tissue of the AB anlage and regenerating nerve cords. The number of such cells gradually increases after evisceration, reaching a maximum after 10-14 dpe, and then drops to the control values. The probable function of Wnt5 is the involvement in the regulation of ECM remodeling and nervous system regeneration. Wnt5 is known to be a regulator of neuronal organization and growth in planarians [212]. In mammals, Wnt5A is involved in the activation of MMPs during the development of skeletal elements [213].