Segmentation during Posterior Regeneration
Segmentation during posterior regeneration has been studied in many species and almost always follows the same general pattern: after the posterior end of the blastema differentiates into the pygidium, morphogenesis of segmentally iterated structures (septa, parapodia, chaetoblasts, ventral nerve cord ganglia, nephridia, etc.) becomes evident at the proximal regions of the blastema, showing a clear anteroposterior developmental gradient. Cell proliferation, which is initially high throughout the entirety of the early blastema, is greatly diminished at the proximal region of the newly formed pygidium but continues at high levels in the newly formed growth zone immediately anterior to the pygidium. Developing segments also show high levels of proliferation until they reach a size and developmental stage similar to that of the segment adjacent to the regenerate. Both cell proliferation and rate of segment formation gradually slow down, until the process can no longer be distinguished from normal posterior growth.
Gene expression patterns during posterior regeneration have been studied in several species. The more complete descriptions so far belong to the errant nereids Platynereis dumerilii and Alitta virens, and the sedentary capitellid Capitella teleta. More circumscribed data are available from other polychaete and clitellate species. With the sole exception of a novel gene discovered in the clitellate Enchytraeus, shown through RNA interference experiments to be necessary for regeneration (Takeo, Yoshida-Noro, and Tochinai 2010), empirical verification of gene function is still lacking. Thus, in most cases, putative gene functions have usually been inferred from temporal and spatial patterns of expression assessed by in situ mRNA hybridization assays.
While expression of any given gene might vary across species, several general patterns hold for most species studied so far. Soon after the initial fast woundhealing response is complete, genes associated with patterning the posterior end of bilaterians (caudal/cdx, wingless/wntl, brachyury/brd) are expressed at the wound epithelium (Figure 10.6) (Ozpolat and Bely 2016; Planques et al. 2018). Other genes expressed at this tissue include even-skipped/evx, engrailed/en, elav, hox2, and hox3 (Figure 10.7). Soon after, GMP genes associated with maintenance of stem cells and germ cell are upregulated, preceding (and likely promoting) blastema formation (Figure 10.6) (Ozpolat and Bely 2016; Planques et al. 2018). These genes include homologues of pixvi, vasa, nanos, PL10, and myc, which are expressed de novo in the wound epithelium and/or the underlying mesoderm, and are thought to promote and sustain local cell dedifferentiation during blastema development. At this point, the blastema shows a tripartite pattern of gene expression: a posterior region that corresponds to non-segmental tissues, an anterior region expressing evx, en, hox2, and hox3, and a narrow ring in between where evx/hox2/hox3 expression overlaps with cdx. The former region gives rise to the pygidium. The latter two regions form the posterior growth zone (PGZ).
GMP genes remain active at the blastema throughout regeneration but become excluded from the pygidium once this terminal structure begins its differentiation (Figure 10.6). Indeed, differentiation of the pygidium is marked by downregulation of most patterning genes found elsewhere in the blastema, except for the posterior markers cdx, bra, and wntl, and genes directing development of pygidial muscles (twist) and innervation (neurogenin/ngn, elav, hoxl, hox4, post2, postl). In species with pygidial appendages, like caudal cirri, their development is marked by expression of distalless/dlx, which promotes appendage outgrowth (Figure 10.6). In general, pygidial gene expression patterns during and after regeneration are markedly different from the region anterior to it, supporting the existence of a fundamental difference between the non-segmental pygidium and the adjacent segmental tissues.
As the blastema grows, the expression domains of hox2, hox3, and evx become restricted to a subterminal narrow ring of endodermal and mesodermal cells adjacent to the anterior boundary of the developing pygidium (Figure 10.6). This ring
FIGURE 10.6 Gene expression patterns during posterior regeneration, as described for the errant nereid Platynereis dumerilii. Modified after Planques et al. (2018) and Kostyuchenko et al. (2019).
becomes the segment addition zone (SAZ), a region showing intense gene expression and cell proliferation that generates cells destined to form segmental tissues. The SAZ behaves as a stem cell niche, and its constituent cells have been proposed to act like the conspicuous stem cells (known as teloblasts) that give rise to segmental tissues during embryonic development in direct developing clitellates like Tubifex tubifex and Helobdella robusta (see Chapters 4 and 7) (Gazave et al. 2013;
Balavoine 2015). While most studies have failed to find sets of clearly larger cells showing asymmetric division within the SAZ in any adult or regenerating annelid, several lines of evidence (including cyclic, coordinated, and oriented division; lower mitotic rate; cyclic gene expression; differential methylation pattern; large nucleus- to-cytoplasm ratio) suggest that cells within the SAZ act like stem cells (Gazave et al. 2013; Niwa et al. 2013; Jong and Seaver 2017; Planques et al. 2018). The hypothesis that the subterminal region adjacent to the pygidium represents a stem cell niche is also supported by data on GMP genes, which shows a strong expression gradient with peak intensity at the SAZ, tapering anteriorly and presenting a sharp posterior boundary adjacent to the pygidial tissues.
As the SAZ proliferates and intercalates new cells, older cells are displaced anteriorly. These latter cells continue proliferating and begin to differentiate into young segmental tissues. This results in the characteristic developmental gradient seen in the PGZ of both posterior regenerates and growing adults. Exactly how segmentation of the nascent tissues is achieved is still unresolved, but experiments on the nereid Perinereis nuntia suggest a boundary-driven segmentation process (see Chapter 4) in which segment boundaries form through a combination of cell cycle synchronization and cell row addition, mediated by the interaction between wntl and hedgehog/hh (Niwa et al. 2013). According to this model, cells at the posteriormost row of the SAZ are located in a zone of cell-cycle synchronization (ZCS; Figure 10.7). Cells at the ZCS enter cyclic mitosis with their mitotic spindles always oriented parallel to the body axis. As a nascent row leaves the ZCS, wntl is expressed and a WNT1 gradient forms, inducing expression of hh in the younger row behind it and setting the future segmental boundary (Figure 10.7A-D). This WNT1 gradient also coordinates cell-cycle entry of the next rows and inhibits them from expressing wntl. As the ZCS keeps adding rows, WNT1 levels fall below a threshold (Figure 10.7E-M), and wntl is expressed in the newest row, which becomes the posterior boundary of the prospective segment and resets the cycle (Figure 10.7N-P). Thus, the number of cell rows incorporated into each nascent segment depends on the strength of wntl expression, a finding supported by the fact that treatment of regenerating P. nuntia with lithium chloride results in an abnormally large number of rows being incorporated into each segment (Niwa et al. 2013). Lithium chloride inhibits GSK3beta, an enzyme that phosphorylates P-catenin, the key intracellular transducer of Wnt signaling; inhibition of GSK3beta thus mimics elevated canonical Wnt signaling. Available data on gene expression and cell proliferation patterns during posterior regeneration in Platynereis dumerilii, Alitta virens, and Capitella teleta are also compatible with this model of segment addition (Novikova et al. 2013; Jong and Seaver 2016; Planques et al. 2018).
Besides wntl and hh, several other genes are expressed in stripes at the PGZ (Figure 10.6), including engrailed, members of the Wnt signaling pathways (frizzled, sfrpl/2/5, tcf), the axonal guidance protein slit, and the prdm3/16 transcription factors (Niwa et al. 2013; Planques et al. 2018). Their roles have not yet been assessed experimentally, but they are likely to participate in the anteroposterior patterning of each segment. Segmentally iterated expression of other genes (including hox cluster members) associated with neurogenesis and myogenesis have also been described
FIGURE 10.7 Cellular model of segment addition and intersegmental boundary specification. Schematic representation of cell organization at the segment addition zone and neighboring pygidial cells. Anterior is to the left. The purple and gray bars represent fixed regions specifying the non-segmental pygidial region and a subterminal zone of cell-cycle coordination. The graded red bar represents a posteriorly oriented gradient of WNT protein generated from stripes of cells expressing wnt (red cells). The red arrows represent initial induction of hedgehog expression by WNT (green cells), and dashed lines represent the intersegmental boundary. Cells with mitotic spindles represent rounds of polarized cell division. See text for details.
(see earlier). There are several genes whose expression has been reported during larval growth and normal posterior growth but their expression during regeneration remains unassessed (Prud’homme et al. 2003; de Rosa, Prud’homme, and Balavoine 2005; Seaver and Kaneshige 2006; Dray et al. 2010; Gazave, Guillou, and Balavoine 2014; Balavoine 2015). Considering the similarities seen in the segment addition process between normal posterior growth and posterior regeneration, it is highly likely that those genes also show similar expression patterns.