Cell Proliferation Indices in Regenerating Alitta virens (Annelida, Errantia).

In recent years, interest in the possible molecular regulators of cell proliferation and differentiation in a wide range of regeneration models has grown significantly, but the cell kinetics of this process remain largely a mystery. Here we try to elucidate the cellular aspects of regeneration by EdU incorporation in intact and posteriorly amputated annelid Alitta virens using quantitative analysis. We found that the main mechanism of blastema formation in A. virens is local dedifferentiation; mitotically active cells of intact segments do not significantly contribute to the blastemal cellular sources. Amputation-induced proliferation occurred predominantly within the epidermal and intestinal epithelium, as well as wound-adjacent muscle fibers, where clusters of cells at the same stage of the cell cycle were found. The resulting regenerative bud had zones of high proliferative activity and consisted of a heterogeneous population of cells that differed in their anterior-posterior positions and in their cell cycle parameters. The data presented allowed for the quantification of cell proliferation in the context of annelid regeneration for the first time. Regenerative cells showed an unprecedentedly high cycle rate and an exceptionally large growth fraction, making this regeneration model especially valuable for studying coordinated cell cycle entry in vivo in response to injury.

Comparative Aspects of Annelid Regeneration: Towards Understanding the Mechanisms of Regeneration.

The question of why animals vary in their ability to regenerate remains one of the most intriguing questions in biology. Annelids are a large and diverse phylum, many members of which are capable of extensive regeneration such as regrowth of a complete head or tail and whole-body regeneration, even from few segments. On the other hand, some representatives of both of the two major annelid clades show very limited tissue regeneration and are completely incapable of segmental regeneration. Here we review experimental and descriptive data on annelid regeneration, obtained at different levels of organization, from data on organs and tissues to intracellular and transcriptomic data. Understanding the variety of the cellular and molecular basis of regeneration in annelids can help one to address important questions about the role of stem/dedifferentiated cells and "molecular morphallaxis" in annelid regeneration as well as the evolution of regeneration in general.

Structural and Functional Characterization of the FGF Signaling Pathway in Regeneration of the Polychaete Worm Alitta virens (Annelida, Errantia).

Epimorphic regeneration of lost body segments is a widespread phenomenon across annelids. However, the molecular inducers of the cell sources for this reparative morphogenesis have not been identified. In this study, we focused on the role of fibroblast growth factor (FGF) signaling in the posterior regeneration of Alitta virens. For the first time, we showed an early activation of FGF ligands and receptor expression in an annelid regenerating after amputation. The expression patterns indicate that the entire regenerative bud is competent to FGFs, whose activity precedes the initiation of cell proliferation. The critical requirement of FGF signaling, especially at early stages, is also supported by inhibitor treatments followed by proliferation assay, demonstrating that induction of blastemal cells depends on FGFs. Our results show that FGF signaling pathway is a key player in regenerative response, while the FGF-positive wound epithelium, ventral nerve cord and some mesodermal cells around the gut could be the inducing tissues. This mechanism resembles reparative regeneration of vertebrate appendages suggesting such a response to the injury may be ancestral for all bilaterians.

FoxA expression pattern in two polychaete species, Alitta virens and Platynereis dumerilii: Examination of the conserved key regulator of the gut development from cleavage through larval life, postlarval growth, and regeneration.

BACKGROUND: foxA orthologs are involved in various processes from embryo patterning to regulation of metabolism. Since foxA conserved role in the development of the gut of errant annelids has never been thoroughly studied, we used a candidate gene approach to unravel the molecular profile of the alimentary canal in two closely related nereid worms with a trochophore-type lecithotrophic larva. RESULTS: The character of foxA expression in the two polychaetes was similar but not identical. The genes were successively activated first in blastoporal cells, then in the stomodeum, the midgut, and hindgut primordia, and in the cells of central and peripheral nervous system. Before the start of active feeding of nectochaetes, we observed a short phase of foxA expression in the entire digestive tract. After amputation of posterior segments, foxA expression was established de novo in the new terminal part of the intestine, and then in the developing hindgut and the anus. CONCLUSIONS: We discovered an early marker of endoderm formation previously unknown in errant annelids. Its expression dynamics provided valuable insights into the gut development. Comparative analysis of foxA activity suggests its primary role in gastrulation morphogenesis independently of its type and in midgut and foregut specification. Developmental Dynamics 248:728-743, 2019. © 2018 Wiley Periodicals, Inc.

Mesoderm patterning and morphogenesis in the polychaete Alitta virens (Spiralia, Annelida): Expression of mesodermal markers Twist, Mox, Evx and functional role for MAP kinase signaling.

Mesoderm represents the evolutionary youngest germ layer and forms numerous novel tissues in bilaterian animals. Despite the established conservation of the gene regulatory networks that drive mesoderm differentiation (e.g. myogenesis), mechanisms of mesoderm specification are highly variable in distant model species. Thus, broader phylogenetic sampling is required to reveal common features of mesoderm formation across bilaterians. Here we focus on a representative of Spiralia, the marine annelid Alitta virens, whose mesoderm development is still poorly investigated on the molecular level. We characterize three novel early mesodermal markers for A. virens - Twist, Mox, and Evx - which are differentially expressed within the mesodermal lineages. The Twist mRNA is ubiquitously distributed in the fertilized egg and exhibits specific expression in endomesodermal- and ectomesodermal-founder cells at gastrulation. Twist is expressed around the blastopore and later in a segmental metameric pattern. We consider this expression to be ancestral, and in support of the enterocoelic hypothesis of mesoderm evolution. We also revealed an early pattern of the MAPK activation in A. virens that is different from the previously reported pattern in spiralians. Inhibition of the MAPK pathway by U0126 disrupts the metameric Twist and Mox expression, indicating an early requirement of the MAPK cascade for proper morphogenesis of endomesodermal tissues.

Vasa, PL10, and Piwi gene expression during caudal regeneration of the polychaete annelid Alitta virens.

Polychaetes are famous for their outstanding ability to regenerate lost body parts. Moreover, these worms possess a number of ancestral features in anatomy, development, and genetics, making them particularly suitable for comparative studies. Thus, fundamental as well as new undisclosed so far features of regenerative processes may be revealed, using polychaetes as a model. In the present work, we aimed to analyze the molecular basis of caudal regeneration in the nereid polychaete Alitta virens (formerly Nereis virens). We focused on homologues genes of RNA helicases Vasa and PL10 and ncRNA-binding proteins Piwi. These markers are suggested to play a significant role in maintenance of undifferentiated state of primordial germ cells and multipotent stem cells across invertebrates. In normal conditions, A. virens homologues of Vasa, PL10, and Piwi were differentially expressed in the subterminal growth zone and germline cells. Caudal amputation induced expression of studied genes de novo, which further accompanies all steps of regeneration. An early appearance of the transcripts in wound epithelium and internal blastemal cells suggests involvement of these genes in the well-known cell dedifferentiation events that assure polychaete regeneration. Provided interpretation of the gene expression dynamics implies the primary restoration of the pygidium and growth zone, which promotes following segment formation. Obtained results are valuable as a molecular fingerprint of the alterations occurring in regulatory state of locally regenerating tissues.

Tools for gene-regulatory analyses in the marine annelid Platynereis dumerilii.

The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid Platynereis, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In Platynereis, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species.