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Enteric nervous regeneration in sea cucumbers

Sea cucumbers are well known for their ability to regenerate their digestive system as they can eviscerate and then regenerate their internal organs, including the gut. After evisceration the remaining mesenteries play a key role in gut regeneration. The laboratory of Jose García-Arrarás has published some reports describing how during regeneration an intestinal primordium develops from a thickening of the mesenterial edge. With time this thickening grows up to the formation of a tube that will become the regenerated gut. One of the components of the sea cucumber gut is the enteric nervous system (ENS) that innervates the gastrointestinal tract. Now, a recent paper from this same laboratory describes for the first time in detail the regeneration of the enteric ENS in the sea cucumber Holothuria glaberrima (

Despite some studies have reported that miss-function of the ENS can cause several neuropathies both neurodegenerative and inflammatory in mammals, very few studies have analyzed the regenerative potential of this system under those contexts. Here, the authors use the sea cucumber to study how the ENS regenerates de novo together with the new gut after evisceration. In a first set of experiments, the authors use a collection of antibodies to label specific neural projections and cells of this ENS. Although, as the authors point out, it is not clear what are the antigens recognized by these antibodies, they are useful to consistently label specific neural fibers and cell populations and see how this pattern is restored during regeneration.

The main components of the ENS were divided depending on their location within the mesothelial layer, the connective tissue and the luminal layer. In the mesothelium they found a fiber network within the muscle layer that innervates the visceral muscle. There is a second mesothelial plexus formed by large nerve bundles that run along the longitudinal axis of the gut, and mostly parallel to the longitudinal muscle fibers. Within the connective tissue there are thick fibers that would correspond to nerves connecting the mesothelial plexus with the connective and luminal layers. There is also a network of small neurons and fine fibers all throughout this connective layer. Finally, the luminal plexus is formed by neuroendocrine-like cells distributed among the luminal epithelial cells. Occasionally, some of these cells display fiber projections extending towards the mesothelium.

Here, the authors used their markers to follow the regeneration process of all these components of the ENS at 3, 5, 7, 10, 14, 21, 28 and 35 dpe (days post evisceration). Summarizing all their stainings ENS regeneration could be divided into several stages: 1) initially there was a neurodegeneration stage consisting in the degradation of the preexisting fibers within the mesentery edge that will give rise to the intestinal primordium (5-7 dpe); 2) during the first days, then, the intestinal primordium lacked any ENS innervation (8-10 dpe); 3) then, re-innervation of this primordium started by 14 dpe. Here, new fibers appeared, mainly proximally in the mesothelium from an extrinsic source, that is, from cells in the mesentery. Moreover, new cells (of intrinsic origin) appeared also within the connective tissue, also mainly in proximal regions; 4) the fourth stage at around 21 dpe, was characterized by the differentiation of large fibers crossing from the mesothelium to the connective tissue as well as for the intrinsic differentiation of new neural cells within the lumen epithelium; 5) by 28 dpe most of the mesothelium was innervated and no differences between proximal and distal areas were observed; finally 6) by 35 dpe the ENS pattern in the regenerated intestine was similar to normal non-eviscerated intestine.

It is important to point out that ENS regeneration occurred in parallel to other important events. Thus, for example, the initial neurodegeneration coincided with the remodeling of the extracellular matrix. Also, fiber regeneration coincided with myogenesis and the incoming neural fibers were possibly re-innervating the newly differentiated muscle fibers. An important question to answer in future experiments concerns the origin of the new ENS cells. The authors discuss that they could originate from the dedifferentiation of muscle cells or coelomic epithelial cells or, alternatively from enteric stem cells or glia present in the intestine or mesentery. In the case of the neuroendocrine cells they seemed to differentiate from the luminal epithelial cells.

In summary, the authors describe here the different stages that characterize the regeneration of the ENS in sea cucumbers. As some events are conserved in mammals following lesions or inflammatory responses as, for example, the observed degeneration-regeneration stages, H. glaberrima can be a good model to understand ENS plasticity in other models. Moreover, there is an obvious interest for bioengineers trying to obtain intestines for transplantation and, as the authors state, studying ENS regeneration could provide insights into the type of cells and timing at which ENS precursors should be added in order to make a properly functional intestine.

Radial glial cells in echinoderm neural regeneration

Neural regeneration is successfully achieved in vertebrate models such as zebrafish and some amphibians. Among non-chordate Deuterostomes, echinoderms can efficiently regenerate their nervous system. A recent paper from the laboratory of José E. García-Arrarás ( describes the important role of radial glial cells in this process. Studying how similarly zebrafish and amphibians regenerate their nervous system compared to the non-chordate echinoderms may provide insights into: i) up to what extend the cellular and molecular mechanisms and events during neural regeneration have been evolutionary conserved among Deuterostomes, and ii) trying to improve the poor regenerative abilities of the mammalian CNS.

Echinoderm and chordate radial glia are similar morphologically and immunocytochemically. In adult echinoderms, radial glial cells retain their proliferative capabilities suggesting a role in neurogenesis. The authors here use sea cucumbers as a model. These animals possess 5 radial nerve cords (RNC) that are joined at the oral side of the body. Each RNC is formed by two bands, the ectoneural and the hyponeural neuroepithelia separated by a thin layer of connective tissue. These neuroepithelia are formed by radial glial cells and interspersed neurons. In their paradigm the authors cut one of the RNC at the mid-body level and analyze how both truncated ends regenerate and get re-connected.

Thus, RNC regeneration is divided into 4 stages: 1) early post-injury phases (days 1-2), 2) late-post injury phase (days 6-8), 3) growth phase (days 8-12) and 4) late regenerate (+21 days). During the early phase the radial glial cells close to the wound start dedifferentiating. These cells lose their basal processes while their cell bodies maintain the epithelial organization in the apical region. During the late phase, the coelomic epithelium migrates over the injury site sealing the wound. Also, at this stage, there is an expansion of the region of the neuroepithelium where the radial glial cells dedifferentiate. Those dedifferentiated radial glial cells do not only show changes in morphology but also show a much-reduced expression of a typical material that is recognized by antibodies against Reissner’s substance (RS), which is typically produced by glial cells also in vertebrates. Next, during the growth phase, the two truncated ends of the RNC at each side of the wound start growing towards each other. The ectoneural and hyponeural bands of the RNC form separate tubular rudiments that grow parallel to each other. Finally, in the late regenerate phase the continuity of the RNC is restored. New radial glial cells re-differentiate adopting their typical morphology and producing again RS-like material. Also, this new neural cord gets populated with neuronal cell bodies and processes.

Then, the authors combined BrdU pulse-chase experiments with labelling with radial glia specific markers. In adult non-regenerating CNS of sea cucumbers a basal level of BrdU incorporation had been previously reported. Now, after injury, no significant increase in BrdU incorporation takes place during the early phase. However, at the late post-injury phase there is a significant increase mainly in the ectoneural part of the RNC. During the growth phase this increase in BrdU-positive cells peaks in both ectoneural and hyponeural cords. From this stage the levels start decreasing until reaching again typical basal levels in the late regenerate phase. Remarkably, most of the BrdU positive cells (90-100%) are positive also for a radial glia-specific marker, indicating the dominant role of radial glia in cell division during RNC regeneration. It is also worthy to mention that whereas in the intact CNS the BrdU-positive cells are randomly scattered during neural regeneration most of the BrdU-positive cells localize in the dedifferentiated glia at the tip of the regenerate.

Finally, the authors show that at the very late regenerate phase (day 51 post-injury) 45% of the BrdU-positive cells are not labelled with the specific radial glia-specific marker and that some of these cells express now different neuronal markers. This suggests that a percentage of the progeny of radial glia differentiate into neurons.

In summary, this paper reports how radial glia is involved not only in mediating the bridging of the wound gap and axonal outgrowth but also acts as a source of new neurons during RNC regeneration in sea cucumber. This leading role for the radial glia resembles very much the role of the chordate radial glia in those regeneration-competent species, which opens the possibility that these conserved mechanisms for neural regeneration are also present in non-regenerating vertebrates but need to be somehow awaken.

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