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In general vertebrates have very limited regenerating capabilities. Among invertebrate chordates however, amphioxus and tunicates show remarkable regenerative abilities. Studying regeneration in these animals is important, as tunicates appear to be the closest living relatives of the vertebrates. Colonial ascidians are capable of whole body regeneration by the activation of stem cells in their vascular system or the epicardium. Solitary ascidians, such as Ciona, show more restricted regenerative capabilities. In addition, it is not clear whether regeneration in these animals depend also on stem cells. Now a recent paper from William R. Jeffery reports on the existence of stem cells in the brachial sac of Ciona, required for regeneration (http://onlinelibrary.wiley.com/doi/10.1002/reg2.26/abstract).
Previous studies had shown that after bisection only basal parts are able to regenerate the distal parts, whereas distal parts are not able to regenerate the missing basal ones. Among the distal parts that can be regenerated researchers have focused mainly on the neural complex and the oral siphon, a muscular tube leading into the pharynx and that contains orange-pigmented sensory organs (OPOs). For both structures regeneration involves the formation of a blastema although the origin of the regenerative cells is not clear.
In this paper, the author focused on the regeneration of the distal oral siphon and its OPOs. First, the author confirmed that only the basal parts were capable of regenerating complete animals. The distal parts did not regenerate any of the proximal (basal) regions and eventually disintegrated. Also, middle body parts were able to regenerate the distal oral siphons with OPOs even in the absence of the basal parts. After the amputation of the oral siphon a blastema containing proliferating cells is formed about 4 days after amputation. Then, he investigated the role of cell proliferation in OPO regeneration. Remarkably, blocking cell division did not affect OPO regeneration. Next, and in order to determine the source of stem cells for distal regeneration two different approaches were used. In a first set of experiments the author labelled dividing cells with EdU for 3, 8, 24 and 48 h after bisection. Control (intact) animals showed EdU labelling in the stomach, intestines and basal stalk. Regenerating animals showed similar levels of EdU labelling in those organs. However, in those regenerating animals a strong EdU labelling was observed from 3 h of regeneration in the transverse vessels of the branchial sac. The labelling was most prominent in the lymph nodes, known to be involved in blood cell renewal. On the other side, the regenerating oral siphon did not show EdU labelling until 48 h of regeneration. Secondly, the location of stem cells was analysed by using alkaline phosphatase (AP) and PIWI markers. In adults, the most intense AP activity was seen in transverse vessels of the branchial sac, where PIWI was also immunodetected. Altogether these results suggest that the transverse vessels of the branchial sac are a potential source of stem cells for distal regeneration.
Next, he carried out several EdU chase experiments to determine the source of the blastema cells. Control and regenerating animals were exposed to EdU for 24 h and then chased without EdU for 5-10 days. Regenerating animals showed an intense labelling in the distal blastema, which was not labelled after 2 days of EdU pulse, which suggests a source of proliferating cells outside the blastema. Then, he transplanted EdU labelled branchial sacs into control hosts that were afterwards bisected at a level leaving the transplanted tissues in the basal part. After 10-15 days of regeneration EdU positive cells were detected in the distally regenerating neural complex and oral siphon. Therefore, it seems that proliferating cells from the branchial sac migrate into the blastema during distal regeneration. Respect to the cells that give rise to the OPOs, experiments with regenerating oral siphons explants suggested that AP labelled stem cells original for the branchial sac would invade the distal areas and differentiate into OPOS during the early stages of regeneration.
The regenerative abilities of Ciona decline with age. So, in a final set of experiments the author wanted to determine whether this decline was related to changes in the stem cells of the branchial sac. He compared the regeneration of young (6 months) and old (12 months) animals. As expected old animals were either unable to regenerate or regenerated partial siphons. Remarkably, the distribution of proliferating cells and the structure of the branchial sacs appeared disorganized in old animals. Moreover, old animals showed very few AP and PIWI labelled cells in the transverse vessels, suggesting that stem cells may be depleted in the branchial sac during aging, being this responsible of their reduced regenerative potential.
In summary, this paper reports on the role of stem cells from the branchial sac in the regeneration of distal structures in Ciona. In these animals the blastema appears to be formed by at least two types of progenitor cells: 1) a subset of branchial sac cells that incorporate EdU very early during regeneration but that they are detected in the blastema after few days (once they migrate there), and 2) another subset of branchial sac cells that migrate to the blastema very early during regeneration and differentiate into the OPOs without undergoing cell division.