Investigating the developmental onset of regenerative potential in the annelid Capitella teleta

An animal's ability to regrow lost tissues or structures can vary greatly during its life cycle. The annelid Capitella teleta exhibits posterior, but not anterior, regeneration as juveniles and adults. In contrast, embryos display only limited replacement of specific tissues. To investigate when during development individuals of C. teleta become capable of regeneration, we assessed the extent to which larvae can regenerate. We hypothesized that larvae exhibit intermediate regeneration potential and demonstrate some features of juvenile regeneration, but do not successfully replace all lost structures. Both anterior and posterior regeneration potential of larvae were evaluated following amputation. We used several methods to analyze wound sites: EdU incorporation to assess cell proliferation; in situ hybridization to assess stem cell and differentiation marker expression; immunohistochemistry and phalloidin staining to determine presence of neurites and muscle fibers, respectively; and observation to assess re-epithelialization and determine regrowth of structures. Wound healing occurred within 6 h of amputation for both anterior and posterior amputations. Cell proliferation at both wound sites was observed for up to 7 days following amputation. In addition, the stem cell marker vasa was expressed at anterior and posterior wound sites. However, growth of new tissue was observed only in posterior amputations. Neurites from the ventral nerve cord were also observed at posterior wound sites. De novo ash expression in the ectoderm of anterior wound sites indicated neuronal cell specification, although the absence of elav expression indicated an inability to progress to neuronal differentiation. In rare instances, cilia and eyes re-formed. Both amputations induced expanded expression of the myogenesis gene MyoD in preexisting tissues. Our results indicate that amputated larvae complete early, but not late, stages of regeneration, which indicates a gradual acquisition of regenerative ability in C. teleta. Furthermore, amputated larvae can metamorphose into burrowing juveniles, including those missing brain and anterior sensory structures. To our knowledge, this is the first study to assess regenerative potential of annelid larvae.     

THE DYNAMICS OF WOUND CLOSURE AND ITS ROLE IN THE PROGRAMMING OF PLANARIAN REGENERATION. II — DISTALIZATION

When the dorsal and ventral epidermal layers join by first intention during the closure of the wound, the cells of their borders (M-cells) do not meet in the same manner in all sections. In anterior sections the dorsal M-cells attach themselves to the ventral basement membrane, so that only the dorsal epidermis is stretched. In posterior sections the dorsal and the ventral M-cells join by their apical edges without being closely apposed to the wound surface. Only the ventral cells are stretched because of their specific motility. In longitudinal sections the dorsal and the ventral M-cells also join by their apical edges, but since they are closely apposed to the wound surface both epidermal layers are stretched. The stretching is a process equivalent to distalization. The junction between the dorsal and the ventral epidermis is shifted ventrally in the anterior wounds (as in the intact heads) and dorsally in the posterior wounds (as in the intact tails). Some abnormalities of wound closure have been observed at levels where heteromorphic regeneration frequently occurs. These findings are consistent with the hypothesis previously advanced (3) that the modalities of wound closure establish the programme for regeneration.     

Ultrastructural Features of Gut Regeneration in Five-Month-Old Pentactulae of the Holothurian Eupentacta fraudatrix

Posterior regeneration of the digestive system after bisection was investigated in the anterior halves of the five-month-old pentactulae of the holothurian Eupentacta fraudatrix using electron microscopy. Three stages of gut restoration were distinguished. The first stage is characterized by degradation of the damaged part of the gut followed by wound healing. Active morphogenetic processes (cell proliferation, dedifferentiation, cell migration, and redifferentiation) are observed at the second stage. During the third (final) stage, the ablated parts differentiate in the posterior portion of the intestine. The cells of the gut remnant tissues were shown to be the cell sources of regeneration. Based on both the data available from the literature and the results of our study, the conclusion was drawn that the mechanisms of gut restoration differ significantly in the pentactulae and adults of E. fraudatrix.     

Cell proliferation changes during pattern regulation in imaginal leg discs of Drosophila melanogaster

Upon fragmentation of a leg imaginal disc, cells near parts of the wounded surface are reprogrammed and form a blastema. This occurs without a change in fate and without the direct contact of the two wounded surfaces (G. H. Karpen and G. Schubiger, Nature (London) 294, 744-747, 1981). Two phases of the cell cycle have now been analyzed for several areas of disc fragments prior to and during wound healing. A mitotic index was used to compare the location of cell division, and autoradiography was used to reveal patterns of DNA synthesis. In contrast to the uniform division pattern in noncultured fragments, more dividing cells were observed near the two wound surfaces after 1 day of in vivo culture. During the second day, wound healing began and mitotic activity increased dramatically near both wound areas, and decreased in distant areas. Three and a half days of culture led to more complete wound closure and only cells on one site continued to show the highest frequency of labeled cells. It is concluded that changes in patterns of DNA synthesis and an increase in cell division begin prior to wound closure. This proliferation is consistent with the morphological changes and regulative behavior observed. In addition, the role of compartmental identity during regulation was tested. After wound closure began an increase in mitotic activity near wounds in the anterior compartment was observed whereas such an increase in division level was not seen in posterior cells near a wound.     

A comparative study of the regenerative processes in a trematode, Philophthalmus gralli, and a planarian, Dugesia dorotocephala

Adults of Philophthalmus gralli, an eyefluke of birds, were laterally amputated mid-way between the anterior testis and the ventral sucker. Worms were sampled at various short time intervals (30 min-6 h) after in vitro culture and long time intervals (1-8 days) after transplantation back to the host. Specimens were fixed for scanning electron microscopy and light microscopy and compared to the planarian, Dugesia dorotocephala, which was laterally transected in the pharyngeal region and maintained in springwater. It was found that wound closure took place by 2 days in the planaria; however, at the end of 8 days wound closure had been initiated but not yet completed in P. gralli. Replacement of major tissues was observed only in D. dorotocephala. Because calcium had been reported to be critical for planarian regeneration, a histochemical stain for calcium distribution was carried out. At the end of the 2-day study, no differences could be found in calcium distribution between the two organisms. No areas of calcium concentration were noted in any tissue important for the regenerative process.     

Regeneration of Soft Tissues Is Promoted by MMP1 Treatment after Digit Amputation in Mice

The ratio of matrix metalloproteinases (MMPs) to the tissue inhibitors of metalloproteinases (TIMPs) in wounded tissues strictly control the protease activity of MMPs, and therefore regulate the progress of wound closure, tissue regeneration and scar formation. Some amphibians (i.e. axolotl/newt) demonstrate complete regeneration of missing or wounded digits and even limbs; MMPs play a critical role during amphibian regeneration. Conversely, mammalian wound healing re-establishes tissue integrity, but at the expense of scar tissue formation. The differences between amphibian regeneration and mammalian wound healing can be attributed to the greater ratio of MMPs to TIMPs in amphibian tissue. Previous studies have demonstrated the ability of MMP1 to effectively promote skeletal muscle regeneration by favoring extracellular matrix (ECM) remodeling to enhance cell proliferation and migration. In this study, MMP1 was administered to the digits amputated at the mid-second phalanx of adult mice to observe its effect on digit regeneration. Results indicated that the regeneration of soft tissue and the rate of wound closure were significantly improved by MMP1 administration, but the elongation of the skeletal tissue was insignificantly affected. During digit regeneration, more mutipotent progenitor cells, capillary vasculature and neuromuscular-related tissues were observed in MMP1 treated tissues; moreover, there was less fibrotic tissue formed in treated digits. In summary, MMP1 was found to be effective in promoting wound healing in amputated digits of adult mice.     

Proliferative response of the stem cell system during regeneration of the rostrum in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Platyhelminthes)

Macrostomum lignano (Platyhelminthes) possesses pluripotent stem cells, also called neoblasts, which power its extraordinary regeneration capacity. We have examined the cellular dynamics of neoblasts during regeneration of the rostrum in M. lignano. First, using live squeeze observations, the growth curve of the rostrum was determined. Second, neoblasts were labelled with 5-bromo-2'-deoxyuridine (BrdU) and an anti-phospho-histone H3 mitosis marker (anti-phos-H3) to analyze their proliferative response to amputation. During the regeneration process, both S- and M-phase cells were present anterior to the eyes, a region that is devoid of proliferating cells during homeostasis. Furthermore, BrdU pulse experiments revealed a biphasic S-phase pattern, different from the pattern known to occur during regeneration of the tail plate in M. lignano. During a first systemic phase, S-phase numbers significantly increased, both in the region adjacent to the wound (the anterior segment) and the region far from the wound (the posterior segment). During the second, spatially restricted phase, S-phase numbers in the anterior segment rose to a peak at 3 to 5 days post-amputation (p-a), while in the posterior segment, S-phase activity approached control values again. A blastema, characterized as a build-up of S- and M-phase cells, was formed 1 day p-a.     

Regeneration of posterior segments and terminal structures in the bearded fireworm,Hermodice carunculata (Annelida: Amphinomidae)

Like many other annelids, bearded fireworms, Hermodice carunculata, are capable of regenerating posterior body segments and terminal structures lost to amputation. Although previous research has examined anterior regeneration in other fireworm species, posterior regenerative ability in fireworms remains poorly studied. As the morphology of the anal lobe (a small, fleshy terminal structure of unknown function) has been used to distinguish East and West Atlantic H. carunculata populations, there is a more imminent need to understand the morphology and organization of tissues in specimens undergoing posterior regeneration, and the timeframe in which significant developmental changes occur. To further investigate this phenomenon, we amputated the posterior segments of living H. carunculata specimens collected from the Gulf of Mexico and monitored posterior regeneration over a 6‐month study period. Although many aspects of posterior regeneration in H. carunculata are consistent with the findings of other annelid regeneration studies, histological analysis revealed that once formed, anal lobe morphology remains relatively unchanged at all stages of posterior regeneration; East Atlantic morphotypes were not observed in the West Atlantic specimens studied here. Additionally, we found that the ventral nerve chord, which is partially responsible for the regeneration of lost body parts in polychaete annelids, terminates within the anal lobe, suggesting that this structure may play a role in the formation of new segments. J. Morphol. 275:1103–1112, 2014. © 2014 Wiley Periodicals, Inc.     

Regeneration and colonization abilities of aquatic plant fragments: effect of disturbance seasonality

The regeneration (regrowth into viable plants or production of propagules, such as turions or buds) and colonization (development of roots and establishment in the sediment) of fragments of six aquatic plant species (Elodea canadensis Michaux, Hippuris vulgaris L., Luronium natans (L.) Rafin., Potamogeton pusillus L., Ranunculus trichophyllus Chaix, Sparganium emersumRehmann) occurring in habitats frequently disturbed by floods, were investigated through laboratory experiments conducted in two seasons, and compared to the recolonization patterns depicted after field experiments. Hypothesis was that differences observed between recolonization patterns after spring and autumn flood disturbances should be related to differences in recolonization (via rooting) and regeneration (via propagules) abilities of species fragments. In May and in August, five types of fragments were collected from the plants. Their development and/or rooting abilities were recorded over 10 weeks in the greenhouse. Fragments from E. canadensisand H. vulgarishad higher regeneration and lower colonization abilities in spring and conversely in autumn. Fragments from R. trichophyllusand S. emersumhad high colonization and low regeneration abilities during both seasons. Fragments from L. natans developed new buds in spring, whereas root development occurred only in autumn. Fragments from P. pusillus never rooted into the sediment, but developed turions in autumn. Differences between recolonization patterns observed in the field at the two seasons can most often be related to differences in regeneration and colonization abilities of species fragments. Species that colonize disturbed areas rapidly whenever the disturbance by flooding occurs have at least one type of vegetative fragment with a high colonization potential; this is called the `always-ready strategy' which appears to be an adaptation of aquatic plants to the unpredictability of flood disturbances.     

THE TIME COURSE OF SIEVE TUBE AND VESSEL REGENERATION AND THEIR RELATION TO PHLOEM ANASTOMOSES IN MATURE INTERNODES OF COLEUS

Quantitative counts of regenerative sieve tubes and vessels were made in a large number of samples of mature internode #5 of C. blumei, with concomitant study of the fine details of vascular regeneration and the occurrence of the normally developing phloem anastomoses. Such anastomoses were found in many of the plants, but their average number in the small regenerating area was low (viz., 0.9 ± 0.2). With the phloem anastomoses excluded from the counts, the time course of regeneration was clear cut—no strands completed their regeneration around the wound until three days after wounding. More regenerative sieve tubes completed their differentiation under all conditions than did regenerative vessels. The number of sieve tubes and vessels regenerated by four days was closely related to the number of preexisting bundles of that type of vascular cell that had been severed by the transverse wound. The ratio of bundles severed by the wound in the phloem to those in the xylem was 2.14, and the ratio of the regenerative sieve tubes to the regenerative vessels was 2.24. For both tracheary and sieve tube cells the initial regeneration was strongly polar (mostly above the wound), as expected from earlier IAA transport data. The path of tracheary regeneration was obviously related to that of the sieve tubes on the other side of the cambium.     

Biology of Pseudopotamilla reniformis (Müller 1771) in the White Sea,with description of asexual reproduction

The tube-dwelling polychaete Pseudopotamilla reniformis (Sabellidae) forms dense and complex aggregations of flexible tubes on hard substrates in the subtidal zone of the White Sea. No sexual reproduction was observed in this study and recruitment appeared to be due to asexual reproduction by architomy in winter, from October to March. The posterior part of the abdomen undergoes spontaneous fission into from 2 to 4 fragments and depending on their position, the fragments regenerate their anterior ends or both anterior and posterior ends. Regeneration in P. reniformis takes place via a combination of epimorphosis (replacement of missing parts by cell proliferation and the growth of new tissue) and morphallaxis (the remodelling of pre-existing structures without cell proliferation). The morphogenetic events during regenerative restoration include de novo formation of branchial crown, formation of thoracic segments and restoration of the posterior end. Asexual reproduction appears to play a crucial role for formation of P. reniformis aggregations and is very important for the population in the White Sea, at the margin of the species’ range.     

The specification of metameric order in the insectCallosobruchus maculatus Fabr. (Coleoptera)

Summary Mechanically dividing an insect egg into anterior and posterior fragments results in a segment gap (Sander 1976), a loss of non-terminal segments in the constricted region. By altering the stage and duration of constriction, we produced different types of egg fragments in the pea beetleCallosobruchus. The patterns formed by these fragments suggest the existence of interactions between anterior and posterior egg regions that influence segment patterning and placement. Segments in excess of the numbers expected on the basis of permanent constrictions were produced in fragments when: (1) the constriction was released before cellularization occurred and (2) in addition the complementary fragment degenerated. Apparently the degenerating fragment induced the formation of excess segments in the developing fragment. Differences in the time and extent of excess segment formation in anterior versus posterior fragments suggest an asymmetric distribution of prerequisites for segment formation. This conclusion is consistent with our finding that a partial reversal of segment sequence (double abdomen formation) can be induced only in posterior fragments by a degenerating fragment, but not in anterior fragments (see companion paper).The formation of excess segments shows that the segment gap observed after permanent separation cannot be due to non-specific damage, caused by the process of constriction as such, to the egg or to localized putative segment precursors.     

Anterior regeneration in the polychaete Marenzelleria viridis (Annelida: Spionidae)

The objective of this study was to examine the regeneration capacity of the spionid polychaete Marenzelleria viridis from Long Island, New York. In the field, ~7% of the worms exhibited regeneration of the anterior end. In the laboratory, worms were ablated at the 10th–50th chaetiger and their regeneration documented. Anterior morphogenesis was similar to that previously reported for spionids, with wound healing, blastema formation, differentiation of segments, and formation of feeding and sensory structures (mouth, palps, nuchal organs) occurring within 14 d. Unlike in some spionids, the segments do not appear to all form simultaneously from the blastema; rather, external differentiation of segments was observed from posterior to anterior on the regenerate. The number of segments replaced was equal to the number ablated for up to 10 segments. A maximum of 17 segments were replaced when 20–30 chaetigers were ablated, and the number replaced decreased to 14 when 40–50 chaetigers were ablated. Survival and normal growth of the worms decreased with more chaetigers ablated; a significantly higher number of worms died or grew abnormally with ≥30 chaetigers ablated, compared to worms with ≤20 chaetigers ablated. Members of M. viridis could be valuable model organisms in the study of the cellular mechanisms involved in regeneration, and further research on regeneration in the field should be completed.     

Regeneration processes in various species of planarians

Blastema growth and functional maturation of the pharynx during regeneration in various planarian species were compared. The intensity of blastema growth was highest in Polycelis tenuis; the lowest, in Schmidtea mediterranea. In the sexual and asexual races of Girardia tigrina blastema growth differed inconsiderably. The function of the pharynx during the regeneration of caudal fragments lacking pharynx was manifested in G. tigrina in the usual amount of time, while in the regeneration of head fragments lacking pharynx, this function occured earlier. In other planarian species of the other two typed, the times of pharynx regeneration had no regular character and took longer compared to the same process in G. tigrina.     

Inhibition of regeneration in the planarian Dugesia polychroa (Schmidt) by treatment with magnesium chloride: a morphological study of wound closure

At a concentration of 0.2% (21 m M) in culture water, magnesium chloride impaired muscle contraction and completely inhibited head regeneration in specimens of Dugesia polychroa cut prepharyngeally. The wound stayed open for nine days, with neoblasts accumulating beneath the wound without any signs of differentiation. Extremely delayed wound closure occurred by spreading epithelial cells, and was completed after 30 days in the magnesium chloride solution. Histological examination confirmed the absence of any regenerated head structures. Interestingly, the inhibitory effect was removed when such headless fragments were cut once more and kept in normal culture water: complete head regeneration then occurred at a normal rate. Among several possible explanations for the failure to regenerate, the following hypothesis is an attractive alternative: direct contact between parenchyma and epithelial cells during the period following injury seems to be an essential stimulus for the start of cell differentiation within the blastema, and the lack of such contact as a result of the drug action prevents normal regeneration. When the wound has eventually closed, a continuous basement membrane separates epithelium from parenchyma. Thus a direct contact between these tissues is never established. This revised version was published online in July 2006 with corrections to the Cover Date.     

Wound healing and regeneration in the imaginal wing disc ofDrosophila

Summary When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In 06 fragments (Bryant 1975a) wound healing has occurred after 1–2 days of culture and the regeneration of missing structures after 2–4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days.When disc fragments are cultured in female abdomens and then exposed to³H-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication.     

Palp growth,regeneration, and longevity of the obligate hermit crab symbiont Dipolydora commensalis (Annelida: Spionidae)

Abstract. The polychaete Dipolydora commensalis is an obligate symbiont of hermit crabs and produces a burrow along the columella of the gastropod shells they inhabit. Adults of D. commensalis have short palps that they use to feed on particles dropped or brought in by the respiratory currents of hosts. To determine whether hermit crabs influence palp length, specimens of D. commensalis were isolated in glass capillary tubes and the growth of palps was measured over a 3‐week period. Palp length was also measured in worms isolated in gastropod shells with or without hermit crabs for 2 weeks. In addition, to determine whether adults of D. commensalis have regeneration capabilities like those of free‐living relatives, worms were cut at the fifth or 15th setiger and then monitored for 35 d. Worms extracted from shells and placed into capillary tubes had initial palp lengths of 1.0±0.4 mm (n=17); after isolation, palps were 40% longer (1.4±0.4 mm, n=17). Worms in gastropod shells with hermit crabs had an average palp length of 0.9±0.4 mm (n=31), whereas worms in shells without hermit crabs had palps that were 33% longer (1.2±0.5 mm, n=40). Adults of D. commensalis are capable of regeneration; 35 d after ablation at setigers 5 or 15, the average number of anterior setigers regenerated was 5 (n=15) and 9±1.3 (n=13), respectively. The average number of posterior setigers regenerated from the 15 setiger anterior fragments was 11±6 (n=10). The findings suggest that the palps (and sometimes anterior ends) of the worms are exposed during feeding and are cut during movement of the hermit crab. In the laboratory worms can live for >4 years, considerably longer than the functional life span of most gastropod shells inhabited by hermit crabs.     

Suppression of Head Regeneration by Accelerated Wound Healing in Hydra

Published evidence suggests that tissue injury is important for head regeneration in hydra [MacWilliams, 1982, 1983a,b; Kobatake and Sugiyama, 1989]. To investigate this problem in more detail, two experimental manipulations, decapitation and mirror-image grafting, were carried out. In the latter, two decapitated polyps were axially grafted to each other to make the wound openings of the two polyps juxtaposed on each other. In normal regenerates, the wound opening closed and healed in 4 to 5 hr, while in mirror-image grafts it healed in about 1 hr. The percentage of head regeneration was lower in mirror-image grafts than that after decapitation. The effect of mirror-image grafting on morphogenetic potential levels was examined using a lateral transplantation technique. Head inhibition levels dropped in both types of regenerates to a similar extent. Head activation levels rose more in normal regenerates than in mirror-image grafts. These results show clearly that the drop in head inhibition level is due to removal of the head and is not affected by grafting. They also show that the increase in head activation levels and in the percentage of head regeneration is affected substantially by the grafting. These observations are consistent with the view that decapitation produced a greater injury effect than mirror-image grafting, and this injury effect raised the head activation level whereas it did not alter the head inhibition level. The fact that the wound remained open for a longer time in normal regenerates than in the grafts suggests that the injury effect depends not on tissue injury itself but on the length of time the wound is open.     

Hyperbaric Oxygen Promotes Proximal Bone Regeneration and Organized Collagen Composition during Digit Regeneration

Oxygen is critical for optimal bone regeneration. While axolotls and salamanders have retained the ability to regenerate whole limbs, mammalian regeneration is restricted to the distal tip of the digit (P3) in mice, primates, and humans. Our previous study revealed the oxygen microenvironment during regeneration is dynamic and temporally influential in building and degrading bone. Given that regeneration is dependent on a dynamic and changing oxygen environment, a better understanding of the effects of oxygen during wounding, scarring, and regeneration, and better ways to artificially generate both hypoxic and oxygen replete microenvironments are essential to promote regeneration beyond wounding or scarring. To explore the influence of increased oxygen on digit regeneration in vivo daily treatments of hyperbaric oxygen were administered to mice during all phases of the entire regenerative process. Micro-Computed Tomography (μCT) and histological analysis showed that the daily application of hyperbaric oxygen elicited the same enhanced bone degradation response as two individual pulses of oxygen applied during the blastema phase. We expand past these findings to show histologically that the continuous application of hyperbaric oxygen during digit regeneration results in delayed blastema formation at a much more proximal location after amputation, and the deposition of better organized collagen fibers during bone formation. The application of sustained hyperbaric oxygen also delays wound closure and enhances bone degradation after digit amputation. Thus, hyperbaric oxygen shows the potential for positive influential control on the various phases of an epimorphic regenerative response.     

Role of Cytokinin in Vessel Regeneration in Wounded Coleus Internodes

Cytokinin was found to be a controlling or limiting factor invessel regeneration around a wound in internodes of Coleus blumeiBenth. in which the endogenous cytokinin level was minimized.The cytokinin was applied in aqueous solution to the base ofexcised, mature internodes that had an active vascular cambium.Each internode also received IAA in lanolin at its apical end.Under low (0.1 %, w/w) or high (10%, w/w) auxin concentrations,the control internodes (without exogenous cytokinin) exhibitedsmall amounts of vessel regeneration. At appropriate concentrationszeatin, kinetin and 6-benzylamino-purine (BAP) induced a significantincrease in vessel regeneration around the wound. The threecytokinins also induced novel patterns of supplementary regenerationfurther from the wound surface. Kinetin and BAP showed the strongestpromoting effect at 5 and 10 µg ml^–1, while zeatinwas most effective at 20 µg ml^–1. At a low (0.1%) auxin level zeatin was the most effective cytokinin, whereaskinetin was the most effective one at high (1 %) auxin. An inhibitoryeffect on vessel regeneration was observed at the highest kinetinconcentration tested (50 µg ml^–1). The regenerationof vessels induced by cytokinin was very polar. Many more regeneratedvessel members differentiated below the wound than above it,and the regeneration process proceeded acropetally from thebase of the internode to its upper parts. Our results implya basipetal polar increase in cambium responsiveness along thestem axis from internode 5 to 7. The possible significance ofsuch a basipetal increase in cambium sensitivity in wood formationin trees is discussed. Auxin, Coleus blumei, cytokinin, vascular differentiation, vessel regeneration, wound xylem