Injury-induced activation of the MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in hydra head regeneration

After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

Disruption of normal forelimb regeneration in adult Notophthalmus viridescens by sublethal concentrations of trypan blue.

The effects of the vital dye trypan blue (TpB) on the regeneration of amputated newt forelimbs were examined. Administration of the dye (10 mug/g body weight) via IP injection during the early wound healing and dedifferentiation phases of regeneration inhibited the normal regenerative response. The accumulation phases of regeneration are similarly halted but only by greater concentrations of TpB (50 mug/g body weight) while redifferentiation and morphogenesis are only affected by still greater concentrations of the dye (100 mug/g body weight). In addition to abolishing the regenerative response, low levels of TpB were also capable of inducing skeletal abnormalities in the regenerates as might be expected from previous reports on the teratogenicity of the dye. The in vitro action of newt hyaluronidase (as well as purified testicular hyaluronidase) on hyaluronate was diminished by TpB, with virtually complete inhibition observed at initial reaction mixture concentrations of 100 mug/ml. The results of this study suggest that TpB acts to disrupt the normal regenerative response by preventing dedifferentiation and remodeling, perhaps by inhibition of various necessary lytic enzyme functions or by interference with normal intercellular communications.     

Analysis of spacing in a periodic pattern

Colonies of hydroids exhibit periodic biological patterns. Polyps form on stolons at fixed distances, obeying distinct rules. The spacing mechanism is based on inhibition emanating from existing polyps, predominantly from the head of the polyp. Removal of polyps from young colonies reduces the distance between initiating polyps and newly formed polyps to 50% of the normal values. Head removal suffices to obtain an almost identical reduction. Polyp enlargement which increases the distance between the inhibition-emitting head and the stolon tissue reduces the intrastolonal range of inhibition. In the stolon tissue, decrease of inhibitory activity occurs. An increase in the stolon/polyp ratio of a colony reduces bud distances. The decay is, in part, due to loss of inhibitory activity into the external medium: if colonies are incubated in conditioned culture medium derived from crowded colonies having normally large interpolyp distances, bud distances increase in test colonies. The effectiveness of transfer of inhibitory activity from tissue into the medium depends on culture conditions. If convection is increased by agitation of the culture medium, the distances between polyp and bud decreases; viscosity enhancement of the culture medium reduces convection and bud distances become larger. This effect is compensated for by additional agitation of the viscosity enhanced culture medium. Our results support the idea that a lateral inhibition mechanism controls polyp spacing in the stolon and that inhibition is based on diffusible inhibitory compounds.     

Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK-3beta inhibitors

In Hydractinia, a colonial marine hydroid representing the basal phylum Cnidaria, Wnt signaling plays a major role in the specification of the primary body axis in embryogenesis and in the establishment of the oral pole during metamorphosis. Here we report supplementing investigations on head regeneration and bud formation in post-metamorphic development. Head and bud formation were accompanied by the expression of Wnt, frizzled and Tcf. Activation of Wnt signaling by blocking GSK-3beta affected regeneration, the patterning of growing polyps and the asexual formation of new polyps in the colony. In the presence of lithium ions or paullones, gastric segments excised from adult polyps showed reversal of tissue polarity as they frequently regenerated heads at both ends. Phorbol myristate acetate, a known activator of protein kinase C increased this effect. Global activation of the Wnt pathway caused growing polyps to form ectopic tentacles and additional heads along their body column. Repeated treatment of colonies evoked the emergence of many and dramatically oversized bud fields along the circumference of the colony. These giant fields fell apart into smaller sub-fields, which gave rise to arrays of multi-headed polyps. We interpret the morphogenetic effects of blocking GSK-3beta as reflecting increase in positional value in terms of positional information and activation of Wnt target genes in molecular terms.     

The level of expression of a protein kinase C gene may be an important component of the patterning process in Hydra

 Several studies have provided strong, but indirect evidence that signalling through pathways involving protein kinase C (PKC) plays an important role in morphogenesis and patterning in Hydra. We have cloned a gene (HvPKC2) from Hydra vulgaris which encodes a member of the nPKC subfamily. In adult polyps, HvPKC2 is expressed at high levels in two locations, the endoderm of the foot and the endoderm of the hypostomal tip. Increased expression of HvPKC2 is an early event during head and foot regeneration, with the rise in expression being restricted to the endodermal cells underlying the regenerating ends. No upregulation is observed if regenerates are cut too close to the head to form a foot. Elevated expression of HvPKC2 is also observed in the endoderm underlying lithium-induced ectopic feet. A dynamic and complex pattern of expression is seen in developing buds. Regeneration of either head or foot is accompanied by an increase in the amount of PKC in both soluble and particulate fractions. An increase in the fraction of PKC activity which is membrane-bound is specifically associated with head regeneration. Taken together these data suggest that patterning of the head and foot in Hydra is controlled in part by the level of HvPKC2 expression, whilst head formation is accompanied by an in vivo activation of both calcium-dependent and independent PKC isoforms. Received: 10 July 1997 / Accepted: 8 November 1997     

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.     

The aberrant,a morphological mutant of Hydra attenuata,has altered inhibition properties

Transplantation experiments have led to the idea that developmental gradients are involved in the patterning processes of hydra. Head activation and head inhibition gradients appear to be involved in head formation. More could be learned about pattern formation if these gradients could be altered in the animal. One approach to this end is to study mutants suspected of having altered gradients. Such mutants may be identified by their altered morphology. In this investigation the aberrant, a morphological mutant of Hydra attenuata, was examined for alterations in its gradients. The aberrant was found to have changes in its inhibition and polarity properties. The inhibition gradient was steeper than normal and the level of inhibition was higher than normal near the head. In addition, the amount of inhibition transmitted from the head was greater than normal, while the amount of inhibition transmitted through the gastric region was less than normal. The polarity of the aberrant gastric region was more difficult than normal to reverse, and the aberrant extremities caused faster polarity reversal than normal extremities. The significance of these changes is discussed in relation to pattern formation.     

Dedifferentiation-specific expression of MMP-9 and the effects of RA on its expression during salamander limb regeneration

The matrix metalloproteinases (MMPs) are well known to responsible for the degradation of extracellular matrix (ECM) during tissue remodelling such as wound healing, metamorphosis, and regeneration. In present study, gelatinase activities were investigated in normal and retinoic acid (RA)-treated limb regenerates. During the early phase of limb regeneration, gelatinase activities increased greatly, and RA caused the enhanced and prolonged gelatinase activities. We also isolated full length of Hynobius MMP-9, and its spatial and temporal expression profiles were examined in normal, RA-treated, and denervated limb regenerates. Whole mount in situ hybridization showed that the expression of MMP-9 increased in the wound epidermis at the wound healing stage and early phase of dedifferentiation stage. In addition, RA enhanced remarkably its expression both in terms of level and duration in the wound epidermis. However, expression signal of MMP-9 was barely detectable in denervated in limb regenerates. Our results may indicate that MMP-9 plays important role(s) in the dedifferentiation process by participating in ECM degradation and enhancement of MMP-9 expression and activity might be closely related to RA-evoked pattern duplication.     

Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation

The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury.     

Patterning of the head in hydra as visualized by a monoclonal antibody. I. Budding and regeneration

A monoclonal antibody, CP8, has been isolated which displays a position-specific binding pattern to epithelial cells of Hydra oligactis. Antibody binding is restricted to the head of adult animals. When a new head develops during the budding process, CP8 binding is present in the area which will form the head well before morphological signs of it. Similarly, following decapitation as a new head regenerates, CP8 label appears covering a domed area at the apical end of the regenerate before tentacles evaginate delineating the head. When bud development or regeneration is complete, CP8 label is restricted to the new head. Experiments indicate the appearance of CP8 label during the formation of a head correlates closely with the patterning events which result in the determination of the tissue to form a head. The usefulness of CP8 as a diagnostic tool for exploring the dynamics of head pattern formation in hydra is discussed.     

Increased phenylalanine incorporation in regenerating skeletal muscle grafts

Skeletal muscle regenerates following grafting, but little is known about protein synthesis and its regulation during regeneration. We determined the sequence of changes in protein synthesis in rat extensor digitorum longus (EDL) muscle by the measurement of phenylalanine (Phe) incorporation into muscle protein at various times after grafting. Compared with control EDL, Phe incorporation in grafts doubled in 1 day, was four- to eight-fold greater from days 2 to 10 after grafting, and then subsided. Tissue mass (wet weight) increased rapidly from days 7 to 20 in EDL grafts. The maximal increase in protein synthesis occurred 7-10 days after grafting, whether or not the nerve was left intact. Autoradiography indicated that incorporated radioactivity was associated with regenerating muscle fibers on day 10. Deficiencies of insulin, pituitary or testicular hormones, or chronic in vivo administration of insulin, growth hormone, testosterone, or tri-iodothyronine did not substantially alter the elevation in incorporation of the Phe into muscle protein 10 days after grafting. The breakdown of EDL protein, measured in vitro simultaneously with protein synthesis, was increased five-fold, and overall protein degradation was elevated six-fold 10 days after grafting. These findings indicate that Phe incorporation is rapidly elevated following grafting of the EDL, and that by days 7-10 reflects synthesis in regenerating muscle fibers. The increase in protein synthesis associated with muscle regeneration at this time appears to be independent of innervation and anabolic hormones.     

Wet wound healing

Wound treatment in a flexible transparent chamber attached to the perimeter of the wound and containing a liquid has been extensively tested in preclinical experiments in pigs and found to offer several advantages. It protects the wound; the liquid medium or saline in the chamber provides in vivo tissue culture-like conditions; and antibiotics, analgesics, and various molecules can be delivered to the wound through the chamber. The wound chamber causes no injury to the wound itself or to the surrounding intact skin. Topical delivery of, for instance, antibiotics can provide very high concentrations at the wound site and with a favorable direction of the concentration gradient. A series of 28 wounds in 20 patients were treated with a wound chamber containing saline and antibiotics. Most patients had significant comorbidity and had not responded to conservative or surgical management with débridement and delayed primary closure or skin grafts. Six wounds had foreign bodies present; four of these were joint prostheses. Seven patients were on corticosteroids for rheumatoid arthritis, lupus, or chronic obstructive pulmonary disease, and four patients had diabetes. Most patients were treated with the wound chamber in preparation for a delayed skin graft or flap procedure, but one was treated with a wound chamber until the wound healed. Twenty-five of the wounds (89 percent) healed, and five wounds (18 percent) required additional conservative management after the initial chamber treatment and grafting procedure. Of the three wounds that did not heal, one healed after additional chamber treatment, one had a skin graft that did not take, and one required reamputation at a higher level. Antibiotic delivery was less than one intravenous dose daily, which avoided the potential for systemic absorption to toxic levels. Antibiotics such as vancomycin and gentamicin could be used in concentrations of up to 10,000 times the minimal inhibitory concentration. Forty-eight hours after application, 20 percent or more of the original antibiotic concentration was present in the wound chamber fluid. In conclusion, the wound chamber provides a safe, powerful tool in the treatment of difficult infected wounds.     

Patterning of the head in hydra as visualized by a monoclonal antibody: III. The dynamics of head regeneration

The dynamics of the early patterning processes leading to the regeneration of a head in tissue excised from the body column of Hydra oligactis were examined by using a monoclonal antibody, CP8. This antibody displays position-specific binding, labeling the head ectodermal epithelial cells. During regeneration of a head, antibody labeling is present well before morphological signs of the head, at a time correlated with the determination of the tissue (Javois et al., Dev. Biol., 117:607-618, '86). By quantifying antibody labeling during regeneration of three different pieces of tissue excised from the body column, it was found that the dynamics of the early patterning processes as visualized by CP8 labeling varied. The pattern of labeling observed as well as the spread of labeled tissue suggested that the amount and geometry of apical tissue in the regenerate played a critical role in the patterning processes. Contrary to the labeling pattern observed in heads which formed during bud development or which regenerated following decapitation (Javois et al., '86), not all the CP8+ tissue was confined to the head structures in these regenerates. Several alternative explanations for this surprising result are presented. The usefulness of these data in refining pattern formation models by more explicitly constraining their parameters is discussed.     

Epidermal cell migration during wound healing in Dugesia lugubris

The epidermal cells that migrate over the surface during the wound closure stage of head regeneration in Dugesia lugubris s.l. were examined by scanning electron microscopy. The effect of cytochalasin B on epidermal cell migration was also examined. During the first few hours after decapitation epidermal cells at the edges of the wound showed significant changes of shape related to the process of migration that was accomplished approximately 10 h after wounding. Flattening of the marginal cells was associated with active epidermal spreading throughout the healing period. Suitable support for migrating cells appeared to be a rhabditic network attached to the wound tissue. Epidermal cell migration was inhibited by cytochalasin B. These results demonstrate that the basis for cell movement in planarians is similar to that of many other systems.     

STK, the src homologue, is responsible for the initial commitment to develop head structures in Hydra

STK, the Src tyrosine kinase homologous of the fresh water polyp hydra, is a key component of the signal transduction system for cell differentiation in this organism. Its activity is strongly increased 6 h after decapitation, and the inhibition of its activity with PP2/AG1879 prevents head development. We generated STK(-) polyps by using double-stranded RNA interference; STK activity of those polyps is blocked through time. STK RNAi silenced animals could not regenerate the head, but the foot, and could not reproduce asexually. The silencing of STK causes the development of ectopic heads in decapitated polyps in the first third of their body. Some head-specific genes, like Ks1, HyTcf, and Hybra1, seem to be regulated by the signaling pathway mediated by STK because their expression is modified in the STK(-) polyps. These findings support an important function for STK in the initial commitment of cells to develop head structures in hydra.     

The influence of hormones and other substances on lens regeneration in vitro

Culturing the dorsal iris epithelium of a newt with a pituitary gland in organ culture greatly enhances the ability of the iris epithelium to produce advanced lens regenerates in vitro. In an attempt to elucidate the mechanism by which the pituitary enhances lens regeneration irido-corneal complexes from adult newts were cultured in medium to which various substances had been added either singly or in numerous combinations. Prolactin, insulin, hydrocortisone, and thyroxine failed to enhance the production of advanced lens regenerates in any of the doses or combinations tested. Similarly, addition of 50 microgram/ml of sodium or calcium ascorbate had no effect on the progress of lens regeneration in vitro. Addition of dibutyryl cyclic-AMP caused an inhibition of depigmentation and regeneration at high doses. The results of these experiments show that the effects of the pituitary cannot be duplicated by hormones which other authors have asserted to be beneficial to limb or tail regenerates in vitro. The results with cyclic AMP suggest that prolonged exposure to high doses of cyclic AMP inhibit regeneration and indicate that further studies on the fluctations in cyclic AMP levels throughout the process of lens regeneration must be done.     

β- and γ-Crystallin Synthesis in Rat Lens Epithelium Explanted with Neural Retina

Culturing the dorsal iris epithelium of a newt with a pituitary gland in organ culture greatly enhances the ability of the iris epithelium to produce advanced lens regenerates in vitro. In an attempt to elucidate the mechanism by which the pituitary enhances lens regeneration irido-corneal complexes from adult newts were cultured in medium to which various substances had been added either singly or in numerous combinations. Prolactin, insulin, hydrocortisone, and thyroxine failed to enhance the production of advanced lens regenerates in any of the doses or combinations tested. Similarly, addition of 50 μg/ml of sodium or calcium ascorbate had no effect on the progress of lens regeneration in vitro. Addition of dibutyryl cyclic-AMP caused an inhibition of depigmentation and regeneration at high doses. The results of these experiments show that the effects of the pituitary cannot be duplicated by hormones which other authors have asserted to be beneficial to limb or tail regenerates in vitro. The results with cyclic AMP suggest that prolonged exposure to high doses of cyclic AMP inhibit regeneration and indicate that further studies on the fluctations in cyclic AMP levels throughout the process of lens regeneration must be done.