Ultrastructural features of the process of wound healing after tail and limb amputation in lizard

Alibardi, L. 2010. Ultrastructural features of the process of wound healing after tail and limb amputation in lizard.—Acta Zoologica (Stockholm)  91 : 306–318 Wound healing and re‐epitelization after amputation of tail and limb in lizard have been studied by electron microscopy to understand the cytological base of immunity to infection in this species. After 2 days post‐amputation in both limb and tail stumps, numerous granulocytes are accumulated over the stump, and participate to the formation of the scab. Bacteria remain confined to the scab or are engulfed by leukocytes and migrating keratinocytes located underneath the scab. Bacteria are degraded within lysosomes present in these cells and are not observed among mesenchymal cells or in blood vessels of the regenerative blastema. Granulocytes, migrating keratinocytes, and later macrophages form an effective barrier responsible for limiting microbe penetration. The innate immunity in lizard is very effective in natural (dirty) condition and impedes the spreading of infection to inner tissues. While the complete re‐epitelization of the tail stump underneath the scab requires 4–7 days, the same process in the limb requires 8–18 or more days post‐amputation, depending from the level of amputation and the persistence of a protruding humerus or femurs on the stump surface. This delay produces the permanence of inflammatory cells such as granulocytes and macrophages in the limb stump for a much longer period than in the tail stump, a process that stimulates scarring.     

A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole

Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp).The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC.We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-β or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation.These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.     

Cell lineage tracing during Xenopus tail regeneration

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures - the spinal cord, the notochord and the segmented myotomes - are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.     

The observation of partial regeneration of the tip of tail in newborn rat pups in a liquid medium

When the wound surface formed as a result of amputation of the tail tip in newborn rats was placed in sterile 0.9% aqueous solution of sodium chloride until full epithelialization of the defect, the epithelium moved onto the blood clot covering the wound. The blood clot was then substituted for connective tissue, into which the traumatized vertebra protruded. The vertebra restored its anatomical integrity, and this led to partial regrowth of the tail. A skin regenerate was formed on the apical tail surface with characteristic features of the intact skin of this locality. In the control animals, no regeneration of the vertebra epiphysis took place and a scar was formed at the stump end.     

Regeneration of the tail bud in Xenopus embryos.

In an attempt to solve some aspect of the long-standing controversy about the regenerative ability of appendages in vertebrate embryos, the tail bud of Xenopus laevis embryos has beenamputated at stage sranging from St. 26 to St. 32 and its ability to regenerate duringa culture period of 2-3 days has been studied. At amputation stages 26-28,the tail bud consisted only undifferentialted mesoderm and ectoderm, but at stage 32 it had afully differentiated neural tube, a vaculotaed notochord and segmented somites. A total of 137amputations at differnt stages gace consistent results: a tail formed in all the operated larvacand it had normal, well-developed axial tissues in most cases. The relatively few cases with abnormal tail struture were stunted, oedematour larvae with defects in the trunk region as well. It is concluded from these experiments that cells near the original tail budare able to differentiate into tialbud tissues and to replace the amputated regoin, even at these late embryoic stages. The implications of these findings for comparative studies on regeneration in vertebrates are discussed.     

Immunolocalization of c‐myc‐positive cells in lizard tail after amputation suggests cell activation and proliferation for tail regeneration

After tail amputation in lizard, a regenerative response is elicited leading to the formation of a new tail. The stimulation of the proliferation process may involve the proto‐oncogene c‐myc. The immunocytochemical analysis detects the c‐myc protein few days after wound in free cells accumulating over the injured tissues of the tail stump. Western blot detects a protein band at 68–70 kDa that is more intense in the regenerating blastema than in normal tail tissues. Nuclei positive for the c‐myc protein are seen in mesenchymal‐like cells located among muscles, connectives and fat tissues of the tail stump 4 days postamputation. Proliferating cells labelled for 5BrdU are seen at 4 days postamputation and are sparse in the mesenchyme of the regenerating blastema formed at 12 days postamputation. Fine immunolocalization of the c‐myc protein shows it is mainly located over euchromatin or poorly condensed chromatin to indicate gene activation. The study correlates the detection of the c‐myc protein with activation of cell division in the injured tissues leading to the formation of the regenerative blastema. The lizard c‐myc protein probably activates a controlled proliferation process through a mechanism that can give information on the uncontrolled process occurring in cancer.     

Immunolocalization of the telomerase‐1 component in cells of the regenerating tail,testis, and intestine of lizards

Using an antibody against a lizard telomerase‐1 component the presence of telomerase has been detected in regenerating lizard tails where numerous cells are proliferating. Immunoblots showed telomerase positive bands at 75–80 kDa in normal tissues and at 50, 75, and 90 kDa in those regenerating. Immunofluorescence and ultrastructural immunolocalization showed telomerase‐immunoreactivity in sparCe (few/diluted) mesenchymal cells of the blastema, early regenerating muscles, perichondrium of the cartilaginous tube, ependyma of the spinal cord, and in the regenerating epidermis. Clusters of gold particles were detected in condensing chromosomes of few mesenchymal and epithelial cells in the regenerating tail, but a low to undetectable labeling in interphase cells. Telomerase‐immunoreactivity was intense in the nucleus and sparCe (few/diluted) in the cytoplasm of spermatogonia and spermatocytes and drastically decreased in early spermatids where some nuclear labeling remains. Some intense immunoreactivity was seen in few cells near the basal membrane of intestinal enterocytes or in leukocytes (likely lymphocytes) of the intestine mucosa. In spermatogonia, spermatids and in enterocytes part of the nuclear labeling formed cluster of gold particles in dense areas identified as Cajal Bodies, suggesting that telomerase is a marker for these stem cells. This therefore suggests that also the sparCe (few/diluted) telomerase positive cells detected in the regenerating tail may represent sparCe (few/diluted) stem cells localized in regenerating tissues where transit amplifying cells are instead preponderant to allow for tail growth. This observation supports previous studies indicating that few stem cells are present in the stump after tail amputation and give rise to transit amplifying cells for tail regeneration. J. Morphol. 276:748–758, 2015. © 2015 Wiley Periodicals, Inc.     

Regeneration of neural crest derivatives in the <Emphasis Type="Italic">Xenopus</Emphasis>tadpole tail

Background  

After amputation of the Xenopus tadpole tail, a functionally competent new tail is regenerated. It contains spinal cord, notochord and muscle, each of which has previously been shown to derive from the corresponding tissue in the stump. The regeneration of the neural crest derivatives has not previously been examined and is described in this paper.     

The role of microtubules in contractile ring function.

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.     

The process of pharynx regeneration in planarians.

To understand the cellular events during planarian regeneration, we analyzed the process of pharynx regeneration in both head and tail pieces using cell-type-specific markers. Interestingly, cells expressing the pharynx-muscle-specific myosin heavy chain gene (DjMHC-A) appeared within 24 h after amputation (prior to the formation of a pharynx rudiment) in the mesenchymal space of the stump, not in the blastema region. These DjMHC-A-positive cells migrated to the midline and formed the pharynx rudiment. Even after formation of the pharynx rudiment, DjMHC-A-positive cells constantly appeared in the mesenchymal space in the region surrounding the pharynx rudiment and participated in the growth of the pharynx rudiment. These observations clearly indicated that the cells involved in pharynx-muscle formation are committed in the mesenchymal space of the stump, rather than in the blastema region or the pharynx rudiment during planarian regeneration. We also analyzed the process of regeneration of the pharynx epithelia using a monoclonal antibody and investigated the origin of the pharynx epithelia.     

Congenital amputation of the toes with replantation at the site of a constriction band in the mid-calf. Case report

We present a case of congenital amputation of the toes with replantation at the site of a constriction band in the mid-calf. The theories of the etiology of constriction bands are discussed, and the importance of this case in supporting the amniotic constriction band theory is emphasized.     

Mechanical feedback and robustness of apical constrictions in Drosophila embryo ventral furrow formation

Formation of the ventral furrow in the Drosophila embryo relies on the apical constriction of cells in the ventral region to produce bending forces that drive tissue invagination. In our recent paper we observed that apical constrictions during the initial phase of ventral furrow formation produce elongated patterns of cellular constriction chains prior to invagination and argued that these are indicative of tensile stress feedback. Here, we quantitatively analyze the constriction patterns preceding ventral furrow formation and find that they are consistent with the predictions of our active-granular-fluid model of a monolayer of mechanically coupled stress-sensitive constricting particles. Our model shows that tensile feedback causes constriction chains to develop along underlying precursor tensile stress chains that gradually strengthen with subsequent cellular constrictions. As seen in both our model and available optogenetic experiments, this mechanism allows constriction chains to penetrate or circumvent zones of reduced cell contractility, thus increasing the robustness of ventral furrow formation to spatial variation of cell contractility by rescuing cellular constrictions in the disrupted regions.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

Acute phase response in amputated tail stumps and neural tissue‐preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene

Regeneration of lost organs involves complex processes, including host defense from infection and rebuilding of lost tissues. We previously reported that Xenopus neuronal pentraxin I (xNP1) is expressed preferentially in regenerating Xenopus laevis tadpole tails. To evaluate xNP1 function in tail regeneration, and also in tail development, we analyzed xNP1 expression in tailbud embryos and regenerating/healing tails following tail amputation in the ‘regeneration’ period, as well as in the ‘refractory’ period, when tadpoles lose their tail regenerative ability. Within 10 h after tail amputation, xNP1 was induced at the amputation site regardless of the tail regenerative ability, suggesting that xNP1 functions in acute phase responses. xNP1 was widely expressed in regenerating tails, but not in the tail buds of tailbud embryos, suggesting its possible role in the immune response/healing after an injury. xNP1 expression was also observed in neural tissues/primordia in tailbud embryos and in the spinal cord in regenerating/healing tails in both periods, implying its possible roles in neural development or function. Moreover, during the first 48 h after amputation, xNP1 expression was sustained at the spinal cord of tails in the ‘regeneration’ period tadpoles, but not in the ‘refractory’ period tadpoles, suggesting that xNP1 expression at the spinal cord correlates with regeneration. Our findings suggest that xNP1 is involved in both acute phase responses and neural development/functions, which is unique compared to mammalian pentraxins whose family members are specialized in either acute phase responses or neural functions.     

Stump currents in regenerating salamanders and newts

We report here that a variety of salamanders and newts from differing habitats all drive a steady ionic electric current out of the forelimb stump tip after forelimb amputation. Several hours after amputation the density of this stump current ranges from about 10 to 100 microA/cm2 in most species, and declines with time. In most cases, the magnitude of the stump current is dependent on the concentration of Na+ in the external medium (an artificial pondwater), suggesting that the well-known Na+ -dependent transcutaneous voltage described in amphibia (particularly frogs) is the EMF for this stump current. These measurements add to those previously reported for the North American red spotted newt (Notophthalmus viridescens), and suggest that electrical changes following amputation of urodele limbs are widespread among members of this group.     

Salvage of amputation stumps by secondary reconstruction utilizing microsurgical free-tissue transfer

During a 2-year period, 15 lower and upper extremity amputees were treated by microsurgical free-tissue transfer in an effort to salvage their amputation stumps. Salvage of length and restoration of contour to aid in prosthetic rehabilitation were the two main indications for reconstruction. Included in the 15 transfers were 3 scapular free flaps, 11 latissimus dorsi musculocutaneous flaps, and 1 groin flap. Thirteen of the patients in this group were refitted with prostheses following reconstruction and did well with no pain or skin breakdown of the resurfaced stumps. The follow-up period on these patients averaged 16 months. One patient, in whom the flap succeeded, underwent stump soft-tissue revision and myodesis. One patient, in whom the flap failed, continued to develop recurrent ulceration in his stump. This clinical experience followed an extensive laboratory study of 12 above-knee amputation patients using noninvasive Doppler ultrasound measurements to determine weight-loading and interface-pressure distribution between the stump and the socket of the prostheses and their relation to stump length and circumference.     

Mitotic activity in the blastema and stump tissues of regenerating hind limbs of Xenopus laevis larvae after amputation at ankle level. An autoradiographic study

Mitotic activity, as indicated by DNA synthesis, was studied by autoradiographic analysis along the proximodistal axis of regenerating limbs in the early and later larval stages 53 and 57 of Xenopus laevis. Wound-healing, dedifferentiation, blastema formation and growth phases were studied. Most of the various stump tissues, as well as the cell mass of the regeneration blastema, were involved. The study showed an increase in DNA synthesis in the stump tissues during their dedifferentiation as well as during blastema formation. The increase was confined mainly to the distal portion (close to the amputation level), so that a proximodistal gradient was discernible. This could be regarded as valid evidence of contribution of the severed stump tissues to the blastema cells. The mesenchymal blastema cells formed after amputation at stage 53 displayed higher mitotic activity than the fibrocytoid blastema cells formed at stage 57. Although the latter were more differentiated than the former, they still showed DNA replication and mitotic division.