Blastema Formation Research Articles

Induction of a noggin-Like Gene by Ectopic DV Interaction during Planarian Regeneration

In previous studies, we have shown that dorsoventral (DV) interaction evokes not only blastema formation, but also morphogenetic events similar to those that occur in regeneration. However, it is still unclear what kinds of signal molecules are involved in the DV interaction. To investigate the signal systems involved in the DV interaction, we focused on a noggin-like gene (Djnlg) identified by the planarian EST project. Djnlg is the first noggin homologue isolated from an invertebrate. In DjNLG, the positions of nine cysteine residues which may be essential for dimer formation were well conserved, but overall, the amino acid sequence of DjNLG did not show high similarity to the sequences of vertebrate Noggins. Expression of Djnlg was observed only in the proximal region of the branch structures in the brain of intact planarians, suggesting that Djnlg may have a role in pattern formation in the brain. Interestingly, transient strong expression of Djnlg was observed in the amputated region of regenerating planarians. Djnlg-expressing cells were detected beneath the muscle 9 h after amputation and were then detected in the ventral subepidermal region of the blastema. The induction of Djnlg expression by amputation was not affected by X-ray irradiation, even though the stem cells were completely eliminated, implying the existence of signal-producing cells which may provide a positional cue to the stem cells. In DV reversed grafting, expression of Djnlg was strongly induced in the DV boundary between the host and donor. These results suggest that ectopic DV interaction may induce expression of Djnlg in the positional cue-producing cells, and that it might be involved in stimulation of blastema formation as well as DV patterning of the body.

Gallium nitrate: effects on cartilage during limb regeneration in the axolotl, Ambystoma mexicanum.

Gallium nitrate, a drug shown to have efficacy in Paget's disease of bone, hypercalcemia of malignancy, and a variety of experimental autoimmune diseases, also inhibits the growth of some types of cancer. We examined dose and timing of administration of gallium nitrate on limb regeneration in the Mexican axolotl, Ambystoma mexicanum. Administered by intraperitoneal injection, gallium nitrate inhibited limb regeneration in a dose-dependent manner. Gallium nitrate initially suppressed epithelial wound healing and subsequently distorted both anterior-posterior and proximo-distal chondrogenic patterns. Gallium nitrate given at three days after amputation severely inhibited regeneration at high doses (6.25 mg/axolotl) and altered the normal patterning of the regenerates at low doses (3.75 mg/axolotl). Administration of 6.25 mg of gallium nitrate at four or 14 days prior to amputation also inhibited regeneration. In amputated limbs of gallium-treated axolotls, the chondrocytes were lost from inside the radius/ulna. Limbs that regenerated after gallium treatment was terminated showed blastema formation preferentially over the ulna. New cartilage of the regenerate often attached to the sides of the existing radius/ulna proximally into the stump and less so to the distal cut ends. J. Exp. Zool. 293:384-394, 2002.

A proliferation gradient between proximal and msxb-expressing distal blastema directs zebrafish fin regeneration.

Previous studies of zebrafish fin regeneration led to the notion that the regeneration blastema is a homogeneous population of proliferating cells. Here, we show that the blastema consists of two components with markedly distinct proliferation properties. During early blastema formation, proliferating cells are evenly distributed. At the onset of regenerative outgrowth, however, blastemal cells are partitioned into two domains. Proximal blastemal cells proliferate at a high rate, shifting from a median G(2) of more than 6 hours to approximately 1 hour. By contrast, the most distal blastemal cells do not proliferate. There is a gradient of proliferation between these extremes. Using bromodeoxyuridine incorporation and anti-phosphohistone H3 labeling, we find a 50-fold difference in proliferation across the gradient that extends approximately 50 microm, or ten cell diameters. We show that during early regeneration, proliferating blastemal cells express msxb, a homeodomain transcriptional repressor. While msxb is widely expressed among proliferating cells during blastema formation, its expression becomes restricted to a small number of non-proliferating, distal blastemal cells during regenerative outgrowth. Bromodeoxyuridine pulse-chase experiments show that distal and proximal blastemal cells are formed from proliferating, msxb-positive blastemal cells, not from preexisting slow-cycling cells. These data support the idea that blastema formation results from dedifferentiation of intraray mesenchymal cells. Based on these findings, we propose a new model of zebrafish fin regeneration in which the function of non-proliferating, msxb-expressing, distal blastemal cells is to specify the boundary of proliferation and provide direction for regenerative outgrowth.

Cell proliferation during blastema formation in the regenerating teleost fin.

Epimorphic regeneration in teleost fins occurs through the establishment of a balanced growth state in which a blastema gives rise to all the mesenchymal cells, whereas definite areas of the epidermis proliferate leading to its extension, thus, allowing the enlargement of the whole structure. This type of regeneration involves specific mechanisms that temporally and spatially regulate cell proliferation. To understand how the blastema is formed and how this growth situation is set up, we investigated cell proliferation patterns in the regenerating fin of the goldfish Carassius auratus from the time of amputation to that of blastema formation by using proliferating cell nuclear antigen immunostaining and bromodeoxyuridine labeling. Wound closure and apical epidermal cap formation took place by epidermal migration and re-arrangement, without the contribution of cell proliferation. As soon as the apical cap had formed, the epidermis started to proliferate at its lateral surfaces, in which all layers maintained cycling for the duration of the studied process. The distal epidermal cap, on the contrary, presented very few cycling cells, and its cytoarchitecture was indicative of continuous remodeling due to ray growth. The basal layer of this epidermal cap showed a typical morphology and remained nonproliferative whilst in contact with the proliferating blastema. Proliferation in the mesenchymal compartment of the ray started far from the amputation plane. Subsequently, cycling cells approached that location, until they formed the blastema in contact with the apical epidermal cap. Differences observed between the epidermis and mesenchyma, regarding activation of the cell cycle and the establishment of proliferative patterns, suggest that differential mechanisms regulate cell proliferation in each of these compartments during the initial stages of regeneration.

Effect of retinoic acid onFgf‐8expression in regenerating urodele amphibian limbs

In our previous study, we have shown that Fgf‐8 is expressed in the basal layer of the apical epithelial cap (AEC) and in the underlying thin layer of mesenchymal tissue of the regenerating limbs of Mexican axolotl, Amby‐stoma mexicanum. Our present RT‐PCR data also demonstrate that Fgf‐8 transcript is localized both in the mesenchymal and epidermal tissues. To understand the effect of retinoic acid (RA) on the expression of Fgf‐8 in the regenerating axolotl limbs, RA was injected intraperitoneally at the dediffer‐entiation stage of limb regeneration. The RA treatment caused a change in the Fgf‐8 expression profile of the regenerating limbs. In RA‐treated limbs, duration of Fgf‐8 expression was prolonged and a high level of expression was maintained during dedifferentiation and blastema formation stages. These results suggest that Fgf‐8 is an important molecule in the process of pattern duplication of regenerating salamander limbs evoked by RA treatment.

Regeneration neurohormones and growth factors in echinoderms

There has been much recent interest in the presence and biological functions of growth regulators in invertebrates. In spite of the different distribution patterns of these molecules in different phyla (from molluscs, insects, and annelids to echinoderms and tunicates), they seem always to be extensively involved in developmental processes, both embryonic and regenerative. Echinoderms are well known for their striking regenerative potential and many can completely regenerate arms that, for example, are lost following self-induced or traumatic amputation. Thus, they provide a valuable experimental model for the study of regenerative processes from the macroscopic to the molecular level. In crinoids as well as probably all ophiuroids, regeneration is rapid and occurs by means of a mechanism that involves blastema formation, known as epimorphosis, where the new tissues arise from undifferentiated cells. In asteroids, morphallaxis is the mechanism employed, replacement cells being derived from existing tissues following differentiation and (or) transdifferentiation. This paper focuses on the possible contribution of neurohormones and growth factors during both repair and regenerative processes. Three different classes of regulatory molecules are proposed as plausible candidates for growth-promoting factors in regeneration: neurotransmitters (monoamines), neuropeptides (substance P, SALMFamides 1 and 2), and growth-factor-like molecules (TGF-β (transforming growth factor β), NGF (nerve growth factor), RGF-2 (basic fibroblast growth factor)).

Nerve-Independence of Limb Regeneration in Larval Xenopus laevis Is Correlated to the Level of fgf-2 mRNA Expression in Limb Tissues

In both larval and adult urodele amphibians, limb blastema formation requires the presence of an adequate nerve supply. In previous research, we demonstrated that the hindlimb of early Xenopus laevis larvae formed a regeneration blastema even when denervated, while the denervated limb of late larvae did not. We hypothesized that the nerve-independence was due to the autonomous synthesis of a mitogenic neurotrophic-like factor by undifferentiated limb bud cells. In this paper, we demonstrate that fgf-2 mRNA is present in larval limb tissues and that its level is correlated to the extent of mesenchymal cells populating the limb: in early limbs, fgf-2 mRNA is present at high levels all over the limb, while, in late limbs, the fgf-2 expression is low and detectable only in the distal autopodium. After denervation, fgf-2 mRNA synthesis increases in amputated early limbs but not in amputated late limbs. The implantation of anti-FGF-2 beads into amputated early limbs hardly lowers the mitotic activity of blastema cells. However, FGF-2 beads implanted into the blastema of late limbs prevent the denervation-induced inhibition of mitosis and oppose blastema regression. Our data indicate that FGF-2 is a good candidate for the endogenous mitogenic factor responsible for blastema formation and growth in amputated and denervated early limbs. However, in amputated late limbs, the very limited fgf-2 expression is not sufficient to promote blastema formation in the absence of nerves.

Expression patterns of Fgf-8 during development and limb regeneration of the axolotl.

Fgf-8 is one of the key signaling molecules implicated in the initiation, outgrowth, and patterning of vertebrate limbs. However, it is not clear whether FGF-8 plays similar role in development and regeneration of urodele limbs. We isolated a Fgf-8 cDNA from the Mexican axolotl (Ambystoma mexicanum) through the screening of an embryo cDNA library. The cloned 1.26-kb cDNA contained an open reading frame encoding 212 amino acid residues with 84%, 86%, and 80% amino acid identities to those of Xenopus, chick, and mouse, respectively. By using the above clone as a probe, we examined the temporal and spatial expression patterns of Fgf-8 in developing embryos and in regenerating larval limbs. In developing embryos, Fgf-8 was expressed in the neural fold, midbrain-hindbrain junction, tail and limb buds, pharyngeal clefts, and primordia of maxilla and mandible. In the developing axolotl limb, Fgf-8 began to be expressed in the prospective forelimb region at pre-limb-bud and limb bud stages. Interestingly, strong expression was detected in the mesenchymal tissue of the limb bud before digit forming stages. In the regenerating limb, Fgf-8 expression was noted in the basal layer of the apical epithelial cap (AEC) and the underlying thin layer of mesenchymal tissue during blastema formation stages. These data suggest that Fgf-8 is involved in the organogenesis of various craniofacial structures, the initiation and outgrowth of limb development, and the blastema formation and outgrowth of regenerating limbs. In the developing limb of axolotl, unlike in Xenopus or in amniotes such as chick and mouse, the Fgf-8 expression domain was localized mainly in the mesenchyme rather than epidermis. The unique expression pattern of Fgf-8 in axolotl suggests that the regulatory mechanism of Fgf-8 expression is different between urodeles and other higher species. The expression of Fgf-8 in the deep layer of the AEC and the thin layer of underlying mesenchymal tissue in the regenerating limbs support the previous notion that the amphibian AEC is a functional equivalent of the AER in amniotes.

Cellular proliferation and neurogenesis in the injured retina of adult zebrafish.

The retinas of adult teleost fish can regenerate neurons following a chemical or mechanical injury. Previous studies have demonstrated that mechanical excision of fish retina induces a hyperplasia within the retinal sheet, including the formation of a proliferative blastema from whence new retinal cells are produced to fill the excision site. The current study was designed to address two issues regarding injury-induced retinal hyperplasia: (1) Retinas of adult zebrafish can regenerate following a surgical excision, but compared to other fish they contain very few proliferative cells: Might retinal injury in adult zebrafish therefore induce minimal, or perhaps no, hyperplasia? (2) The fate of injury-induced, proliferative retinal cells outside surgical excision sites has yet to be determined. Do such cells produce retinal neurons? Evidence is presented that mechanical injury to the adult zebrafish retina induces a dramatic increase in the number of proliferative cells both within and external to the lesion site, and some of these cells apparently migrate within the radial dimension of the retina. Evidence is also presented that injury-induced proliferative cells outside a lesion site can produce retinal neurons--including cone photoreceptors, interplexiform cells, and amacrine cells--that are incorporated into the extant retina. The results suggest that the adult zebrafish retina contains a latent population of cells that is induced to proliferate following retinal injury, and that these cells might represent a novel avenue for pluripotent neurogenesis within the intact adult teleost retina.

Induction of lef1 during zebrafish fin regeneration.

Because the transcription factor Lef1 is important for development of several vertebrate organs but has not been investigated for involvement in epimorphic regeneration, we examined its expression during regeneration of amputated adult zebrafish caudal fins. We found that lef1 is markedly up-regulated in the newly formed wound epidermis of the fin regenerate and is maintained in the basal epidermal layer during formation of the regeneration blastema. During regenerative outgrowth, lef1 expression is strongest in epidermal cells adjacent to newly aligned scleroblasts that secrete bone matrix, while it is low or undetectable in epidermis adjacent to mesenchymal areas with either mature bone or proliferative distal blastema cells. This localization is similar to that of the putative fin ray patterning signal Shh. In addition, brief treatments of fin regenerates with retinoic acid or the synthetic Fgfr1 inhibitor SU5402 down-regulate epidermal lef1, similar to their effects on shh. These results suggest a role for Lef1 in scleroblast alignment analogous to that proposed for Shh. Other Wnt signaling pathway members wnt3a, wnt5, and beta-catenin are also expressed in the fin regenerate. Our data suggest that Lef1 has specific roles in inducing and patterning vertebrate regenerating tissue.

Roles for Fgf Signaling during Zebrafish Fin Regeneration

Following amputation of a urodele limb or teleost fin, the formation of a blastema is a crucial step in facilitating subsequent regeneration. Using the zebrafish caudal fin regeneration model, we have examined the hypothesis that fibroblast growth factors (Fgfs) initiate blastema formation from fin mesenchyme. We find that fibroblast growth factor receptor 1 (fgfr1) is expressed in mesenchymal cells underlying the wound epidermis during blastema formation and in distal blastemal tissue during regenerative outgrowth. fgfr1 transcripts colocalize with those of msxb and msxc, putative markers for undifferentiated, proliferating cells. A zebrafish Fgf member, designated wfgf, is expressed in the regeneration epidermis during outgrowth. Furthermore, we show that a specific inhibitor of Fgfr1 applied immediately following fin amputation blocks blastema formation, without obvious effects on wound healing. This inhibitor blocks the proliferation of blastemal cells and the onset of msx gene transcription. Inhibition of Fgf signaling during ongoing fin regeneration prevents further outgrowth while downregulating the established expression of blastemal msx genes and epidermal sonic hedgehog. Our findings indicate that zebrafish fin blastema formation and regenerative outgrowth require Fgf signaling.

Analysis of Gene Expressions during Xenopus Forelimb Regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.

Extent of ossification at the amputation plane is correlated with the decline of blastema formation and regeneration in Xenopus laevis hindlimbs.

Xenopus laevis larvae gradually lose the ability to regenerate lost hindlimb structures as they progress through metamorphosis. Previous studies have suggested that this loss of regenerative capacity occurs in a proximal-to-distal fashion. We assessed the quality of overall regeneration and early bud blastema formation in order to evaluate previous explanations for this loss of regenerative ability in Xenopus. We further examined the extent to which epidermis, basement membrane, dermis, cartilage, bone, periosteum, and accumulated mesenchyme within the blastema are involved in the decline of regenerative abilities during mid-metamorphic stages of development. Each tissue was scored based on its contributions to the regeneration blastema, in accordance with previously reported blastemal descriptions. Tadpoles amputated at the ankle and tarsal-metatarsal joints scored objectively higher within the overall regeneration and blastema quality rating systems. Both joint sites met more criteria associated with regeneration-capable blastemas than tadpoles amputated through the middle of the tarsus, especially at later stages of metamorphosis. The three amputation sites studied began to vary in their ability to regenerate skeletal elements and to generate productive blastemas during the same stages at which we initially observed ossification of the tarsus. These results suggest that the decline of Xenopus hindlimb regeneration does not occur in a strictly proximal-to-distal fashion but rather is dependent at later stages on the state of ossification of the structure through which amputation occurs. Our morphological and cellular observations reveal specific times and places during Xenopus hindlimb development at which further investigations into tissue-specific molecular events during early regeneration should be focused.

Fragmenting oligochaete Enchytraeus japonensis: a new material for regeneration study.

Enchytraeus japonensis, a recently described terrestrial oligochaete, reproduces asexually by fragmentation and subsequent regeneration. Taking notice of its high potential as a new material for regeneration study, detailed studies were undertaken on the regeneration and reproduction of E. japonensis. The full-grown body divided into 6-13 fragments that regenerated into complete individuals in 4 days, grew to full length in 10 days, and then fragmented again. Regeneration of the head and tail was epimorphic, involving blastema formation, while old segments in the regenerating fragment morphallactically transformed into the appropriate segments to retain the proper body proportions, which could be visualized by histochemistry for alkaline phosphatase. Artificially cut fragments regenerated either normally or into dicephalic monsters with biaxial heads depending on the conditions. Fragmentation could be induced by decapitation, and sexual reproduction was also found inducible in the laboratory. These findings, together with its simple metameric morphology and ease of culture and handling, suggest that E. japonensis is an excellent material for studying animal regeneration.

RA regulation of keratin expression and myogenesis suggests different ways of regenerating muscle in adult amphibian limbs.

Formation of a regeneration blastema following limb amputation is believed to occur through a process of dedifferentiation. It has been suggested, however, that the cells contributed to the blastema by the stump muscle are satellite-like cells, rather than cells originated by dedifferentiation. We have previously shown that simple epithelial keratins 8 and 18 are expressed in the mesenchymal progenitor cells of the regenerating amphibian limb and in cultured cells with myogenic potential, and that their expression appears to be causally related to changes in proliferation and differentiation. We show here that retinoic acid (RA) affects the expression of these keratins differently in myogenic cells originated from normal limb and limb blastema. Furthermore, we find that the effects of RA on proliferation, myogenic differentiation and adhesion of these cells also differ. In fact, whereas RA does not affect keratin expression, proliferation or myogenic differentiation in blastemal cells, it does decrease keratin levels and thymidine incorporation and increase myogenesis in cells from normal limb. Conversely, RA increases cell adhesion only in blastemal cells. Significantly, these effects of RA on cultured cells are consistent with those observed in vivo. Overall the results presented here suggest that in the urodele limb there are two distinct cell populations with myogenic potential, one originating from dedifferentiation and one equivalent to the satellite cells of the mammalian muscle, which are likely to be primarily involved in blastema formation and muscle repair, respectively.

The role of dorsoventral interaction in the onset of planarian regeneration.

During planarian regeneration, dorsal and ventral tissues adhere to each other as a result of wound closure. To investigate the role of dorsoventral (DV) interaction in regeneration, dorsal and ventral portions were ectopically reoriented relative to each other by grafting. We sucked a small piece of planarian body into a Pasteur pipette, and then grafted the piece to the original position in DV-reversed orientation. 4 days after grafting, a blastema-like white region was formed on the boundary between the host and the graft. The region grew and formed a cup-shaped projection on both the dorsal and ventral sides of the worm after about 1 week. Histological analysis and in situ hybridization using dorsal and ventral markers revealed that the grafted piece maintained its original DV polarity. Analysis using DV boundary markers clearly indicated that a DV axis was newly established within the projection. Chimeric analysis using a strain-specific marker demonstrated that the projection grew on the DV boundary. Further analysis, using a marker specific for neoblasts and differentiating cells, showed that the projection was composed of neoblasts and/or neoblast-derived cells. Thus, ectopic DV interaction can cause blastema formation, outgrowth and establishment of a DV axis. In addition, staining with tissue- and region-specific markers showed that the projection appears to be identical to the structure from the grafted position to the most anterior or posterior tip. These results suggest that DV interaction evoked by wound closure has a important role in the onset of regeneration, especially for blastema formation and establishment of the DV axis during planarian regeneration.

Inhibition of Chondrogenesis byWntGene Expressionin Vivoandin Vitro

The Wnt family of secreted signaling proteins are implicated in regulating morphogenesis and tissue patterning in a wide variety of organ systems. SeveralWntgenes are expressed in the developing limbs and head, implying roles in skeletal development. To explore these functions, we have used retroviral gene transfer to expressWnt-1 ectopically in the limb buds and craniofacial region of chick embryos. Infection of wing buds at stage 17 and tissues in the head at stage 10 resulted in skeletal abnormalities whose most consistent defects suggested a localized failure of cartilage formation. To test this hypothesis, we infected micromass cultures of prechondrogenic mesenchymein vitroand found that expression ofWnt-1 caused a severe block in chondrogenesis.Wnt-7a, a gene endogenously expressed in the limb and facial ectoderm, had a similar inhibitory effect. Further analysis of this phenomenonin vitroshowed thatWnt-1 andWnt-7a had mitogenic effects only in early prechondrogenic mesenchyme, that cell aggregation and formation of the prechondrogenic blastema occurred normally, and that the block to differentiation was at the late-blastema/early-chondroblast stage. These results indicate that Wnt signals can have specific inhibitory effects on cytodifferentiation and suggest that one function of endogenous Wnt proteins in the limbs and face may be to influence skeletal morphology by localized inhibition of chondrogenesis.

Expression of integrins during axolotl limb regeneration.

Limb regeneration in urodeles is achieved through the dedifferentiation of tissues at the amputation plane and through the production of the blastema. This tissue breakdown is possible by extensive alterations in molecules of the extracellular matrix. In this respect we describe the regulation of several integrins during such events. It was found that alpha 1 and beta 1 integrins were down-regulated as blastema formation proceeded. In contrast, the expression of alpha 3, alpha 6 and alpha v integrins were upregulated in the blastema. These data are consistent with the roles of integrins in developmental phenomena and are discussed in light of the mechanisms of dedifferentiation.

Expression of type XII collagen by wound epithelial, mesenchymal, and ependymal cells during blastema formation in regenerating newt (Notophthalmus viridescens) tails.

Previously we showed that type XII collagen (col XII) is highly upregulated in the regenerating newt (Notophthalmus viridescens) forelimb. Here, using immunohistochemistry and in situ hybridization, we studied the pattern of expression of col XII during early stages of adult newt tail regeneration. The results show that immunoreactivity of col XII is first seen as a thin layer beneath the wound epithelium (WE) at 3 days after amputation. Reactivity associated with the mesenchyme becomes obvious at day 4 and increases considerably between days 6 and 7 after amputation. In situ hybridization indicates that the early WE-associated reactivity and later mesenchymal reactivity are due to increased col XII gene expression by the WE and mesenchyme, respectively. At 7 days after tail amputation both wound epithelial and mesenchymal cells exhibit a strong riboprobe signal. Interestingly, a distinct riboprobe signal is also seen in the cells of the outgrowing ependymal tube at day 7 but little if any col XII immunoreactivity is present. The spatial pattern of col XII gene expression changes by day 14 after amputation in that transcription in mesenchyme is maintained at a high level, in the WE it is reduced, and in ependyma it ceases to be detectable. Local deprivation of the spinal cord significantly lowers the level of col XII message in the mesenchyme. Much of this decrease in transcription is due to minimal mesenchymal cell accumulation secondary to spinal cord ablation. The temporal and spatial patterns of expression of the col XII gene in the WE, mesenchyme, and ependyma during tail regeneration strongly suggest a role for col XII in regulating both spinal cord outgrowth and spinal cord-dependent tail regeneration.

Perspective: a suggested role for basement membrane structures during newt limb regeneration.

Interactions between epithelium and mesenchyme, which occur across a basement membrane (BM) zone, are essential to generate a growth bud, or blastema, from which a new limb regenerates. An intact BM at that interface is believed to inhibit regeneration, but that mechanism of inhibition is not understood. Interference contrast microscopy and antibodies to laminin have been used to describe reformation of the BM and the basal lamina (BL) and their relationships to wound epithelium and mesenchyme in successive stages of blastema formation. The BL is initially absent from the amputation surface and is reestablished to continuity by the late bud stage of regeneration. It forms generally from base to apex, precedes reticular lamina (RL) formation, and is absent beneath most of the wound epithelium. Our inability to correlate mesenchymal cell accumulation exclusively with the area lacking BL apically and postaxially prompted rethinking of the significance of the BL. Consistent with these and other observations, we suggest that the BL, when it forms during blastema formation, appears to function as in other developing systems to stabilize the phenotype of adjacent cells. Thus, epithelium becomes epidermis and adjacent mesenchyme synthesizes RL and becomes dermis. Accordingly, the feature that distinguishes regenerating from nonregenerating appendages is the ability of regenerating appendages to delay BL closure until after a critical mass of mesenchymal cells has accumulated.