Reduction in cadmium-induced toxicity by c-Jun modulation in mouse embryo limb bud cells.

BACKGROUND: While it is known that cadmium-exposed embryonic cells have increased activation of c-Jun N-terminal kinase (JNK), the role of this stress signaling pathway in the embryotoxic response is not clear. Thus, the effects of modification of the transcription factor c-Jun, one of the downstream targets of JNK, on cadmium-induced embryotoxicity were investigated in primary cultures of mouse embryo limb bud cells. METHODS: Cultures of limb bud cells harvested on day 11 of gestation were pretreated with antisense oligonucleotides (ASO) to c-Jun to reduce its expression, and then incubated with cadmium in the form of cadmium chloride. Toxicity was measured through assessments of cell proliferation and differentiation, while the effectiveness of the ASO in reducing c-Jun was assessed through Western blotting using phosphorylation-specific antibodies. RESULTS: When cells were treated with ASO c-Jun, the total amounts of c-Jun and also cadmium-induced c-Jun activation were diminished. Cadmium-induced cytotoxicity, indicated by reduced cell numbers and differentiation, was found to decrease when cells were exposed to the antisense oligonucleotides to c-Jun. In addition, limb cell numbers and differentiation were also enhanced by exposure to ASO in the absence of cadmium. CONCLUSION: The JNK pathway, and particularly the downstream effector c-Jun, appears to play an important role in regulating cell survival and differentiation in mouse embryo limb bud cells both in the presence and absence of the toxic metal cadmium.

Transglutaminase 2 regulates early chondrogenesis and glycosaminoglycan synthesis.

The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-ß activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering.

Coordinated and sequential activation of neutral and acidic DNases during interdigital cell death in the embryonic limb.

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.

Functional analysis of chick heparan sulfate 6-O-sulfotransferases in limb bud development.

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O-sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O-sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS-6-O-sulfotransferases (HS6ST-1 and HS6ST-2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf-8 and Fgf-10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST-2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf-8 and Fgf-10 expressions than that achieved with HS6ST-1 RNAi. HS6ST-1 RNAi and HS6ST-2 RNAi caused a significant but distinct reduction in the levels of different 6-O-sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6-O-sulfation of HS play essential roles in chick limb bud development.

Alterations in the temporal expression and function of cadherin-7 inhibit cell migration and condensation during chondrogenesis of chick limb mesenchymal cells in vitro.

Endochondral bone formation requires a complex interplay among immature mesenchymal progenitor cells to form the cartilaginous anlagen, which involves migration, aggregation and condensation. Even though condensation of chondrogenic progenitors is an essential step in this process, its mechanism(s) has not been well studied. Here, we show that cadherin-7 plays a central role in cellular condensation by modulating cell motility and migration. In this study, many mesenchymal cells failed to migrate, and precartilage condensation was inhibited, after knockdown of endogenous cadherin-7 levels. Exposure of mesenchymal cells to SB203580 (a specific inhibitor of p38MAPK), LiCl (an inhibitor of GSK-3beta) or overexpression of beta-catenin resulted in inhibition of cadherin-7 levels and, subsequently, suppression of cell migration. Collectively, our results suggest that cadherin-7 controls cell migration in chick limb bud mesenchymal cells, and that p38MAPK and GSK signals are responsible for regulating cadherin-7-mediated cell migration.

Thalidomide induces limb anomalies by PTEN stabilization, Akt suppression, and stimulation of caspase-dependent cell death.

Thalidomide, a drug used for the treatment of multiple myeloma and inflammatory diseases, is also a teratogen that causes birth defects, such as limb truncations and microphthalmia, in humans. Thalidomide-induced limb truncations result from increased cell death during embryonic limb development and consequential disturbance of limb outgrowth. Here we demonstrate in primary human embryonic cells and in the chicken embryo that thalidomide-induced signaling through bone morphogenetic proteins (Bmps) protects active PTEN from proteasomal degradation, resulting in suppression of Akt signaling. As a consequence, caspase-dependent cell death is stimulated by the intrinsic and Fas death receptor apoptotic pathway. Most importantly, thalidomide-induced limb deformities and microphthalmia in chicken embryos could be rescued by a pharmacological PTEN inhibitor as well as by insulin, a stimulant of Akt signaling. We therefore conclude that perturbation of PTEN/Akt signaling and stimulation of caspase activity is central to the teratogenic effects of thalidomide.

Essential role of heparan sulfate 2-O-sulfotransferase in chick limb bud patterning and development.

The interactions of heparan sulfate (HS) with heparin-binding growth factors, such as fibroblast growth factors (FGFs), depend greatly on the chain structures. O-Sulfations at various positions on the chain are major factors determining HS structure; therefore, O-sulfation patterns may play a crucial role in controlling the developmental and morphogenetic processes of various tissues and organs by spatiotemporally regulating the activities of heparin-binding growth factors. In a previous study, we found that HS-2-O-sulfotransferase is strongly expressed throughout the mesoderm of chick limb buds during the early stages of development. Here we show that inhibition of HS-2-O-sulfotransferase in the prospective limb region by small inhibitory RNA resulted in the truncation of limb buds and reduced Fgf-8 expression in the apical ectodermal ridge. The treatment also reduced Fgf-10 expression in the mesenchyme. Moreover 2-O-sulfated HS, normally abundant in the basement membranes and mesoderm under ectoderm in limb buds, was significantly reduced in the treated buds. Phosphorylation levels of ERK and Akt were up-regulated in such truncated buds. Thus, we have shown for the first time that 2-O-sulfation of HS is essential for the FGF signaling required for limb bud development and outgrowth.

Cathepsin D gene expression outlines the areas of physiological cell death during embryonic development.

The implication of lysosomes in the activation of physiological cell death (PCD) was proposed some decades ago. In this work, we show that the expression of the lysosomal enzyme cathepsin D is up-regulated in developing tissues undergoing apoptosis. By comparing vital and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) labeling patterns with in situ hybridization for this gene in a variety of tissues and organs, we show that this procedure constitutes a reliable technique to map the regions of PCD in the embryo. Using this methodological approach, we report the occurrence of two new areas of PCD in the developing limb.

IkappaB kinase-alpha acts in the epidermis to control skeletal and craniofacial morphogenesis.

IkappaB kinase-alpha (IKK-alpha) exhibits protein-kinase-dependent and -independent functions. Its kinase activity is required for lymphoid organogenesis and mammary gland development, whereas a kinase-independent activity is required for epidermal keratinocyte differentiation. In addition to failed epidermal differentiation, IKK-alpha-deficient mice exhibit abnormal skeletal and craniofacial morphogenesis. As similar defects are not exhibited by mice that experience systemic inhibition of NF-kappaB, we postulated that the morphogenetic defects in IKK-alpha-deficient mice are not caused by reduced NF-kappaB activity but instead are due to failed epidermal differentiation that disrupts proper epidermal-mesodermal interactions. We tested this hypothesis by introducing an epidermal-specific Ikka (also known as Chuk) transgene into IKK-alpha-deficient mice. Mice lacking IKK-alpha in all cell types including bone and cartilage, but not in basal epidermal keratinocytes, exhibit normal epidermal differentiation and skeletal morphology. Thus, epidermal differentiation is required for proper morphogenesis of mesodermally derived skeletal elements. One way by which IKK-alpha controls skeletal and craniofacial morphogenesis is by repressing expression of fibroblast growth factor (FGF) family members, such as FGF8, whose expression is specifically elevated in the limb bud ectoderm of IKK-alpha-deficient mice.

Protein phosphatase 2A as a new target for morphogenetic studies in the chick limb.

The family of ser/thr protein phosphatases 2A (PP2A) is a major regulator of cell proliferation and cell death and is critically involved in the maintenance of homeostasis. In order to analyse the importance of PP2A proteins in apoptotic and developmental processes, this review focuses on previous studies concerning the role of PP2A in morphogenesis. We first analyse wing formation in Drosophila, a model for invertebrates, then chick limb bud, a model for vertebrates. We also present a pioneer experiment to illustrate the potential relevance of PP2A studies in BMP signalling during chicken development and we finally discuss the BMP downstream signalling pathways.

Embryonic expression of cholesterogenic genes is restricted to distinct domains and colocalizes with apoptotic regions in mice.

Cholesterol biosynthesis has been assumed to be an ubiquitous process in vertebrate organisms. Here we present data demonstrating that expression of key enzymes of cholesterol biosynthesis is restricted to specific tissues during embryonic development. Distinct expression starts in the dorsal neural tube at embryonic day 8 and is later detected in dorsal root and cephalic ganglia, in the pharyngeal pouches and limb buds. In the limb, expression becomes progressively restricted to interdigital regions during differentiation. Caspase3 whole mount immunostaining revealed that cholesterol biosynthesis colocalizes with apoptotic regions that are targets of the morphogenic signal Sonic hedgehog. This expression pattern correlates closely with the shared phenotypic features of cholesterol biosynthesis and hedgehog mutants.

Caspase-3 mediates retinoid-induced apoptosis in the organogenesis-stage mouse limb.

BACKGROUND: Caspases are key mediators in the regulation and execution of apoptosis, a crucial part of the morphogenetic process during limb development. Caspase-8 and -9 are upstream caspases. Caspase-8 mediates the extrinsic pathway of apoptosis triggered by signaling through TNF-R1 family receptors. Caspase-9 is activated during the intrinsic pathway downstream of mitochondria. Caspase-3 is an effector caspase that initiates degradation of the cell in the final stages of apoptosis. Vitamin A is a potent teratogen that causes limb reduction defects in embryos exposed during organogenesis. Previous in vitro studies have shown that exposure of the organogenesis-stage murine limb to vitamin A results in excessive levels of apoptosis. The goal of this work was to characterize the involvement of caspase-3, -8, and -9, as well as cytochrome-c release from the mitochondria, in the apoptotic cascade induced by vitamin A. METHODS: Limb buds from gestational day 12 CD-1 mice were cultured in a chemically defined medium in the absence or presence of vitamin A. Cultures were terminated after 6 days to examine the effect of the drug on gross morphology. Apoptosis was detected by TUNEL staining after culture for 24 hr. Caspase activation was determined by Western blotting and localized by immunohistochemistry of control and treated limbs. The release of cytochrome-c into the cytoplasm was assessed by Western blotting after cell-fractionation. RESULTS: Limbs cultured in the presence of vitamin A showed a dose-dependent growth reduction and dysmorphogenesis of the cartilaginous anlagen. Apoptosis was increased in the interdigital, anterior, and posterior marginal zones and in the apical ectodermal ridge. Western-blotting confirmed the presence of activated caspase-3 that increased with time in culture and vitamin A concentration. Cleaved caspase-3 immunoreactivity colocalized with TUNEL stained limb regions and increased dramatically with increasing drug concentrations. In contrast, procaspase-8 and -9 were not activated. Exposure to high concentrations of vitamin A did, however, increase cytoplasmic cytochrome-c, suggesting mitochondrial involvement. CONCLUSIONS: Caspase-3 is a key effector caspase in the apoptotic pathway induced by Vitamin A. While caspases-8 and -9 are not responsible for the activation of caspase-3 in response to the drug, cytochrome-c release from mitochondria may play an upstream role.

Expression of ADAMTS1 during murine development.

ADAMTS1/METH1 belongs to the ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteins that currently comprises 18 members. Targeted inactivation of the ADAMTS1 gene results in morphological defects in the kidney, adrenal gland, and adipose tissue in addition to growth retardation and infertility in females. To gain further insight on the biology of ADAMTS1, we examined its expression pattern in the developing mouse from embryonic day 10 (E10) to E18. Expression analysis by RNase protection assays revealed detectable levels of ADAMTS1 transcripts in E10-E18 yolk sac, placenta, brain, heart, lung, limb bud, liver, spleen, and kidney, with much lower levels in the adult. Using in situ hybridization, we have localized ADAMTS1 transcripts predominantly to the epithelium of the developing lung, pancreas, kidney and to a subset of neurons in a temporally restricted manner. Expression was also detected in the tunica media of the aorta, pulmonary, and hepatic vessels.

Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development.

BACKGROUND: Glutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter gamma-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development. RESULTS: Our analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds. CONCLUSIONS: Some of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated.

Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development.

During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located approximately 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.

The SH2 tyrosine phosphatase shp2 is required for mammalian limb development.

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.

Limb and skin abnormalities in mice lacking IKKalpha.

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.

Cytochemical localization of adenylate cyclase in the limb buds of Bufo bufo.

The importance of cyclic nucleotides in the regulation of the processes of differentiation and embryonic development is known. The possible role that cyclic adenosine monophosphate (cAMP) plays during the development of the posterior limb of Bufo bufo is studied by the cytochemical localization of adenylate cyclase (AC), an enzyme that catalyzes the synthesis of the cyclic nucleotide. The method is based on the reaction between the enzyme AC and its specific substrate AMP-PNP (5'-adenylylimidodiphosphate) in the presence of lead. The lead precipitates that form as secondary reaction products are evidence of enzymatic activity. Reaction products are present only at the epithelial level in the limb bud; initially, such products are visible only at the base of the bud, particularly on the epithelial fascia located at the boundary with the body. During successive elongation and toe formation, AC activity is only present on the cells of the proximal portion of each new segment. Enzymatic activity is never present in correspondence to the ectodermal apical crest. cAMP is probably not involved in the processes of cellular proliferation but, rather, in the processes of inducing differentiation of the internal mesenchymal cells.

A comparative review of the sites of histoenzymatic dephosphorylating activities and of gene expression in developing limb buds of mammalian and avian species.

The sites of dephosphorylating activities histochemically demonstrated in developing limb buds of mammalian and avian species have been reviewed and compared with the pattern of gene expression or of other biochemical properties reported at similar stages in the corresponding sites. Alkaline phosphatase, acid phosphatase, 5' nucleotidase and ATP-phosphohydrolase reactions were studied in mouse, rat and chick embryos. Alkaline phosphatase only was detected in mole limb buds whereas only 5' nucleotidase and ATP-phosphohydrolase were revealed in limb rudiments of macacus rhesus embryos. Five decisive periods or events of limb morphogenesis have been considered successively: (1) the early stages during which the prospective limb constituents acquire limb forming properties and give rise to the young limb buds, (2) the invasion of the limb bud mesoderm by myogenic cells of somitic origin, (3) the ectoderm-mesoderm interactions with particular emphasis on the properties displayed by the apical ectodermal ridge and by the underlying subridge mesoderm of the progress zone, (4) the period of growth and pattern formation along the proximo-distal, anterior-posterior and dorso-ventral axes, with special attention to the properties of the zone of polarizing activity, and (5) the period of tissular predifferentiation particularly as concerns prospective skeletal, musculo-tendinous and connective tissues, with brief comments about growing nerves and blood vessels. At least during the morphogenetic period, most dephosphorylating properties appear independently associated with gene expression or other regional biochemical properties. Many sites of dephosphorylating activity may therefore be considered as interesting markers of ungoing morphogenetic events among which tissue interaction and signalling are frequently concerned.

Healing of incisional wounds in the embryonic chick wing bud: characterization of the actin purse-string and demonstration of a requirement for Rho activation.

Small skin wounds in the chick embryo do not heal by lamellipodial crawling of cells at the wound edge as a skin wound does in the adult, but rather by contraction of an actin purse-string that rapidly assembles in the front row of epidermal cells (Martin, P., and J. Lewis. 1992. Nature (Lond.). 360:179-183). To observe the early time course of actin purse-string assembly and to characterize other cytoskeletal components of the contractile machinery, we have followed the healing of incisional or slash wounds on the dorsum of the chick wing; these wounds take only seconds to create and heal within approximately 6 h. Healing of the epithelium depends on a combination of purse-string contraction and zipper-like closure of the gap between the cut edges of the epithelium. Confocal laser scanning microscope studies show that actin initially aligns into a cable at the wound margin in the basal layer of the epidermis within approximately 2 min of wounding. Coincident with actin cable assembly, we see localization of cadherins into clusters at the wound margin, presumably marking the sites where segments of the cable in adjacent cells are linked via adherens junctions. A few minutes later we also see localization of myosin II at the wound margin, as expected if myosin is being recruited into the cable to generate a contractile force for wound healing. At the time of wounding, cells at the wound edge become transiently leaky, allowing us to load them with reagents that block the function of two small GTPases, Rho and Rac, which recently have been shown to play key roles in reorganiztion of the actin cytoskeleton in tissue-culture cells (Hall, A. 1994. Annu. Rev. Cell Biol. 10:31-54). Loading wound edge epidermal cells with C3 transferase, a bacterial exoenzyme that inactivates endogenous Rho, prevents assembly of an actin cable and causes a failure of healing. No such effects are seen with N17rac, a dominant inhibitory mutant Rac protein. These findings support the view that in this system the actin cable is required for healing-both the purse-string contraction and the zipping up-and that Rho is required for formation of the actin cable.