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Home , Laminin, Tenascin C

Journal of Science 112, 3847-3853 (1999) 3847 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0725

COMMENTARY The -C knockout revisited

Eleanor J. Mackie1 and Richard P. Tucker2,* 1School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia 2Department of Cell Biology and Human Anatomy, University of California at Davis, Davis, California 95616, USA *Author for correspondence ([email protected])

Published on WWW 3 November 1999

SUMMARY

In the past seven years, two groups have independently healing after suture injury of . In both skin and produced tenascin-C-knockout mice. These mice are born corneal wounds, fibronectin expression is abnormally low alive and, originally, were described as showing no in tenascin-C-knockout mice. Finally, in vitro studies abnormalities. More recent studies, many involving indicate that haemopoietic activity is defective in bone pathological intervention, have shown that tenascin-C- marrow from these mice. When examined together, these knockout mice have several defects. The mice exhibit studies provide evidence for precise functions for tenascin- abnormal behaviour, as well as abnormalities in brain C, as well as an explanation for why the sequence of chemistry. They also show defects in structure and repair tenascin-C is so highly phylogenetically conserved. of neuromuscular junctions, in the ability to recover from snake-venom-induced glomerulonephritis and in chemically induced dermatitis. Healing of skin wounds is Key words: Homologous recombination, Phenotype, Extracellular morphologically normal, but the mice exhibit defects in matrix

INTRODUCTION found almost exclusively in the central nervous system (CNS), and tenascin-X, a mammalian tenascin found in some In the early 1980s several labs independently discovered and connective and around blood vessels. Tenascin-C was characterized the that eventually became known cloned from zebrafish, amphibians, birds and mammals, and its as tenascin. Immunohistochemistry revealed tenascin in highly conserved sequences provided further evidence for its everyone’s favorite : around motile cells, fundamental function (Fig. 1; Chiquet-Ehrismann et al., 1994a; at hot spots of proliferation and at sites of branching Erickson, 1994). The precise function(s), however, remained , near inductive events, in cartilage, tendons elusive. Cell culture studies showed that tenascin-C promotes and quite prominently in the developing nervous system. both adhesion and detachment, and can stimulate and inhibit Tenascin was also shown to be upregulated at the margins of cell division, and that its ability to bind to classic healing wounds and in the stroma of many tumours (for depends on the species from which the tenascin-C was isolated reviews see Erickson, 1993a; Tucker, 1994; Chiquet- (for reviews see Erickson, 1993a; Faissner et al., 1994). Some Ehrismann, 1995; Mackie, 1997). The results of in vitro of the differences are probably related to the type of cell under studies were just as titillating. In contrast to most substrate- investigation or the bioassay used. It also seemed likely that at adhesion molecules, purified tenascin has anti-adhesive least some of the contradictory observations would be properties. Most cells can attach weakly to tenascin-coated explained by studies of the many tenascin-C splice variants substrata, but they remain rounded and particularly motile (e.g. Mackie and Tucker, 1992). Many of us were shocked to (e.g. Lotz et al., 1989; Halfter et al., 1989). Tenascin was learn that tenascin-C-knockout mice, among the first to be touted as a ‘magic bullet’, potentially responsible for key made by homologous recombination, were indistinguishable events in systems as diverse as neural-crest morphogenesis from their wild-type littermates (Saga et al., 1992): the and tumour metastasis. tenascin-C-knockout mice were the same size as the controls; The cloning and sequencing of tenascin eventually revealed they were fertile; and cursory histological examinations a family of molecules in which tenascin’s distinctive EGF-like revealed no gross deficits in neuroarchitecture or principal repeats and fibrinogen-like C terminus are separated by a series organ systems (Fig. 2). Not only had -knockout of fibronectin type III repeats (Fig. 1; reviewed by Chiquet- technology failed to clarify a function for tenascin-C, it pointed Ehrismann et al., 1994a). The tenascin discovered first was to a minor or even completely redundant role for this named tenascin-C to distinguish it from tenascin-R, which is (Erickson, 1993b). 3848 E. J. Mackie and R. P. Tucker

EGF-like repeats type III repeats fbg-like domain A

Fig. 2. An adult female tenascin-C-knockout mouse. Although phenotypically normal, tenascin-C-knockout mice have abnormal behaviour and responses to stress and trauma.

B knockout mice. Forsberg et al. (1996) settled the controversy once and for all. They independently constructed a second

Mouse KVEGYSGTAGDSMNYHNGRSFSTYDKDTDSAITNCALSYKGAFWYKNCHR tenascin-C-null mouse that was, like the first, phenotypically Human ...... A...... F...... R.... normal. Pig ...... A...... F...... Chicken R.D...... T...... F...N...... Evidence for the upregulation of other forms of tenascin to Xenopus ...R...... N...... Zebrafish H.G...... T..H..P.....N.N.I.V...... compensate for the missing tenascin-C has not been forthcoming. Saga et al. (1992) considered this possibility and looked for upregulation of tenascin-X (which they called Mouse VNLMGRYGDNNHSQGVNWFHWKGHEYSIQFAEMKLRPSNFRNLEGRRKRA Human ...... H...... tenascin-MHC) by northern blotting: the levels of tenascin-X Pig ...... S...... mRNAs appeared to be as those in the wild-type mice. Cell- Chicken ...... S...... Xenopus ...... TS....I...... culture studies (Sakai et al., 1996) and immunohistochemical C Zebrafish ..I...... S..K...... H.VE.....I..A....F...K..S analysis of tenascin-R in the brains of tenascin-C-knockout Fig. 1. (A) Murine tenascin-C is a modular glycoprotein, composed mice (Steindler et al., 1995) have yielded similar results. Even of 14 EGF-like repeats (red diamonds), a series of fibronectin type III some of tenascin-C’s most adamant supporters have concluded repeats (blue ovals) and a globular fibrinogen-like domain at its C from these studies that mice lacking tenascin-C do indeed terminus. One or more additional fibronectin type III repeats are develop in at least a grossly normal fashion and that, if found in some alternatively spliced isoforms (yellow oval). anything is compensating for the loss of tenascin-C function in (B) Tenascin-C exists in the extracellular matrix as a hexamer in these mice, it is probably not one of the other known . which six chains are covalently linked near their N-termini. We’re back to square one: what does tenascin-C do? Steps (C) Putative functional domains within tenascin-C are highly toward understanding non-redundant functions of tenascin-C, conserved between species, which implies fundamental roles in and explanations for its phylogenetic conservation, have finally development, disease or responses to stress. Here, the 100 residues at come from more-detailed analysis of the behaviour of the the C terminus of mouse (GenBank accession no. JQ1322), human (NP 002151), pig (S19694), chicken (212748) and zebrafish knockout mice and their responses to trauma (Table 1). (CAA61489) tenascin-C are aligned. The partial sequence of a Xenopus tenascin-C sequence (I51647) is included. The human and pig sequences share 99% similarity with mouse, the chicken 98%, TENASCIN-C-KNOCKOUT MICE HAVE ABNORMAL Xenopus 95%, and zebrafish 91%. BEHAVIOUR

Tenascin-C-knockout mice are indeed born alive, fertile and THE FIRST STEP OF KNOCKOUT GRIEF: DENIAL outwardly normal. Their behaviour, however, is markedly abnormal. Fukamauchi et al. (1996) first reported this abnormal The knockout results were sufficiently unexpected that many behaviour four years after the development of the knockout. researchers suspected that the mice described by Saga et al. The delay was largely due to the time required to backcross the (1992) still express some form of tenascin-C or that the animals into a strain that was suitable for such analysis. Normal tenascin-R and tenascin-X are upregulated and compensate for C57BL/6N mice, as well as heterozygous littermates, can swim tenascin-C in the knockouts. Mitrovic and Schachner (1995) for three minutes in a water tank without training. In contrast, published results supporting the former hypothesis. Using many of the tenascin-C-knockouts fail the test and must be immunohistochemistry and in situ hybridization, they detected rescued before drowning. The mutant mice also have aberrant tenascin-C immunoreactivity and transcripts in the brains of behaviour on dry land. Normal mice move about their cages the tenascin-C-knockout mice. The signal was reduced in most frequently during the dark cycle. The tenascin-C- comparison with that evident in wild-type animals, and the knockout mice move about their cages almost incessantly, immunoreactivity appeared to be intracellular and not regardless of the dark-light cycle. In a follow up study extracellular, but the authors reasonably concluded that even Fukamauchi et al. (1998b) showed that tenascin-C-null mice traces of residual tenascin-C could account for the absence of retain exploratory activity in an open-field test. Such phenotypic abnormalities in the knockouts. Unfortunately, this hyperactive behaviour would certainly expose them to hopeful explanation did not withstand further scrutiny: Settles predation or put them in other life-threatening situations more et al. (1997) used a battery of tenascin-C antibodies to frequently; this might explain why tenascin-C has been demonstrate the utter absence of tenascin-C protein in the conserved through vertebrate evolution. Tenascin-C knockout revisited 3849

Table 1. Summary of tenascin-C-knockout mouse phenotypes Study Phenotype Reference Behaviour Hyperlocomotion Fukamauchi et al. (1997b) Poor swimming Fukamauchi et al. (1996) Abnormal circadian rhythm Fukamauchi et al. (1996) Neurochemistry Reduced tyrosine hydroxylase Fukamauchi et al. (1997a) Reduced Y Fukamauchi et al. (1998a) Increased preprotachykinin A Fukamauchi and Kusukabe (1997) Increased Fukamauchi and Kusukabe (1997) Peripheral nerves and Abnormal peripheral nerves Cifuentes-Diaz et al. (1998) nerve regeneration Abnormal Cifuentes-Diaz et al. (1998) Reduced sprouting after BoTx-A Cifuentes-Diaz et al. (1998) CNS injury More in * Steindler et al. (1995) Glomerulonephritis Failure to regenerate Nakao et al. (1998) Dermatitis Increased severity of hapten- Koyama et al. (1998) induced dermatitis Wound healing Reduced fibronectin (skin) Forsberg et al. (1996) Reduced fibronectin () Matsuda et al. (1999) Wounds compressed with fewer Matsuda et al. (1999) migrating keratocytes (cornea) Tumorigenesis Increased monocytes/macrophages Talts et al. (1999) Altered tumour organization Talts et al. (1999) Haemopoiesis Reduced haemopoiesis in vitro Ohta et al. (1998)

*Presented as preliminary evidence.

What insights do these studies give us into the function of barrels do, in fact, form in the absence of tenascin-C (Steindler tenascin-C? Given that the gross architecture of the CNS is et al., 1995). Steindler et al. (1995) also observed the normal in these animals, the problem probably lies at a development of glial scars following a cortical stab wound molecular, and not a morphological, level. In fact, Fukamauchi in the tenascin-C knockout. Tenascin-C immunoreactivity and colleagues (1996, 1997a,b,c, 1998a,b; Fukamauchi and increases dramatically around astroglial scars (Laywell et al., Kusakabe, 1997) have shown that the levels of transcripts 1992). In sections through the stab wounds in the tenascin-C- encoding certain neurotransmitters are altered in the mutants Ð knockout mouse, the authors report that ‘more astrocytes may the levels of tyrosine hydroxylase mRNAs are reduced, and be associated with the wounds in the P13 knockout,’ but they the levels of preprotachykinin A and cholecystokinin failed to present quantitative data. We are left with just a hint mRNAs are increased Ð and that the abnormal behaviour of of a phenotype: an early indication that a basic role for the mice can be temporarily diminished through appropriate tenascin-C is associated with tissue repair. pharmacological approaches. Thus, tenascin-C seems to play a role in the development or maintenance of brain chemistry. Srinivasan et al. (1998) found that the extracellular domain of NERVE LESIONS AND THE NEUROMUSCULAR the β2 subunit of the type IIA brain sodium channel binds to JUNCTION tenascin-C. Their findings suggest that tenascin-C localizes and concentrates these sodium channels; the disruption of brain Tenascin-C is abundant in the developing nervous system (see chemistry in the knockouts isn’t therefore surprising. Tucker et al., 1994). It also persists in parts of the adult CNS associated with and plasticity (e.g. Miragall et al., 1990; Gates et al., 1995) and in the mature peripheral nervous ABNORMAL GLIAL SCARRING? system at nodes of Ranvier (Rieger et al., 1986) and at the neuromuscular junction (NMJ; Daniloff et al., 1989). Steindler et al. (1995) were actually the first to report an Following nerve damage tenascin-C expression increases both abnormal phenotype, albeit an apparently mild one, in the around muscle and in the nerve itself (Sanes et al., 1986; tenascin-C-knockout mice. Given its ability in vitro to inhibit Daniloff et al., 1989; Martini et al., 1990). Given these migration of some cell types but promote the migration of observations as well as reports that antibodies to tenascin-C others (see Faissner, 1997, for a review), tenascin-C might help can inhibit re-innervation of muscle (Mege et al., 1992; establish selective barriers to . The most striking Langenfeld-Oster et al., 1994), it seemed likely that peripheral example of this may be the somatosensory barrel fields found nerves would regenerate and re-innervate muscle less in the mouse cortex. Tenascin-C is abundant in the matrix efficiently in the tenascin-C-knockout mice. bordering the five rows of neuron clusters, or barrels, that are Two groups, working independently but with knockout mice found in the developing cerebral cortex and correspond to the provided by the same laboratory, have studied peripheral nerve five rows of whiskers (Steindler et al., 1989; Crossin et al., regeneration and development in the tenascin-C-null 1990). The knockout mice made it possible to test the mice in considerable detail. Remarkably, Moscoso et al. (1998) hypothesis that the tenascin-C found in the matrix surrounding concluded that tenascin-C is ‘dispensable for major aspects of each barrel is crucial to barrel development. Examination of synaptic development and regeneration,’ whereas Cifuentes- the developing cortex with fluorescent peanut agglutinin, Diaz et al. (1998) concluded that ‘tenascin-C is involved not another marker of these boundaries, revealed that normal only in myelination and outgrowth but also in the 3850 E. J. Mackie and R. P. Tucker formation and stabilization of the NMJ.’ How can we explain kidney (Aufderheide et al., 1987), and its expression is strongly these apparently contradictory conclusions? induced in a variety of forms of glomerulonephritis (Assad Moscoso et al. (1998) carefully describe the size and shape et al., 1993). It was a logical step to investigate the of end plates, the ultrastructure of the nerve terminal, and the pathophysiology of glomerulonephritis in mice lacking presence of typical synaptic antigens in the tenascin-C- tenascin-C. Habu snake venom can be used to induce a model knockout mice and age-matched controls. In each case, the of regenerative glomerulonephritis that involves destruction of tenascin-C-null animals were indistinguishable from the mesangial cells (the specialized glomerular vascular smooth controls. To study the role of tenascin-C in regeneration, they muscle cells) followed about one month later by recovery. used forceps to crush the nerve to the sternocleidomastoid near Nakao et al. (1998) used this approach in tenascin-C-knockout the muscle. After 3, 4, 5 and 7 days, they removed and mice of three different genetic backgrounds and found striking sectioned the muscle, and determined the percentage of end differences, the degree of which varied with background strain. plates that had been reinnervated. The numbers were In the most extreme case (the GRS/A background), the indistinguishable in the controls and mutants. Finally, the progression of disease was irreversible: all of the tenascin-C- authors determined the number of sprouts that developed from knockout animals died within four months because of renal the end plates of partially denervated muscles; once again the failure. tenascin-C-knockout mice resembled the controls. One of the problems with the use of in vivo studies of Cifuentes-Diaz et al. (1998) studied the structure of sciatic knockout mice to elucidate protein function is that the nerves and reported that control mice have fewer unmyelinated biological processes found to be defective are usually complex: fibers than do tenascin-C-null mice. They found that the they involve not only multiple biochemical pathways but also unmyelinated fibers in the knockout mice are often in direct diverse cell types. Thus, mere observation of a pathological contact with one another instead of Schwann cells. End plates phenotype in a knockout animal does not necessarily pinpoint from the levator auris longus (LAL) are also abnormal in the the precise stage of the process at which the missing protein is knockouts. 32% of the end plates examined had unusual critical. This is certainly the case with observations on terminal arborizations, or features associated with degeneration glomerulonephritis in tenascin-C-knockout mice. Glomeruli or regeneration. Cifuentes-Diaz et al. (1998) then injected are simple but highly organized vascular structures. In addition botulinum type-A toxin, which blocks acetylcholine release to the vascular and supporting cells present in normal and induces terminal sprouting, near the LAL of wild-type and glomeruli, infiltrating polymorphonuclear cells and tenascin-C-knockout mice. Only 16% of the nerve terminals macrophages are present in the inflamed . Nakao et examined from the knockout mice exhibited sprouting, in al. (1998) carried out detailed histological and in vitro contrast to 47% in the controls. investigations to determine which of these cell types is affected Many factors might have contributed to the differing by the absence of tenascin-C. Histological investigation of conclusions obtained in the two studies, most notably the fact glomeruli in kidneys from tenascin-C-null mice demonstrated that the two groups used different methods to induce sprouting. early apoptosis of mesangial cells, a deficiency in mesangial Distinct cohorts of are likely to be upregulated in the two proliferation and, subsequently, excessive deposition of models; tenascin-C might be compensated for in one case but extracellular matrix. Cultured mesangial cells from tenascin- not the other. The lessons to be learned from these studies C-null mice showed a reduced rate of proliferation, which include the importance of using multiple approaches to induce could be restored by addition of exogenous tenascin-C. The injury (see the discussion of chemically induced dermatitis and levels of platelet-derived growth factor BB and transforming corneal wound healing below) as well as a need for cautious growth factor β in the inflamed glomeruli of tenascin-C-null interpretation of both negative and positive results. Tenascin- mice are also abnormal, as are the responses of cultured C does something in adult peripheral nerves and at the tenascin-C-deficient mesangial cells to these factors. These regenerating NMJ, and its role might be important enough that observations led to the conclusion that the cellular defect it has contributed to the gene becoming highly conserved in resulting in lack of resolution of glomerulonephritis in diverse species; however, its unique function is subtle enough tenascin-C-null mice is a defect in mesangial cell proliferation, that one has to look in exactly the right place and use the right which might be a consequence of dependence on tenascin-C kind of tools to find it. for normal responses to growth factors.

GLOMERULONEPHRITIS DERMATITIS

Tenascin-C is also expressed in many non-neural tissues, where Several groups have also observed strong induction of expression is often associated with pathological conditions tenascin-C in a variety of inflammatory skin conditions such as neoplasia and inflammation. Researchers have (Mackie et al., 1988; Schalkwijk et al., 1991; Koyama et al., speculated on the role of tenascin-C in inflammatory 1996). Koyama et al. (1998) subjected tenascin-C-knockout conditions, but descriptive studies on expression patterns mice to chemically induced dermatitis to determine whether cannot provide definitive information on function. The tenascin-C plays a critical role in inflammation of the skin. availability of tenascin-C-null mouse strains has allowed Application of 2,4-dinitrofluorobenzene (DNFB) to the skin of investigation of the requirement for this protein in the the external ear of normal mice results in a significant progression of inflammatory conditions in several tissues; such thickening of the skin for five days after treatment, which is studies have yielded some of the most striking phenotypes. followed by a return to normal thickness. The response to Tenascin-C is present in a restricted distribution in normal DNFB involves an early, transient, polymorphonuclear-cell Tenascin-C knockout revisited 3851 infiltration of the dermis, and subsequent infiltration of stroma of wild-type but not tenascin-C-deficient corneas, mononuclear cells by day 5. In tenascin-C-knockout mice the which suggests that tenascin-C plays a role in keratocyte thickening of the ear was significantly greater than in wild-type migration or survival. In wild-type animals, induction of mice, polymorphonuclear cells were still abundant at day 5, fibronectin expression occurred in both models of corneal and aberrant disorganized deposits of extracellular matrix were damage; in contrast, in tenascin-C-deficient animals, although present. induction of fibronectin was observed in perforation wounds, As in the glomerulonephritis model, the magnitude of the it was absent from suture wounds. These results indicate that difference in response between wild-type and tenascin-C- tenascin-C is required for normal recovery from corneal insults knockout animals varies with genetic background. These in which tenascin-C expression is induced. In such wounds, as results might imply that tenascin-C limits the recruitment or in healing skin wounds, tenascin-C appears to influence either survival of polymorphonuclear cells in tissues subjected to a expression of fibronectin or its retention in the extracellular chemical insult. Such a conclusion is overly simplistic, given matrix. The effects of tenascin-C on keratocytes in corneal that after application of 12-O-tetradecanoylphorbol 13-acetate suture wounds might depend on its effect on the abundance of tenascin-C-knockout mice and wild-type mice show an fibronectin. equivalent infiltration by polymorphonuclear cells. The target cell for tenascin-C function in the DNFB model is probably a cell responsible for release of chemoattractant cytokines (e.g. TUMORIGENESIS the keratinocyte), rather than the polymorphonuclear cell itself. Thus, as in the case of glomerulonephritis, the induction of Much of the early inspiration to study tenascin-C resulted from tenascin-C in chemically induced dermatitis appears to reflect its abundant expression in tumours (e.g. Bourdon et al., 1983; a functional role for this protein. In both cases, tenascin-C Chiquet-Ehrismann et al., 1986), and the list of tumour types appears to limit the severity of disease, but in neither case does in which it is identified continues to expand. Considerable it appear to have a direct effect on the inflammatory cells speculation as to the role of tenascin-C in neoplasia has themselves. accompanied the publications on this subject, but once again the availability of tenascin-C-null mice has allowed the first in vivo functional studies to be carried out. Talts et al. (1999) WOUND HEALING IN SKIN AND CORNEA crossed tenascin-C-knockout mice with a strain that spontaneously develops mammary tumours following When, several years ago, investigators observed the dramatic induction of the polyoma middle T oncogene; there was no increase in tenascin-C expression in a variety of tissues difference in any of several parameters relating to rate and undergoing wound repair, it was proposed that tenascin-C degree of development of primary tumours and of metastases plays a critical role in this process (Mackie et al., 1988; Chuong between tenascin-C-null and wild-type animals. The and Chen, 1991; Tervo et al., 1991). Forsberg et al. (1996) organization of the tumour stroma, however, did differ: smaller tested this hypothesis in the tenascin-C-knockout mouse. They tumour cell-nests were evident in tenascin-C-null animals. prepared full-thickness skin wounds by excision of a small There was also a significantly greater infiltration of cells of the circular area of skin, and described wounds as undergoing monocyte/macrophage lineage in the tumour stroma of normal repair on the basis of the morphological appearance of knockout animals. Although these results indicate that (at least cryostat sections. Proliferation, migration and apoptosis rates in this particular mouse model where mice have a poor chance of the important cell types (epidermal keratinocytes, fibroblasts of fighting tumours) tenascin-C does not regulate the and macrophages) appeared to be normal. Deposition of progression of tumorigenesis, a common theme appears to be fibronectin in the wounds was, however, very much reduced in emerging in which tenascin-C modulates matrix organization the granulation tissue of wounds from tenascin-C-null mice. in many tissues. This might indicate that there is a fundamental alteration in matrix organization in wounds from tenascin-C-knockout mice, but the authors did not carry out mechanical testing of HAEMOPOIESIS these wounds to determine whether such an alteration affects the strength of the scar. Tenascin-C is expressed in bone marrow (Mackie et al., 1987); Matsuda et al. (1999) have investigated repair of two it provides an adhesive substratum for haemopoietic cells and different types of corneal wounds in tenascin-C knockout mice: stimulates their proliferation in vitro (Klein et al., 1993; linear perforation wounds, and wounds produced by placement Seiffert et al., 1998). Bone marrow cells from tenascin-C-null of sutures in the cornea. During healing of the perforation mice cultured under conditions favoring either myeloid or wounds, only weak induction of tenascin-C was seen in lymphoid colony formation showed reduced haemopoietic corneas from wild-type mice, and healing in tenascin-C-null activity (Ohta et al., 1998). Given that in vitro haemopoiesis is mice was indistinguishable from that of wild-type mice. In dependent on both the haemopoietic progenitors and the suture wounds, however, tenascin-C induction at the wound supporting stromal cells, Ohta et al. (1998) used crossover margins in wild-type corneas was significantly greater than that cultures to investigate the requirements for tenascin-C in perforation wounds, and there were clear morphological expression in these cell types. They found that tenascin-C differences between wild-type and tenascin-C-null corneas. production by stromal cells is most important, but that Corneas from wild-type animals were thickened at the suture production by both cell types is required for optimal site, whereas tenascin-deficient corneas were compressed. haemopoiesis. Exogenous tenascin-C in the medium caused a Corneal stromal cells (keratocytes) were present in the wound dose-dependent compensation for the lack of endogenous 3852 E. J. Mackie and R. P. Tucker tenascin-C, and the target cells for this effect appear to be glomerulopathies. Virchows Arch. B Cell Pathol. Include. Mol. Pathol. 63, stromal cells rather than haemopoietic progenitors. Despite 307-316. these observations on haemopoiesis in vitro, there is no Aufderheide, E., Chiquet-Ehrismann, R. and Ekblom, P. (1987). Epithelial- mesenchymal interactions in the developing kidney lead to expression of evidence for aberrant haemopoiesis in tenascin-C-null mice in tenascin in the mesenchyme. J. Cell Biol. 105, 599-608. vivo (Ohta et al., 1998). Defects might only become apparent Bourdon, M. A., Wikstrand, C. J., Furthmayer, H., Matthews, T. J. and if the haemopoietic system is placed under stress, for example, Bigner, D. D. (1983). Human -mesenchymal extracellular matrix if there is an increased requirement for blood-derived cells. The antigen defined by monoclonal antibody. Cancer Res. 43, 2796-2805. Chiquet-Ehrismann, R., Mackie, E. J., Adams Pearson, C. and Sakakura, conditions involving inflammation mentioned above are T. (1986). Tenascin: an extracellular matrix protein involved in tissue examples of such conditions, but in no case does the interactions during fetal development and oncogenesis. Cell 47, 131-139. fundamental defect in tenascin-C-deficient animals appear to Chiquet-Ehrismann, R., Kalla, P., Pearson, C. A., Beck, K. and Chiquet, be an inability to recruit blood-derived inflammatory cells. In M. (1988). Tenascin interferes with fibronectin action. Cell 53, 383-390. Chiquet-Ehrismann, R., Hagios, C., and Matsumoto, K. (1994a). The addition, spontaneous mammary tumours in tenascin-C- tenascin gene family. Perspect. Dev. Neurobiol. 2, 3-7. knockout mice contain more cells of the monocyte lineage than Chiquet-Ehrismann, R., Tannheimer, M., Koch, M., Brunner, A., Spring, do those of wild-type mice, the opposite of what would be J., Martin, D., Baumgartner, S. and Chiquet, M. (1994b). Tenascin-C expected if recruitment of this haemopoietic lineage were expression by fibroblasts is elevated in stressed gels. J. Cell Biol. compromised. Thus, there is not yet any evidence that the 127, 2093-2101. Chiquet-Ehrismann, R. (1995). Tenascins, a growing family of extracellular deficiency in haemopoiesis in culture has any significance in matrix . Experientia 51, 853-862. vivo. Chuong, C. M. and Chen, H. M. (1991). Enhanced expression of neural molecules and tenascin (cytotactin) during wound healing. Am. J. Pathol. 138, 427-440. Cifuentes-Diaz, C., Velasco, E., Meunier, F. A,, Goudou, D., Belkadi, L., CONCLUSION Faille, L., Murawsky, M., Angaut-Petit, D., Molgo, J., Schachner, M., Saga, Y., Aizawa, S. and Rieger, F. (1998). The peripheral nerve and the Is tenascin-C a superfluous and redundant protein? At many neuromuscular junction are affected in the tenascin-C-deficient mouse. Cell sites of expression, perhaps it is. However, detailed analysis of Mol. Biol. (Noisy-le-grand) 44, 357-379. Crossin, K. L., Prieto, A. L., Hoffman, S., Jones, F. S. and Friedlander, D. the tenascin-C-knockout mice indicates that tenascin-C plays R. (1990). Expression of adhesion molecules and the establishment of a critical role in the development and/or maintenance of proper boundaries during embryonic and neural development. Exp. Neurol. 109, 6- brain chemistry, a role that is perhaps related to the recent 18. observation that tenascin-C binds to a specific sodium channel. Daniloff, J. K., Crossin, K. L., Pincon-Raymond, M., Murawsky, M., In vitro studies have suggested (e.g. Chiquet-Ehrismann et al., Rieger, F. and Edelman, G. M. (1989). Expression of cytotactin in the normal and regenerating neuromuscular system. J. Cell Biol. 108, 625-635. 1988; Probstmeier and Pesheva, 1999) that tenascin-C plays a Erickson, H. P. (1993a). Tenascin-C, tenascin-R and tenascin-X: a family of role as an anti-adhesive molecule that can balance the adhesive talented proteins in search of functions. Curr. Opin. Cell Biol. 5, 869-876. properties of fibronectin to optimize cell-matrix interactions. Erickson, H. P. (1993b). Gene knockouts of c-src, transforming growth factor In fact, tenascin-C expression itself is linked to mechanical beta 1, and tenascin suggest superfluous, nonfunctional expression of proteins. J. Cell Biol. 120, 1079-1081. stress in vitro (Chiquet-Ehrismann et al., 1994b), and it has Erickson, H. P. (1994). Evolution of the tenascin family Ð implications for recently been shown to be highly elastic (Oberhauser et al., function of the C-terminal fibrinogen-like domain. Perspect. Dev. Neurobiol. 1998). The knockouts confirm this. Both in skin lesions and in 2, 9-19. corneal scars, levels of fibronectin are reduced in the tenascin- Faissner, A., Scholze, A., and Gotz, B. (1994). Tenascin in developing neural tissues: only decoration? Perspect. Dev. Neurobiol. 2, 53- C knockouts, when compared with controls: the knockout mice 66. seem to detect the absence of anti-adhesive tenascin-C and Faissner, A. (1997). The tenascin gene family in axon growth and guidance. compensate for the imbalance in adhesive forces by reducing Cell Tissue Res. 290, 331-341. the expression of an adhesive molecule. Similar changes in Forsberg, E., Hirsch, E., Frohlich, L., Meyer, M., Ekblom, P., Aszodi, A., Werner, S. and Fassler, R. (1996). Skin wounds and severed nerves heal matrix expression could be reported in future studies of neural- normally in mice lacking tenascin-C. Proc. Nat. Acad. Sci. USA 93, 6594- crest cell migration, branching morphogenesis or 6599. chondrogenesis, just a few of the places where tenascin-C is Fukamauchi, F., Mataga, N., Wang, Y. J., Sato, S., Youshiki, A. and expressed in the embryo. Finally, the knockout mice have Kusakabe, M. (1996). Abnormal behavior and neurotransmissions of revealed potential roles for tenascin-C in haemopoiesis, tissue tenascin gene knockout mouse. Biochem. Biophys. Res. Commun. 221, 151- 156. repair and the organization of tumour stroma. 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