Single-stranded antisense RNA probes have been used to study the expression of the metalloproteinase inhibitor TIMP (tissue inhibitor of metalloproteinases), during mouse embryogenesis and in adult tissues. Using a sensitive RNase protection assay, low levels of transcript can be detected in a variety of tissues, including maternal deciduum, embryonic kidney, lung and amnion. Higher levels are seen in osteogenic tissues such as calvaria, while the highest level in any tissue is found in the ovary, though even here expression is an order of magnitude below that observed in growth factor-treated fibroblasts in vitro. Using the technique of in situ hybridization, TIMP transcripts can first be detected in osteogenic tissues in the head and limb at about 15-5 days post coitum, and increase in amount until birth. The high levels of TIMP RNA in the ovary are localized to cells of the corpora lutea.

Collagen is the most abundant protein in vertebrates, being the major constituent of connective tissue extracellular matrices in skin, tendon, cartilage and bone. These tissues, and others, are dynamic structures that undergo extensive remodelling, particularly during embryogenesis and in the adult in response to wound healing, or during breast and uterine involution. The synthesis, assembly and turnover of the extracellular matrix (ECM) must therefore be tightly controlled in order to maintain correct tissue organization (Mullins & Rohrlich, 1983). Furthermore, aberrant turnover of collagenous matrices is associated with disease states such as rheumatoid arthritis, osteoporosis and invasion of tumour cells through basement membranes.

The rate of ECM turnover is determined by the net activity of secreted proteases, which is controlled at three levels: (1) the rate of production of latent enzymes, (2) the activation of latent enzymes by proteolytic processing and (3) the production of specific protease inhibitors (Krane, 1985).

The Mr = 28000 secreted glycoprotein TIMP (tissue inhibitor of metalloproteinases) plays a key role in modulating the local activity of a number of metalloproteinases, including collagenase, stromelysin (proteoglycanase, transin) and gelatinase (Cawston et al. 1981; Murphy & Reynolds, 1985). It inactivates metalloproteinases by forming a tight 1:1 complex with the enzyme molecule (Cawston et al. 1983). The importance of TIMP action in local inhibition of collagenolysis and connective tissue breakdown has been demonstrated by the ability of exogenously added TIMP to reduce the invasiveness of B16/BL6 melanoma cells in amnion invasion assays (Mignatti et al. 1986) and by the observation that antibodies against TIMP promote the ability of chondrocytes and endothelial cells to degrade type I collagen films in vitro (Gavrilovic et al. 1987).

Recent work has shown that expression of metalloproteinases and TIMP in vitro is controlled by diverse extracellular stimuli, including growth factors (Edwards et al. 1985, 1987; Chua et al. 1985; Bauer et al. 1985) tumour-promoting phorbol esters (Murphy et al. 1985; Clark et al. 1985; Brinckerhoff et al. 1986; Edwards et al. 1986; Herrlich et al. 1986; Angel et al. 1987a,b), hormones (Sakyo et al. 1986) and steroids (Clark et al. 1987). In particular, enhanced production of collagenase and TIMP by human MRC-5 fibroblasts in response to basic fibroblast growth factor (bFGF) was found to involve increases in gene transcription measured in nuclear run-off assays (Edwards et al. 1987). An additional agent that may be of considerable importance in the regulation of extracellular matrix turnover in vivo is transforming growth factor beta (TGF-β) which modulates the effects of bFGF, resulting in a reciprocal repression of collagenase expression and enhancement of TIMP transcription (Edwards et al. 1987). This TGF-β-induced alteration of the extracellular proteolytic balance may make a significant contribution towards the overall action of this growth modulator in the promotion of matrix deposition in vitro and in vivo (Ignotz & Massague, 1986; Roberts et al. 1986).

TIMP is encoded by a single gene located on the X chromosome in the mouse (Jackson et al. 1987) and can be expressed by a range of cell types in vitro, including fibroblasts, monocytes and macrophages (Welgus & Stricklin, 1983; Bar-Shavit et al. 1985; Campbell et al. 1987). Cell-type-specific patterns of TIMP expression and inducibility are also observed in the differentiated derivatives of murine embryonic stem cells in vitro (Edwards et al. unpublished). In this paper, we extend this analysis by examining the expression of TIMP transcripts in vivo by RNase protection of embryonic RNAs and in situ hybridization techniques. A comparison of the patterns of expression of genes for specific proteases and protease inhibitors such as TIMP, with those for their inducing agents such as TGF-/J (Heine et al. 1987) and cachectin/tumour necrosis factor and IL-1 (Vlassara et al. 1988) may facilitate an understanding of the way in which these agents cooperate in vivo to regulate morphogenesis and remodelling of the extracellular matrix.

Our results indicate that whilst TIMP is expressed at a low level in many tissues during embryogenesis, transcripts are more abundant in certain tissues undergoing extracellular matrix deposition and remodelling such as sites of osteogenesis. In addition, very high levels of TIMP transcripts are observed in the corpora lutea of the ovary, suggesting that TIMP expression may be important for some aspect of follicular physiology.

Embryos and adult tissues

Embryos and adult tissues were derived from either CBA, (CBAxC57BL/6) or ICR (Harland Sprague Dawley) mice. No significant differences between strains were observed. Noon on the day of vaginal plug is 0-5 days (d) post coitum. For in situ hybridization, both embryos and adult organs were fixed in 4% paraformaldehyde in phosphate-buffered saline, pH 7-4, at 4°C for 16 h, dehydrated and embedded in paraffin wax as described (Nomura et al. 1988). Total RNA was prepared either by a modification of the method of Auffray & Rougenon (1980), or the guanidinium isothiocyanate/cesium chloride method described by Maniatis et al. (1982). Ovaries were pooled from CBA females between 8-5 and 18-5 d of pregnancy and from adult nonpregnant females at all stages of the ovulatory cycle.

Single-stranded RNA probes for nuclease protection and in situ hybridization

The 331 bp Sau3A fragment of the mouse TIMP cDNA clone 16C8a (Edwards et al. 1986) was subcloned into pGEMl. Antisense RNA was generated with T7 polymerase after linearization with Win dill, while sense strand was made with SP6 polymerase after linearization with EcoRI. The murine glyceraldehyde 3-phosphate dehydrogenase (MGAP) probe was derived from a 280 bp HindUl/Psti fragment cloned into pGEM3, using T7 polymerase after linearization with HmdIII.

RNase protection assays

RNase protection assays were performed essentially as described by Zinn et al. (1983). Hybridizations used 10 pg of total RNA isolated from mouse tissues or 2−10 µg of cytoplasmic RNA from Swiss 3T3 fibroblasts along with 2−4×105 ctsmin−1 of TIMP riboprobe and, in some experiments, 104ctsmin−1 of MGAP riboprobe. Probe specific activity was 3×108ctsmin−1µgl. In all assays, 25µg yeast tRNA was included and hybridizations were carried out at 50°C for 18 h. Ribonuclease treatments used 40 µg ml−1 RNase A (Sigma) and 5 units ml−1 RNase T1 (Pharmacia) at 30°C for 30min. Protected fragments were analysed on a 5% polyacrylamide/urea sequencing gel and after drying the gel, autoradiography was carried out using preflashed Kodak XAR-5 film at −70°C for 16 h. Two protected fragments of 245 bases and approximately 255−275 bases were obtained using the MGAP riboprobe. Since the probe was derived from the 3’ end of the MGAP CDNA clone and contained a run of T residues, it is likely that the upper more heterogeneous protected band results from polyadenylated MGAP transcripts and the lower discrete band from sequences that lack polyA tails.

In situ hybridization

This was performed as described (Nomura et al. 1988) with the following modifications: the 35S-labelled probe was used at a concentration of 2×105 ctsmin−1µl−1, RNase treatment was increased to 20 µg ml−1 at 37°C for 30min, and exposure time was increased to 3 weeks (negative strand) and 5 weeks (positive strand control). With these long exposure times some nonspecific hybridization is seen to fetal red blood cells. However, these could be clearly distinguished from surrounding mesenchyme by phase-contrast microscopy.

RNase protection analysis of TIMP expression in embryonic and maternal RNA

Initial experiments used Northern blot analysis to study TIMP expression in a variety of murine embryonic and extraembryonic tissues from which total RNA had been prepared (data not shown). A single 0·9−10kilobase size class of TIMP transcripts was detectable in several tissues, e.g. deciduum and calvaria, but amounts were considerably lower than in mitogen-stimulated fibroblasts (Edwards et al. 1985, 1987) and required long autoradiographic exposure times. We therefore decided to perform RNase protection assays to increase the sensitivity of detection. The results of these experiments are shown in Figs 1 and 2.

Fig. 1

Distribution of TIMP transcripts in embryonic mouse tissues as revealed by RNase protection assays. Assays were performed as described in the Methods section using 10 ng total RNA in each annealing reaction. The dating of the embryonic tissues is to the nearest whole day. An arrow indicates the position of the protected probe fragment. Lane A, undigested riboprobe; lanes B-D, assays using RNA from Swiss 3T3 fibroblasts, which were either quiescent and confluent (lane B), stimulated for 6h with 10 ng ml−1 bFGF (lane C), or stimulated for 6 h with 10 ng ml−1 each of bFGF and TGF-β (lane D); lane E is a control reaction using 25 μg of yeast tRNA. The bottom panel shows a short exposure of the centre panel, in which signals from Swiss 3T3 RNA are within the linear range of film response.

Fig. 1

Distribution of TIMP transcripts in embryonic mouse tissues as revealed by RNase protection assays. Assays were performed as described in the Methods section using 10 ng total RNA in each annealing reaction. The dating of the embryonic tissues is to the nearest whole day. An arrow indicates the position of the protected probe fragment. Lane A, undigested riboprobe; lanes B-D, assays using RNA from Swiss 3T3 fibroblasts, which were either quiescent and confluent (lane B), stimulated for 6h with 10 ng ml−1 bFGF (lane C), or stimulated for 6 h with 10 ng ml−1 each of bFGF and TGF-β (lane D); lane E is a control reaction using 25 μg of yeast tRNA. The bottom panel shows a short exposure of the centre panel, in which signals from Swiss 3T3 RNA are within the linear range of film response.

Fig. 2

Comparison of TIMP transcript levels in selected embryonic, neonatal and maternal tissues as revealed by RNase protection assays. Assays were carried out as described in the legend to Fig. 1, except that MGAP riboprobe was included along with the TIMP probe to act as an internal standard. Lane A, a control reaction using 25 μg yeast tRNA; lanes B and C, assays using 2 ng of cytoplasmic RNA from confluent, quiescent Swiss 3T3 fibroblasts (lane B) and from cells stimulated for 6 h with 10 ng ml−1 bFGF (lane C). Arrows indicate the positions of protected fragments of the TIMP probe (1), and the MGAP probe (2 and 3). Size markers were an AluI digest of pGEM4Z.

Fig. 2

Comparison of TIMP transcript levels in selected embryonic, neonatal and maternal tissues as revealed by RNase protection assays. Assays were carried out as described in the legend to Fig. 1, except that MGAP riboprobe was included along with the TIMP probe to act as an internal standard. Lane A, a control reaction using 25 μg yeast tRNA; lanes B and C, assays using 2 ng of cytoplasmic RNA from confluent, quiescent Swiss 3T3 fibroblasts (lane B) and from cells stimulated for 6 h with 10 ng ml−1 bFGF (lane C). Arrows indicate the positions of protected fragments of the TIMP probe (1), and the MGAP probe (2 and 3). Size markers were an AluI digest of pGEM4Z.

As can be seen from Fig. 1, TIMP mRNA is widely distributed in the tissues examined, but there is evidence of both significant tissue-specificity and temporal regulation of expression. The highest levels of expression were detected in the amnion, calvaria and deciduum (which represents the maternally derived portion of the implantation site before the formation of the definitive placenta), and to a lesser extent in the kidney. In all of these tissues, the level of expression exceeds that detectable in quiescent Swiss 3T3 fibroblasts (lane B), but it is at least an order of magnitude less than that observed in bFGF-stimulated fibroblasts (lane C), or in fibroblasts treated with bFGF and TGF-β, which results in a superinduction of TIMP (lane D), [Edwards et al. 1987], The earliest embryonic stage tissue shown in this figure is the 10·5-day yolk sac, but in other assays we have detected TIMP message by RNase protection in the 7·5- and 8·5·day whole embryo (data not shown). ‘The temporal pattern of TIMP expression in the tissues examined can be divided into three basic classes: first, those in which TIMP expression increases during embryonic development, exemplified by amnion, calvaria, placenta and lung; second, those in which expression appears to decrease, such as kidney, and third, those in which expression appears to change little or not at all during the course of development, e.g. yolk sac, brain and gut. In other tissues such as liver, TIMP expression is negligible.

Fig. 2 shows the expression of TIMP in other embryonic tissues and certain maternal and neonatal structures. In these experiments, a second probe that detected transcripts of mouse glyceraldehyde 3-phosphate dehydrogenase (MGAP), was included as a control for the amount of RNA used in each hybridization. Earlier studies have shown that MGAP is expressed at similar levels in all embryonic tissues studied (Nomura et al. 1988). High levels of TIMP expression were observed in the adult adrenal and in the deciduum, which along with the Swiss 3T3 RNA samples, provide a comparison to Fig. 1. Expression in the 14-5 d uterus and 18·5d limb was also high. However, the highest level of TIMP expression was seen in the ovaries of both pregnant and nonpregnant adult mice. The RNA sample from the ovaries of pregnant mice was partially degraded as judged by agarose gel electrophoresis, leading to reduced signals for both MGAP and TIMP transcripts: normalization of MGAP transcript abundance to that observed in all the other samples indicates that TIMP mRNA is equally abundant in both of the preparations from ovaries.

Overall, the results of Figs 1 and 2 reveal that TIMP expression, in addition to being modulated by growth factors in vitro, is subject to differential spatial and temporal regulation in a variety of tissues during normal development.

The experiments described above do not detect sites of local expression within tissues. The identity of particular cell types expressing TIMP transcripts within complex tissues such as developing bone and ovary was accordingly further analysed using in situ hybridization techniques.

Analysis of TIMP expression by in situ hybridization Developing bone

The earliest that TIMP transcripts could be detected by in situ hybridization in sections of embryonic heads and limbs was about 15·5 d p.c. By comparison with the signal observed in adjacent sections hybridized with antisense RNA to 2ar (osteopontin) and SPARC (osteonectin) (Nomura et al. 1988), the level of TIMP RNA was very low and required exposure of sections to photographic emulsion for at least three weeks for clear visualization. Within the limits of detection, TIMP RNA was first seen in cells associated with the formation of membrane bone around cartilaginous elements in the head, and in the calvaria. The level of TIMP RNA in these regions of collagen deposition and bone formation increased between 16·5 d p.c. and birth (Figs 3 and 4, and data not shown).

Fig. 3

TIMP mRNA distribution in developing membrane bone. (A) Detail of a section through the upper part of the head of a 16·5 d p.c. embryo hybridized with negative-strand probe. Note cartilage (CT) surrounded by developing membrane bone. Also shown is a tooth bud (TB) and a whisker follicle (W). Bright-field illumination. (B) Same field as A, photographed under dark-ground illumination. Note high density of silver grains in the region of membrane bone formation. (C) Detail of tooth bud in lower jaw of the same section as A. Bright-field illumination. (D) Same field as C, photographed under dark-ground illumination. Note high density of silver grains in the membrane bone surrounding the tooth bud. Scale bar, 50 μm.

Fig. 3

TIMP mRNA distribution in developing membrane bone. (A) Detail of a section through the upper part of the head of a 16·5 d p.c. embryo hybridized with negative-strand probe. Note cartilage (CT) surrounded by developing membrane bone. Also shown is a tooth bud (TB) and a whisker follicle (W). Bright-field illumination. (B) Same field as A, photographed under dark-ground illumination. Note high density of silver grains in the region of membrane bone formation. (C) Detail of tooth bud in lower jaw of the same section as A. Bright-field illumination. (D) Same field as C, photographed under dark-ground illumination. Note high density of silver grains in the membrane bone surrounding the tooth bud. Scale bar, 50 μm.

Fig. 4

TIMP distribution in calvarial bone of 18·5d p.c. embryo. Detail of a section through the head of 18·5 d p.c. embryo hybridized with negative-strand TIMP probe. Darkground illumination. Note high density of silver grains in the developing calvarium (C). BR, brain. Scale bar, 50 μm.

Fig. 4

TIMP distribution in calvarial bone of 18·5d p.c. embryo. Detail of a section through the head of 18·5 d p.c. embryo hybridized with negative-strand TIMP probe. Darkground illumination. Note high density of silver grains in the developing calvarium (C). BR, brain. Scale bar, 50 μm.

In the embryonic limb, TIMP RNA was first detected by in situ hybridization at about 16·5d p.c. in the ossification collar around the cartilage rudiment and in the region of the primary marrow cavity and initial sites of endochondral bone formation (Figs 3, 4, and 5). This distribution pattern is very similar to that observed with antisense SPARC (Holland et al. 1987; Nomura et al. 1988) with the exception that at no stage could significant levels of TIMP RNAs be detected in proliferating or hypertrophic cartilage.

Fig. 5

TIMP mRNA distribution in the developing limb of a 16·5 d p.c. embryo. (A and B) Detail of a section through the limb of a 16·5 d p.c. embryo hybridized with negative-strand (A) and positive-strand (B) TIMP RNA probe. HC, hypertrophic cartilage. Bright-field illumination. (C and D) Same as A and B but dark-ground illumination. (E and F) Higher power details of C and D. Note high densities of hybridization grains with negative-strand probe in theprimary ossification collar and sites of endochondnal bone formation within the marrow cavity. Only background grains are seen in the region of hypertrophic cartilage. Scale bar, 100 μm (A-D), 50 μm (E and F).

Fig. 5

TIMP mRNA distribution in the developing limb of a 16·5 d p.c. embryo. (A and B) Detail of a section through the limb of a 16·5 d p.c. embryo hybridized with negative-strand (A) and positive-strand (B) TIMP RNA probe. HC, hypertrophic cartilage. Bright-field illumination. (C and D) Same as A and B but dark-ground illumination. (E and F) Higher power details of C and D. Note high densities of hybridization grains with negative-strand probe in theprimary ossification collar and sites of endochondnal bone formation within the marrow cavity. Only background grains are seen in the region of hypertrophic cartilage. Scale bar, 100 μm (A-D), 50 μm (E and F).

Ovary

High levels of TIMP RNA were detected in the corpus luteum of the ovary (Fig. 6), which is an extensively vascularized structure composed of mature granulosa cells specialized for the synthesis and secretion of steroids. Sections of ovary from both adult cycling and pregnant females showed similar patterns of TIMP expression, except that more corpora lutea were present in the ovaries of pregnant animals (Fig. 6). No significant hybridization to thecal cells with antisense TIMP RNA could be detected at any stage. Neither immature nor antral follicles contained detectable levels of TIMP transcripts, and the density of hybridization grains in oocytes was similar with both positive- and negative-strand RNA probes.

Fig. 6

TIMP RNA distribution in the pregnant and nonpregnant ovary. (A and B) Detail of a section through the ovary of a nonpregnant, adult female hybridized with negative-strand TIMP probe and photographed under bright-field (A) and darkground (B) illumination. Note corpus luteum (CL), antral follicle (AF) and two immature follicles. A high density of hybridization grains is seen uniformly throughout the corpus luteum. (C and D) Higher power detail of A and B showing only background levels of grains in the thecal layer (arrows) and oocyte (O). (E) Detail of a section through the ovary of a pregnant female showing several corpora lutea (CL) and an antral follicle (AF). (F) Detail of a section through the ovary of a nonpregnant female hybridized with negative-strand probe. Scale bar: 100 μm (A,B,E and F); 50 μm (C and D).

Fig. 6

TIMP RNA distribution in the pregnant and nonpregnant ovary. (A and B) Detail of a section through the ovary of a nonpregnant, adult female hybridized with negative-strand TIMP probe and photographed under bright-field (A) and darkground (B) illumination. Note corpus luteum (CL), antral follicle (AF) and two immature follicles. A high density of hybridization grains is seen uniformly throughout the corpus luteum. (C and D) Higher power detail of A and B showing only background levels of grains in the thecal layer (arrows) and oocyte (O). (E) Detail of a section through the ovary of a pregnant female showing several corpora lutea (CL) and an antral follicle (AF). (F) Detail of a section through the ovary of a nonpregnant female hybridized with negative-strand probe. Scale bar: 100 μm (A,B,E and F); 50 μm (C and D).

Other embryonic and adult tissues

Sections of a variety of other embryonic and adult tissues were hybridized with both positive- and negative-strand TIMP RNAs. These include tissues such as deciduum, kidney and amnion that gave positive signals in the RNase protection assays (Figs 1 & 2). In these cases, the overall grain density appeared higher in sections hybridized with negative-strand probes compared to positive-strand probes, but no clear localization to a phenotypically distinct subpopulation of cells could be observed.

Two major conclusions can be drawn from the observations described in this paper. First, RNase protection assays show that the TIMP gene is expressed in a number of embryonic tissues, including amnion, kidney, skin and lung as well as the maternal deciduum. However, the steady-state levels of RNA in these tissues are very low; less than or equal to levels observed in unstimulated fibroblasts and at least an order of magnitude less than that observed in stimulated fibroblasts (Figs 1 & 2). Since in situ hybridization failed to localize a specific signal in sections of these tissues, it suggests that most cells contain some TIMP RNA and expression is neither confined to, nor overly expressed in, a subpopulation of cells such as infiltrating macrophages or granulated metrial gland cells. This situation contrasts with that of another growth factorinducible gene 2ar/oesteopontin (Nomura et al. 1988) where, in the deciduum, for example, a minor subpopulation of granulated metrial gland cells express very high levels of transcript within a surrounding population of cells in which transcripts cannot be detected. Attempts to increase the sensitivity of the in situ hybridizations with the TIMP probes that we employed led to increased overall background, with no overall increase in signal-to-noise ratio. The reasons for this are not clear, but may reflect the base composition or nucleotide sequence of the TIMP probe.

The relatively low level expression of TIMP in many tissues may help to maintain a balance between extracellular matrix deposition versus degradation and turnover. Indeed, matrix deposition coupled with growth and expansion is a feature of organs such as the embryonic kidney, in which both the glomerular and tubular basement membranes are being elaborated, and the deciduum in which a pericellular matrix is assembled around the maternal cells (Wewer et al. 1986). The mesodermal and ectodermal components of the murine amnion are also separated by a prominent basement membrane (Hogan et al. 1986). Significant levels of TIMP protein are found in amniotic fluid (Cawston et al. 1981; Stricklin et al. 1986) and this may be derived from the amnion itself during gestation. Nevertheless, these findings do not exclude the possibility that TIMP expression in these tissues may become specifically elevated by local release of inducing agents such as growth factors and TGF-β in response, for example, to wounding or other pathological situations.

The second conclusion to be reached from these studies is that certain tissues contain sufficiently high levels of TIMP transcripts to be detected by RNase protection and localized by in situ hybridization. An important example of this is seen in mesenchymal cells in areas of membrane bone formation in the 16·5 d p.c. embryonic head (Figs 3 and 4) and the ossification collar surrounding the cartilage of the developing limb (Fig. 5). Expression is also detected at later stages in regions of endochondrial bone formation such as the centre of the cartilaginous model of the developing limb (Fig. 5). Bone is a tissue in which matrix deposition and remodelling is particularly important and in which it might be supposed that stringent local control of metalloproteinase activity is required. The resolution of the in situ hybridization technique, and the paucity of appropriate markers does not allow us unambiguously to identify the cell type(s) expressing TIMP in developing bone. The distribution is, however, clearly different from that observed for 2ar/osteopontin (Nomura et al. 1988). It is of interest that recent studies employing both in situ hybridization (Sandberg et al. 1988) and immunohistochemical (Heine et al. 1988) methods have shown that TGF is expressed and localized in regions of endochondrial and membrane bone formation in the mouse and human embryo. This raises the possibility that this local expression of TGF-βduring normal osteogenesis may contribute to coincident elevated levels of TIMP expression.

It is important to note, however, that there is no overall absolute correlation between elevated TIMP levels and sites of TGFβ expression. In particular, TGF-β deposition is marked in tissues such as the dermis (Heine et al. 1988) where TIMP expression is comparatively low. It is clear from our studies of TIMP expression in fibroblastic cells in vitro that TGF-β modulates the actions of other growth factors (Edwards et al. 1987). Alone, TGF-β is a very poor inducer of TIMP, and even this low level and delayed activation may be a secondary consequence of its ability to induce the expression of other growth factors such as platelet-derived growth factor (Leof et al. 1986). Thus, the lack of TIMP expression in vivo in some sites of high TGF-β localization may reflect the absence of other positiveacting growth factor stimuli. Alternatively, tissue- or cell-specific mechanisms may override growth factor signals to silence TIMP expression.

An unexpected finding in this study was the high levels of TIMP transcripts expressed in the corpora lutea of the ovary (Fig. 6). In situ localization suggests that expression is predominant in granulosa cells and must be initiated after ovulation since large antral follicles and thecal cells do not express detectable levels of TIMP. It is conceivable that TIMP protein is required in this situation to prevent premature destruction (luteolysis) of the corpora lutea and may therefore play an important role in the maintenance of pregnancy. It will be of interest to identify whether ovarian reproductive hormones or growth factors are involved in the induction of TIMP expression following ovulation.

Taken together with our previous study of the embryonic expression of another growth factor-inducible gene 2ar/osteopontin, the results presented here demonstrate that genes which are coordinately expressed in vitro in response to growth factor stimulation display specific and distinct patterns of expression in vivo. The analysis of TIMP expression reported here will, however, provide a basis for investigating the regulation of TIMP expression during pathological situations such as osteoporosis and arthritis.

We gratefully acknowledge the advice of Dr A. Saito, Vanderbilt School of Medicine for photography and Gary Paterno for help with RNase protection assays. A.J.W. was supported by a Medical Research Council UK research studentship. J.K.H. and D.R.E. acknowledge financial support from the Cancer Research Campaign. S.N. and B.L.M.H. acknowledge support from the Medical Research Council of Great Britain for the portion of the work carried out at the National Institute of Medical Research, Mill Hill, London.

     
  • bFGF

    basic fibroblast growth factor

  •  
  • ECM

    extracellular matrix

  •  
  • MGAP

    mouse glyceraldehyde 3-phosphate dehydrogenase

  •  
  • TGF-β

    transforming growth factor beta

  •  
  • RNase

    ribonuclease

  •  
  • TIMP

    tissue inhibitor of metalloproteinase

  •  
  • d p.c.

    days post coitum

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