Expression of the IGF-ll/mannose-6-phosphate receptor mRNA and protein in the developing rat

Insulin-like growth factors (IGFs) are peptides that have been implicated in embryonic, fetal and postnatal growth (Sara and Hall, 1984). In the rat, IGF-I, produced by the liver, predominates in postnatal and adult life and its production is principally controlled by growth hormone (Sara and Hall, 1984; Salmon and Daughaday, 1957; Gluckman, 1986; Beck et al. 1987). IGF-II is produced in embryonic, fetal and early neonatal life, being strongly expressed in the visceral yolk sac, in a range of mesodermally derived embryonic tissues and organs and in the liver and choroid plexus (Beck et al. 1987; Beck et al. 1988a,b; Stylianopoulou et al. 1988a,b). In vitro IGF-II has been shown to induce DNA synthesis (Sara and Hall, 1984), promote cell survival (Biddle et al. 1988) and to stimulate sulphate incorporation into heparan and chondroitin sulphate (Salmon and Daughaday, 1957; Zapf et al. 1978). IGF-II may have a major role in the development of embryonic tissues, particularly those of mesodermal origin (Sara and Hall, 1984).

Little is known about the control of IGF-II expression during embryonic life, although placental lactogen (Adams et al. 1983) and glucose levels (Gluckman et al. 1985) have been shown to influence expression in vitro. IGF-II mRNA expression is switched off before adulthood in all tissues except the choroid plexus (Beck et al. 1988a,b; Stylianopoulou et al. 1988b). The increase in circulating levels of corticosteroids that occurs at weaning (Walker et al. 1986) appears to be involved in down-regulating expression, at least in the liver, as treatment of pre-weaning rats with cortisone or dexamethasone quickly and completely switches off hepatic IGF-II synthesis (Beck et al. 19886).

IGF-II can exert insulin-like effects via the insulin and the type-I (IGF-I) receptors (Rechler, 1985). These share considerable structural similarities: both are tyrosine kinases with an α β-dimer structure and substantial homology at the protein and nucleic acid level (Ullrich et al. 1986). IGF-II has lower binding affinity for the insulin receptor than for the IGF-I receptor but it has greatest affinity for a distinct receptor, the type II (IGF-II) receptor (Sara and Hall, 1984; Rechler, 1985; Gammeltoft, 1989), a single chain polypeptide (M_r ∼250k) with a short cytoplasmic domain lacking tyrosine kinase activity (Morgan et al. 1987; Roth, 1988). Cloning and sequencing of cDNAs encoding the type-II receptor revealed close homology with the bovine mannose-6-phosphate receptor and it is accepted that, in both rats and humans, the IGF-II receptor and the cation-independent mannose-6-phosphate receptor are identical (Roth, 1988; MacDonald et al. 1988).

The mannose-6-phosphate receptor is involved in the intracellular transport of lysosomal enzymes through the Golgi apparatus and the scavenging of extracellular lysosomal enzymes (von Figura and Haslik, 1986). Mannose-6-phosphate and IGF-II bind to separate sites on the receptor and binding of mannose-6-phosphate potentiates IGF-II binding (MacDonald et al. 1988). Insulin (Roth, 1988), IGF-I and EGF, (Braulke et al. 1989) acting via their own receptors, cause redistribution of the IGF-II/M-6-P receptor to the cell surface. The function of the receptor with respect to IGF-II activity remains to be elucidated. There is conflicting evidence as to whether the receptor can participate in signal transduction (reviewed by Roth, 1988; Czech, 1989; Gammeltoft, 1989) and it has been suggested that IGF-II exerts metabolic and mitogenic effects solely via the IGF-I receptor (Mottola and Czech, 1984). It has been recently reported that neither the chicken nor the frog mannose-6-phosphate receptors will bind IGF-II and it is likely that in these species the effects of IGF-II are exerted via the type I IGF receptor (Clairmont and Czech; 1989). Studies dissecting the functional domains of the IGF-II/M-6-P receptor have begun. It has been demonstrated that mutations in specific regions of the cytoplasmic tail of the molecule affect the cellular localisation and function of the receptor (Glickman et al. 1989; Lobel et al. 1989) but as yet no ability of the cytoplasmic domain to activate any intracellular signalling pathways has been reported. It may well be that the receptor has a function in the uptake and metabolism of IGF-II by tissues that produce and respond to the peptide (Czech, 1989), thus controlling its local concentration and availability. Another possibility is that the receptor functions as an extracellular carrier protein. A truncated form of the protein is detected in fetal and neonatal serum; this appears to be produced by proteolytic cleavage of the C-terminal of the molecule proximal to the membrane-binding domain as a separate mRNA has not been detected (Kiess et al. 1987; MacDonald et al. 1989). Whilst the serum levels of the truncated receptor are considered to be too low for it to function as a major binding protein (MacDonald et al. 1989), it is possible that it functions locally in tissues with a high level of IGF-II and receptor production.

If the IGF-II/mannose-6-phosphate receptor is important in the role of IGF-II then it would be expected to be expressed at high levels in tissues considered to be targets for IGF-II action. Since IGF-II is the predominant fetal somatomedin in the rat, but is virtually absent except in the central nervous system in the adult (Beck et al. 1987; Beck et al. 1988a,b; Stylianopoulou et al. 1988a,b), we have studied the temporal and spatial distribution of the IGF-II/mannose-6-phosphate receptor during rat embryonic and neonatal development using in situ hybridization (ISH) and immunocytochemistry. We show that the mRNA and protein is expressed at high levels during embryonic development. Expression is principally localised to tissues expressing high levels of IGF-II mRNA and commences at the same time as IGF-II expression.

For Northern blotting, a 2.4 kb cDNA for the rat IGF-II/Mannose-6-phosphate receptor (clone K3. Morgan et al. 1987) was used and a 0.467 kb probe for the mouse 7S ribosomal gene, which should be expressed at an equal level in all cells (Balmain et al. 1982), was used as a control for RNA loading. Probes (10 ng) were labelled by the random priming method (Feinberg and Vogelstein, 1983) to ∼2×10⁹disints min^-lμg^-1. For ISH, the 2.4kb IGF-II/ mannose-6-phosphate receptor cDNA or a 500bp AluI subfragment was cloned into Bluescript (Stratagene) and plasmid prepared by the CsCl method (Maniatis et al. 1982). Linearised template was transcribed using ³²P-UTP (0.5 and 2.4kb template) or ³⁵S-UTP (0.5 kb template) as previously described (Senior et al. 1988). Both sense (–ve control) and antisense transcripts were produced to a specific activity of 0.5–2×10⁹ disints min^-1 μg^-1. Their mean length was reduced to 100–200 bases by alkaline hydrolysis (Cox et al. 1984).

Total RNA was prepared by a modification of the method of Auffray and Rougeon (1980; Beck et al. 1987). Samples of RNA were electrophoresed through 1 % agarose/formaldehyde gels (Maniatis et al. 1982) and transferred to Hybond N (Amersham). The membranes were hybridised for 18h at 42°C in 5×SSPE/50% formamide/l×Denhardt’s/ 0.1 mg ml^-1 salmon sperm DNA/0.5×SDS/6% polyethylene glycol 6000 and washed down to 0.l×SSPE/0.lxSDS at 65°C and autoradiographed at –70°C. For quantitation of mRNA, the filters were stripped by incubation in 5mM Tris–HCl pH7.5/2mM EDTA pH8.0/0.1×Denhardt’s at 65°C, then reprobed under the above conditions with the 7S ribosomal probe. RNA sizes were estimated using BRL RNA markers run on the same gel, transferred to the filter and stained by the methylene blue method (Maniatis et al. 1982).

Conceptuses (pregnancy timed from midnight preceding the appearance of the copulation plug) from 5.5 days (immediately postimplantation) to 20.5 days (immediately prenatal) and a range of tissues and organs from neonatal and adult animals were collected. For cryostat sectioning, the material was orientated in moulds, submerged in OCT cryomountant (R.A.Lamb), frozen at the surface of hexane/dry ice and stored at –40°C. For wax sections, the tissue was fixed for 18–48 h (depending on size of block) in 4% paraformaldehyde in PBS at room temperature, stored in 0.5 M sucrose in PBS then processed and wax embedded by standard methods.

Cryostat sections were cut (9μm), taken up onto subbed slides (Rentrop et al. 1986) and laid on dry ice for >20 min, fixed in 4% paraformaldehyde in PBS at room temperature (20min), rinsed, dehydrated though graded alcohols and dried. Wax sections (5 μm) were dewaxed in xylene, rehydrated through graded alcohols and digested for 10 min at 37°C in 0.125μg ml^-1 Pronase E (Sigma, autodigested for 4h prior to use) in 50 mM Tris–HCl pH 7.5/5 mM EDTA pH 8.0, rinsed in PBS/0.1% glycine, post-fixed in 4% paraformaldehyde in PBS (10 min at room temperature) and dehydrated through graded alcohols and dried. Sections were hybridised for 18 h at 50°C in 0.3 M NaCl/10mM Na₂PO₄ pH 6.8/10mM Tris–HCl pH7.5/5mM EDTA pH8.0/l×Denhardts’/l mg ml^-1 yeast RNA/50% formamide/10% dextran sulphate with the probe at ∼100 ng ml^-1 under coverslips. The slides were washed for 4h in 3 changes of the hybridization buffer without probe, yeast RNA or dextran sulphate, digested for 1h at 37°C with 150μg ml^-1 RNase A (boiled for 1 min prior to addition) in 0.5 M NaCl/10mM Tris–HCl pH 7.5/1 mM EDTA pH 8.0, washed twice for 30min in 2×SSC at 65°C then dehydrated through graded alcohols and dried. ³²P-labelled slides were exposed to Kodak X-Omat film overnight to assess the level and tissue distribution of labelling. The slides were dipped in Ilford K5 emulsion diluted 5 to 3 w/v in water containing 1 ml of glycerol in 60 ml at 45°C. The slides were allowed to dry upright in the dark for at least 2h then exposed desiccated at 4°C for 4–14 days. The slides were developed at 15°C by sequential immersion in Kodak D19 (4min at 160g 1^-1), 1 % acetic acid (1 min), Ilford Hypam fixer (4 min 1 in 5 dilution) and then washed in water. The emulsion was fixed for 10 min in 10% formol-saline, the slides were stained with haematoxylin, dehydrated and cleared and coverslips applied. They were then examined under bright- and dark-field illumination.

Wax-embedded sections prepared as above were dewaxed and rehydrated. They were digested with 0.025% pepsin in 0.01M HC1 for 30–45 min at 37°C and blocked with swine serum (diluted 1 in 5) for 1 h. Rabbit polyclonal anti-IGF-Il/ mannose-6-phosphate receptor antibody (MacDonald et al. 1989) was applied at a 1 in 100 to 1 in 500 dilution in 50 mM Tris–HCl pH 7.5 and the slides were incubated for 72 h at 4°C. The antiserum was washed off in several changes of 50 mM Tris-HCl pH 7.5. The antibody was detected using the biotin/avidin/peroxidase/DAB method (Dakopatts) and the slides were counterstained with haematoxylin, dehydrated, cleared and coverslips mounted with DPX (Lamb). Control sections were stained as above but a) the primary antiserum was omitted, b) the primary antiserum was replaced with normal rabbit serum or c) a rabbit antibody to ACTH (Dakopatts) was used in place of the lGF-Il/mannose-6-phosphate receptor antibody.

Northern blotting using the 2.4 kb cDNA for the rat IGF-II/M-6-P receptor (Morgan et al. 1987) was used to establish whether the mRNA was expressed during gestation and to determine the size of the mRNA. RN A extracted from 9.5 day egg cylinders (which comprised ectoplacental cone, extra-embryonic membranes and early head-fold embryo), 11.5, 14.5 and 16.5-day-old rat embryos and RNA from visceral yolk sac at 12.5, and 14.5 days gestation was used. In all samples, a single ∼9 kb band was observed (Fig. 1), which agrees with the published size for the rat IGF-II/M-6-P mRNA (Morgan et al. 1987).

Receptor mRNA was undetectable by in situ hybridization until ∼7 days gestation). It first appeared in the region of the visceral yolk-sac endoderm overlying the ectoplacental cone (which was itself unlabelled) at 7.5 days with little labelling over the embryonic pole of the egg cylinder or in any of the other extra-embryonic membranes. Immunocytochemical examination indicated that translation had occurred in visceral yolk sac at this stage and there was also evidence of staining in the primary endoderm adjacent to the embryonic ectoderm.

At 8.5 days, the strongest labelling was again seen in the region of the visceral yolk-sac endoderm but not over any of the other extra-embryonic membranes. Hybridization was now apparent, though at a lower level, over the primitive streak embryo (Fig. 2A,B). Immunocytochemistry for the protein showed a similar pattern to that for mRNA (Fig. 2C) with strongest staining in visceral yolk-sac endoderm. Cells that reacted positively showed a low level of diffuse cytoplasmic staining accompanied by more intense membrane staining and presence of a strongly positive paranuclear reaction presumably corresponding to the location of the Golgi apparatus (Fig. 2C), a site where the IGF-II/mannose-6-phosphate receptor is known to be concentrated (von Figura and Haslik, 1986; Valentino et al. 1988).

At 9.5 days of gestation the three chambered egg-cylinder shows distinct hybridization over the amnion, chorion, allantoic mesoderm and visceral yolk-sac endoderm. Detectable hybridization was absent from the glycogen-laden cells of the ectoplacental cone overlying the collapsing epamniotic cavity (i.e. the anlage of the spongiotrophoblast). In the embryo itself, there was distinct hybridization over the primary endoderm and in the mesoderm moving out from the primitive streak itself though the ectoderm including the neural folds remained negative. Staining for the receptor protein shows an identical pattern of distribution and intensity indicating that translation was taking place at the sites of gene transcription.

Throughout the period between 7.5 and 9.5 days, the parietal yolk-sac and the mural trophoblastic giant cells showed no discernible hybridization or protein staining, neither did the decidual cells surrounding the implantation chamber. Some hybridization and protein staining was evident, however, in the actively decidualising zone surrounding this region.

Throughout development of the extra-embryonic membranes, the visceral yolk-sac endoderm and mesoderm continue to express IGF-II/M-6-P mRNA and stain for protein but at all stages the message is undetectable in parietal yolk sac. When the chorio-allantoic placenta becomes established, (at about 11.5 days) IGF-II/ mannose 6-phosphate receptor mRNA is detectable in the developing labyrinth and expression persists throughout the remainder of gestation. Hybridization appears to be associated with the endothelium of the fetal capillaries rather than the differentiating elements of the trophoblast. Precise localisation is impossible at the level of resolution achievable with ³²P- and ³⁵S-labelled probes (Fig. 3A, B) but, following immunocytochemistry, protein staining is strongest in the fetal vessel endothelium (Fig. 3C). Strong labelling and protein staining of the walls of the major umbilical vessels, particularly the arteries is also seen. Labelling and protein staining was absent from junctional zone (spongiotrophoblast, Fig. 3A, B) but occasional giant cells showed evidence of both receptor mRNA and protein.

In embryonic and fetal tissues, the most striking region of transcription and translation of the IGF-Il/M-6-P receptor gene is in the cardiovascular system. At 10.5 days, this is already apparent in the endothelium of the heart tube (the heart is functional from ∼11 days), the cells of the myo-epicardial mantle and the developing blood vessels throughout the embryo (Fig. 4A,B,C). It persists throughout gestation in all endothelial cells as well as in cardiac muscle and in smooth muscle cells associated with developing muscular arteries and arterioles (Fig. 5A,B,C).

At the head-fold stage, mRNA and protein are demonstrable in the thickened epithelium constituting the floor of the foregut and moderately low levels of activity persist in the gut and respiratory endoderm throughout gestation. With the onset of histo-differentiation IGF-II/M-6-P mRNA and protein is evident in developing skeletal muscle as well as in the perichondrium and periosteum of the skeletal system, but is absent from mature cartilage and bone cells (Fig. 6A,B,C). Smooth muscle shows a range of levels of expression of mRNA and protein; the highest levels being, as discussed above, in developing blood vessels: lower levels are present in the bronchi (Fig. 5A,B,C) and both mRNA and protein are virtually absent from the gut wall. Hybridization was also seen in the liver but at a lower level than in muscle; however, protein staining was quite marked in the later stages of hepatic development and was clearly seen to be restricted to parenchymal cells (Fig. 7). Variable but distinct levels of hybridization are detectable throughout the mesenchyme during embryonic and fetal development. In particular, the mesenchyme of the lung is strongly reactive (Fig. 5A,B).

Throughout gestation none of the ectodermal tissues of the embryo and fetus or their derivatives (including the nervous system) showed evidence of hybridization. Occasional groups of nerve cells in the central nervous system and dorsal root ganglia showed equivocal diffuse cytoplasmic staining for protein but this was not associated with a strongly positive paranuclear deposit. On such evidence and in the absence of positive hybridization, these cells cannot be regarded as definitively demonstrating the presence of the protein. Levels of receptor mRNA were scarcely above background in the choroid plexus but protein staining was evident in the endothelium of blood vessels in this structure.

In all tissues examined, the level of receptor mRNA declines immediately after birth and is below the level detectable by ISH in the adult even in tissues such as liver and spleen that have high levels of lysosomal enzymes. The same is true of the heart and blood vessels, which express the gene at high levels during development. This decline in mRNA levels in the heart is confirmed by Northern blotting (Fig. 8).

Occasional cortical areas of the adult cerebellum showed equivocal evidence of hybridization and receptor protein. A complete topographical study of the central nervous system was outside the scope of this investigation and the presence of the receptor mRNA in neural tube/neural crest derivatives awaits confirmation.

Expression of the IGF-Il/mannose-6-phosphate receptor commences in the extra-embryonic membranes at about 7.5 days gestation. The earliest detectable expression of IGF-II mRNA is also seen at this time both in the extra-embryonic membranes and in the ectopiacental cone. Whilst embryonic expression of the receptor is evident at ∼8.5 days embryonic expression of IGF-II mRNA is not evident until ∼9.5 days (R. Florance, manuscript in preparation), suggesting that the receptor may mediate the interaction of the primitive streak embryo with extra-embryonically produced IGF-II during this period.

Embryonic and fetal expression of receptor mRNA is seen at sites where there are also high levels IGF-II mRNA expression, such as the developing cardiovascular system, skeletal muscle and perichondrium (Beck et al. 1987). A paracrine/autocrine role for IGF-II has been suggested in these tissues (Sara and Hall, 1984; Underwood and D’Ercole, 1984; Han et al. 1987). The co-ordinate expression of IGF-II and the IGF-II/ mannose-6-phosphate receptor in these tissues both spatially and temporally suggests a role for the receptor during this period when IGF-II is playing a crucial but still undefined local role in development, possibly as part of the putative paracrine/autocrine loop. On the other hand, the expression of the receptor in the embryo does not correlate with tissues that have high levels of lysosomal activity, the expression being highest in cardiac and skeletal muscle and perichondrium where lysosomal activity is low.

The liver, which expresses high levels of IGF-II mRNA during the fetal and pre-weaning period (Beck et al. 1987; Beck et al. 1988a; Stylianopoulou et al. 1988a; Brown et al. 1987), has relatively low levels of IGF-II/M-6-P receptor mRNA and this organ may be involved primarily in the secretion of IGF-II into the fetal circulation in an endocrine manner. However, the demonstration of the receptor protein in fetal hepatic parenchymal cells may indicate that the turnover of the receptor could be much lower than in other fetal tissues. IGF-11 mRNA is also expressed at a high level in choroid plexus in fetal, neonatal and adult life. However, expression of receptor mRNA is low in choroid plexus at every stage examined. This organ appears to secrete IGF-II into the cerebro-spinal fluid for use elsewhere in the central nervous system and a role for IGF-II in the adult brain has been postulated (Stylianopoulou et al. 19886).

A significant finding was the high level of hybridization and protein staining in developing heart and blood vessels which suggest that there is some interaction between these structures and circulating IGF-II. Circulating IGF-II is bound to carrier proteins which limit its bio-availability and access to the extracellular fluid (Daughaday et al. 1980; Zapf et al. 1985). The developing cardiovascular endothelium could regulate the levels of IGF-II in the circulation by mopping up excess unbound IGF-II.

We were unable to demonstrate convincingly IGF-II/ M-6-P receptor mRNA or protein in the developing central or peripheral nervous system. Though other studies have demonstrated the presence of the receptor in adult brain both by immunocytochemistry (Valentino et al. 1988) and radio-ligand binding (Mendelsohn, 1987) our limited observations on adult rat brain have not shown localisation of mRNA synthesis at a level detectable by ISH. This may indicate that IGF-II involved in CNS development operates via a pathway that differs from that in other tissues.

In all tissues, the level of receptor mRNA is down-regulated after birth and declines more rapidly than the mRNA for IGF-II. In adult tissues, even those containing cells with large concentrations of lysosomes, mRNA was not detected above background by ISH, suggesting that the regulation of mRNA transcription differs between adult and fetal cells. The mannose-6-phosphate receptor is known to be extensively recycled (von Figura and Haslik, 1986) in adult cells but the kinetics and turnover of the protein and mRNA in embryonic cells remain to be determined. Recently, the expression of IGF-II and its receptor has been studied in vitro in cultures of mouse skeletal muscle (Tollefsen et al. 1989), IGF-II expression was found to be up-regulated with the induction of differentiation as was the level of receptor (as assessed by binding studies).

The recent finding that the mannose-6-phosphate receptor in the chicken and the frog does not bind IGF-II has been taken as further evidence against a direct signal transduction role since both chicken and mammalian cell lines show similar in vitro responses to IGF-II (Czech, 1989). The question remains as to the role of the IGF-Il/mannose-6-phosphate receptor in mammals. It has been suggested that the IGF-II/mannose-6-phosphate receptor may participate in the metabolism of IGF-II (Czech, 1989). Our results are consistent with a hypothesis that high levels of IGF-II receptor present in embryonic and fetal tissues serve to stabilise local concentrations of IGF-II at required values by endocy-tosing excessive amounts of locally synthesised growth factor. Such a mechanism would be particularly important in regions where IGF-II played an autocrine or paracrine role in morphogenesis. This situation is now generally accepted to be the case in mesodermally derived structures. In the neonatal period, IGF-II ceases to have an important physiological role outside the central nervous system (Beck et al. 1988a); it is reasonable therefore that the expression of its receptor should be down-regulated but not entirely extinguished since it continues to function in the intracellular transport of lysosomal enzymes.

We are grateful to Drs W. Rutter and D. O. Morgan of the Hormone Research Institute and Department of Biochemistry and Biophysics, University of California for supplying the cDNA used in this study and to Dr M. Czech of the Dept of Biochemistry, University of Massachusetts, for providing the receptor antibody. The work was supported by a grant from The Wellcome Trust (UK). Dr P. V. Senior is a Wellcome Research Fellow.