Development 109, 67-73 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

67

Expression of the IGF-II mannose-6-phosphate receptor mRNA and protein

in the developing rat

PAUL V. SENIOR1*, SIMON BYRNE1, WILLIAM J. BRAMMAR2 and FELIX BECK1*

'Department of Anatomy, Medical Science Bldg, University of Leicester, University Rd, Leicester LEI 9NH, UK2Department of Biochemistry, Adrian Bldg, University of Leicester, University Rd, Leicester LEI 7NH, UK

•Current address to which correspondence should be sent: Howard Florey Institute for Experimental Physiology and Medicine, Universityof Melbourne, Parkville, Victoria 3052, Australia

Summary

The insulin-like growth factor II (IGF-II) receptor isidentical to the mannose-6-phosphate receptor (M-6-P),but its role as a somatomedin transducer is uncertain.IGF-II/M-6-P receptor expression was studied by in situhybridization (ISH) in the developing rat. Expressionoccurs in extra-embryonic membranes at the time ofIGF-II mRNA induction and later at paracrine/autocrine sites of IGF-II action (skeletal muscle andperichondrium) in the embryo. Highest levels of recep-

tor mRNA occur in heart and major vessels. Postnatallytranscription is strongly down-regulated. This suggests arole for the IGF-II/M-6-P receptor in IGF-II action orturnover during development distinct from its role inlysosomal transport.

Key words: insulin-like growth factors, IGF-II, receptor, ratembryo, mannose-6-phosphate, mRNA, protein.

Introduction

Insulin-like growth factors (IGFs) are peptides thathave been implicated in embryonic, fetal and postnatalgrowth (Sara and Hall, 1984). In the rat, IGF-I,produced by the liver, predominates in postnatal andadult life and its production is principally controlled bygrowth hormone (Sara and Hall, 1984; Salmon andDaughaday, 1957; Gluckman, 1986; Beck et al. 1987).IGF-II is produced in embryonic, fetal and earlyneonatal life, being strongly expressed in the visceralyolk sac, in a range of mesodermally derived embryonictissues and organs and in the liver and choroid plexus(Beck etal. 1987; Beck et al. 1988a,6; Stylianopoulou etal. 1988a ,6). In vitro IGF-II has been shown to induceDNA synthesis (Sara and Hall, 1984), promote cellsurvival (Biddle et al. 1988) and to stimulate sulphateincorporation into heparan and chondroitin sulphate(Salmon and Daughaday, 1957; Zapf et al. 1978). IGF-II may have a major role in the development ofembryonic tissues, particularly those of mesodermalorigin (Sara and Hall, 1984).

Little is known about the control of IGF-II ex-pression during embryonic life, although placental lac-togen (Adams etal. 1983) and glucose levels (Gluckmanet al. 1985) have been shown to influence expression invitro. IGF-II mRNA expression is switched off beforeadulthood in all tissues except the choroid plexus (Becket al. 1988a,b; Stylianopoulou et al. 19886). The in-

crease in circulating levels of corticosteroids that occursat weaning (Walker et al. 1986) appears to be involvedin down-regulating expression, at least in the liver, astreatment of pre-weaning rats with cortisone or dexa-methasone quickly and completely switches off hepaticIGF-II synthesis (Beck et al. 19886).

IGF-II can exert insulin-like effects via the insulinand the type-I (IGF-I) receptors (Rechler, 1985). Theseshare considerable structural similarities: both are tyro-sine kinases with an a-/3-dimer structure and substantialhomology at the protein and nucleic acid level (Ullrichet al. 1986). IGF-II has lower binding affinity for theinsulin receptor than for the IGF-I receptor but it hasgreatest affinity for a distinct receptor, the type II (IGF-II) receptor (Sara and Hall, 1984; Rechler, 1985;Gammeltoft, 1989), a single chain polypeptide (Mr~250k) with a short cytoplasmic domain lacking tyro-sine kinase activity (Morgan et al. 1987; Roth, 1988).Cloning and sequencing of cDNAs encoding the type-IIreceptor revealed close homology with the bovinemannose-6-phosphate receptor and it is accepted that,in both rats and humans, the IGF-II receptor and thecation-independent mannose-6-phosphate receptor areidentical (Roth, 1988; MacDonald et al. 1988).

The mannose-6-phosphate receptor is involved in theintracellular transport of lysosomal enzymes throughthe Golgi apparatus and the scavenging of extracellularlysosomal enzymes (von Figura and Haslik, 1986).Mannose-6-phosphate and IGF-II bind to separate sites

68 P. V. Senior and others

on the receptor and binding of mannose-6-phosphatepotentiates IGF-II binding (MacDonald et al. 1988).Insulin (Roth, 1988), IGF-I and EGF, (Braulke et al.1989) acting via their own receptors, cause redistri-bution of the IGF-II/M-6-P receptor to the cell surface.The function of the receptor with respect to IGF-IIactivity remains to be elucidated. There is conflictingevidence as to whether the receptor can participate insignal transduction (reviewed by Roth, 1988; Czech,1989; Gammeltoft, 1989) and it has been suggested thatIGF-II exerts metabolic and mitogenic effects solely viathe IGF-I receptor (Mottola and Czech, 1984). It hasbeen recently reported that neither the chicken nor thefrog mannose-6-phosphate receptors will bind IGF-IIand it is likely that in these species the effects of IGF-IIare exerted via the type I IGF receptor (Clairmont andCzech; 1989). Studies dissecting the functional domainsof the IGF-II/M-6-P receptor have begun. It has beendemonstrated that mutations in specific regions of thecytoplasmic tail of the molecule affect the cellularlocalisation and function of the receptor (Glickmanet al. 1989; Lobel et al. 1989) but as yet no ability of thecytoplasmic domain to activate any intracellular signal-ling pathways has been reported. It may well be that thereceptor has a function in the uptake and metabolism ofIGF-II by tissues that produce and respond to thepeptide (Czech, 1989), thus controlling its local concen-tration and availability. Another possibility is that thereceptor functions as an extracellular carrier protein. Atruncated form of the protein is detected in fetal andneonatal serum; this appears to be produced by proteo-lytic cleavage of the C-terminal of the molecule proxi-mal to the membrane-binding domain as a separatemRNA has not been detected (Kiess et al. 1987;MacDonald et al. 1989). Whilst the serum levels of thetruncated receptor are considered to be too low for it tofunction as a major binding protein (MacDonald et al.1989), it is possible that it functions locally in tissueswith a high level of IGF-II and receptor production.

If the IGF-Il/mannose-6-phosphate receptor is im-portant in the role of IGF-II then it would be expectedto be expressed at high levels in tissues considered to betargets for IGF-II action. Since IGF-II is the predomi-nant fetal somatomedin in the rat, but is virtually absentexcept in the central nervous system in the adult (Becket al. 1987; Beck et al. 198&2,b; Stylianopoulou et al.1988a,b), we have studied the temporal and spatialdistribution of the IGF-II/mannose-6-phosphate recep-tor during rat embryonic and neonatal developmentusing in situ hybridization (ISH) and immunocytochem-istry. We show that the mRNA and protein is expressedat high levels during embryonic development. Ex-pression is principally localised to tissues expressinghigh levels of IGF-II mRNA and commences at thesame time as IGF-II expression.

Materials and methods

Probes and labellingFor 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 7Sribosomal gene, which should be expressed at an equal levelin all cells (Balmain et al. 1982), was used as a control forRNA loading. Probes (10 ng) were labelled by the randompriming method (Feinberg and Vogelstein, 1983) to~2xlO*disints min~Vg~'- For ISH, the 2.4kb IGF-II/mannose-6-phosphate receptor cDNA or a 500bp Alul sub-fragment was cloned into Bluescript (Stratagene) and plasmidprepared by the CsCl method (Maniatis et al. 1982). Linear-ised template was transcribed using 32P-UTP (0.5 and 2.4kbtemplate) or 35S-UTP (0.5 kb template) as previously de-scribed (Senior et al. 1988). Both sense (-ve control) andantisense transcripts were produced to a specific activity of0.5-2xl09disints min~' j<g~'. Their mean length was reducedto 100-200 bases by alkaline hydrolysis (Cox et al. 1984).

Northern blottingTotal RNA was prepared by a modification of the method ofAuffray and Rougeon (1980; Beck et al. 1987). Samples ofRNA were electrophoresed through 1 % agarose/formaldehyde gels (Maniatis et al. 1982) and transferred toHybond N (Amersham). The membranes were hybridised for18h at 42°C in 5xSSPE/50% formamide/lxDenhardt's/O.lmgmr1 salmon sperm DNA/0.5xSDS/6% polyethyleneglycol 6000 and washed down to O.lxSSPE/O.lxSDS at 65°Cand autoradiographed at —70°C. For quantitation of mRNA,the filters were stripped by incubation in 5 mM Tris-HClpH 7.5/2 mM EDTA pH 8.0/0. lxDenhardt's at 65°C, thenreprobed under the above conditions with the 7S ribosomalprobe. RNA sizes were estimated using BRL RNA markersrun on the same gel, transferred to the filter and stained by themethylene blue method (Maniatis etal. 1982).

Tissue preparationConceptuses (pregnancy timed from midnight preceding theappearance of the copulation plug) from 5.5 days (immedi-ately postimplantation) to 20.5 days (immediately prenatal)and a range of tissues and organs from neonatal and adultanimals were collected. For cryostat sectioning, the materialwas orientated in moulds, submerged in OCT cryomountant(R.A.Lamb), frozen at the surface of hexane/dry ice andstored at —40°C. For wax sections, the tissue was fixed for18-48 h (depending on size of block) in 4% paraformal-dehyde in PBS at room temperature, stored in 0.5 M sucrose inPBS then processed and wax embedded by standard methods.

In Situ hybridizationCryostat sections were cut (9//m), taken up onto subbed slides(Rentrop et al. 1986) and laid on dry ice for >20 min, fixed in4% paraformaldehyde in PBS at room temperature (20min),rinsed, dehydrated though graded alcohols and dried. Waxsections (5jnn) were dewaxed in xylene, rehydrated throughgraded alcohols and digested for 10 min at 37°C in0.125 jig ml"' Pronase E (Sigma, autodigested for 4h prior touse) in 50 mM Tris-HCl pH 7.5/5 mM EDTA pH 8.0, rinsed inPBS/0.1% glycine, post-fixed in 4% paraformaldehyde inPBS (10 min at room temperature) and dehydrated throughgraded alcohols and dried. Sections were hybridised for 18 hat 50°C in 0.3 M NaCI/10 mM Na2PO4 pH 6.8/10 mM Tris-HClpH7.5/5mM EDTA pHS.O/lxDenhardts'/l mgrnl"' yeastRNA/50% formamide/10% dextran sulphate with the probeat —lOOngml"1 under coverslips. The slides were washed for4h in 3 changes of the hybridization buffer without probe,yeast RNA or dextran sulphate, digested for 1 h at 37°C with150;/gml~' RNase A (boiled for 1 min prior to addition) in0.5 M NaCl/lOmM Tris-HCl pH 7.5/1 mM EDTA pH8.0,

IGF-II receptor expression in rat embryos 69

washed twice for 30min in 2xSSC at 65°C then dehydratedthrough graded alcohols and dried. 32P-labelled slides wereexposed to Kodak X-Omat film overnight to assess the leveland tissue distribution of labelling. The slides were dipped inIlford K5 emulsion diluted 5 to 3 w/v in water containing 1 mlof glycerol in 60 ml at 45°C. The slides were allowed to dryupright in the dark for at least 2h then exposed desiccated at4°C for 4-14 days. The slides were developed at 15°C bysequential immersion in Kodak D19 (4min at 160gr') , 1%acetic acid (1 min), Ilford Hypam fixer (4min 1 in 5 dilution)and then washed in water. The emulsion was fixed for 10 minin 10% formol-saline, the slides were stained with haema-toxylin, dehydrated and cleared and coverslips applied. Theywere then examined under bright- and dark-field illumination.

ImmunocyiochemistryWax-embedded sections prepared as above were dewaxedand rehydrated. They were digested with 0.025% pepsin in0.01 M HC1 for 30-45 min at 37°C and blocked with swineserum (diluted 1 in 5) for lh. 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 mMTris-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 mMTris-HCl pH7.5. The antibody was detected using thebiotin/avidin/peroxidase/DAB method (Dakopatts) and theslides were counterstained with haematoxylin, dehydrated,cleared and coverslips mounted with DPX (Lamb). Controlsections were stained as above but a) the primary antiserumwas omitted, b) the primary antiserum was replaced withnormal rabbit serum or c) a rabbit antibody to ACTH(Dakopatts) was used in place of the lGF-Il/mannose-6-phosphate receptor antibody.

Results

Expression of IGF-II/mannose-6-phosphate mRNAduring gestation by Northern BlottingNorthern blotting using the 2.4 kb cDNA for the ratIGF-II/M-6-P receptor (Morgan etal. 1987) was used toestablish whether the mRNA was expressed duringgestation and to determine the size of the mRNA. RNAextracted from 9.5 day egg cylinders (which comprisedectoplacental cone, extra-embryonic membranes andearly head-fold embryo), 11.5, 14.5 and 16.5-day-oldrat embryos and RNA from visceral yolk sac at 12.5,and 14.5 days gestation was used. In all samples, asingle ~9kb band was observed (Fig. 1), which agreeswith the published size for the rat IGF-II/M-6-PmRNA (Morgan et al. 1987).

Expression of IGF-lljmannose-6-phosphate receptormRNA and protein in the early postimplantationembryo (5.5-9.5 days post coitum)Receptor mRNA was undetectable by in situ hybridiz-ation until ~7 days gestation). It first appeared in theregion of the visceral yolk-sac endoderm overlying theectoplacental cone (which was itself unlabelled) at 7.5days with little labelling over the embryonic pole of theegg cylinder or in any of the other extra-embryonicmembranes. Immunocytochemical examination indi-cated that translation had occurred in visceral yolk sacat this stage and there was also evidence of staining in

P u Y o l k

tmbryos sac

in m in mo> —" <o

in in

kB9.5 -7.5 -

Fig. 1. Northern blot; 100/jg of total RNA from 9.5 day rategg-cylinder and from 11.5 day 14.5 day and 16.5 day wholeembryo and 12.5 day and 14.5 day visceral yolk-sac wererun. The filter was hybridized with the 32P-labelled 2.4kbIGF-II/M-6-P receptor cDNA probe. The blot was exposedto Fuji X-ray film at —70°C for 4 days withoutintensification screens. A strong band of ~9kb is evident inall lanes. O=origin.

the primary endoderm adjacent to the embryonic ecto-derm.

At 8.5 days, the strongest labelling was again seen inthe region of the visceral yolk-sac endoderm but notover any of the other extra-embryonic membranes.Hybridization was now apparent, though at a lowerlevel, over the primitive streak embryo (Fig. 2A,B).Immunocytochemistry for the protein showed a similarpattern to that for mRNA (Fig. 2C) with strongeststaining in visceral yolk-sac endoderm. Cells thatreacted positively showed a low level of diffuse cyto-plasmic staining accompanied by more intense mem-brane staining and presence of a strongly positiveparanuclear reaction presumably corresponding to thelocation of the Golgi apparatus (Fig. 2C), a site wherethe IGF-Il/mannose-6-phosphate receptor is known tobe concentrated (von Figura and Haslik, 1986; Valen-tino 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 en-doderm. Detectable hybridization was absent from theglycogen-laden cells of the ectoplacental cone overlyingthe collapsing epamniotic cavity (i.e. the anlage of thespongiotrophoblast). In the embryo itself, there wasdistinct hybridization over the primary endoderm andin the mesoderm moving out from the primitive streakitself though the ectoderm including the neural foldsremained negative. Staining for the receptor proteinshows an identical pattern of distribution and intensityindicating that translation was taking place at the sitesof gene transcription.

Throughout the period between 7.5 and 9.5 days, the

70 P. V. Senior and others

parietal yolk-sac and the mural trophoblastic giant cellsshowed no discernible hybridization or protein staining,neither did the decidual cells surrounding the implan-tation chamber. Some hybridization and protein stain-ing was evident, however, in the actively decidualisingzone surrounding this region.

Receptor mRNA and protein expression from mid-gestation to birth (10.5 to 21.5 days)Throughout development of the extra-embryonic mem-branes, the visceral yolk-sac endoderm and mesodermcontinue to express IGF-II/M-6-P mRNA and stain forprotein but at all stages the message is undetectable inparietal yolk sac. When the chorio-allantoic placentabecomes established, (at about 11.5 days) IGF-II/mannose 6-phosphate receptor mRNA is detectable inthe developing labyrinth and expression persiststhroughout the remainder of gestation. Hybridizationappears to be associated with the endothelium of thefetal capillaries rather than the differentiating elementsof the trophoblast. Precise localisation is impossible atthe level of resolution achievable with 32P- and 35S-labelled probes (Fig. 3A,B) but, following immuno-cytochemistry, protein staining is strongest in the fetalvessel endothelium (Fig. 3C). Strong labelling andprotein staining of the walls of the major umbilicalvessels, particularly the arteries is also seen. Labellingand protein staining was absent from junctional zone(spongiotrophoblast, Fig. 3A,B) but occasional giantcells showed evidence of both receptor mRNA andprotein.

In embryonic and fetal tissues, the most strikingregion of transcription and translation of the IGF-Il/M-6-P receptor gene is in the cardiovascular system. At10.5 days, this is already apparent in the endothelium ofthe heart tube (the heart is functional from ~11 days),the cells of the myo-epicardial mantle and the develop-ing blood vessels throughout the embryo(Fig. 4A,B,C). It persists throughout gestation in allendothelial cells as well as in cardiac muscle and insmooth muscle cells associated with developing muscu-lar arteries and arterioles (Fig. 5A,B,C).

At the head-fold stage, mRNA and protein aredemonstrable in the thickened epithelium constitutingthe floor of the foregut and moderately low levels ofactivity persist in the gut and respiratory endodermthroughout gestation. With the onset of histo-differen-tiation IGF-II/M-6-P mRNA and protein is evident indeveloping skeletal muscle as well as in the perichon-drium and periosteum of the skeletal system, but isabsent from mature cartilage and bone cells(Fig. 6A,B,C). Smooth muscle shows a range of levelsof expression of mRNA and protein; the highest levelsbeing, 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 fromthe gut wall. Hybridization was also seen in the liver butat a lower level than in muscle; however, proteinstaining was quite marked in the later stages of hepaticdevelopment and was clearly seen to be restricted toparenchymal cells (Fig. 7). Variable but distinct levels

of hybridization are detectable throughout the mesen-chyme during embryonic and fetal development. Inparticular, the mesenchyme of the lung is stronglyreactive (Fig. 5A,B).

Throughout gestation none of the ectodermal tissuesof the embryo and fetus or their derivatives (includingthe nervous system) showed evidence of hybridization.Occasional groups of nerve cells in the central nervoussystem and dorsal root ganglia showed equivocal diffusecytoplasmic staining for protein but this was not associ-ated with a strongly positive paranuclear deposit. Onsuch evidence and in the absence of positive hybridiz-ation, these cells cannot be regarded as definitivelydemonstrating the presence of the protein. Levels ofreceptor mRNA were scarcely above background in thechoroid plexus but protein staining was evident in theendothelium of blood vessels in this structure.

Expression of receptor mRNA and protein in thepostnatal period and in the adultIn all tissues examined, the level of receptor mRNAdeclines immediately after birth and is below the leveldetectable by ISH in the adult even in tissues such asliver and spleen that have high levels of lysosomalenzymes. The same is true of the heart and bloodvessels, which express the gene at high levels duringdevelopment. This decline in mRNA levels in the heartis confirmed by Northern blotting (Fig. 8).

Occasional cortical areas of the adult cerebellumshowed equivocal evidence of hybridization and recep-tor protein. A complete topographical study of thecentral nervous system was outside the scope of thisinvestigation and the presence of the receptor mRNAin neural tube/neural crest derivatives awaits confir-mation.

Discussion

Expression of the IGF-Il/mannose-6-phosphate recep-tor commences in the extra-embryonic membranes atabout 7.5 days gestation. The earliest detectable ex-pression of IGF-II mRNA is also seen at this time bothin the extra-embryonic membranes and in the ectopla-cental cone. Whilst embryonic expression of the recep-tor is evident at ~8.5 days embryonic expression ofIGF-II mRNA is not evident until -9.5 days (R.Florance, manuscript in preparation), suggesting thatthe receptor may mediate the interaction of the primi-tive streak embryo with extra-embryonically producedIGF-II during this period.

Embryonic and fetal expression of receptor mRNA isseen at sites where there are also high levels IGF-IImRNA expression, such as the developing cardiovascu-lar system, skeletal muscle and perichondrium (Beck etal. 1987). A paracrine/autocrine role for IGF-II hasbeen suggested in these tissues (Sara and Hall, 1984;Underwood and D'Ercole, 1984; Han et al. 1987). Theco-ordinate expression of IGF-II and the IGF-II/mannose-6-phosphate receptor in these tissues bothspatially and temporally suggests a role for the receptor

2C 3C

Fig. 2. Implantation chamber at 8.5 days gestation showing the egg-cylinder cutlongitudinally. (A) 1SH bright-field and (B) dark-field; there is strong hybridization of the500 bp ^P-labelled 1GF-II/M-6-P receptor probe to visceral yolk-sac endoderm (v) with alower level of labelling evident in the embryonic region (e). The ectoplacental cone (ec),and parietal yolk-sac (arrowed) are negative, scale bar=100/mi. (C) Immunocyto-chemistry with anti-IGF-Il/M-6-P receptor antibody. There is strong staining of visceralyolk-sac (v) with intracellular inclusions (example arrowed), there is less staining of theembryonic pole (e) and the parietal yolk-sac (p) and trophoblastic giant cells (t) arenegative. Scale bar=50/mi.Fig. 3. 19.5 day placenta; (A) ISH bright-field and (B) dark-field, section hybridized with500 base 35S-labelled probe. Labelling is restricted to the placental labyrinth (1) where it isassociated with the fetal endothelial cells rather than with the trophoblast. The junctionalzone (j) comprising mainly of spongiotrophoblast shows no hybridization abovebackground, scale bar=100/<m. (C) Immunocytochemistry with the receptor antibodydemonstrates that protein is present in endothelial cells of the capillaries (c) rather than introphoblast cells (t), scale bar=50/an.Fig. 4. 10.5 day embryo; (A) ISH bright-field and (B) dark-field of a section hybridizedwith the 500 base 32P-labelled receptor probe. Strongest labelling is evident in the hearttube (h). Visceral yolk-sac (v), somites (s) and general mesoderm (md) are also labelledbut less intensely. (C) Immunocytochemistry with anti-receptor antibody show highestlevels of protein in the heart tube (h). The foregut (fg) and visceral yolk-sac (v) are alsostained as are cells in the mesoderm (md) but less staining is evident in neural plate (n),scale bar=100^m.

• >

4A

n

md

fg

V

4C

A77S

• . ^ . . j ^ ^ ^ pa

Fig. 5. Lung from a 19.5 day fetus; (A) ISH bright-field and (B) dark-fieldof a section hybridized with the 35S-labelled 500 base receptor probe.Strongest hybridization is seen in the muscular wall of the pulmonaryartery (pa) and is less intense in the wall of the branching vessel (b). Theendotheliurn (en) of small blood vessels is also labelled. A lower level oflabelling is seen in the smooth-muscle of the bronchiole (br) and in thelung mesenchyme (ms) but the bronchial epithelium is negative, scalebar=100/an. (C) immunocytochemistry with the anti-receptor antibodyreveals the highest levels of protein in the smooth-muscle component ofthe wall of the pulmonary artery (pa) with very little staining of thesmooth-muscle of the bronchi (br) there is also staining of cells in thepulmonary mesenchyme (ms), scale bar=50/*m.Fig. 6. 19.5 day fetus, developing vertebral bodies and their associatedskeletal muscle; (A) ISH bright-field and (B) dark-field of a sectionhybridized with the 35S-labelled receptor probe demonstrates stronglabelling of perichondrium (pc) and skeletal muscle (s) but labelling isabsent from mature chondrocytes (c) and neural tube (n), scalebar=100/an. (C) Immunocytochemistry with the anti-receptor antibodyreveals high levels of protein in the perichondrium of the developing rib(pc) and and intercostal (skeletal) muscle (s) but less staining of maturechondrocytes (c), scale bar=50/an.Fig. 7. Immunocytochemistry with the anti-receptor antibody showsprotein within parenchymal cells (pr) in a section of 19.5 day fetal liver butnot in haematopoietic cells (he), scale bar=25/an.

IGF-II receptor expression in rat embryos 71

1 2 3 4 5 6 7

kb

9-5-III*

0-36- i MB

Fig. 8. Northern blot, -30ng of total RNA from 18.5 (1)and 19.5 day (2) fetal heart and 1.5 day (3) and 6 day (4)neonatal heart together with 3 samples of adult heart (5-7)total RNA (30, 35 & 40 pg) were electrophoresed andtransferred as described. (A) filter probed with 32P-labelled2.4 kb receptor cDNA, (B) filter stripped and reprobed with32P-labelled 7S ribosomal cDNA. Levels of IGF-II/M-6-Preceptor mRNA are greatly reduced in the immediatepostnatal period, a result which cannot be due todifferences in RNA loading on the gel. The level ofreceptor mRNA in adult heart is almost undetectabledespite the larger amounts of adult RNA loaded.

during this period when IGF-II is playing a crucial butstill undefined local role in development, possibly aspart of the putative paracrine/autocrine loop. On theother hand, the expression of the receptor in theembryo does not correlate with tissues that have highlevels of lysosomal activity, the expression beinghighest in cardiac and skeletal muscle and perichon-drium where lysosomal activity is low.

The liver, which expresses high levels of IGF-IImRNA during the fetal and pre-weaning period (Becket al. 1987; Beck et al. 1988a; Stylianopoulou et al.1988a; Brown et al. 1987), has relatively low levels ofIGF-II/M-6-P receptor mRNA and this organ may beinvolved primarily in the secretion of IGF-II into thefetal circulation in an endocrine manner. However, thedemonstration of the receptor protein in fetal hepaticparenchymal cells may indicate that the turnover of thereceptor could be much lower than in other fetal tissues.IGF-II mRNA is also expressed at a high level inchoroid plexus in fetal, neonatal and adult life. How-

ever, expression of receptor mRNA is low in choroidplexus at every stage examined. This organ appears tosecrete IGF-II into the cerebro-spinal fluid for useelsewhere in the central nervous system and a role forIGF-II in the adult brain has been postulated (Styliano-poulou et al. 1988b).

A significant finding was the high level of hybridiz-ation and protein staining in developing heart andblood vessels which suggest that there is some interac-tion between these structures and circulating IGF-II.Circulating IGF-II is bound to carrier proteins whichlimit its bio-availability and access to the extracellularfluid (Daughaday et al. 1980; Zapf et al. 1985). Thedeveloping cardiovascular endothelium could regulatethe levels of IGF-II in the circulation by mopping upexcess unbound IGF-II.

We were unable to demonstrate convincingly IGF-II/M-6-P receptor mRNA or protein in the developingcentral or peripheral nervous system. Though otherstudies have demonstrated the presence of the receptorin adult brain both by immunocytochemistry (Valentinoet al. 1988) and radio-ligand binding (Mendelsohn,1987) our limited observations on adult rat brain havenot shown localisation of mRNA synthesis at a leveldetectable by ISH. This may indicate that IGF-IIinvolved in CNS development operates via a pathwaythat differs from that in other tissues.

In all tissues, the level of receptor mRNA is down-regulated after birth and declines more rapidly than themRNA for IGF-II. In adult tissues, even those contain-ing cells with large concentrations of lysosomes, mRNAwas not detected above background by ISH, suggestingthat the regulation of mRNA transcription differsbetween adult and fetal cells. The mannose-6-phos-phate receptor is known to be extensively recycled (vonFigura and Haslik, 1986) in adult cells but the kineticsand turnover of the protein and mRNA in embryoniccells remain to be determined. Recently, the expressionof IGF-II and its receptor has been studied in vitro incultures of mouse skeletal muscle (Tollefsen et al.1989), IGF-II expression was found to be up-regulatedwith the induction of differentiation as was the level ofreceptor (as assessed by binding studies).

The recent finding that the mannose-6-phosphatereceptor in the chicken and the frog does not bind IGF-II has been taken as further evidence against a directsignal transduction role since both chicken and mam-malian cell lines show similar in vitro responses to IGF-II (Czech, 1989). The question remains as to the role ofthe IGF-Il/mannose-6-phosphate receptor in mam-mals. It has been suggested that the IGF-Il/mannose-6-phosphate receptor may participate in the metabolismof IGF-II (Czech, 1989). Our results are consistent witha hypothesis that high levels of IGF-II receptor presentin embryonic and fetal tissues serve to stabilise localconcentrations of IGF-II at required values by endocy-tosing excessive amounts of locally synthesised growthfactor. Such a mechanism would be particularly import-ant in regions where IGF-II played an autocrine orparacrine role in morphogenesis. This situation is nowgenerally accepted to be the case in mesodermally

72 P. V. Senior and others

derived structures. In the neonatal period, IGF-IIceases to have an important physiological role outsidethe central nervous system (Beck et al. 1988a); it isreasonable therefore that the expression of its receptorshould be down-regulated but not entirely extinguishedsince it continues to function in the intracellular trans-port of lysosomal enzymes.

We are grateful to Drs W. Rutter and D. O. Morgan of theHormone Research Institute and Department of Biochemis-try and Biophysics, University of California for supplying thecDNA used in this study and to Dr M. Czech of the Dept ofBiochemistry, University of Massachusetts, for providing thereceptor antibody. The work was supported by a grant fromThe Wellcome Trust (UK). Dr P. V. Senior is a WellcomeResearch Fellow.

References

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(Accepted 8 February 1990)