fetal development and organogenesis · classical studies of experimental embryology demonstrated...

INTRODUCTION

Classical studies of experimental embryology demonstratedthat inductive interactions between epithelial and mesenchy-mal tissues lead to the determination of cell fate and the sub-sequent formation of tissues and organs (Gurdon, 1992; Saxén,1972; Saxén et al., 1980). The molecular changes associatedwith tissue interactions have been analyzed during recentyears, and regulatory roles have been proposed for growthfactors, extracellular matrix molecules and various cell surfacecomponents (Bernfield et al., 1992; Chiquet-Ehrismann et al.,1986; Jalkanen et al., 1993; Thesleff et al., 1991; Vainio et al.,1989, 1993). Heparin-binding growth factors are expressed indeveloping organs (Gonzalez et al., 1990; Niswander and

Martin, 1992; Wilkinson et al., 1988) and they appeasignaling roles in embryonic induction (Niswande1993).

Midkine (MK) is a heparin-binding molecule thainduced in some cell types by treatment with retinoic a(Huang et al., 1990; Kadomatsu et al., 1988; Matsuba1990). This 13

×103

Mr secreted protein is unusuallycysteine and basic amino acids, and promotes the gneuronal and PC12 cells (Kadomatsu et al., 1988; Miet al., 1993; Muramatsu, 1993; Tomomura et al.Another heparin-binding protein with a molecular about 18×103, the heparin binding-growth associated m(HB-GAM), has been isolated from perinatal r(Rauvala, 1989). HB-GAM has 50% sequence iden

DevelopmPrinted in

Midkin th amolecu titutfamily in tlation o mats

Int. J. inityantibod yze ttributio nt. F14.5 da re decentral ial plimb bu e, urand sk welocalize cellsbaseme pitheenchym ein dconside as Hstaining hybrresults K trathat was not always consistent with the distribution of MKprotein in developing tissues. In many epithelio-mesenchy-

tributimb bdecanjacenGAMaran MK audiedthe penchyssionB-GAorphin e

r inte

Key words: midkine, pleiotrophin, HB-GAM, syndecan-1, racid, embryonic development, epithelial-mesenchymal inter

SUMM

Expr din AM

(plei ith dur

fetal ge

Thimio a2,3, auvaEero L ma 1Institu and2Depar of Tu3Turku rku, 4Depar , Na5Institu Hels6Depar Hels

*Author fo partm en

37

ed withuds and-1 weret prolif- boundsulfate-nd HB- in vitroroteins.

mal cell. Taken

M mayogenesispithelio-ractions

ing

la5,

ssociatede a newhe regu-u (1993)-purifiedheir dis-rom 9 totected inrocesses,ogenital,re often and inlial-mes-ecreasedB-GAM

idizationnscripts

mal organs MK and HB-GAM were codissyndecan-1, a cell surface proteoglycan. In lfacial processes, MK, HB-GAM, and synlocalized to the apical epithelium and the aderating mesenchyme. Both MK and HB-syndecan-1 in solid-phase assays in a hepdependent manner. The biological effects of GAM on limb and facial mesenchyme were stby application of beads preloaded with Neither MK nor HB-GAM stimulated mesproliferation or induced syndecan-1 expretogether these results indicate that MK and Hplay regulatory roles in differentiation and mof the vertebrate embryo, particularly mesenchymal organs, and suggest moleculawith syndecan-1.

g cytokines, midkine (MK) and HB-G

epithelial-mesenchymal interactions

nesis

Takashi Muramatsu4, Hisako Muramatsu4, Heikki RThesleff1

Orthodontics, University of Helsinki, Helsinki, Finlandrku, Turku, FinlandTurku, Finlandgoya University, Nagoya, Japaninki, Finland inki, Finland

ent of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Swed

ent 121, 37-51 (1995)Great Britain © The Company of Biologists Limited 1995

ession of the heparin-bin

otrophin) is associated w

development and organo

s A. Mitsiadis1,*, Markku Salmivirtehtonen6, Markku Jalkanen3 and Ir

te of Dentistry, Department of Pedodonticstment of Medical Biochemistry, University Centre for Biotechnology, University of Tutment of Biochemistry, School of Medicinete of Biotechnology, University of Helsinki,tment of Pathology, University of Helsinki,

e (MK) and heparin binding-growle (HB-GAM or pleiotrophin), consof heparin-binding proteins implicatedf growth and differentiation (T. MuraDev. Biol. 37, 183-188). We used affies against MK and HB-GAM to analn during mouse embryonic developmey post-coitum (dpc), both proteins we and peripheral nervous systems, facds, sense organs, respiratory, digestiveletal systems. MK and HB-GAMd on the surface of differentiating nt membranes of organs undergoing eal interactions. The levels of MK protrably in the 16.5 dpc embryo, where persisted in many tissues. Our in situ

revealed a widespread expression of M

ARY

r correspondence (present address): T. Mitsiadis, De

r to haver et al.,

t can becid (RA)ra et al., rich in

rowth ofchikawa, 1990).mass ofolecule

at braintity with

etinoicactions

38

MK (MoutgrowRauvala,et al., 19GAM thRIHB (another p1989). R1991), eoutgrowinduced chick coheparin-fibroblas

In situchemicaRIHB aembryonMuramaet al., 19et al., 1functionMK, HBof effecleukemiaMaruta e1992; RaWe decidopment, mouse dTo addredevelopmapplied t

Syndeteoglycaheparin-al., 1992binds gmolecule(Jalkanemore, thmesenchorgans (al., 1989to syndesyndecan

MATER

Tissue pHybrid membryoniappearanc9-16.5 dphyde (PFAand embesilanized

AntibodMK, HB-tochemist

T

erenmies and Rauvala, 1990), exhibits neuriteth activity (Hampton et al., 1992; Raulo et al., 1992; 1989), and may be a mitogen for some cell types (Li90). Because of the suspected widespread role of HB-e term pleiotrophin has been proposed (Li et al., 1990).retinoic acid-induced heparin-binding protein) isrotein isolated from the chicken embryo (Vigny et al.,IHB has 65% sequence identity with MK (Urios et al.,xhibits mitogenic activity and enhances neurite

th in PC12 cells (Raulais et al., 1991). Because of RA-expression, it has been proposed that RIHB is theunterpart of MK. All members of this new family ofbinding proteins have entirely distinct sequences fromt growth factors (FGFs). hybridization, slot blot analysis, and immunohisto-

l studies have demonstrated that MK, HB-GAM andre expressed in a variety of tissues during the

anti-mouse MK polyclonal antibody (aMK), rabbit anti-rat HB-GAMpolyclonal antibody (aHB-GAM), and rat anti-mouse syndecan mono-clonal antibody 281-2 have been described earlier (Jalkanen et al., 1985;Muramatsu et al., 1993; Rauvala, 1989). Specific binding of aMK(Muramatsu et al., 1993) and aHB-GAM (Rauvala, 1989) has been pre-viously demonstrated using crude extracts of tissues. To confirm thatthe antibodies do not cross-react, purified MK and HB-GAM proteinswere analyzed by western blotting using both antibodies.

ImmunohistochemistryImmunohistochemistry was performed as previously described(Mitsiadis et al., 1992). Briefly, sections were incubated overnight at4°C with aMK (concentrations 3.3-4 µg/ml), aHB-GAM (concentra-tions 1-2 µg/ml), and rat monoclonal antibody 281-2 against the coreprotein of syndecan-1 (concentrations 0.75-1 µg/ml) in 0.2% bovineserum albumin (BSA)/2% normal goat serum (NGS)/phosphate-buffered saline (PBS), pH 7.4. Control sections were incubated eitherwith 2% BSA/PBS or with normal rabbit serum. Sections were thenincubated with either biotinylated secondary goat anti-rabbit antibody

. A. Mitsiadis and others

ic development of mouse (Kadomatsu et al., 1990; (dilution 1:250 in PBS) or rat anti-mouse antibody for the detection
tsu et al., 1993; Nakamoto et al., 1992), rat (Rauvala94; Vanderwinden et al., 1992), and chicken (Duprez993; Vigny et al., 1989), suggesting developmentals for these molecules. Functional studies with purified-GAM and RIHB proteins have reported a wide rangets on several cell lines including teratocarcinoma,, and PC12 cells (Hampton et al., 1992; Li et al., 1990;t al., 1993; Muramatsu et al., 1993; Nurcombe et al.,ulais et al. 1991; Raulo et al., 1992; Rauvala, 1989).ed to analyze the roles of MK and HB-GAM in devel-by following their distribution during embryonic
evelopment (9-16.5 dpc) by immunohistochemistry.ss the biological roles of MK and HB-GAM in organent, the proteins were incorporated into beads and

o limb and jaw mesenchyme in vitro.cans are a family of cell surface heparan sulfate pro-ns (HSPG) regulating the biological effects of severalbinding molecules (for recent reviews, see Bernfield et; Jalkanen et al., 1993; Rapraeger, 1993). Syndecan-1rowth factors and several extracellular matrixs, such as type Ι collagen, fibronectin and tenascin

n et al., 1993; Salmivirta et al., 1991, 1992). Further-e expression of syndecan-1 is regulated by epithelial-ymal interactions during the development of various

of syndecan (Jackson, USA; dilution 1:1,000), and then washed andincubated with avidin-biotin-peroxidase complex (Vector Laborato-ries Inc., Burlingame, USA). Peroxidase was revealed by incubationwith 3-amino-9-ethylcarbazole (AEC) containing 1% H2O2.

Heparitinase treatment. Some sections were incubated with 2µg/ml heparitinase in NaCl/Pi containing 1 mM CaCO3 for 1 hour at37°C. After several washes the immunological localization of MKwas performed as described above.

Probes and in situ hybridization For in situ hybridization studies, a 629 bp fragment of MK mousecDNA was subcloned into pGEM3 plasmid. 35S-UTP-labeled (1,000Ci/nmol, Amersham) single-stranded sense (pSP64) and antisense(pSP65) RNA probes were prepared by standard procedures. ThepSP64 and pSP65 plasmid vectors were linearized with EcoRI andHindIII restriction enzymes respectively (Promega). The labeledprobes were ethanol-precipitated, resuspended in 100 mM DTT,diluted in hybridization solution (60% deionized formamide, 20 mMTris-HCl, 5 mM EDTA, pH 8, 0.3 M NaCl, 1× Denhardt’s, 0.5 mg/mlyeast RNA, 10% dextran sulfate), and used at 50,000-60,000 cpm/µl.

In situ hybridization was performed according to the method ofWilkinson and Green (1990). Autoradiography was performed bydipping the slides in autoradiographic emulsion (NTB2 Kodak), andexposing them for 10 days at 4°C. Exposed slides were developed inD-19 (Kodak).

Solursh et al., 1990; Trautman et al., 1991; Vainio et). We compared the distribution of MK and HB-GAMcan-1 and studied the binding of these proteins to-1.

IALS AND METHODS

reparationice (CBA×C57BL and CBA×NMRI) were used at

c stages. Embryonic age was determined from the firste of the vaginal plug (0 dpc) and by morphological criteria.c mouse embryos were fixed overnight in 4% paraformalde-) in PBS, pH 7.4. The whole embryos were then dehydrated

dded in paraffin wax. 7 µm serial sections were mounted onslides, dried overnight, and stored in air-tight boxes at 4°C.

iesGAM, and syndecan-1 antibodies were used for immunohis-ry. Preparation and characterization of affinity purified rabbit

Syndecan-1 binding assayMetabolically 35SO4-labeled syndecan-1 ectodomain (the extracellu-lar part of the syndecan-1) was isolated from mouse mammary epithe-lial (NmuMG) cells as previously described (Salmivirta et al., 1991).Bovine type Ι collagen (Boehringer), recombinant murine MK(Muramatsu and Muramatsu, 1991) and rat HB-GAM (Raulo et al.,1992) were used in binding studies. Blot-quality BSA was used as anegative control.

To assess syndecan-1 binding to proteins, equimolar amounts oftype Ι collagen, MK and HB-GAM diluted in PBS were immobilizedonto nitrocellulose membranes (0.45 µm, Schleicher & Schuell) asdescribed previously (Salmivirta et al., 1991). The membranes weresubsequently rinsed with PBS and incubated in PBS containing 1%BSA for 1 hour at 20°C to block non-specific binding sites. Themembranes were then incubated in PBS containing 35SO4-syndecan-1 (20,000 cpm/ml) at 4°C for 14 hours followed by three washes withPBS. In order to study the binding mechanism, 50 µg/ml of freeheparan sulfate (HS, Sigma) or chondroitin sulfate (CS, Sigma) wasadded to PBS containing 35SO4-syndecan-1. Dried membranes werecovered with plastic foil and the membrane-bound radioactivity wasdetected using a CS-250 Molecular Imager phosphor imaging and

M

high sensitivity imaging screens (BioRad, Hercules, CA). The resultswere quantified using PhosphorAnalyst software and are expressed aspercentages of syndecan-1 bound to MK or HB-GAM in comparisonto radioactivity bound to an equimolar amount of type I collagen.

BrdU labeling and immunostaining of explants as wholemounts The distal parts of the forming hindlimb and facial processes of 11-12 dpc mouse embryos were dissected and incubated for 3 minutes in2.25% trypsin/0.75% pancreatin on ice. Epithelial and mesenchymaltissues were separated under a stereomicroscope. Facial and limbepithelia were recombined to mesenchyme of the same tissue andembryonic age. The tissues were placed on pieces of Nuclepore filters(pore size, 0.1 µm), supported by metal grids (Trowell-type). Affi-gelblue agarose beads (100-200 mesh, 75-150 µm diameter; Bio-Rad)preloaded with MK or HB-GAM protein (diluted into 0.1% BSA toconcentrations of 5-100 ng/µl per 5 µl per 50 beads) were then placedon top of the mesenchyme (according to Vainio et al., 1993). Controlbeads were incubated in 0.1% BSA in PBS. The recombinants werecultured for 16-32 hours in Dulbecco´s minimal essential medium(DMEM) supplemented with 10% fetal calf serum (FCS; Gibco) in ahumidified atmosphere of 5% CO2 in air at 37°C. Explants werelabeled for 1-2 hours with bromodeoxyuridine (BrdU) according tothe manufacturer’s instructions (Amersham and BoehringerMannheim) and fixed in 4% PFA overnight at 4°C. Explants wereincubated with mouse anti-BrdU monoclonal antibody for 1 hour at37°C, then with biotinylated goat anti-mouse IgG diluted 1:600(Jackson Labs, USA) for 30 minutes at 37°C, and finally with avidin-biotin-peroxidase complex (Vector, USA). An antibody againstsyndecan-1 was also used in whole-mount staining.

RESULTS

Comparative analysis of MK and HB-GAM proteindistributionOur western-blot analysis demonstrated that the anti-MKantibody reacts only with the MK protein, and that the HB-

GAM proantibody (FHB-GAM at 9-10 dpcwith ectodderived ceHB-GAM 12.5 to 14.restricted atissues, seorgans, an

Fig. 1. Western blots of MK and HB-GAM. For both A and B; lane1, 10 ng MK; lane 2, 100 ng MK; lane 3, 10 ng HB-GAM; lane 4,100 ng HB-GAM. The proteins were transferred to nitrocellulosefrom 5-20% gradient SDS-PAGE and detected with affinity-purifiedanti-HB-GAM (0.25 µg/ml) for the samples in A and with affinity-purified anti-MK (0.15 µg/ml) for those in B. The positions ofprotein markers are shown on the right (as Mr×10−3).

Table

Whisker folli

Nose

Inner ear

Eye

Tooth

Salivary glan

Lung

Esophagus

Stomach

Intestine

Kidney

Ureter

Pancreas

HeartLiverTestisAdrenal glanCentral nervo

Neopallial MidbrainDiencephaChoroid plNeural tub

Peripheral neDorsal rooNeuronal p

Dermal epithOral epitheliuTongue epithFacial mesenDigital mesenCartilageImmature bonOsteoblasts/oMusclesEndothelia

(−) no stainstaining; (NSmusculature.

39idkine and HB-GAM in mouse embryos

tein is recognized only by the anti-HB-GAMig. 1). A pattern of widespread yet specific MK andstaining was observed in embryonic mouse tissues (data not shown). Immunoreactivity was associatedermal structures, mesoderm- and neuroectoderm-lls, and their extracellular matrices. The strongeststaining was found in the developing heart. From5 dpc, the MK and HB-GAM staining became morend was detected in ectodermal structures, neuronalnse organs, facial and limb processes, internald skeletal tissues (see Table 1 and Figs 2A,B, 3, 4,

1. Immunocalization of MK, HB-GAM, andsyndecan-1 in 14.5 dpc mouse embryos

MK HB-GAM Syn-1

cles ep *** *** ***mes *** - ***
ep (olfactory) *** ** −
mes * - −ep *** ** −

mes * −/* *ep *** * NS

mes ** −/* NSep *** ** ***

mes *** ** ***ds ep *** ** ***

mes * −/* **ep *** * *

mes * −/* *ep * * ***

mes − − −ep *** ** **

mes *** * −musc ** * *

ep *** ** ***mes ** − −ep *** ** −

mes * − −ep ** * **

mes * − −/*ep ** ** **

mes * −/* −/*−/* ** ** ** *** * **

d * *** NSus systemcortex *** *** −

** *** −lon * ** −exus − −/* −e − ** −rvous systemt ganglia − *** −rojections (tail) * *** **elium *** *** ***m * * ***elium *** *** ***chyme *** −/* ***chyme *** * ***

*** ** −e *** *** −steocytes −/* *** −

** *** −/** * ***

ing; (*) weak staining; (**) moderate staining; (***) strong) not studied. ep, epithelium; mes, mesenchyme; musc,

40

5). At 16in manypersistedsections (data notextracellsulfate ch

NervousMK and lium froshown). the dorsaHB-GAMtube. GaHB-GAMin the cecorpus stalso obsenerves. Ithe roof tricular zanuclear moleculeity was flobe, whpositivelmordiumGAM. Indorsal arnuclear situated iwidely d

T

Fig. 2. ImAbbreviatBar, 600 µ


.5 dpc, MK protein distibution decreased considerably developing organs, whereas the HB-GAM staining in a wide variety of tissues. Heparitinase treatment ofreduced or completely abolished the MK staining shown), suggesting that the protein is bound to theular matrix and the cell surfaces through heparanains of proteoglycans.

systemHB-GAM proteins were localized in the neural epithe-m the earliest stage analyzed (9-10 dpc; data notMK was widely expressed in the brain vesicles and inl part of the neural tube, whereas the distribution of was restricted in some areas of the brain and neural

nglia and neuronal projections were positive only for. From 12.5 to 14.5 dpc, both proteins were present

rebral hemispheres, telencephalon, diencephalon andriatum (Figs 2A,B, 3B, 4A). HB-GAM staining wasrved in the motor nuclei of the trigeminal and facial

ntense expression of MK and HB-GAM was found inof the neopallial cortex, in the intermediate and ven-one, whereas only MK staining was observed in thelayer. The meninges were intensely stained for boths (Fig. 5Ca,Cb). Intense MK and HB-GAM reactiv-ound in the roof of the midbrain and in the olfactoryereas in the medulla oblongata only some cells werey stained. In the choroid plexus and the cerebral pri- a very weak staining was observed only for HB- the neural tube, MK was restricted to one part of theea, whereas the HB-GAM staining was diffuse. A

HB-GAM staining was occasionally found in cellsn the ventral part of the tube (Fig. 4F). HB-GAM wasistributed in the peripheral nervous system (Figs

g was evident in the dorsaleripheral neurons (Figs 3I,AM staining was faint andeloping brain (e.g. cerebralng HB-GAM reactivity wasstem.

ity was found in the devel-ining was moderate. Fromes showed MK reactivity, widely distributed in thee atrium (Fig. 2B). In blood

munohistochemical localization of MK (A), HB-GAM (B), a 14.5 dpc mouse embryo.ions: f, facial process; o, olfactory organ; b, brain; d, forming ural tube; v, vertebrae; l, lung.m.

4B,C,E, 5Jb), whereas MK staininroots of the ganglia and in some p7Ea). At 16.5 dpc, MK and HB-Gfound in restricted areas of the devprimordium for MK), whereas strofound in the peripheral nervous sy

Cardiovascular systemAt 9 dpc, strong HB-GAM reactivoping heart, whereas the MK sta12.5 to 14.5 dpc, only the valvwhereas HB-GAM reactivity wasmusculature of the ventricle and th

nd syndecan-1 (C), in sagittal sections of a digits; h, heart; li, liver; i, intestine; nt, ne

Fig. 3. Distribution of MK in different organs at differentdevelopmental stages. A cell surface staining is often observed inboth epithelial and mesenchymal cells. The staining frequentlydecorates basement membranes separating epithelial andmesenchymal components. (A) Kidney (k), testis (t), gut (g), liver (l),and pancreas (p) of a 13 dpc mouse embryo. (B) Intense staining (redcolor) in the neopallial cortex and the cephalic epithelium of a 14dpc embryo. Note that the choroid plexus (arrow) is not labeled.(C) Prevertebrae (ribs) at 13 dpc. (D) Intense staining in the activelyproliferating mesenchyme of the mandibular process and tooth (tb),as well as in Meckel’s cartilage (arrowhead) at 13.5 dpc. Note thestrong reactivity in the epithelium of the tongue (tg). (E) Labeling(red color) in Meckel’s cartilage of a 14 dpc embryo. Note thepositive reaction of the forming bone (arrow). (F) Inner ear andcartilaginous otic capsule (c) of a 14 dpc embryo. (G) Stomach andsmooth muscle layer (arrow) at 14.5 dpc. (H) Submandibular glandof a 14.5 dpc embryo. (I) Olfactory epithelium of a 13.5 dpc embryo.Note the staining in nerve fibers. (J) Actively proliferatingmesenchyme of the mandibular process at 14.5 dpc. (K) Positivesingle cells in the dermis of a 16.5 dpc embryo. Bars, 50 µm.

41Midkine and HB-GAM in mouse embryos

42

vessels, a weak MK and HB-GAM staining was observed inendothelial cells and basement membranes (Fig. 7Da). At 16.5dpc, only HB-GAM protein was localized in heart tissue. Atthis developmental stage, astrong MK and HB-GAM reac-tivity was detected in singlecells in the dermis (Fig. 3K),presumably macrophages.

Skeletal systemAt 12.5 dpc, MK protein wasdetected in precartilaginousmesenchymal condensations ofthe otic and nose capsule, in thesclerotome-derived precartilage(prevertebrae; Fig. 3C), and inMeckel’s cartilage. At 14.5 dpc,the skeletal elements consist ofcartilage, the first ossificationcenters appear, and the muscu-lature is differentiating. MK andHB-GAM staining waslocalized in cells involved inendochondral ossification and indifferentiating muscles (Figs2A,B, 5Aa,b). The perichon-drium and the hypertrophiedchondrocytes were intenselystained for MK (Figs 3D,E,5Da). HB-GAM immunoreac-tivity was absent from perichon-drium (Fig. 5Db). In the devel-oping sternum, the cartilage waspositive only for HB-GAM.Intramembranous ossificationimplies the direct conversion ofmesenchymal cells intoosteoblasts. Cells involved inthis process forming mandibu-lar, maxillary and cranial bones,exhibited MK and HB-GAMstaining (Figs 2A,B, 4A, 5Ca,b).

T. A. Mitsiadis and others

Fig. 4. Immunolocalization of HB-GAM in different tissues at 14.5dpc. At this embryonic stage thestaining is mainly observed inepithelial and neuronal cells,whereas mesenchymal cells andbasement membranes are rarelylabeled. (A) Intense staining in theneopalliar cortex, diencephalon,and forming cranial bone (arrow).(B) Dorsal root ganglion.(C) Maxillary process. Note thestrong staining of nerve fibers.(D) Developing whisker follicle.(E) Neurons innervating thewhisker follicle (arrow).(F) Intracellular staining in cells ofthe neural tube. (G) Lung.(H) Submandibular gland. (I) Innerear. Bars, 50 µm.

Differentiated osteoblasts and osteocytes were positive onlyfor HB-GAM (Fig. 5Ba,b). MK and HB-GAM appeared to bepresent in immature bone matrix, whereas mature bone was


Fig. 5. Comparisonof the distribution ofMK (Xa) and HB-GAM (Xb) indifferent tissues of13-16.5 dpc mouseembryos.(A) Striated musclesat 14.5 dpc.(B) Maxillary boneat 14.5 dpc.(C) Meninges andforming cranial boneat 14.5 dpc.(D) Cartilage of theribs at 14.5 dpc.(E) Liver at 14.5 dpc.(F) Lung at 13 dpc.(G) Kidney at 13dpc. (H) Intestine at14.5 dpc. (I) Adrenalgland at 14.5 dpc.(J) Digits at 16.5dpc. Bar, 25 µm.

44

devoid of these proteins. At 16.5 dpc, MK and HB-Gstaining persisted in chodrocytes of several cartilagenous sttures, whereas in osteoblasts and muscles only HB-GAM rtivity was found.

Organs and tissues undergoing epithelial-mesenchyminteractions

Sense organsIn the developing eye, MK staining was detected in the neretina, in lens, and in the condensed mesenchyme aroundeye cup (data not shown). In the developing inner ear (114.5 dpc), MK and HB-GAM staining was observed inthickened part of the vestibular epithelium, responsible forsensory function, in the surrounding mesenchyme, and in nfibers (Figs 2A,B, 3F, 4I). At 16.5 dpc, a very faint staining was observed in sensory epithelium, whereas GAM protein was present in mesenchyme (data not shownthe developing olfactory organ (from 12.5 to 13.5 dpc), the and HB-GAM proteins were distributed in the olfactory epilium (containing the sensory receptor cells) and the surrouing mesenchyme (Fig. 3I). In its respiratory part, the stainwas weaker in epithelium and absent from mesenchyDuring subsequent developmental stages (14.5 dpc), the tribution of MK and HB-GAM became more restricted andstaining was localized only in parts of the olfactory epithel(Fig. 2A,B). As elsewere, the levels of the MK prodecreased by 16.5 dpc, whereas a faint HB-GAM staining detected in epithelium.

Hair and whisker folliclesIntense MK and HB-GAM staining was observed on surfaces of both epidermal and mesenchymal cells locatethe sites of the hair and whisker follicles (Figs 2A,B, 7DWhen the epithelium started to invaginate the underlying cdensing mesenchyme, MK staining was present in both cponents of the developing whisker follicles (Figs 2A, 7whereas the HB-GAM protein was located only in ectodercells (Figs 2B, 4C,D). At this developmental stage (14.5 dnerve endings surrounding the whisker follicles were posifor HB-GAM (Fig. 4E). At 16.5 dpc, a very weak MK rtivity was found only in the mesenchymal component ofwhisker follicle. Faint HB-GAM staining was detected inepithelium and in some mesenchymal cells.


Fig. 6. Localization of syndecan-1 in different tissues of 12.5-13 neuronal cells. (A) Meninges (m) and cranial epithelium (ce). (B)

AMruc-eac-

al

ural the2.5- the theerveMKHB-). In

Respiratory systemAt 12 dpc, the endoderm-derived tracheal epithelium and theadjacent mesenchyme exhibited strong MK immunoreactivity,whereas the staining with HB-GAM was weak in epitheliumand absent from mesenchyme (data not shown). From 13 to16.5 dpc, MK protein was distributed on the surfaces of epithe-lial cells of the intrapulmonary segmental bronchi, bronchioli,alveolar ducts, and surrounding lung parenchyma (Figs 2B,5Fa). The distribution pattern of MK in bronchial epitheliumwas related to its differentiation status: the protein wasexpressed in differentiating bronchial epithelial cells, but itdiminished and disappeared completely from differentiatedcells. In contrast, faint HB-GAM staining was observed inbronchial epithelial cells, whereas the surrounding lungparenchyma was negative (Figs 2B, 4G, 5Fb).

Digestive system
MK From 12 to 14.5 dpc, MK and HB-GAM proteins were found the-nd-ingme.dis- theiumteinwas
thed at

a).on-

om-Ca),malpc),tiveeac- the the

at sites of the oral epithelium where palatal rugae are formed(Figs 2A,B, 7Fa). The epithelium of the tongue exhibited alsostrong MK and HB-GAM staining (Figs 2A,B, 3D, 7Fa). In thedeveloping submandibular salivary gland, both proteins werelocalized on the cell surfaces of the branching epithelial buds,whereas only MK staining was found in basement membranesand in mesenchyme (Figs 3H, 4H, 7Ba). In the esophagus, MKand HB-GAM staining were detected on the surfaces of epithe-lial cells, whereas the mesenchymal cells and the basementmembranes were negative (Fig. 2A,B). In the stomach andintestine, MK protein was found in both mucosal epitheliumand mesenchyme (Figs 2A, 3A,G, 5Ha). The distribution ofHB-GAM protein was almost similar to that of MK in thedeveloping stomach but the staining was weaker, whereas amoderate staining was observed only in the mucosal epithe-lium of intestine (Figs 2B, 5Hb). MK and HB-GAM reactiv-ity were also observed in the outer mesenchymal layer of thestomach and the intestine, which gives rise to the smoothmuscle layer (Fig. 3G). At 16.5 dpc, the MK staining persistedin the esophagus, stomach, and intestine (data not shown). Inthe duodenum, the MK immunoreactivity was distributed inboth epithelium and mesenchyme, whereas in midgut thestaining was found in mesenchymal cells surrounding themucosa. In contrast, HB-GAM staining was observed only in
epithelial cells of intestinal mucosa.
dpc mouse embryos. The staining is detected in epithelial, mesenchymal, and Prevertebrae. (C) Lung. (D) Liver. Bar, 25 µm.


Urogenital systemFrom 12.5 to 13.5 dpc, both MK and HB-GAM proteins werepresent in the mesenchyme and tubules of the developingmetanephros (Figs 3A, 5Ga,b). From 14.5 to 16.5 dpc, MK wasdistributed in parenchyma, basement membrane and epithe-lium of nephrogenic tubules of the kidney, whereas the HB-GAM staining was absent from the mesenchymal cells sur-rounding the developing metanephros. Both proteins werelocalized on the surfaces of epithelial cells and in the periph-eral and central core mesenchyme of the genital tubercle (datanot shown). At 16.5 dpc, only HB-GAM staining persisted inthe genital organ.

Facial processes and limb budsIn facial processes and developing limbs (from 10 to 14.5 dpc),strong MK and HB-GAM reactivities were detected in epithe-lium and underlying proliferating mesenchyme (Figs 2A,B,3D, 7Aa), whereas the underlying proliferating mesenchymeand the basement membrane exhibited strong staining only forMK (Fig. 3J). At 16.5 dpc, MK was absent from the facialprocesses (data not shown), whereas in limbs the staining wasrestricted to the mesenchyme situated at the distal part of thedeveloping digits (Fig. 5Ja). Weak HB-GAM reactivity wasobserved only in the surface epithelium of the facial processesand digits (Fig. 5Jb).

Fig. 7. Comparison of the distribution of MK (Xa) and syndecan-1 (Xb) in different tissues of 13-14.5 dpc mouse embryos. (A) Limbs at 13dpc. (B) Submandibular gland at 14 dpc. (C) Developing whisker follicles at 13.5 dpc. (D) Developing hair follicles and blood vessels (arrows)at 14.5 dpc. (E) Dorsal root ganglia (gg) at 13 dpc. (F) Palatal rugae (arrows) and tongue (tg) epithelium from 14.5 dpc embryo. Abbreviation:oe, oral epithelium. Bars, 50 µm.

46

Immunolocalization of syndecan-1 in mouseembryosPrevious data have demonstrated syndecan-1 expression inembryonic mouse tissues undergoing epithelial-mesenchymalinteractions, such as the developing limb buds (Solursh et al.,1990), kidney (Vainio et al., 1989) and whisker follicles(Trautman et al., 1991). We studied the expression ofsyndecan-1 in 12.5-14.5 dpc mouse embryos. At 12.5 and 13.5dpc, the patterns of syndecan-1 distribution in developingtissues and organs corresponded largely to those of MK andHB-GAM. The proteoglycan was widely distributed inepithelia of the skin, sensory organs, tongue, esophagus,stomach, gut, pancreas, and ureter (Figs 6C, 7Bb,Cb,Db,Fb).Syndecan-1 staining was found in both epithelium and mes-enchyme of several organs undergoing epithelial-mesenchy-mal interactions, such as whisker follicles, teeth, salivaryglands, lung and kidney. The mesenchyme of the facialprocesses, limb buds, genital tubercle and tail was alsointensely stained (Fig. 7Ab). Furthermore, strong staining wasdetected in the perichondrium of the cartilage of the ribs (Fig.6B). The liver (Fig. 6C) and endothelial cells (Fig. 7Db) werealso positive. Interestingly, syndecan-1 was detected in struc-tures of the central and peripheral nervous systems at thisdevelopmental stage. Earlier findings suggest that syndecan-1is not expressed in adult central nervous system. However,syndecan-1 mRNA transcript of unusual size; (4.5×103 Mr,usually 3.4 and 2.6×103 Mr) has been found in mouse brain(Saunders et al., 1989). A diffuse staining was observed on cellsurfaces of the developing brain (neopallial cortex, midbrain,diencephalon) and neural tube. Intense syndecan-1 stainingwas also found in the meninges (Fig. 6A). In the peripheralnervous system, the staining was evident in the roots of the

ganglia and in neuronganglia were negative

At 14.5 dpc, syndecand several previouslyThis distribution correand HB-GAM proteireactivity persisted inand gut epithelium, wonly the respiratory pof organs and tissuinteractions (e.g. whisdetected in both epithwith the distributionsyndecan-1 staining wand stomach. FurthermHB-GAM reactivity wwas absent from kidn

Localization of MKOur in situ hybridizaKadomatsu et al. (1913 dpc mouse embrythe developing orgacorrelate to those of tembryos, MK transcrthe facial processes (F(Fig. 8E). In organs uactions such as the oand gut (Fig. 8F) MKlium and mesenchym(Fig. 8A,D) and neuthe signal was absent


Fig. 8. MK mRNA expression in various developing tissues and organs in sagittal sections of 13 anprocess, tongue (tg), and brain (b) at 13 dpc. The signal is absent from the trigeminal gaglia (gg). (and tongue (tg) at 13 dpc. (C) Section of the same area as in B, labeled with a sense probe. (D) BraNote that the choroid plexus (arrow) is labeled. (E) Genital tubercle (gt) and tail (tl) at 14.5 dpc. (FBar, 200 µm.

al projections, whereas the bodies of the (Fig. 7Eb).an-1 distribution became more restricted stained organs were negative (Fig. 2C).sponded in part with the patterns of MKn expression (see table 1). Syndecan-1 skin, palate, tongue, esophagus, stomachhereas in the olfactory organ epithelium,ortion was positive. In the vast majorityes undergoing epithelial-mesenchymalker follicles, teeth) a strong staining waselium and mesenchyme, corresponding of MK and HB-GAM. In contrast,as faint and punctuated in lung, heart,ore, several tissues expressing MK and

ere negative for syndecan-1: the stainingey and neuronal structures.

transcripts in mouse embryostion results confirm the previous data of90) on MK mRNA expression in 11 andos, and show that in the vast majority ofns the patterns of mRNA expressionhe MK protein. In 13 to 14.5 dpc mouseipts were detected in the mesenchyme ofig. 8A,B), limbs, genital tubercle and tailndergoing epithelial-mesenchymal inter-lfactory organ (Fig. 8B), inner ear, lung,transcripts were expressed in both epithe-e. Neuronal tissues including the brain

ral tube expressed MK mRNA, whereasfrom cell bodies in the ganglia (Fig. 8A).

d 14.5 dpc mouse embryos. (A) MaxillaryB) Facial processes (f), olfactory organ (o),in (neopallial cortex) of a 14.5 dpc embryo.) Intestine (i) and pancreas (pn) at 14.5 dpc.

47in mouse embryos

The cintensespecifimental

At mKadomsignal analyshead oresults16.5 d(Fig. 9kidneywere ntochemsuch as

MK andepenMK anheparininvolveproteinsurfacetherefo1 in aproteinmouse and HSyndecequimo(200 psyndec

Fig. 9.illuminBars, 2

Midkine and HB-GAM

10A). HS (50 µg/ml) effec-h protein (Fig. 10B). CS, in of syndecan-1 to collagenB-GAM by about 60% (Fig.

ce syndecan-1n in limb and facial

y whole-mount immunohis-ion of this molecule around-GAM protein (Fig. 11A).ecan-1 was induced in thebined epithelium (arrows).

mination (A-C) and dark-fieldfactory organ (o).

horoid plexus, which was negative for MK protein,ly expressed MK transcripts (Fig. 8D, arrow). Noc signal was detected with sense probe at any develop- stage throughout the study (Fig. 8C).

ore advanced developmental stages (15 to 19 dpc),atsu et al. (1990) reported that the MK hybridizationwas detectable only in the kidney. However, slot blotis has shown that MK mRNA is also expressed in thef later-stage embryos (Nakamoto et al., 1992). Our

show that the MK mRNA signal persists, at least untilpc, in the brain, facial processes (Fig. 9D), soft palateE), olfactory organ (Fig. 9F), teeth, limbs, intestine, and genital tubercle (data not shown). Interestingly, weot able to detect MK protein expression by immunohis-

compared to type I collagen (Fig.tively reversed the binding to eacturn, had no effect on the bindingbut inhibited binding to MK and H10B).

MK and HB-GAM do not induexpression or cell proliferatiomesenchymeAnalysis of syndecan expression btochemistry did not reveal inductthe beads releasing MK or HBHowever, the expression of syndmesenchyme contacting the recom

Sagittal sections of several developing tissues at 16.5 dpc. MK mRNA expression seen in bright-field illuation (D,E,F). (A,D) Mandibular process (mp), whisker follicles (arrows). (B,E) Soft palate (sp). (C,F) Ol00 µm.

istry in several tissues expressing the MK transcripts in facial processes and soft palate.

d HB-GAM bind to syndecan-1 in a dose-dent mannerd HB-GAM are growth-associated molecules that bind, and it is thus likely that cell surface HSPGs ared in the regulation of cellular responses to theses. This hypothesis relies on the assumption that cell HSPGs are able to recognize MK and HB-GAM. Were tested the binding of MK and HB-GAM to syndecan- solid-phase assay using nitrocellulose-immobilizeds and metabolically 35SO4-labeled syndecan-1 frommammary epithelial cells. Syndecan-1 bound both MKB-GAM in a dose-dependent manner (Fig. 10A).an-1 bound MK and HB-GAM very similarly aslar amounts of type I collagen. The largest amounts

mol) of proteins resulted in about 2- and 5-fold morean-1 being bound to HB-GAM and MK respectively, as

Cell proliferation was analysed by labeling the explants withbromodeoxyuridine (BrdU). In recombinants of epithelium andmesenchyme of the hindlimb or jaw, the epithelium inducedcell proliferation in mesenchyme (arrows), whereas the beadsreleasing either MK or HB-GAM were not able to stimulatecell proliferation (Fig. 11B,C).

DISCUSSION

Developmental roles of MK and HB-GAMOur immunohistochemical studies demonstrated characteristicdistribution patterns for midkine (MK; Kadomatsu et al., 1988)and heparin binding-growth associated molecule (HB-GAM orpleiotrophin; Rauvala, 1989), members of a novel family ofheparin-binding molecules (Muramatsu, 1993), during mouseembryonic development (9 to 16.5 dpc). Both proteins areexpressed in the central and peripheral nervous systems.Several in vitro experiments have shown that MK and HB-

48

GAM mneuronalMuramaRaulo et

The aorgans ugland, lutiation, sdifferent

Another interesting aspect of MK and HB-GAM distributionwas their specific localization in the basement membranes ofdeveloping organs. At 16.5 dpc, weak MK staining decoratedthe basement membranes of several organs (e.g. salivary gland,lung), which ceased to express MK mRNA, suggesting that theembryonic basement membranes may serve as sites of storagefor the protein.

MK and HB-GAM expression was often detected at sites ofcartilage and bone formation. The first stage of endochondralbone formation involving the transformation from mes-enchyme to cartilage correlated with the expression of MK onthe surfaces of the condensed mesenchymal cells. Furthermore,direct conversion of mesenchymal cells into osteoprogenitors(intramembranous ossification) correlated with the expressionof both MK and HB-GAM. Hence, these molecules may playa regulatory role during initiation of cartilage and bone devel-opment.

T

Fig. 10. (collagen (filters usi35SO4-labwith phoss.e.m. of of bindingsubtractedglycosamand type Ito proteinpresence or both (HHS or CSbinding abound rad


a t p

nui

Comparison of the expression pattern of MK mRNA and the

AC

neptw

i so

.n

corresponding protein in developing organs are in line withboth autocrine and paracrine modes of action. In some casesthe MK immunoreactivity was absent from sites of mRNAexpression (e.g. choroid plexus; see arrows at Figs 3B, 8D),suggesting either paracrine effects or inability to translate it.Our immunohistochemical analysis showed that HB-GAM wasdistributed principally in basement membranes and epithelialcell surfaces, whereas, in an earlier study, Vanderwinden et al.(1992) showed that the HB-GAM transcripts were mainlylocalized in neuroectodermal and mesenchymal cell lineages.Hence, it is apparent that the expression of the mRNA does notalways correspond to that of the protein, suggesting a paracrinemechanism of action for HB-GAM in several developingorgans. In the developing nervous system, however, both HB-GAM transcripts and the protein seem to localize to the sameareas, suggesting an autocrine mechanism of action. For moredefinitive clues on how MK and HB-GAM might act it isessential to indentify their cell surface receptors.

It is not clear at present whether the biochemical modes ofaction of MK and HB-GAM are similar. Most of the resultsobtained to date support the notion that MK and HB-GAMfunction autonomously, but this does not exlude the possibil-ity that they could interact as well. MK and HB-GAM werefrequently localized in the same developing organ, often with

) Binding of syndecan-1 to MK, HB-GAM, and type IOL I). The proteins were immobilized onto nitrocellulose

g a vacuum blotting apparatus. The filter was incubated inled syndecan-1. Protein-bound syndecan-1 was detectedhor imaging equipment. Results represent the mean ±o independent analyses and are expressed as percentages

to type I collagen. Amounts binding to BSA have been

y initiate and maintain the differentiated state ofcells (Li et al., 1990; Michikawa et al., 1993;

su and Muramatsu, 1991; Muramatsu et al., 1993;al., 1992; Rauvala, 1989; Rauvala et al., 1994).pearance of MK and HB-GAM staining in many

ndergoing branching morphogenesis (e.g. salivaryg, kidney) was concomitant with epithelial differen-ggesting that these molecules may be involved in theation process of several epithelial cell lineages.

overlapping patterns of expression. RA induces the expressionof MK (Matsubara et al., 1994; Muramatsu, 1993) whereasHB-GAM expression is not affected by RA (Merenmies,1992). Furthermore, no available information exists about‘cross-talk’ between the individual genes (i.e. whether HB-GAM could be induced by MK or vice versa). Analysis intransgenic animals of either loss- (knock out) or gain-of-function (i.e. ectopic expression) mutation, may contribute toour understanding of MK and HB-GAM function inembryonic development.

Association of MK and HB-GAM with epithelial-mesenchymal interactions during organdevelopment Studies in experimental embryology have demonstrated thatorgan development depends on sequential and reciprocal inter-actions between epithelial and mesenchymal tissues(Gumbiner, 1992; Saxén, 1972; Saxén et al., 1980). MK andHB-GAM were expressed in many developing organs, such as

from the values. (B) Effect of the freenoglycans on the binding of syndecan-1 to MK, HB-GAM,collagen (COL). The binding of 35SO4-labeled syndecan-1 immobilized onto nitrocellulose filters was assessed in thef 50 µg/ml heparan sulfate (HS), chondroitin sulfate (CS),S + CS). Control represents the binding in the absence of Values are expressed as percentages of uninhibitedd are obtained using phosphor imaging analysis of filter-ioactivity.

49ryos

glands, expressiinstanceand HBepitheliulying menchymthe mRNin the mal., 1992gesting

In dewere locand the tion patopment upon i(Richmahas beethat the trolling WeddenNiswandenchymbuds. TaHB-GAenchym

SyndecMK andSyndecaexpresseduring tfolliclesbe regulorgans (al., 1989also in flocaliza

Fig. 11. ( s soaked inMK prot .(B,C) Ef ether withepitheliu phase S).The epithAbbrevia

Midkine and HB-GAM in mouse emb

A) Effects of MK on syndecan-1 expression. Jaw mesenchyme and epithelium (11 dpc) were cultured together with beadein (100 ng/µl) for 16-32 hours. Syndecan-1 expression in mesenchyme is induced by epithelium (arrows) but not by MKfects of MK or HB-GAM protein on cell proliferation. Limb (B) or jaw (C) mesenchyme was cultured for 16-32 hours togm and MK (B) or HB-GAM (C) beads. Proliferations were detected by immunostaining BrdU-incorporating cells (cells in

k

-

ee

pv

t

n

ne

a

Ma

h,a

at

e, although notdicate that MK do not inducenchyme wheninfluences theence it may acten et al., 1993;simultaneouslyctin and type Irexpression ofoth FGF-1 andyndecan-1 maymultiple waysi, 1991), and it exist for MK

syndecan-1 areonzalez et al.,t results), sug-the binding of

elium induced cell proliferation in the adjacent mesenchyme (arrows), whereas MK and HB-GAM had no effect on cell proliferation.

idney, lung, gut, whisker follicles and teeth, and theiron correlated with inductive tissue interactions. For, in the whisker follicle (sensory hair or vibrissa), MKGAM were first intensely expressed in the thickenedm. When the epithelium started to invade the under-senchyme, cells of the prospective root papilla mes- started to express MK immunoreactivity. AlthoughA expression for HB-GAM has been reported to occuresenchymal sheet of the whiskers (Vanderwinden et), the protein was detected in epithelium, again sug-aracrine action.eloping limb buds, both MK and HB-GAM proteinsalized in the epithelium of the apical ectodermal ridgemesenchyme of the progress zone. A similar distibu-ern was observed in the facial processes. The devel-of the limb buds and facial processes is dependentteraction between mesenchyme and epitheliumn and Tickle, 1989; Tickle, 1991; Wedden, 1987). It demonstrated by tissue recombination experimentspithelium regulates facial and limb outgrowth by con-both the maintenance (Richman and Tickle, 1989;

ulations, and HB-GAM was expressed at somall, of these sites (see Table 1). Our studies inand HB-GAM bind to syndecan-1, but that theysyndecan expression in facial and limb mesereleased from agarose beads. Syndecan-1 binding and mitogenic activity of FGF-2 and has a low affinity growth factor receptor (JalkanSalmivirta et al., 1992). Syndecan-1 can bind to FGF-2 and ECM molecules such as fibronecollagen (Salmivirta et al., 1992), and ovesyndecan-1 inhibits the biological effects of bFGF-2 in 3T3 cells (Mali et al., 1993). Hence, sregulate the biological functions of FGFs in during organogenesis (Ruoslahti and Yamaguchis possible that similar regulatory mechanismsand HB-GAM.

It is apparent that MK, HB-GAM, FGFs and frequently colocalized in embryonic tissues (G1990; Niswander and Martin, 1992; our presengesting molecular interactions. For example,

tions: b, agarose bead; e, epithelium; m, mesenchyme. Bar, 200 µm.

, 1987) and the proliferation (Minkoff, 1991;er et al., 1993; Reiter and Solursh, 1982) of mes-l cells at the apex of the facial processes and limbken together, these observations suggest that MK and

may be involved in signalling during epithelial-mes-l interactions.

an-1 may regulate the biological effects of HB-GAMn-1, a cell surface heparan sulfate proteoglycan, isd in morphogenetically active mesenchymal cellse development of facial processes, limb buds, whisker kidney, and tooth. Its expression has been shown toted by epithelial-mesenchymal interactions in several

Bernfield et al., 1992; Thesleff et al., 1991; Vainio et) and our present results indicate that this is the casecial processes and limb buds. MK showed striking co-ion with syndecan-1 in many mesenchymal cell pop-

MK and HB-GAM to syndecan-1 in embryonic mesenchymecould considerably reduce the free heparan sulfate sites of theproteoglycan, thereby affecting the binding and biologicalactivities of FGFs. In our assay system, MK and HB-GAMproteins that were released from the beads in limb and jaw mes-enchyme did not stimulate cell proliferation, whereas FGFs aremitogens for these cells. However, expression of MK and HB-GAM in these tissues correlates with active cell division. Theeffect of MK and HB-GAM on cell proliferation appears todepend on cell type: in several cell lines they do not affect themitogenic activity, whereas they may be mitogens for someother cell populations (Hampton et al., 1992; Li et al., 1990;Muramatsu and Muramatsu, 1991; Nurcombe et al., 1992;Raulo et al., 1992). Taken together these findings suggest thatmolecular interactions may exist between FGFs, MK, HB-GAM and syndecan-1 during development, and that thesemolecules participate in the spatial and temporal control ofmorphogenesis.

50

We are(Unité de630, ParisMs Anja Tskilful tecThis workScience F

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