Expression of the human NOV gene in first trimester fetal tissues

Abstract NOV, located on human chromosome 8q24.1,was originally cloned following discovery of its avian homolog as a consequence of over-expression in virallyinduced nephroblastoma. The gene product is a secreted,modular, protein and a member of the CCN gene family.Evidence to date indicates that the expression of the wildtype protein is associated with cellular quiescence in normal embryonic fibroblasts yet produces growth stim-ulatory effects on established murine NIH 3T3 cells.Here we report the expression of NOV in the first trimes-ter of human embryogenesis, between 5 and 10 weeks.In situ hybridisation and immunohistochemistry revealwidespread expression in derivatives of all three germlayers. The most abundant sites of expression are in themotor neurons and floor plate of the spinal cord, adrenalcortex, fusing skeletal, and smooth muscle, the urogeni-tal system and the developing heart. Additionally, ex-pression is seen in the cranial ganglia, differentiatingchondrocytes, gonads, and lung. The sites of expressionsuggest strongly that autocrine or paracrine expression ofNOV is associated with the process of cell differentia-tion.

Keywords NOV · Cell differentiation · Muscle · Motorneurone · Ganglia · CNS · Human · Fetus · CCN

Introduction

The CCN (Cyr61, Ctgf, NOV) family of growth regula-tors comprises a group of structurally conserved peptidesthat are predominantly matrix-associated heparin-bind-ing proteins, playing roles in fundamental biological pro-cesses such as wound healing, differentiation, angiogen-esis and tumorigenesis.

NOV is one of the prototypic members of the CCNfamily cloned as a consequence of its overexpression inretrovirally induced nephroblastoma (Joliot et al. 1992).Forced expression of NOV in normal secondary chickenembryo fiboblasts (CEF) is associated with quiescenceand is downregulated upon oncogenic v-src induced trans-formation (Scholtz et al. 1996). Overexpression of an ami-no-truncated form of NOV was shown to be transformingwhereas overexpression of a full length NOV was inhibi-tory to CEF growth (Joliot et al., 1992). These observa-tions suggested that NOV encodes a negative cell-growthregulator. More recently, purified human NOV protein wasreported to stimulate growth of NIH 3T3 cells in culture(Liu et al. 1999), indicating that the biological activity ofthe NOV protein might be dependent upon the cell typeand environmental factors. Similar context-dependent actions have been reported for WISP1 (see below).

The chicken, human, mouse and frog NOV geneshave been cloned and are highly conserved, both in in-tron/exon organisation and peptide sequence (Martinerieet al. 1992; Perbal 1995; Snaith et al. 1996; Ying andKing 1996). The human and mouse genes are present assingle copies on chromosome 8q24.1 and chromosome15 between D15 Mit153 and 183, respectively.

The CCN family now comprises 6 members, WISP1–3 (Wnt induced secreted protein), Cyr61, CTGF andNOV. The members of this family are characterised by38 conserved cysteine residues and are modular in construction, being composed of combinations of four

S. Kocialkowski · P.N. Schofield (✉ )Laboratory of Stem Cell Biology, Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DY,UKe-mail: PS@ mole.bio.cam.ac.ukTel.: +44-1223-333893, Fax: 44-1223-333786

H. YegerDepartment of Pathology, Hospital for Sick Children and University of Toronto, Toronto,Canada

J. KingdomMaternal-Fetal Medicine, Mount Sinai Hospital, Suite 775B, University of Toronto, 600 University Avenue, Toronto, Ontario,Canada M5G 1X5

B. PerbalLaboratoire d’Oncologie Virale et Moleculaire, UFR de Biochimie, Université Paris 7, D. Diderot, 75005 Paris,France

Anat Embryol (2001) 203:417–427 © Springer-Verlag 2001

O R I G I N A L A RT I C L E

Sylvia Kocialkowski · Herman Yeger · John KingdomBernard Perbal · Paul N. Schofield

Expression of the human NOV gene in first trimester fetal tissues

Accepted: 28 February 2001

domains each containing a structural motif recognized inproteins with key biological functions. The first motif(GCGCCXXC), is found in the IGF binding proteins, thesecond motif (VWC) is identical to the von Willebrandfactor C oligomerisation domain, the third motif (TSP-1)extracellular matrix binding domain is found in thrombo-spondin and the fourth (CT) domain contains a cystineknot motif characteristic of some growth factors includ-ing PDGF, NGF and TGF-β (Bork, 1993). Structure pre-diction carried out using the PhD and TOPITS programs(unpublished data) indicated a predominantly β sheetcomposition forming a helical propeller structure. CTGF(Kim et al. 1997) and NOV (Burren et al. 1999) bind IGFin vitro with a 100–1000 fold lower affinity than classi-cal IGF binding proteins. However, in other studies, noIGF binding to NOV was observed under standard ligandblotting assay conditions (Chevalier et al. 1998; Liu etal. 1999; Perbal, unpublished; P.N. Schofield, unpub-lished), and consequently the low affinity binding forIGF remains controversial and the inclusion of the CCNfamily as members of the IGFBP superfamily has recent-ly been questioned (Grotendorst et al. 2000).

The common heparin binding motifs, which are wellconserved within the family, have been shown to be essen-tial for binding of fibroblasts to Cyr61, where there is evi-dence for differential requirements for HSPGs in bindingvia different integrins (Chen et al. 2000; α6β1 in fibroblastsand αvβ3 in endothelial cells). One exception to this isWISP2, which lacks the C-terminal domain bearing theHSPG binding motifs, leading to the suggestion that WISP2might be able to affect the binding of other CCN familymembers to matrix in a dominant negative and cell type-de-pendent manner (Kumar et al 1999, Chen et al. 2000).

The CTGF (connective tissue growth factor) and Cyr61(cysteine rich 61) proteins are encoded by immediate earlygenes (O’Brien, et al. 1990; Bradham, et al. 1991). Con-nective tissue growth factor, a cysteine-rich mitogen se-creted by human vascular endothelial cells, is related tothe SRC-induced immediate early gene product CEF-10(Bradham et al. 1991). Both have been reported to pro-mote and mediate cell adhesion, migration, proliferationand survival (Kireeva, et al. 1996; Grotendorst 1997;Brigstock 1999; Lau and Lam, 1999), induce angiogenesisin vivo, chondrogenesis in vitro, and represent novel ligands for integrins αVβ3 and αIIbβ3 (Kireeva et al. 1998)and α6β1 (Chen et al. 2000). CTGF was also demonstrat-ed to mediate mitogenic and matrigenic activities of TGFβ(Kothapalli et al. 1997, 1998; Duncan et al. 1999).

While Cyr61 and WISP1 were reported to promote tumor growth (Babic et al. 1998; Xu et al. 2000),Elm1/WISP1 and rCOP-1/WISP2 have been shown to inhibit tumor growth (Hashimoto et al. 1998; Zhang et al. 1998), therefore confirming that, in spite of theirhighly related structural organization, each of the CCNproteins may display quite specific biological propertiesand that this may change according to context. Recentlymutations in WISP3, found in patients suffering from pro-gressive pseudorheumatoid dysplasia (PPD; MIM208230),have been shown to interfere with normal post-natal carti-

lage growth (Hurvitz et al. 1999), reinforcing the evidencefor a pivotal role for members of the CCN family in tissuedifferentiation and homoeostasis.

The functions of CCN family members in develop-ment are currently under intense investigation. Expres-sion during chondro/osteogenesis, angiogenesis and thedevelopment of the CNS has been reported for some fam-ily members, and whilst the role of NOV in neurogenesisis not yet understood Cyr61 is induced as a response to the stimulation of muscarinic acetyl chloline receptorsin adult rat brain and primary hippocampal neuronal culture suggesting a potential role in neuronal plasticity(Albrecht et al. 2000). CTGF is expressed at very highlevels in hypertrophic chonodrocytes during endochon-dral ossification (Nakanishi et al. 2000) and also stimu-lates angiogenesis. Three family members are known tobe induced in response to Wnt signalling: WISP1 (Elm1),WISP2 (rCOP-1) and WISP3. Expression of WISP1 hasbeen shown to be dependent on β-catenin (Xu et al. 2000)as expected from its Wnt responsiveness. The recent im-plication of WISP3 in the inherited human PPD syn-drome, associated with abnormal organisation and differ-entiation of growth zone chondrocytes is powerful evi-dence for an important role of at least this family memberin maintenance of normal post natal chondrocyte growthand differentiation (Hurvitz et al. 1999).

In normal human embryos expression of NOVhas been associated with differentiation of the kidney (Chevalier et al. 1998) and central nervous system (Su et al. 1998). In Wilms’ tumors, expression of NOV wasshown not to be restricted to growth arrested cells andwas a marker of heterotypic differentiation (Chevalier et al. 1998). To date, the expression of NOV in the orga-nogenetic phase of development has not been reportedand in order to establish processes and organ systems inwhich NOV might be important we have undertaken anexpression study of NOV protein and mRNA in early human embryos between 5 and 10 weeks of gestation.

We demonstrate by in situ hybridisation and immuno-histochemistry that NOV is widely expressed in deriva-tives of all germ layers. In addition to high levels of ex-pression in the adrenal cortex, NOV was expressed athigh levels in specific areas of the CNS, cartilage, and infusing skeletal muscle, suggesting a role in the terminaldifferentiation of specific tissues.

Materials and methods

Collection and staging of human embryonic material

Permission for this study was granted by the ethics committees ofCambridge Regional Health Authority and University CollegeHospital London. Human embryonic and early fetal tissues wereobtained at the time of legal termination of pregnancy followingmaternal consent. All human fetal material was obtained by directaspiration termination of pregnancy under ultrasound guidance topreserve embryo integrity (Soothill and Rodeck 1994). Fetal mate-rial was washed thoroughly in ice cold phosphate buffered saline(PBS; Dulbecco and Vogt 1954) and fixed in cold fresh 4% (w/v)paraformaldehyde (PFA) in PBS within 5 min of aspiration. Onlymorphologically normal material was used in this study. Fetal age

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was estimated by foot length where appropriate and otherwise bycombination of crown/rump length and, following sectioning, byCarnegie histological criteria. Embryos used in this study were 5,6, 8 (×2), 10 (×2) weeks of gestation. Complete embryos were ob-tained at each of the time points, and partially disrupted specimensat 8 and 10 weeks. Results shown in the figures are from embryosEGA9, EGA13 and EGA26, which were precisely staged by Carnegie criteria to eight (EGA9) and ten (EGA13 and 26) weeks.

RNA samples were additionally obtained from individual firsttrimester fetuses obtained by aspiration termination through theMRC tissue bank. RNA was extracted from first trimester fetal tissues using the method of Chomzynski and Sacchi (1987). Fetalage was estimated from the crown rump length and foot lengthwhen appropriate.

In situ hybridisation

Fetal tissues were wax-embedded in a mixture of waxes giving amelting point of 56°C to optimise RNA preservation. Sectionswere cut at 4 µm thickness and placed on either vectabond treated(Vector laboratories) or superfrost (BDH) slides. In situ hybridisa-tion was performed according to (Wilkinson and Nieto 1993).Briefly, paraffin-embedded sections were deparaffinised in xylene,re-hydrated by passage through a graded ethanol series. The slideswere then dipped successively for 5 min into 0.9% (w/v) NaCl solution then in PBS, and incubated for 20 min in 4% (w/v) para-formaldehyde (PFA) pH 7.2. The slides were washed twice in PBSfor 5 min and treated with 20 µg/ml proteinase K (Boehringer) diluted in 50 mM TRIS, 5 mM EDTA pH 7.4 for 10 min at roomtemperature. The slides were briefly washed in distilled watertreated with 0.1 M triethanolamine-HCl (TEA) pH 8.0 and thenplaced in fresh 0.1 M TEA containing 0.25% (v/v) acetic anhy-dride for 10 min at room temperature. The sections were thenwashed for 5 min in PBS, 0.9% NaCl and dehydrated through anethanol series. Following prehybridisaton in hybridisation bufferfor 6 h, the radioactive probe was added to the slides and hybrid-ised at 55°C overnight. Autoradiography was performed by dip-ping the slides in autoradiographic emulsion (Ilford, K5 emulsionin gel form) and exposing them for 10 days at 4°C. Exposed slideswere developed in Kodak D19 developer, counterstained with either neutral red or methylene blue, dehydrated and mounted.

Probes

For in situ hybridisation studies several DNA fragments of the hu-man NOV genomic DNA sequence were subcloned into transcribablevectors. Each fragment corresponds to a different exon of the NOVgene. A 143 bp fragment corresponding to 65 nucleotides of 5’ UTR and exon 1 was subcloned into pCRII™ (InVitrogen). A 120 bp fragment corresponding to exon 4 was subcloned in topCRIITM (InVitrogen). Finally a 1081 nucleotide fragment corre-sponding to exon 5 and the 3’ UTR region (EX) was subcloned intopBS-KSII+ (Stratagene). The plasmids were linearised with an ap-propriate restriction endonuclease located in the polylinker immedi-ately adjacent to the insert at the opposite end of the promoter andsingle stranded sense or antisense RNA probes were prepared by run-off transcription in the presence of 35S-UTP (1300 Ci/mmol, DupontN.E.N.). The labeled probes were ethanol-precipitated twice, resus-pended in 50 mM DTT and diluted in hybridisation solution (50%deionised formamide, 0.3 M NaCl, 20 mM TRIS-HCl pH 8.0. 5 mMEDTA, 10 mM sodium phosphate pH 8.0, 10% w/v dextran sulphate,1×Denhardt’s solution, 0.5 mg/ml yeast tRNA and 10 mM DTT).

Immunohistochemistry

Immunohistochemistry experiments were performed on 5 µm formalin-fixed paraffin-embedded sections as previously described(Kim et al. 1996) with the following modifications: after dewaxingand rehydration steps, microwave assisted antigen retrieval was per-formed first with 200 ml of 0.01 M sodium citrate pH 6.0 for 3 cy-cles of 5 min heating followed by cooling at room temperature, then

into a pressure cooker with one liter of 0.01 M sodium citrate pH 6.0heated for 20 min. After blocking of the endogenous peroxidase ac-tivity with 3% v/v hydrogen peroxide in water for 30 min, non-spe-cific antigenic sites were saturated by incubation in 5% v/v normalgoat serum in antibody diluting buffer (Dimension Laboratories) for30 min. For immunodetection of NOV protein, sections were then in-cubated with K19 M antibody (Chevalier et al. 1998) at 1:200 at 4°Covernight, washed three times in wash buffer (30 mM TRIS pH 7.5;150 mM NaCl; 1% w/v BSA; 0.05% v/v Triton X-100), incubatedwith biotinylated goat anti-rabbit secondary antibody (Molecularprobes) at 1:250 in diluting buffer for 1 h at room temperature, thenwashed three times in wash buffer and incubated in avidin-peroxi-dase (Dimension Laboratories) at 1:1000 for 1 h at room tempera-ture. After final washes in wash buffer, peroxidase enzyme reactionwas developed for 3–7 min in DAB solution (Research Genetics),and sections were counterstained with hematoxylin-ammonia blue-ing and mounted with Permount after dehydration through graded alcohols. Controls were performed both by incubation leaving outthe primary antibody or overnight preincubation of the K19 M antibody with 10 µg/ml of specific peptide at 4°C.

Northern blotting

Northern blotting of 20 µg of total fetal tissue RNA was carried outas described in (Brice et al. 1989), and probed with probe (EX) de-scribed above labelled with 32P-dCTP by random hexanucleotidepriming (Amersham Megaprime kit, Amersham, UK) according tothe manufacturers instructions. Probe specificity was confirmed byRNAse protection and each exon-specific probe hybridised to thesame mRNA species on Northern blots (data not shown).

Results

NOV expression in the first trimester embryo

RNA from available fetal tissues was subjected to Northern blotting with probe EX demonstrating the pres-ence of the previously reported 2.5 kb mRNA species in allexpressing tissues (Fig. 1). NOV RNA could be detected

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Fig 1 Expression of NOV mRNA in first trimester fetal tissues:RNA was prepared from fetal tissues between 8 and 10 weeks of ageas described in Materials and methods. Northern blotting (A, B) wascarried out as described in Materials and methods and filters probedsucessively with probe EX (A) and GAPDH (B) as a loading control.Markers used were GIBCO/BRL RNA molecular weight markers

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os covering the period 5–10 weeks of gestation. Two em-bryos were fragmented and consequently no data wasavailable for the head or thoracic regions for these speci-mens; however, within the time window examined, all ofthe fetuses showed consistent patterns of expressionevolving with development of individual tissues. Most ofthe sites of NOV protein expression correspond to sitesof RNA synthesis, indicating that the protein is probablyacting on site, predominantly in an autocrine or paracrinefashion. Immunoreactivity is associated with the cyto-plasm of all expressing cell types, with characteristic appearance in the ECM/pericytoplasmic region in someplaces. Immunoreactivity was clearly present in a fibril-lar pattern within muscle fibres and nerve axons. Expres-sion was seen in derivatives of all of the primitive germlayers as follows:

Ectoderm

The major site of NOV expression in the first trimesterfetus is the central nervous system. Neurons of both thespinal cord and the brain express significant amounts ofNOV mRNA and protein (Fig. 2C, Fig. 4J, N, L).

mainly in the adrenal gland, limb bud, heart, and meta-nephros. A weak signal was observed for spinal cord; liverand lung showed no detectable expression at this stage.

In situ hybridisation was carried out using all threeprobes described in Materials and methods; whilst simi-lar distributions of signal were obtained, the longer exon5 probe (EX) gave quantitatively the best signal and datapresented below were obtained using this probe. Previ-ous studies examining the expression of the gene and exon specific expression have indicated no evidence foralternative splicing in the NOV gene of human, chick ormouse to date. Investigation was made on seven embry-

Fig. 2A–G In situ hybridisation to NOV mRNA in human fetal tis-sues at ten weeks of age: all of the tissues shown here are from the same conceptus. A Transverse section of metanephric kidneyshowing transmitted light image of autoradiogram following hybridisation to probe EX as described in Materials and methods, counterstained with neutral red (g glomerulus, c condens-ing metanephric mesenchyme, t differentiating nephrogenic epithe-lia, cd collecting ducts). B Control section for the same kidney hybridised with a sense probe and counterstained with neutral red.C Lumbar region of the spinal cord (fp floor plate, vh ventral horn),D Dorsal root ganglion. E Gut. mucosa (m). F Foregut and pan-creas. G Fusing axial skeletal muscle. A, C, F ×200; B×220; D, E,G×320

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Fig. 3 In situ hybridisation to NOV mRNA in human fetal tissuesat 8 and 10 weeks of age: in situ hybridisation to NOV mRNA wascarried out on sagittal (A–C, E, F) and transverse, (D) sections ofa ten-week-old and an eight-week-old human embryo respectively.Sections were hybridised with an antisense (A, D, E) NOV probe(EX) as described in Materials and methods or a sense (control)probe (C) and photographed under dark field illumination. B andF are bright field images of fields A and E. A Cranial regionshowing trigeminal ganglion (tg), auditory apparatus (a) and cranial mesenchyme. B transmitted light image of A; capsule ofotic vesicle labelled (o). C Adjacent section to A hybridised withsense probe (control). D Transverse section through adrenal gland(ad), gonadal ridge (gr), and mesonephros (mn) showing hybrid-isation to the adrenal cortex (no medulla present at this time), interstitium of the gonadal ridge, mesonephric ducts and the meso-thelium surrounding the mesonephros. E Sagittal section throughdorsum showing dorsal root ganglia (drg) and vertebral bodies(vb). F Light field image of E. A–C×100; D×200; E, F×25

Within the brain, levels are similar through the neurectoderm of the fore, mid and hind brains at all stages of development seen, though in the later fetus themidbrain and particularly the presumptive corpus stria-tum show slightly higher labelling. The choroid plexusand leptomeninges are rich sources of NOV (Fig 4K).

Within the head, the cranial ganglia are major sites ofNOV mRNA and protein expression, notably ganglion V,the trigeminal (Fig. 3A). It was not possible to determinewhether this expression is in neurectoderm-derived or neu-ral crest-derived neurons in the material examined. Othercranial ganglia, notably the vagal (X), IX, VIII and VII, theauditory complex ganglia, are major sites of expression andhigh levels are seen in the neuroepithelial lining of the oticvesicle and its later derivative, the saccule (Figs. 3A, 4L).

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Within the spinal cord low levels of NOV RNA andprotein are seen throughout. In the earliest fetus this isaccompanied by slightly increased labeling of the floorplate, but in later material the floor plate labeling be-comes very strong and in the caudal (lumbar) portion ofthe spinal cord is accompanied by labeling of the cellbodies of neurons in the ventral horns, anatomically con-sistent with the cell bodies of motor neurons (Fig. 2C).Labeling of motor neurons was absent in the rostral spinal cord. High levels of signal are seen in the neuralcrest-derived dorsal root ganglia at all levels (Figs. 2D,3E, 4I), and in all of the ages examined. Other neuralcrest derived structures such as the adrenal medulla areeither not present at the stages examined or did not express NOV RNA. Cranial mesenchyme derived fromneural crest does however show NOV RNA expressionparticularly in the first branchial arch derivatives. Theperiderm shows consistent low level labeling.

NOV protein is present in both spinal nerves, and dorsal root ganglia (Fig. 4I), but immunoreactivityspreads into the marginal layer of the spinal cord, and ispresent throughout the ependymal layer. This pattern isbroader than that predicted from RNA expression. Theoverall higher level of NOV protein throughout the spinal cord may mask that predicted in the ventral hornneurons from in situ hybridisation. No significant ex-pression is seen in Rathke’s pouch.

Mesoderm

The predominant mesodermal component expressingNOV is muscle (Figs. 2G, 4F), and expression is seen incardiac, smooth and skeletal muscle. Skeletal muscle isthe predominant site of muscle NOV RNA expression,and appears to be specific for myotubes and fusing myo-blasts. The smooth muscle of the gut and cardiac muscleappear to express lower levels of NOV RNA in compari-son to skeletal muscle (Fig. 2E, G), but in skeletal andcardiac muscle RNA and protein levels are consistently

high (Fig. 4E, F, H, O). This may be explained by accu-mulation as a consequence of the relatively long half lifeof the protein (Chevalier et al. 1998).

Vascular endothelia show variable expression of NOV;there is staining of large vessel, but not capillary endothe-lia, in some places. The resolution provided by immuno-histochemistry indicates that the endocardium is negativein the heart (Fig. 4E) suggesting that although it is in inti-mate contact with the myocardium, an abundant source of NOV, the protein is not secondarily accumulated. Theendocardial cushions are completely negative (Fig. 4G).

Chondrocytes are generally negative, for exampleMeckel’s cartilage, but in the older embryo there is labeling of the perichondium and hypertrophic chondro-cytes in the distal upper limb and to a lesser extent in the ribs (Fig. 4P). Protein is also detected in the peri-chondrium of the vertebral bodies. Cranial mesenchymeshows consistent expression with higher levels in the otic capsule (Fig. 3A, B).

The meso- and metanephroi are major sites of expres-sion, with highest levels in the epithelial component ofboth structures (Figs. 2A, 4D). In the mesonephros themesonephric and paramesonephric ducts show NOVRNA expression as do the stromal and other tubularcomponents; however the mesonephric glomeruli havemarkedly reduced expression. In the metanephros themetanephric blastema shows a high level of expressionin induced mesenchyme. This increases with increasingdifferentiation in both the S-shaped and comma-shapedbodies but then drops dramatically with terminal differ-entiation into glomeruli. Signal is evident in the primarysex cords of the gonad, the fetal adrenal cortex (Fig. 3D),and in the mesonephric duct-derived collecting ducts(Fig. 2A)at all stages examined.

In the developing urogenital tract there are markeddiscrepancies between protein and RNA in several celltypes. The relative expression of NOV protein in the condensed metanephric mesenchyme is reduced fromwhat would be expected by in situ hybridisation, andwhereas the developing epithelia are consistent, the lev-els of NOV protein in the podocytes of both the meso-nephric and metanephric glomeruli are extremely high;NOV mRNA was scarcely detectable in these structures(Fig. 4A, C, D). This is consistent with previous datafrom Wilms’ tumours (Chevalier et al. 1998).

There is slight immunoreactivity in the liver, but it is not clear if this is below the level of background staining. Vascular endothelia of small vessels penetratingthe adrenals, lungs and mesentery also show labeling.

Endoderm

There is abundant mRNA for NOV in the mucosa of the gut (Fig. 2E) at all levels and it is present in the epithelium of the bronchioles and pancreatic ducts (Fig. 2F). Levels of NOV protein are low in both the gutmucosa (Fig. 4H) and the bronchial epithelia (Fig. 4M)but still label above background consistent with in situ

Fig. 4 Embryonic distribution of NOV protein. Immunocytochem-istry performed with antibody K19 M on sagittal sections of aneight week embryo, counterstained with haematoxylin as de-scribed in Materials and methods. A–C Metanephric glomeruli(g); A stained to show immunoreactivity in podocytes, ×400; B control using preadsorbed antibody as described in Materialsand methods, ×400; C accumulation of NOV in podocytes com-mencing in the S-shaped body phase of glomerular development(S), ×200. D Mesonephric nephrons (g glomerulus, mnt meso-nephric tubules) ×400. E ventricular muscle of the heart, ×400 (m cardiac muscle, ec endocardium). F Tongue showing striatedmuscle staining ×50. G Presumptive cardiac valve showing endo-cardial cushions (ecc) ×100. H Foregut ×100. I Dorsal root gangli-on ×200. J Cortex of forebrain ×100 Pial surface toward bottom ofpanel. K Choroid plexus ×100. L Auditory apparatus, showing thesaccule (S) and the auditory ganglion (VII, ag). ×200. M Lungshowing major bronchiolar epithelium (be) ×100 N Ventral aspectof lumbar region of the spinal cord showing floor plate (fp)×100.O Striated muscle of the body wall ×100). P Cartilage model ofthe distal forelimb showing perichondrium, condensing mesen-chyme, muscle (m), and hypertrophic chondrocytes (h) ×100

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hybridisation. Protein is clearly seen in the pancreaticducts.

The liver and haematopoietic tissue show signal at the margins of detection at all time points investigated.

Discussion

The distribution of NOV protein and mRNA is wide-spread in the first trimester embryo, and although only ashort time window was examined in this study, taking inthe early events of organogenesis (between 5 and tenweeks), there seem to be few evidently time-dependentchanges within individual tissues with the exception ofincreased labeling of the floor plate after 8 weeks. Thisconclusion is, however, limited to the specimens avail-able and therefore further more complete studies onmore accessible embryos such as the mouse might beuseful to confirm our findings.

This descriptive study indicates organ systems andprocesses that may be particularly dependent on NOVexpression for their controlled proliferation and differen-tiation during key phases of organogenesis. The predom-inant sites of expression of NOV are in the nervoussystem and muscle. In both cases the gene is active interminally differentiating cells, notably ventral horn neu-rons, cranial and sympathetic ganglia, and fusing skeletalmuscle.

The pattern of expression of NOV in the embryo isclosely reminiscent of several of the other CCN familymembers, suggesting either closely related functions andtherefore possible redundancy, or a combinatorial mech-anism of action, possibly through heterodimerisation orcompetition for key molecular sites of action. Cyr61 andCTGF are both expressed by smooth muscle, cardiacmuscle and the vascular endothelium like NOV, andwithin the bronchiolar epithelium. Cyr61 is found insome neural crest derivatives – the dorsal root gangliaand cranial mesenchyme; cartilage and axial skeletalmuscle, but despite these similarities with NOV it ishighly expressed in simple epithelia, the periderm, glos-sal and oral epithelium, and is not found in the CNS.CTGF is expressed in the collecting tubules of the kid-ney but not in any of the structures derived from meta-nephric mesenchyme and therefore only partially over-laps with NOV expression (Kireeva et al, 1997). Thereare striking similarities between the expression of NOVand IGFBP-5. The predominant sites of IGFBP-5 expres-sion are differentiating skeletal muscle, the adrenalgland, spinal cord, brain and perichondrium, althoughthe two are not entirely congruent (Cerro et al. 1993,Delhanty et al. 1993, Schuller et al. 1993, Green et al.1994, Han et al. 1996).

NOV in the musculoskeletal system

The developing musculo-skeletal system is emerging asa common site of expression for the CCN family mem-

bers, reinforcing the conclusion that they are importantin both pre- and post-natal control of growth and differ-entiation of cartilage bone and muscle. NOV expressionis very strong in fusing skeletal and cardiac muscle, sug-gesting that it may be associated with the terminal stagesof myogenic differentiation. It has recently been reportedthat NOV is expressed in the progenitors of the hypaxialmusculature in the mouse dermomyotome (Natarajan et al. 2000). The presence of an E box motif in the pro-moter of NOV suggests that the expression of NOV inmuscle may be a consequence of activation by bHLHproteins involved in myogenesis (Rudnicki and Jaenisch1995).

NOV is now added to the list of family members ex-pressed during cartilage development. Cyr61 is ex-pressed during murine chondrogenesis and (O’Brien andLau 1992; Wong et al. 1997) transiently expressed dur-ing repair of postnatal bone lesions in both osteocytesand chondrocytes (Hadjiargyrou et al. 2000), CTGF isimportant during chondrocyte hypertrophy (Kubota et al.2000) and WISP3 mutation is associated with profounddysregulation of the normal control of post-natal chon-drocyte growth and differentiation (Hurvitz et al. 2000).The conclusion of Hurvitz et al. that cartilage is a site ofnon-overlapping function for one member of the familybased on the appearance of a recessive phenotype in thehuman syndrome, begs the question of whether otherspecific stages of post-natal or pre-natal cartilage differ-entiation may require the expression of more than onefamily member.

NOV in the nervous system

The data presented here suggest that there is a role forNOV in the development of the central nervous system.At the early stages studied, the pattern of expression of NOV in the developing spinal cord is very specific and is limited to the floor plate and the ventral horns.The expression in the ventral horns is clearly neuronaland from anatomical criteria appears to be in motor neurons. A study of NOV expression in the human central nervous system at much later developmentalstages lead to similar conclusions (Su et al. 1998). Thisstudy established that NOV is mainly expressed in somato-motor neurons in the spinal cord at 16 weeks and in the higher central nervous system (notably motornuclei and cranial ganglia) at later stages (up to 38weeks gestation). Expression is much stronger in thelumbar region than in the thoracic region of the sameembryo, suggesting that maturation or possibly success-ful target finding increases NOV expression or stabilisesits RNA. Interestingly the presence of NOV protein isvery much more widespread, possibly as a result of uptake of paracrine NOV, and the significance of this isunclear.

NOV is distantly related to the Drosophila morpho-genic genes twisted gastrulation and short gastrulation(Mason et al. 1997), the homolog of the vertebrate

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chordin gene that is involved in dorso/ventral specifica-tion of the neural tube (Holley et al. 1995). All of thesefactors interact with Dpp/BMP4 members of the TGFβfamily suggesting that NOV too might be TGFβ respon-sive. Whilst NOV was not found to respond to TGFβ( B. Perbal, unpublished) both Cyr61 and CTGF are bothhighly TGFβ-responsive, at least in some cell lines (Grotendorst et al. 1996). There is little overlap betweenthe expression of TGF β isoforms in the human embryoand NOV (Gatherer et al. 1990) except in the ventralhorns of the spinal cord. The high expression of NOVin the floor plate cells which proliferate quite slowly(Jessell et al. 1989) is consistent with expression of NOVas a proliferation-inhibiting factor at this site.

It is notable that members of the Wnt family, which in-duce expression of the CCN members WISP1–3, also reg-ulate distinct aspects of cell proliferation and differentia-tion and are important in musculoskeletal, renal, and neu-ronal development (Cossu and Borello 1999; Ikeya et al.1997; Kispert et al. 1998; Lee et al. 2000; Parr et al.1998; Vainio et al. 1999). The expression of NOV in theseembryonic and fetal tissues suggests that NOV may alsobe dependent directly or indirectly on Wnt expression andmay constitute an effector of Wnt-mediated signals, a hy-pothesis that may now be addressed.

Cellular distribution of NOV and function

NOV is a secreted, glycosylated, heparin binding protein,containing motifs expected to promote accumulation inthe extracellular matrix (Perbal 1995). However, the im-munohistochemistry presented here confirms that, as re-ported previously (Chevalier et al. 1998) NOV protein ispredominantly cytoplasmic, being found in the cyto-plasm of all the cells expressing the mRNA, includingnerves. Whilst some ECM staining is seen in this andother studies (Chevalier et al. 1998; Perbal 1999), it is quantitatively relatively weak. Recent reports havealso suggested that there is nuclear accumulation of anamino-truncated isoform of NOV in some cell types (Perbal 1999), but in the tissues examined here this phe-nomenon was not seen. Comparison with Fisp12, the murine homolog of human CTGF, and Cyr61 shows asimilar cellular and subcellular localisation to that re-ported here with NOV. As all of these family memberspossess a canonical amino-terminal secretion signal, thissuggests that they are either inefficiently secreted or arebeing accumulated by re-uptake. This phenomenon hasbeen demonstrated for IGFBP-3, and 5, where a highmolecular weight cell-surface receptor has been impli-cated (Leal et al. 1997; Andress 1998; Schedlich et al.1998), and in some cell types is associated with translo-cation to the nucleus (Schedlich et al. 1998). It is intrigu-ing that mature podocytes demonstrate high levels ofNOV protein in their cytoplasm but express very littlemRNA, suggesting that, in these post-mitotic cells,(Abrahamson et al. 1991) the protein is either greatlystabilised or it is being accumulated from secreted pep-

tide in an autocrine or paracrine fashion. Similar accu-mulation is seen in glomeruloids associated with tripha-sic Wilms’ tumour, indicating that structural integrity ofthe nephron is not necessary for this phenomenon to occur (Chevalier et al. 1998).

A role for NOV in cell-adhesion signaling hasemerged from results obtained with the two-hybridsystem in which interaction of NOV with fibulin 1C wasrecently established (Perbal et al. 1999). The interactionsof CTGF, Cyr61 and NOV with the integrin-mediatedpathways also imply a role for the CCN proteins in cellgrowth signaling (Bork 1993; Grotendorst 1997; Kireevaet al. 1997). The presence of an IGFBP-related motif atthe N-terminus of the CCN family members has beentaken to suggest that they may also act as either IGF- orpossibly insulin-binding factors, thus modulating cellproliferation in a way analogous to that seen with theIGFBPs. So far very weak IGF binding has been demon-strated for CTGF, and NOV (Kim et al. 1997;Burren et al. 1999), but our own unpublished findings and thoseof Perbal and co-workers (Chevalier et al. 1998) suggestthat NOV does not bind IGFs or insulin with detectableaffinity (see Grotendorst et al. 2000).

The CCN family and control of proliferation

Members of the CCN family are associated with bothpromotion and suppression of cell proliferation. In nor-mal chicken fibroblastic cells, NOV expression was re-ported to be associated with cell quiescence, in contrastto the mitogenic properties of CTGF and Cyr61. Inter-estingly, expression of the WISP1/Elm1 and WISP2/rCOP-1 genes are associated with decreased metastaticpotential of melanoma cells (Hashimoto et al. 1998) andabrogation of transformation (Zhang et al. 1998) respec-tively, indicating that these members may be more simi-lar to NOV in their action. The recent description of pro-liferative activity of human NOV on mouse NIH3T3cells is suggestive of NOV having biological functionsthat might be modulated by, or depend on, cell-specificfactors (Liu et al. 1999). Our previous observation thatNOV is also detected in highly proliferating blastemalcells in Wilms’ tumors (Chevalier et al. 1998) is inagreement with the observation that NOV expression isnot necessarily associated with cell quiescence, andtherefore in itself is not sufficient to induce or maintainquiescence. It is clear from this study that expression ofNOV per se is not necessarily associated with lack ofproliferative competence, as cells in the fetal adrenalcortex, perichondrium, mesonephric tubules and meta-nephric blastema for example, are all engaged in activedivision at this stage and all express significant levels ofNOV. The known biological functions of NOV (see In-troduction) are consistent with a role in induction of cellular quiescence as well as stimulation of prolifera-tion as discussed above, and this might account for itsexpression both in terminally differentiating and prolif-erating cells.

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Acknowledgements This work was funded by the National Kid-ney Research Fund. B. Perbal acknowledges the support of LigueNationale Contre le Cancer (Comités National, du Cher et de l’Indre), AFM (Association Française contre les Myopathies),Matra- Hachette, and FRM (Fondation Pour la Recherche Médic-ale) and H. Yeger the Canadian Cancer Society and the NationalCancer Institute, Canada. We thank Prof. Charles Rodeck for hisvaluable support, Dr. Leslie Wong, MRC tissue bank, CatherineBoulter and Elisabetta Andermarcher for comments on the manu-script. Cecile Martinerie is acknowledged for technical assistanceand derivation of probes for the study.

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