Promoter Function of the Angiogenic Inducer Cyr61Gene in Transgenic Mice: Tissue Specificity, InducibilityDuring Wound Healing, and Role of the SerumResponse Element*

BRANKO V. LATINKIC†, FAN-E MO, JEFFREY A. GREENSPAN,NEAL G. COPELAND, DEBRA J. GILBERT, NANCY A. JENKINS, SUSAN R. ROSS,AND LESTER F. LAU

Department of Molecular Genetics (B.V.L., F.-E.M., L.F.L.), University of Illinois at Chicago College ofMedicine, Chicago, Illinois 60607-7170; Munin Corporation (J.A.G.), Chicago, Illinois 60612; MouseCancer Genetics Program (N.G.C., D.J.G., N.A.J.), National Cancer Institute-Frederick, Frederick,Maryland 21702; and Department of Microbiology (S.R.R.), University of Pennsylvania School ofMedicine, Philadelphia, Pennsylvania 19104-6142

ABSTRACTThe cysteine-rich angiogenic protein 61 (Cyr61) is an extracellular

matrix-associated, heparin-binding protein that mediates cell adhe-sion, stimulates cell migration, and enhances growth factor-inducedcell proliferation. Cyr61 also promotes chondrogenic differentiationand induces neovascularization. In this study, we show that a 2-kbfragment of the Cyr61 promoter, which confers growth factor-induc-ible expression in cultured fibroblasts, is able to drive accurate ex-pression of the reporter gene lacZ in transgenic mice. Thus, transgeneexpression was observed in the developing placenta and embryoniccardiovascular, skeletal, and central and peripheral nervous systems.The sites of transgene expression are consistent with those observedof the endogenous Cyr61 gene as determined by in situ hybridizationand immunohistochemistry. The transgene expression in the cardio-vascular system does not require the serum response element, apromoter sequence essential for transcriptional activation of Cyr61 by

serum growth factors in cultured fibroblasts. Because the serum re-sponse element contains the CArG box, a sequence element impli-cated in cardiovascular-specific gene expression, the nonessentialnature of this sequence for cardiovascular expression of Cyr61 isunexpected. Furthermore, the Cyr61 promoter-driven lacZ expres-sion is inducible in granulation tissue during wound healing, as issynthesis of the endogenous Cyr61 protein, suggesting a role forCyr61 in wound healing. Consistent with this finding, purified Cyr61protein promotes the healing of a wounded fibroblast monolayer inculture. In addition, we mapped the mouse Cyr61 gene to the distalregion of chromosome 3. Together, these results define the functionalCyr61 promoter in vivo, and suggest a role of Cyr61 in wound healingthrough its demonstrated angiogenic activities upon endothelial cellsand its chemotactic and growth promoting activities upon fibroblasts.(Endocrinology 142: 2549–2557, 2001)

THE ANGIOGENIC INDUCER cysteine-rich angiogenicprotein 61 (Cyr61) is an extracellular matrix (ECM)-

associated signaling protein capable of multiple functions (1).Cyr61 is a member of the Cyr61/connective tissue growth factor(CTGF)/nephroblastoma overexpressed (CCN) protein family,which currently consists of six members: Cyr61, CTGF, nephro-blastoma overexpressed, Wnt-induced secreted protein(WISP)-1, WISP-2, and WISP-3; the first three genes discoveredprovide the acronym for the protein family (1–3). Structurally,Cyr61 and CCN proteins share four conserved modular do-

mains with sequence similarities to insulin-like growth factorbinding proteins, von Willebrand factor, thrombospondin, andgrowth factor cysteine knots. Each of these domains is encodedby a separate conserved exon, suggesting that CCN genes arosethrough exon shuffling of preexisting genes. Orthologs of thisfamily have been found across vertebrate species from Xenopusto human.

Encoded by a growth factor-induced immediate-early gene,Cyr61 is a 40-kDa secreted cysteine-rich protein (4, 5). Due to itsstrong heparin-binding activity, Cyr61 is tightly associated withthe ECM and the cell surface immediately after secretion and isnot found in the cell culture medium (5). Purified Cyr61 proteinpromotes the adhesion, migration, and proliferation of bothendothelial cells and fibroblasts (6, 7). The expression of Cyr61during mouse embryogenesis is tightly correlated with mesen-chymal condensations as they differentiate into chondrocytesand with the developing vasculature (8). Consistent with thesefindings, purified Cyr61 promotes chondrogenic differentiationin mouse limb bud mesenchymal micromass cultures (9) andinduces neovascularization in vivo (10). Moreover, expression ofCyr61 enhances the tumorigenicity of human tumor cells inimmunodeficient mice by increasing tumor size and vasculardensity. These observations indicate that Cyr61 may play im-

Received January 10, 2001.Address all correspondence and requests for reprints to: Lester F. Lau,

Ph.D., Department of Molecular Genetics, University of Illinois at Chi-cago College of Medicine, 900 South Ashland Avenue, Chicago, Illinois60607-7170. E-mail: lflau@uic.edu.

* This work was supported by grants from the NIH (CA52220 andCA46565; to L.F.L.), a Small Business Innovation Research Grant(CA78044; to J.A.G.), and by the National Cancer Institute, Departmentof Health and Human Services (National Cancer Institute-FrederickCancer Research and Development Center) (to N.G.C., D.J.G., andN.A.J.).

† Present address: Division of Developmental Biology, National In-stitute for Medical Research, Mill Hill, London NW7 1AA, UnitedKingdom

0013-7227/01/$03.00/0 Vol. 142, No. 6Endocrinology Printed in U.S.A.Copyright © 2001 by The Endocrine Society

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portant roles in angiogenesis and chondrogenesis during em-bryonic development.

Cyr61 is a ligand of, and binds directly to, the integrin aVb3on endothelial cells and the integrin aIIbb3 on blood platelets(11, 12). In fibroblasts, Cyr61 mediates cell adhesion throughthe integrin a6b1 with the requirement of cell surface heparansulfate proteoglycans as coreceptors (13). Fibroblast adhe-sion to immobilized Cyr61 induces an array of adhesivesignaling events, including actin cytoskeleton reorganiza-tion, formation of filopodia and lamellipodia concomitantwith formation of integrin a6b1-containing focal complexes,activation of intracellular signaling molecules including focaladhesion kinase, Rac, p42/p44 mitogen-activated protein ki-nases, and up-regulation of matrix metalloproteinases 1 and3 (14). The finding that Cyr61 interacts with multiple inte-grins put its functional diversity into a mechanistic context.Integrins are heterodimeric, cell surface adhesion receptorsthat are capable of transducing extracellular signals into in-tracellular responses that regulate cell adhesion, migration,proliferation, differentiation, and survival (15, 16). Thus,many of the activities of Cyr61 may be explained throughintegrin-mediated signaling pathways.

An immediate-early gene, Cyr61 is transcriptionally acti-vated rapidly in fibroblasts by serum, basic fibroblast growthfactor, platelet-derived growth factor, and transforminggrowth factor-b1 without requiring de novo protein synthesis(4, 17–19). The mouse Cyr61 promoter has been studied incultured fibroblasts in transient transfection assays (20). Itwas found that a serum response element (SRE) locatedapproximately 2 kb upstream of the transcription start site isnecessary and sufficient to confer inducibility by serum, ba-sic fibroblast growth factor and platelet-derived growth fac-tor (20). The Cyr61 SRE includes a canonical CArG box, whichhas been implicated in expression of genes specific to thecardiovascular system (21, 22).

In this study, we have defined the functional Cyr61 pro-moter in vivo, mapped the mouse Cyr61 gene, and charac-terized the expression of Cyr61 during embryogenesis andwound healing. Our results show Cyr61 expression in thedeveloping placenta and cardiovascular, skeletal, and ner-vous systems, and revealed the nonessential nature of theCyr61 SRE for expression in the cardiovascular system. Fur-ther, a role for Cyr61 in cutaneous wound repair is supportedby its induced expression in granulation tissue and ability topromote healing of a wounded monolayer in culture. To-gether with the demonstrated activities of Cyr61 (6, 9, 10, 14),these results provide insights into the biological functions ofCyr61 in vivo.

Materials and MethodsTransgenic mice

Outbred Swiss Webster mice were used in this study. Transgenic micewere generated using established protocols (23). The 22065/165 frag-ment of the murine Cyr61 promoter (20) was used to replace the pgk-1promoter in the plasmid pgk/b-gal (24) by blunt-end ligation, resultingin the construct 2lacZ with the Escherichia coli lacZ gene under the controlof the 2 kb Cyr61 promoter, and has a polyadenylation signal derivedfrom the bovine GH gene. A 1.4lacZ construct was derived from the 2lacZconstruct by deleting the Cyr61 promoter sequence distal to the AflII site.All animal research procedures were conducted with prior approval ofthe University of Illinois Committee on Animal Care and in accordance

with standards set forth in the NIH guidelines for the care and use oflaboratory animals.

Histological analysis

Imunohistochemistry was carried out as described (7) and staining forb-galactosidase activity was performed as described (25).

Wound healing assays

To identify animals carrying the transgene, tails of mice were excisedfor extraction of DNA for Southern blotting (25). The tail wounds thuscreated were also used to monitor expression of the transgene duringwound healing by histological analysis. Similar results were also ob-tained in excisional back wounds.

Monolayers of fibroblasts were wounded as described (26, 27) to sim-ulate aspects of the healing process in cell culture. Cyr61 protein waspurified as described (6). Tissue culture dishes (60-mm diameter) werecoated overnight at 4 C with either 10 mg/ml Cyr61, 10 mg/ml fibronectin(Life Technologies, Inc., Rockland, MD), or Cyr61 storage buffer as de-scribed previously, and blocked with 1% BSA for 1 h as described (6). NIH3T3 cells were plated at 3 3 106 cells per dish in DMEM containing 10% calfserum, and incubated at 37 C, 10% CO2 to allow them to form confluentmonolayers. After reaching confluence, the cell medium was changed toserum-free DMEM. Confluent monolayers were wounded with a razorblade to create a linear wound and denuding cells on one side of the woundedge, as described previously (26, 27). After wounding, the cells were refedDMEM containing 0.2% FBS and incubated 20 h at 37 C, 10% CO2. Cellswere then fixed with absolute methanol and stained with Giemsa-Wrightstain (Harleco, Kansas City, MO). Cell migration into the simulated woundarea was quantified by counting (under 3100 magnification in three dif-ferent fields) and summing the number of cells at various distances fromthe wound edge as described (28). The data reported were obtained fromfour independent experiments.

Interspecific mouse backcross mapping

Interspecific backcross progeny were generated by mating (C57BL/6J 3Mus spretus) F1 females and C57BL/6J males as described (29). A total of205 N2 mice were used to map the Cyr61 locus (see Results). DNA isolation,restriction enzyme digestion, agarose gel electrophoresis, Southern blottransfer, and hybridization were performed essentially as described (30).All blots were prepared with Hybond-N1 nylon membrane (AmershamPharmacia Biotech). The probe, an approximately 6.6 EcoRI fragment ofmouse genomic DNA (20) was labeled with [a-32P]dCTP using a nicktranslation labeling kit (Roche Molecular Biochemicals, Indianapolis, IN);washing was performed to a final stringency of 0.8 3 single-strand con-formational polymorphism, 0.1% SDS, 65 C. Fragments of 4.0, 2.3, 1.9, 1.7,0.8, and 0.6 kb were detected in HincII-digested C57BL/6J DNA and frag-ments of 6.0, 4.0, 1.9, and 1.5 kb were detected in HincII-digested M. spretusDNA. The presence or absence of the 6.0- and 1.5-kb HincII M. spretus-specific fragments, which cosegregated, was followed in backcross mice. Adescription of the probes and RFLPs for the loci linked to Cyr61 includingLmo4 and Rabggtb has been reported previously (31, 32). Recombinationdistances were calculated using Map Manager, version 2.6.5 from theRoswell Park Cancer Institute, Buffalo, NY, http://mapmgr.roswellpark.org/classic.html. Gene order was determined by minimizing the numberof recombination events required to explain the allele distribution patterns.

Results

The 2-kb Cyr61 promoter recapitulates endogenous expression intransgenic mice. We have previously shown that the 22065/165 fragment of the Cyr61 promoter is sufficient to confertranscriptional inducibility by serum growth factors in cul-tured fibroblasts (20). To investigate the regulatory functionof this promoter fragment in the whole organism, a construct(2lacZ) fusing this fragment to the E. coli lacZ gene was usedto create transgenic mice. Three independent establishedlines and five transient transgenic lines were generated andanalyzed. Established lines were analyzed for transgene ex-

2550 PROMOTER FUNCTION OF Cyr61 GENE IN TRANSGENIC MICE Endo • 2001Vol. 142 • No. 6

pression at various developmental stages from E8.5 throughadulthood; transient lines were analyzed at E12.5.

All 2lacZ transgenic mice, both established and transientlines, show expression in developing cardiovascular and ner-vous systems (Fig. 1, A and B). In addition, expression of thetransgene was detected in the developing skeleton in two ofthe three established lines. All tissues that express the trans-gene have been confirmed to express endogenous Cyr61 inprevious studies (7, 8) and in this study, and all loci of lacZexpression in this study have been confirmed by immuno-histochemical staining of the endogenous Cyr61 protein(Figs. 2C, 3, and 4, and data not shown). In no case wasinappropriate expression observed.

At the earliest developmental stage examined, E8.5, ex-pression was observed in the primitive streak and the heart(data not shown). A day later, at E9.5, expression was alsodetectable in the notochord, somites, and the brain (Fig. 1A).Expression of the transgene continued to follow develop-ment of the cardiovascular system and somites (Fig. 1B). Inthe E11.5 embryo (Fig. 1B), expression was evident in themajor blood vessels and the heart; expression in these tissuespersisted throughout embryonic development (data notshown). Highly dynamic and restricted expression of thetransgene was also observed in the nervous system. At E9.5,transgene expression was detected in the midbrain and at thebase of telencephalon, consistent with data obtained by in situhybridization (8) (Fig. 1A). By E11.5, expression in the midbrainwas down-regulated while the forebrain expressed a high levelof Cyr61 promoter activity, which was sustained throughoutembryonic development (Fig. 1B and data not shown). By E14.5,the ventral spinal cord, dorsal ganglia, and nerves innervatinglimbs all expressed b-galactosidase (Fig. 2B). EndogenousCyr61 protein was also detected in the ventral spinal cord asjudged by immunohistochemical staining (Fig. 2C).

Expression of the transgene in the skeletal system beganin the developing notochord (Fig. 1A). By E11.5, expressionof the transgene in precursors of the axial skeleton, the scle-rotomes, is evident (Fig. 1B). Precursors of the appendicular

skeleton also express the transgene, for example, the con-densing mesenchyme of the E12.5 embryo forming the digitsin the forelimb (Fig. 2, D and E). At E16.5, the transgene wasexpressed in newly formed bones as well (data not shown).

Expression of the transgene in embryos was particularlyprominent in the placenta (Fig. 3A), consistent with levels ofCyr61 messenger RNA and protein (7, 8). In the placenta,transgene expression was localized in the trophoblastic giantcells (Fig. 3B) and in endothelial cells of developing bloodvessels (Fig. 3C). Because the placenta is an intensely angio-genic organ whereby maternal and embryonic blood mustexchange nutrients, oxygen and waste, the angiogenic ac-tivities of Cyr61 may play a role in the development of theplacental vasculature.

Taken together, these data show that the 22065/165 frag-ment of the Cyr61 promoter is sufficient to mediate expres-sion of the reporter gene in a manner that mirrors the ex-pression pattern of endogenous Cyr61 in vivo. This includesextraembryonic expression in the placenta, and embryonicexpression in the cardiovascular, skeletal, and central andperipheral nervous systems.

The SRE is not required for cardiovascular expressionof Cyr61

The SRE located at nucleotide 21933/21921 was identi-fied as an essential regulatory element in the Cyr61 promoterfor transcriptional activation by serum growth factors incultured fibroblasts (20). To test the function of this sequencein vivo, a construct was prepared (termed 1.4lacZ) that con-tains 1.4 kb of the Cyr61 promoter, deleting about 0.6 kb ofuptream DNA from the 2lacZ construct and thus removingthe SRE. Two transgenic lines carrying this construct wereanalyzed. Both lines expressed the transgene in the cardio-vascular and central nervous systems in a pattern similar tothat of the 2lacZ lines (Fig. 1C). Therefore, the 1.4-kb fragmentof the Cyr61 promoter is sufficient to direct tissue-specifictransgene expression in the cardiovascular and central ner-

FIG. 1. Pattern of Cyr61 promoter-driven lacZ expression in transgenic mouse embryos. A, E9.5 embryo of 2lacZ line 2 stained for b-galac-tosidase activity. Arrows point to the notochord and midbrain. Also note staining in the tail somites and heart. B, E11.5 embryo of 2lacZ line2 stained for b-galactosidase activity. Although expression in the midbrain is being down-regulated, expression in the forebrain becomesprominent. Expression in sclerotomes of somites (dorsal segmented precursors of the vertebral column) is also evident. C, E12.5 embryo of 1.4lacZline #1 stained for b-galactosidase activity. Expression in the cardiovascular system and at least some aspects of expression in the nervous systemare similar to the 2lacZ lines. bv, Blood vessel; h, heart; f, forebrain; n, notochord; m, midbrain.

PROMOTER FUNCTION OF Cyr61 GENE IN TRANSGENIC MICE 2551

vous systems. Because the CArG box sequence within the SREhas been implicated in mediating cardiovascular gene expres-sion in addition to its role in growth factor-mediated activation(21, 22), it is somewhat unexpected that the CArG box is dis-pensable for Cyr61 expression in the cardiovascular system.

Neither 1.4lacZ transgenic line expressed the transgene inthe skeletal system (Fig. 1C). Because only two of the threeestablished 2lacZ lines exhibited skeletal expression of thetransgene, possibly due to integration effects, we cannot

conclude whether the distal 0.6-kb promoter sequence isrequired for skeletal expression.

Cyr61 expression is induced in granulation tissue duringwound repair

The angiogenic, chemotactic, and proliferation-enhancingactivities of Cyr61 suggest its possible function in woundrepair. To investigate whether Cyr61 promoter activity is

FIG. 2. cyr61 promoter fragment-driventransgene expression and endogenousCyr61 localization in nervous systemand developing skeleton. A, Diagramshowing the plane of view of sampleshown in B. B, Whole mount of E14.5embryo of 2lacZ line 3 stained for b-ga-lactosidase activity. The plane of view isperpendicular to the long body axis atthe shoulder level, as illustrated in A.Note expression in the ventral half ofthe spinal cord (sc) and the dorsal rootganglia (drg) flanking both sides.Nerves innervating the limbs emanat-ing from the dorsal root ganglia alsoexpress the transgene. C, Transversesection of an E14.5 embryo showing thedorsal region, including the spinal cord,and stained with anti-Cyr61 antibodies;3200 magnification. In addition to thespinal cord, Cyr61 staining is seen inthe dorsal root ganglia (drg) and in axialmuscle (m). D, Left front limb of E12.5embryo of 2lacZ line 2, showing b-galactosidase staining in mesenchymalcondensations before the formation ofcartilage of the digits. Expression is alsoseen in a blood vessel (bv). E, Cross sec-tion of sample in D, stained for b-galac-tosidase activity and counterstainedwith nuclear fast red; 3100 magnifica-tion.

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induced in response to wounding, cutaneous wounds wereinflicted on 2lacZ transgenic mice, and transgene expressionwas assessed at various times after wounding. Transgeneexpression was not detected in the unwounded dermis, butbecame detectable by 3 days after wounding (data notshown). The number of dermal fibroblasts expressing thetransgene steadily rose and peaked 10–12 days post wound-ing (Fig. 4B). As the wound healed by day 21, expression wasdown-regulated and only a few fibroblasts were still ex-pressing the transgene (data not shown). Most of these trans-gene-expressing cells were in the area of newly formed ECMas detected by histochemical staining for connective tissue,suggesting that induction of Cyr61 is associated with theECM remodeling phase of wound healing. We confirmedthat the Cyr61 protein is induced in healing wounds byimmunohistochemical staining (Fig. 4D). It is notable that at1 day post wounding, Cyr61 was not detected in the layer ofkeratinocytes migrating to reepithelialize the wounded area,and in keratinocytes immediately adjacent to the wounds(Fig. 4C).

Purified Cyr61 promotes fibroblast migration into awounded monolayer

As noted above, the biological activities of Cyr61 suggestits possible roles in wound healing. We investigated thispossibility further using a cell culture system to assess mi-gration of fibroblasts into a simulated wound (27, 28). NIH3T3 fibroblasts were grown to confluence on plates precoatedwith Cyr61 or fibronectin. A fibroblast monolayer waswounded using a razor blade to mark the dish and to denudecells on one side of the wound edge. We then measured thenumber of cells migrating over various distances across thewounded edge 20 h after wounding (Fig. 5). Like fibronectin,Cyr61 was able to promote the migration of fibroblasts fromthe wound edge to the denuded area (Fig. 5E).

Chromosomal location of the mouse Cyr61 gene

The mouse chromosomal location of Cyr61 was determinedby interspecific backcross analysis using progeny derived frommatings of [(C57BL/6J 3 M. spretus) F1 3 C57BL/6J] mice. Themapping results indicate that Cyr61 is located in the distalregion of mouse chromosome 3 linked to Lmo4 and Rabggtb.Although 139 mice were analyzed for every marker andare shown in the segregation analysis (Fig. 6), up to 165mice were typed for some pairs of markers. Each locus wasanalyzed in pairwise combinations for recombination fre-quencies using the additional data. The ratios of the totalnumber of mice exhibiting recombinant chromosomes tothe total number of mice analyzed for each pair of loci andthe most likely gene order are as follows: centromere-Lmo4-2/165-Cyr61-4/160-Rabggtb. The recombination fre-quencies (expressed as genetic distance in centimorgans 6se) are as follows: Lmo4-1.2 6 0.9-Cyr61-2.4 6 1.2-Rabggtb.The distal region of mouse chromosome 3 shares a regionof homology with human chromosome 1p, consistent withthe reported localization of the human ortholog of CYR61to lp31–1p22 (33).

Discussion

In this study, we report the first in vivo analysis of the Cyr61promoter using transgenic mice. We show that a 2-kb up-stream sequence is sufficient to confer accurate expression ofCyr61 in transgenic mice, including correct expression in theplacenta and developing cardiovascular, skeletal, and ner-vous systems during embryogenesis, and inducible expres-sion in dermal fibroblasts of granulation tissue duringwound healing. Consistent with the notion that Cyr61 playsa role in healing, we also show that purified Cyr61 proteinpromotes healing of a denuded area of a monolayer in culture.

Previous studies identified a 2-kb Cyr61 promoter frag-ment that is necessary and sufficient for serum inducibility

FIG. 3. cyr61–2lacZ transgene expression in the placenta. A, Whole placenta of E12.5 embryo of 2lacZ line 1 stained for b-galactosidase activity,showing extensive staining in blood vessels. View from the maternal side is shown. B, Section of sample in A showing strong staining introphoblastic giant cells. C, Section of sample in A revealing expression in endothelial cells of blood vessels in the placenta.

PROMOTER FUNCTION OF Cyr61 GENE IN TRANSGENIC MICE 2553

in transient transfection assays in fibroblasts (20). Within thispromoter fragment, an SRE containing a CArG box locatedat 21933/21921 was shown to mediate transcriptional re-sponse to serum, fibroblast growth factor, and platelet-derived growth factor (20). The CArG box is known to beinvolved in both growth factor-induced transcriptional ac-tivation and in cardiovascular-specific gene expression (21,22). Surprisingly, deletion of this CArG box sequence fromthe Cyr61 promoter did not alter expression of the transgenein the cardiovascular system (Fig. 1C). Clearly, sequencesother than the CArG box are sufficient to mediate Cyr61expression in the heart and blood vessels. Identification ofthese sequences in future studies will help to understand thediversity of regulatory elements that can specify geneexpression in the cardiovascular system.

The major sites of Cyr61 expression as determined by insitu messenger RNA hybridization (8) and immunohisto-chemistry (7), including the placenta and developing em-bryonic blood vessels and cartilage, were also detected assites of transgene expression in the present study. In addi-

tion, because of the high sensitivity of the b-galactosidaseassay, this study revealed details of spatial and temporalregulation of Cyr61 that were not observed in previous stud-ies. Briefly summarized, Cyr61 promoter-driven expressionin 2lacZ transgenic lines could be detected in a wide range oftissues, including the following: 1) the placenta, includingtrophoblastic giant cells and placental vessels (Fig. 3); 2) theentire cardiovascular system including the heart, major ar-teries, and veins (Figs. 1 and 3), as well as the lung (data notshown); 3) the embryonic skeletal system, including the no-tochord, sclerotomes, limbs (Figs. 1 and 2), and developingbone (data not shown); 4) the developing nervous system,including the ventral spinal cord, dorsal root ganglia, partsof the mesencephalon and telencephalon (Figs. 1 and 2), andthe olfactory bulb (data not shown); and 5) the embryonicand adult epidermis, in the inner root sheath of hair follicles(Fig. 4 and data not shown), and in granulation tissue duringcutaneous wound healing (Fig. 4).

What are the functional implications of this complex Cyr61expression pattern? It has been shown that Cyr61 promotes

FIG. 4. Inducible cyr61 expression during cutaneous wound repair. 2lacZ transgenic mice were analyzed for lacZ expression following tailwounding as described in the text. A, Section of the 1-day-old wound, stained for b-galactosidase activity and counterstained. No lacZ stainingwas detected. B, Section of the 12-day-old wound, stained for b-galactosidase activity and counterstained with the van Gieson trichrome methodfor connective tissue. Arrowheads point to dermal fibroblasts expressing b-galactosidase activity. Arrows point to the junction between newlyformed dermis of the healing wound and the uninjured dermis rich in collagen, which gives rise to the strong red staining. C, Section of 1-day-oldtail wound stained with anti-Cyr61 antibodies. Note lack of Cyr61 staining at the site of wounding (w), compared with strong Cyr61 stainingin the epidermis (white E) more distal to the wound. Arrow indicates the junction between uninjured epidermis and the wounded area; arrowheadpoints to the epidermis where strong Cyr61 immunostaining is observed. Strong staining for Cyr61 is also seen in the hair follicles (hf). D, Sectionof 12-day-old tail wound stained with anti-Cyr61 antibodies. Whorl-like pattern of anti-Cyr61 antibody-stained cells is characteristic ofmyofibroblasts (arrowheads). Arrows point to the junction between newly formed dermis of the healing wound and the uninjured dermis. White E,Epidermis; 3100 magnification in all sections shown.

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endothelial cell adhesion, migration, and proliferation in cul-ture, and induces neovascularization in vivo (6, 7, 10). Thus,Cyr61 is an angiogenic factor that may play important rolesin the development of the embryonic and placental vascu-lature. The expression of Cyr61 in endothelial cells is sus-tained after the blood vessels are formed, suggesting a rolefor Cyr61 in vessel maintenance. Moreover, the angiogenicactivity of Cyr61 may be important in tissue remodeling inthe adult, as indicated by the expression of Cyr61 in gran-ulation tissue during cutaneous wound healing (Fig. 4). The2-kb Cyr61 promoter is sufficient to drive this inducible ex-pression in transgenic mice. In addition, purified Cyr61 pro-tein promotes the migration of fibroblasts into the woundedarea of a monolayer (Fig. 5), supporting a role in woundhealing. In this context, Cyr61 has been shown to induceangiogenesis, to up-regulate expression of metalloprotein-ases (14), to induce chemotaxis, and to promote mitogenesisin endothelial cells and fibroblasts (6, 7, 10). Thus, it is pos-sible to propose that Cyr61 may play important roles inwound healing by inducing angiogenesis, stimulating matrixdegradation and remodeling, and promoting fibroblast che-motaxis and proliferation in the granulation tissue.

Fibroplasia, a key component of the wound healing pro-cess in vivo, includes the migration of fibroblasts into thewound area. In this regard, it is noteworthy that Cyr61 ex-

pression increases steadily during the early stages of woundhealing, peaks around day 12 post wounding during a stagecharacterized by extensive fibroplasia, and gradually de-clines to the basal (uninduced) level by 21 days post wound-ing, by which time fibroplasia has been essentially completed(Fig. 4 and data not shown). Serial sections revealed thatduring the peak of Cyr61 expression most of the dermalfibroblasts in the granulation tissue expressed the protein,and that the subsequent decline in Cyr61 expression wasmirrored by a decline in the number of dermal fibroblasts inthe granulation tissue that expressed the protein. This ob-servation suggests that Cyr61 is involved in a function com-mon to most of the dermal fibroblasts in the granulationtissue during fibroplasia. It is interesting to note that anothermember of the CCN family, CTGF, has also been shown tobe induced in the granulation tissues of healing wounds (34)and to promote fibrosis (35). Although we detected Cyr61expression in normal epidermis, this expression is down-regulated during the early phase of reepithelialization (Fig.4C). Whether Cyr61 secreted by some other cell type pro-motes keratinocyte migration or whether Cyr61 is not in-volved in this process, or even negatively regulates the pro-cess, remains to be elucidated.

We have demonstrated previously that Cyr61 acts as achondrogenic differentiation factor in micromass cultures of

FIG. 5. Fibroblast migration into a simulated wound area in precoated dishes. Confluent monolayers of NIH 3T3 cells were wounded using arazor blade to scrape off cells and mark the site of wounding. A, Cell monolayer immediately after wounding. Cells treated similarly but grownon culture dishes precoated with either BSA (B), 10 mg/ml Cyr61 (C), or 10 mg/ml fibronectin (D) were further incubated for 20 h before fixationand photography (350 magnification), showing migration of cells into the denuded area. E, Numbers of cells migrating across the wound edgewere quantified at increments of 0.1 mm from the wound edge. For each experiment the sum of the total number of migrating cells in threefields of vision was calculated for each migrated distance. Data presented are the averages of four independent experiments.

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limb bud mesenchymal cells (9). As noted above, we detectedCyr61 expression during skeletal development (Figs. 1 and 2,and data not shown). Thus, Cyr61 is expressed in mesodermthat gives rise to cartilage and bone, indicating that Cyr61likely plays a critical role in chondrogenesis in vivo. Theproposed role of Cyr61 in the development of cartilage andbone likely reflects several of its known activities. For ex-ample, its ability to promote cell-matrix adhesion and/orcell-cell aggregation of cultured limb bud mesenchymal cells(9) could play a role in the initiation of the chondrogenicdifferentiation pathway. Cyr61 protein is also found in hy-pertrophic cartilage and ossified bone (7), suggesting that theangiogenic activity of Cyr61 may be necessary for endochon-dro-ossification at later stages of skeletal development (10).In this context, it is noteworthy that Cyr61 expression isinduced following bone fracture repair (36), again indicatinga role for Cyr61 in the formation of the skeleton.

This study has also revealed prominent expression ofCyr61 in the developing embryonic central and peripheral

nervous systems (Figs. 1 and 2). However, the specific ac-tivity of Cyr61 upon neuronal cells has not yet been eluci-dated. Neuronal cells of the developing central and periph-eral nervous systems undergo extensive directed migration,a process critically dependent on the ECM that interacts withthese cells (37, 38). Given the known adhesive and chemo-tactic activities of Cyr61, it is tempting to speculate that Cyr61may mediate neuronal cell-ECM interactions and play a rolein directing axonal outgrowth and neuronal migration (14).In this regard, it is interesting to note that the carboxy-terminal domain of the Cyr61 protein exhibits 25% aminoacid sequence homology with the Drosophila protein Slit,which is a key regulator of axon guidance, axonal branching,and cell migration (39–41). Cyr61 is also induced duringdifferentiation of the immortalized hippocampal neuronalcell line H19-7 in culture (42), suggesting a role for Cyr61 inneuronal differentiation. Furthermore, Cyr61 expression hasbeen detected in the cerebral cortex of the adult rat (43) andmouse (Latinkic, B. V., and L. F. Lau, unpublished data)brain, and is induced by muscarinic acetylcholine receptorsignaling in culture (43). Together, these findings suggestthat Cyr61 may play a role in both embryonic neuronaldevelopment and cholinergic regulation of synaptic plastic-ity in the adult. However, the specific roles of Cyr61 in theseprocesses remain to be determined.

We have mapped Cyr61 to the distal end of chromosome3 in mice (Fig. 6). We have compared our interspecific mapof chromosome 3 with a composite mouse linkage map thatreports the map location of many uncloned mouse mutations(provided by Mouse Genome Database, a computerized da-tabase maintained at The Jackson Laboratory, Bar Harbor,ME). Cyr61 mapped in a region of the composite map thatlacks mouse mutations with a phenotype that might be ex-pected for an alteration in this locus (data now shown).

In summary, we have identified the Cyr61 promoter se-quence necessary and sufficient for accurate expression intransgenic mice. The specific sites of Cyr61 expression can beinterpreted in terms of the known activities of the Cyr61protein as determined in various assays, suggesting thatthese activities contribute to specific functional roles forCyr61 in the development of the cardiovascular, skeletal, andneuronal systems during embryogenesis, as well as in tissuereconstruction such as wound healing. Further analysis us-ing mutant mice with targeted disruption of the Cyr61 genewill likely provide new insights into the biological functionsof Cyr61 in these processes.

Acknowledgment

We thank members of our laboratories for helpful discussions andMary Barnstead for excellent technical assistance.

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FIG. 6. Cyr61 maps in the distal region of mouse chromosome 3.Cyr61 was placed on mouse chromosome 3 by interspecific backcrossanalysis. The segregation patterns of Cyr61 and flanking genes in 139backcross animals that were typed for all loci are shown at the top ofthe figure. For individual pairs of loci, more than 139 animals weretyped (see text). Each column represents the chromosome identifiedin the backcross progeny that was inherited from the (C57BL/6J 3 M.spretus) F1 parent. The black boxes represent the presence of aC57BL/6J allele and white boxes represent the presence of a M. spre-tus allele. The number of offspring inheriting each type of chromo-some is listed at the bottom of each column. A partial chromosome 3linkage map showing the location of Cyr61 in relation to linked genesis shown at the bottom of the figure. Recombination distances betweenloci in centimorgans are shown to the left of the chromosome and thepositions of loci in human chromosomes, where known, are shown tothe right.

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