during mouse embryogenesis - Development · mouse embryo cDNA library (Novagen) at high stringency...

12
INTRODUCTION Members of the basic helix-loop-helix (bHLH) family of tran- scription factors have been shown to play important roles in the control of cell differentiation, proliferation and oncogene- sis (for review, see Kadesch 1993). The bHLH motif is composed of a basic region followed by two predicted amphi- pathic α-helices separated by an unstructured loop (Murre et al., 1989a). The HLH region mediates dimerization and brings together the basic regions of bHLH proteins to form a bipartite DNA-binding domain that binds a subset of sequences sharing the consensus CANNTG, known as an E-box (Murre et al., 1989a,b). There are several subclasses of bHLH proteins, distinguish- able by their dimerization specificities, DNA-binding prefer- ences and expression patterns. Class A bHLH proteins are widely expressed and dimerize preferentially with Class B bHLH proteins, which are cell-type-specific (Murre et al., 1989a,b). Examples of Class A bHLH proteins are the E2A gene products E12 and E47 (Murre et al., 1989a), HEB (Hu et al., 1992), and the Drosophila daughterless gene product (Caudy et al., 1988). Among the most well characterized Class B bHLH proteins are the myogenic regulatory factors MyoD, myogenin, myf5 and MRF4; the Drosophila and mammalian achaete scute gene products, which regulate neurogenesis; and twist, which controls mesoderm formation (reviewed in Jan and Jan, 1993). Although Class A and B bHLH proteins can homodimerize, heterodimerization is preferred and is likely to be responsible for the biological activity of these proteins in vivo (Murre et al., 1989a; Davis et al., 1991; Lassar et al., 1991; Chakraborty et al., 1991). The widespread expression of Class A bHLH proteins and the presence of functional E-boxes in the control regions of numerous cell-type-specific genes suggest the existence of cell-specific bHLH proteins in many cell types. Although members of the bHLH family share conserved amino acids within the bHLH region, the extent of nucleotide homology among the genes encoding these proteins is in most cases insuf- ficient to allow cross-hybridization between divergent family members. Therefore, to search for novel cell-type-specific bHLH proteins, we sought to exploit the ability of cell-specific bHLH proteins to dimerize selectively with ubiquitous Class 1099 Development 121, 1099-1110 (1995) Printed in Great Britain © The Company of Biologists Limited 1995 Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to regulate growth and differentiation of numerous cell types. Cell-type- specific bHLH proteins typically form heterodimers with ubiquitous bHLH proteins, such as E12, and bind a DNA consensus sequence known as an E-box. We used the yeast two-hybrid system to screen mouse embryo cDNA libraries for cDNAs encoding novel cell-type-specific bHLH proteins that dimerize with E12. One of the cDNAs isolated encoded a novel bHLH protein, called scleraxis. During mouse embryogenesis, scleraxis transcripts were first detected between day 9.5 and 10.5 post coitum (p.c.) in the sclero- tome of the somites and in mesenchymal cells in the body wall and limb buds. Subsequently, scleraxis was expressed at high levels within mesenchymal precursors of the axial and appendicular skeleton and in cranial mesenchyme in advance of chondrogenesis; its expression pattern in these cell types foreshadowed the developing skeleton. Prior to formation of the embryonic cartilaginous skeleton, scleraxis expression declined to low levels. As development proceeded, high levels of scleraxis expression became restricted to regions where cartilage and connective tissue formation take place. Scleraxis bound the E-box consensus sequence as a heterodimer with E12 and activated tran- scription of a reporter gene linked to its DNA-binding site. The expression pattern, DNA-binding properties and tran- scriptional activity of scleraxis suggest that it is a regulator of gene expression within mesenchymal cell lineages that give rise to cartilage and connective tissue. Key words: bHLH protein, sclerotome, somites, chondrogenesis, mouse, scleraxis, skeletal formation SUMMARY Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis Peter Cserjesi 1 , Doris Brown 1 , Keith L. Ligon 1 , Gary E. Lyons 2 , Neal G. Copeland 3 , Debra J. Gilbert 3 , Nancy A. Jenkins 3 and Eric N. Olson 1, * 1 Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA 2 Department of Anatomy, The University of Wisconsin Medical School, Madison, WI 53706, USA 3 Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA *Author for correspondence

Transcript of during mouse embryogenesis - Development · mouse embryo cDNA library (Novagen) at high stringency...

Page 1: during mouse embryogenesis - Development · mouse embryo cDNA library (Novagen) at high stringency with a 400-bp fragment from the 5 ′ end of the scleraxis cDNA obtained from the

INTRO

Members of the basic helix-loop-helix (bHLH) family of tran-scription factors have been shown to play important roles inthe control of cell differentiation, proliferation and oncogene-sis (for review, see Kadesch 1993). The bHLH motif iscomposed of a basic region followed by two predicted amphi-pathic

α-helices separated by an unstructured loop (Murre etal., 1989a). The HLH region mediates dimerization and bringstogether the basic regions of bHLH proteins to form a bipartiteDNA-binding domain that binds a subset of sequences sharingthe consensus CANNTG, known as an E-box (Murre et al.,1989a,b).

There are several subclasses of bHLH proteins, distinguish-able by their dimerization specificities, DNA-binding prefer-ences and expression patterns. Class A bHLH proteins arewidely expressed and dimerize preferentially with Class BbHLH proteins, which are cell-type-specific (Murre et al.,1989a,b). Examples of Class A bHLH proteins are the

E2Agene products E12 and E47 (Murre et al., 1989a), HEB (Hu etal., 1992), and the Drosophila daughterless gene product

Ca Class b yoD,

myogenin, myf5 and MRF4; the Drosophila and mammalianachaete scute gene products, which regulate neurogenesis; andtwist, which controls mesoderm formation (reviewed in Janand Jan, 1993). Although Class A and B bHLH proteins canhomodimerize, heterodimerization is preferred and is likely tobe responsible for the biological activity of these proteins invivo (Murre et al., 1989a; Davis et al., 1991; Lassar et al.,1991; Chakraborty et al., 1991).

The widespread expression of Class A bHLH proteins andthe presence of functional E-boxes in the control regions ofnumerous cell-type-specific genes suggest the existence ofcell-specific bHLH proteins in many cell types. Althoughmembers of the bHLH family share conserved amino acidswithin the bHLH region, the extent of nucleotide homologyamong the genes encoding these proteins is in most cases insuf-ficient to allow cross-hybridization between divergent familymembers. Therefore, to search for novel cell-type-specificbHLH proteins, we sought to exploit the ability of cell-specificbHLH proteins to dimerize selectively with ubiquitous Class

099DevelopmPrinted in

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udy et al., 1988). Among the most well characterized HLH proteins are the myogenic regulatory factors M

1

ance of chondrogenesis; its expression pattern in types foreshadowed the developing skeleton. Pri

ation of the embryonic cartilaginous skelraxis expression declined to low levels. As developceeded, high levels of scleraxis expression becricted to regions where cartilage and connective tation take place. Scleraxis bound the E-box conse

uence as a heterodimer with E12 and activated ption of a reporter gene linked to its DNA-binding expression pattern, DNA-binding properties and ptional activity of scleraxis suggest that it is a reguene expression within mesenchymal cell lineages rise to cartilage and connective tissue.

words: bHLH protein, sclerotome, somites, chondrogenesisse, scleraxis, skeletal formation

hat prefigures skeletal formation

ns2, Neal G. Copeland3, Debra J. Gilbert3,

s M. D. Anderson Cancer Center, 1515 Holcombe Blvd

Madison, WI 53706, USAFrederick Cancer Research and Development Center,

ent 121, 1099-1110 (1995) Great Britain © The Company of Biologists Limited 1995

raxis: a basic helix-loop-helix protein

g mouse embryogenesis

Cserjesi1, Doris Brown1, Keith L. Ligon1, Gary E. L A. Jenkins3 and Eric N. Olson1,*

tment of Biochemistry and Molecular Biology, University of Ten, TX 77030, USAtment of Anatomy, The University of Wisconsin Medical Schoalian Genetics Laboratory, ABL-Basic Research Program, NC

ck, MD 21702, USA

or correspondence

DUCTION (B

rs of the basic helix-loop-helix (bHLH) family ofiption factors have been shown to regulate growthfferentiation of numerous cell types. Cell-type- bHLH proteins typically form heterodimers withous bHLH proteins, such as E12, and bind a DNAus sequence known as an E-box. We used the yeastrid system to screen mouse embryo cDNA librariesAs encoding novel cell-type-specific bHLH proteinserize with E12. One of the cDNAs isolated encoded

l bHLH protein, called scleraxis. During mousegenesis, scleraxis transcripts were first detected day 9.5 and 10.5 post coitum (p.c.) in the sclero- the somites and in mesenchymal cells in the bodyd limb buds. Subsequently, scleraxis was expressed levels within mesenchymal precursors of the axialpendicular skeleton and in cranial mesenchyme in

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1100

A bHLHand Soexpressiwith E1a novel expressesubsequand cardimerizeand actibinding binding scleraxislineages

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Yeast eThe GAinsertingal., 1989(Chevraywhich ambinding dintroducethe transresultingblot anallibrary, cmouse eChevray and Nathscription

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DNA secDNA idigestiongene). Boof nesteddideoxy accordingcarried o

To obtmouse e400-bp frthe yeast

In situ hSense an1.2-kb scRNA po

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proteins. Using the yeast two-hybrid system (Fieldsng, 1989), we screened a mouse embryo cDNAon library for cDNAs whose products could dimerize2. Here we describe the isolation of a cDNA encodingbHLH protein, referred to as scleraxis, which was firstd in the sclerotomal compartment of the somites andently appeared in mesenchymal primordia of bonestilage, and in tendons and ligaments. Scleraxisd and bound the E-box consensus sequence with E12

vated transcription throughout its multimerized DNA-site in transfected cells. Its expression pattern, DNA-properties and transcriptional activity suggest that may regulate gene expression within chondrogenic during embryogenesis.

IALS AND METHODS

hybridization was described by Deutsch et al. (1988). In situ protocolswere as previously described (Edmondson et al., 1994).

In vitro transcription and translation and DNA-bindingassaysA glutathione-S-transferase (GST)-E12 fusion protein was created bycloning a portion of the E12 cDNA encompassing amino acids 210-648, which includes the bHLH region, in-frame with GST(Chakraborty et al., 1991). A polyhistidine-scleraxis fusion proteinwas created by cloning a portion of the scleraxis cDNA encompass-ing amino acids 9-207 in-frame with the metal-binding domain ofpRSET (Invitrogen). GST-E12 was purified from bacterial extractsusing glutathione-agarose beads as described (Chakraborty et al.,1991). Polyhistidine-scleraxis was purified by metal affinity chro-matography according to manufacturer’s instructions (Invitrogen).

In vitro transcription and translation of scleraxis and E12 wasperformed using the TnT kit (Promega) according to manufacturer’sinstructions. Following translation, 5 µl of lysate was used in gelmobility shift assays. Parallel translations were performed in the

. Cserjesi and others

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35

pression vectors L4(DBD)-E12bHLH fusion vector was constructed bythe region encoding amino acids 502-648 of E12 (Murre et) into the SmaI site of the yeast expression plasmid pPC62 and Nathans, 1992). This generates a chimeric cDNA inino acids 1-147 of yeast GAL4, which encompass the DNA-omain (DBD), are in-frame with E12. The region of E12d into this vector encompasses the bHLH region, but it lacksription activation domain. Expression and stability of theGAL4(DBD)-E12 fusion protein was confirmed by westernysis (data not shown). A GAL4(TAD) fusion expressiononstructed from cDNA from day 14.5 post coitum (p.c.)bryonic poly(A)+ RNA, was generously provided by P.

(Johns Hopkins Medical School, Baltimore, MD)(Chevrayans, 1992). This library contains cDNAs fused to the tran-activation domain (TAD) of GAL4.

rains and methodsain PCY2 (MATα∆gal4∆gal80 URA::3GAL1-lacZ lys2-his3-∆200 trp1-∆63 leu2 ade2-101ochre) (Chevray and1992) was used in an initial screening of ~5×105 colonies.sformed with GAL4(DBD)-E12 bHLH were grown under

tion, transformed with 10 µg of embryonic library DNA, and SD-Trp-Leu plates. es with reconstituted GAL4 activity were identified by

for β-galactosidase as described previously

presence of [ S]methionine to ensure that the proteins were synthe-sized.

A double-stranded oligonucleotide probe corresponding to the leftE-box from the mouse muscle creatine kinase (MCK) enhancer(Sternberg et al., 1988; Lassar et al., 1989) was labeled with 32P andused as a probe for gel mobility shift assays. The sequence of theprobe was CCCAGACATGTGGCTGCTCCC. For competitionexperiments, a 100-fold molar excess of unlabeled oligonucleotidewas included in the binding reaction. DNA-binding assays wereperformed as described (Chakraborty et al., 1991) using aliquots of invitro translated proteins or 500 ng of the indicated bacterial fusionproteins.

Transfection assaysTo test the transcriptional activity of scleraxis, four tandem copies ofan E-box with the following sequence (CCGAACACATGTGC-CCGC) were inserted upstream of the thymidine kinase genepromoter and the chloramphemicol acetyltransferase (CAT) reporter.This E-box binds scleraxis/E12 heterodimers with high affinity (D.B., unpublished results). The scleraxis cDNA was cloned into thevector pECE-Flag (Ellis et al., 1986) such that the 9 amino acid recog-nition sequence for hemagglutinin monoclonal antibody was fused in-frame to amino acid 8 of scleraxis. This expression vector containsthe SV40 promoter and enhancer. The 3′ untranslated region of themouse protamine gene was inserted downstream of the scleraxiscDNA.

COS-1 cells were transiently transfected as described previously

er et al., 1993). Plasmids from β-galactosidase positiveere isolated and transformed into Escherichia coli Electro-10B cells (GIBCO/BRL).

uencing and analysisserts were excised from the GAL4(TAD) plasmids bywith NotI plus SalI and subcloned into pBSKII+ (Strata-th strands of plasmids were sequenced using a combination deletions and oligonucleotide primers in conjunction withsequencing using Sequenase (United States Biochemical) to manufacturer’s instructions. Sequence analysis wast using the GCG sequence analysis software package.

ain full-length scleraxis cDNAs, we screened a day 13 p.c.bryo cDNA library (Novagen) at high stringency with a

agment from the 5′ end of the scleraxis cDNA obtained fromscreen.

ybridizationd anti-sense 35S-labeled probes were synthesized from aleraxis cDNA insert cloned into pBSKII+ using T3 and T7ymerases, respectively. The Pax-1 probe used for in situ

(Sternberg et al., 1987) with 5 µg of tk-CAT or 4E-tkCAT and 5 µgof scleraxis expression vector, empty expression vector, or EMSV-MyoD (Davis et al., 1987). 48 hours after transfection, cells wereharvested and CAT activity was determined in cell extracts.

Interspecific mouse backcross mappingInterspecific backcross progeny were generated by mating(C57BL/6J×M. spretus)F1 females and C57BL/6J males as described(Copeland and Jenkins, 1991). A total of 205 N2 mice were used tomap the Scx locus (see text for details). DNA isolation, restrictionenzyme digestion, agarose gel electrophoresis, Southern blot transferand hybridization were performed essentially as described (Jenkins etal., 1982). All blots were prepared with Hybond–N+ nylon membrane(Amersham). The probe, an ~600 bp StuI/NotI fragment of mousescleraxis, cDNA was labeled with [α32P]dCTP using a random primedlabeling kit (Stratagene); washing was done to a final stringency of0.8× SSCP, 0.1% SDS, 65°C. Fragments of 10.1 and 2.5 kb weredetected in EcoRI-digested C57BL/6J DNA, and fragments of 6.0 and2.5 kb were detected in EcoRI-digested M. spretus DNA. Thepresence or absence of the 6.0 kb M. spretus-specific EcoRI fragmentwas followed in backcross mice.

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1101Scleraxis and skeletal formation in mouse

scription of the probes and RFLPs for the loci linked to Scxg thyroglobulin (Tgn), platelet-derived growth factor beta and wingless related MMTV integration site 1 (Wnt1) hasiven previously (Brannan et al., 1992). Recombinations were calculated as described (Green, 1981) using the

er program SPRETUS MADNESS. Gene order was deter-y minimizing the number of recombination events required tothe allele distribution patterns.

LTS

ning for E12 dimerization partners using thetwo-hybrid system ch for novel bHLH proteins that dimerize with E12, wee yeast two-hybrid system (Fields and Song, 1989),relies on protein-protein interactions to reconstitute anal transcription factor from two separate proteins, onech carries a DNA-binding domain (DBD), the other a

Because the yeast two-hybrid system selects for fusionproteins in which the cloned protein is fused in-frame to aminoacid 147 of yeast GAL4, the rescued cDNAs are truncatedwithin the protein coding region. We therefore used the originalscleraxis cDNA to rescreen an embryo cDNA library forcDNAs encompassing the complete open reading frame. Thecomposite nucleotide sequence obtained from several overlap-ping scleraxis cDNAs was 1140 bp in length and contained apolyadenylation signal followed by a poly(A) tail at its 3′ end(Fig. 1A). The size of the cloned cDNA corresponds to that ofthe scleraxis transcript detected by northern blot (data notshown), suggesting that the cDNA encompasses most or all ofthe transcript. The cDNA sequence predicts that scleraxis is a207-amino acid protein with a Mr=22,238 (pI=10.9), which isslightly smaller than the size of the in vitro translation productobtained from the scleraxis mRNA (see below).

The homology between scleraxis and other bHLH proteinsis shown in Fig. 1C. Within the bHLH region, scleraxis showed

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ption activation domain (TAD). For this screen, a was created in which the bHLH region of E12 was the DBD of yeast GAL4. We showed previously thatL4-E12 fusion protein fails to activate the expressioner genes under control of the GAL4 DNA-binding sitet. However, when the GAL4-E12 fusion protein wasssed with a plasmid encoding the bHLH region offused to the GAL4 TAD, GAL4-dependent markerere activated, as a consequence of dimerization of E12oD (Staudinger et al., 1993; see also Chakraborty et al.,Thus, it appeared that the GAL4-E12 fusion proteinprovide a sensitive and specific means of identifying encoding novel dimerization partners for E12 fromexpression libraries in yeast.creen for E12 dimerization partners, we transformed atrain harboring a GAL4-dependent lacZ reporter genee GAL4-E12 expression vector and a cDNA libraryy 14.5 p.c. mouse embryos in which cDNAs were fusedAL4-TAD (see Materials and Methods). Screening ofindependent transformants yielded three cDNAs

of activating lacZ expression. Specificity of the inter- between E12 and the proteins encoded by the positiveclones was tested by rescuing the activating plasmidsast and retransforming them into the same yeast strain

61% identity to mouse twist (Wolf et al., 1991). Thehematopoietic bHLH proteins NSCL-1 (Begley et al., 1992),lyl-1 (Mellentin et al., 1989) and tal-2 (Xia et al., 1991) alsoshowed greater than 50% identity to scleraxis within the bHLHregion. Notably, all of the conserved amino acids that definethe bHLH family of transcription factors were conserved inscleraxis, including the basic amino acids that have beenshown to mediate DNA binding of other bHLH proteins (Daviset al., 1991; Brennan et al., 1991). Other residues in the basicregion are divergent, suggesting that scleraxis may bind asubset of E-boxes distinct from that of other bHLH proteins.Scleraxis also lacks the residues in the basic region that havebeen shown to mediate muscle gene activation by myogenicbHLH factors (Davis et al., 1991; Brennan et al., 1991).

Outside the bHLH region, scleraxis showed no significanthomology to other known bHLH proteins. Other notablefeatures of scleraxis include a proline-rich region near thecarboxyl-terminus (Fig. 1A,B) similar to transcription activa-tion domains in several transactivator proteins (Mermod et al.,1989). There are consensus sites for phosphorylation by caseinkinase II (residues 24 and 34), protein kinase A (residues 115and 206), and protein kinase C (residue 119) (Kemp andPearson, 1990).

Scleraxis transcripts are expressed in the

r with expression plasmids encoding unrelated proteins the GAL4 DBD. None of the activating plasmids wereactivate the lacZ marker gene in the absence of GAL4-sed on the time required to induce lacZ activity and the

y of lacZ staining, the activating clones appeared to bepotent than the MyoD-activating plasmid

nger et al., 1993) in inducing expression of the lacZ gene.nucleotide sequences and deduced open reading framesactivating cDNAs were determined by DNA sequenc-o of the cDNAs were overlapping and the other was

. A search of the sequences against the database showed cDNAs encoded novel bHLH proteins. We chose ton the two cDNAs that were overlapping. The proteind by these cDNAs was called scleraxis, because it wasressed in the sclerotome and in progenitors of the axial

n during mouse embryogenesis (see below). The prop-f the other novel bHLH protein will be described

ere (P. Cserjesi and E. Olson, unpublished data).

sclerotome, the developing skeleton, andconnective tissuesThe expression pattern of scleraxis transcripts during mousedevelopment was determined by in situ hybridization toembryos that were 7.5 through 16.5 days p.c. We detected verylow basal levels of scleraxis transcripts throughout develop-ment. However, the first significant expression that waslocalized to specific cell types was observed between days 9.5and 10.5 p.c. in the lateral sclerotome and in mesenchymalcells of the limb buds and body wall (Fig. 2B). Scleraxis tran-scripts were not detected in the somites prior to compartmen-talization nor were they present at significant levels in themyotomes or dermatomes of the somites.

The paired box gene Pax-1 is expressed in a portion of thesclerotome beginning at about day 9 p.c. (Deutsch et al., 1988)and has been implicated in sclerotome differentiation (Wallinet al., 1994). To determine whether Pax-1 and scleraxis definethe same regions of the sclerotome, we compared the

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++ Fig. 1. Nucleotide sequence ofscleraxis cDNA and deduced openreading frame of scleraxis protein.(A) Nucleotide sequence andpredicted open reading frame ofscleraxis. The bHLH region,indicated in bold, encompassesresidues 78-131. A proline-richregion lies between residues 161-170. (B) Schematic of scleraxisprotein. ++, basic region. (C) Homologies between the

GCCGCCACCACCACTGG P P P P L ACTTCTGCCTCAGCAACC F C L S N QAAGTTAGAAGGAGGAGG S * CCACTGAAGAGTCACGGCACGCTCACCAGGCCAGCAGGCGGGTGATGGCTTCCATGCTGGCTCTCTACAAAGTCTGGAAATTTTG

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n patterns of the two genes by in situ hybridization tol sections of embryos at day 10.5 p.c. Scleraxisn at this stage was highest in the ventrolateral regionerotome, whereas Pax-1 expression was concentratedentromedial region between the notochord and thebe (Fig. 2B and C), as described previously (Deutsch988; Wallin et al., 1994). Whereas scleraxis was

expressed initially in the lateral region of the sclerotome,scleraxis expression eventually extended into the ventromedialregion of the sclerotome (Fig. 3A). Thus, scleraxis and Pax-1show distinct temporal and spatial expression patterns anddemarcate different regions of the sclerotome.

Parasagittal sections showed high levels of scleraxisexpression in a metameric pattern extending along the length

T--VM--V---Q--Q-L-E-RA---KI---L-S-.-----Q--K--AR--DF-Y

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bHLH region of HLH2 and otherbHLH proteins. A dash indicatesamino acid identity at that position.Sources of sequences are Wolf etal. (1991), mouse twist; Begley etal. (1992), human NSCL-1;Mellentin et al. (1989), human lyl-1; Xia et al. (1991), human tal-2;Edmondson and Olson (1989),mouse myogenin; Johnson et al.(1990), rat MASH-1.

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1103Scleraxis and skeletal formation in mouse

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1104

of the r4A). Atscleraxiand thetomes oat this srib primbuds anbase of the hyexpressing cell

By dvertebra4B). Frexpressbones othe axiabones band lonto the periphecentral

Sclercalvariacells in verse seshowedlateral tomes ahighly scleraxirevealedthe cauembryowere alsof the diaphra(Figs 4Aconnectwith cexpressthe musdiaphrathe casenective

Withanlagenexpress(Fig. anlagenrestrictethe emband epiing limin regiotissue sof theligamenmaticalthe high

ostrocaudal axis of the embryo at day 11.0 p.c. (Fig. the rostral end of the embryo, these condensations ofs expression represent the precursors of the vertebrae intervertebral discs. Scleraxis expression in the sclero-f the less mature caudal somites could also be observedtage. Scleraxis transcripts were also expressed in theordia, as well as within mesenchymal cells in the limbd frontonasal region. Mesenchymal cell masses at thethe tongue, which give rise to the thyroid cartilage andoid bone, also showed high levels of scleraxision. The pericardium also contained scleraxis-express-s (Fig. 4A).ay 11.5 p.c., expression of scleraxis in the developinge and intervertebral discs had extended caudally (Fig.

ontal sections of day 11.5 p.c. embryos revealed highion of scleraxis transcripts in precursors of the ribs andf the limbs (Fig. 5B). Its expression in progenitors ofl and appendicular skeleton preceded ossification of the

were restricted predominantly to regions that develop intodense connective tissues such as tendons, ligaments andcartilage. Within the developing face and skull, scleraxis wasexpressed in the facial region during early development, but itwas never detected in regions of intramembranous boneformation in the cranium. Scleraxis was also not expressed inthe irregular dense connective tissue of the skin.

While scleraxis expression was clearly concentrated indeveloping connective tissue and cartilage, very low levels ofscleraxis expression could be detected in a wide range of adulttissues by northern analysis (data not shown). Since manytissues contain a connective component, scleraxis expressionmay reflect the presence of connective cells.

Scleraxis binds DNA as a heterodimer with E12The presence of conserved basic amino acids in the basicregion of scleraxis suggested that it had the potential to bindDNA. To test for DNA-binding activity, we expressed

P. Cserjesi and others

y at least 2 days. Cross-sections through the future ribsg bones showed that scleraxis expression was confinedchondroblasts and chondroblast precursors at the

ry, with no expression in mature chondrocytes in thecore of these presumptive bones (Fig. 5B). axis transcripts were not detected in precursors of thel bones, but were present at high levels in mesenchymalthe frontonasal region. Trans-ctions through the somites also scleraxis expression in theregions of the rostral sclero-t day 11.5 p.c. (Fig. 5C). The

localized expression ofs in the somites was also by tangential sections through

dal region of a day 12.5 p.c. (Fig. 3B). Scleraxis transcriptso present in the sternal region

chest wall, as well as in thegm, tongue and heart valves

-C, 5C), all of which containive tissue. We cannot stateertainty that scleraxis is

ed in connective tissue withincular tissues of the tongue andgm, but we believe this to be based on its restriction to con-tissue and chondrogenic cells.in 48 hours after the rib were first detected, scleraxision declined to basal levels4B,C). Expression in the of the long bones becamed to the less mature regions ofryo, such as the pelvic region

physeal plates of the develop-bs. Concurrently, expressionns of future dense connectiveuch as the diaphragm, bronchi lungs, heart valves andts of the spine increased dra-

ly. By day 14.5 p.c. (Fig. 5D), levels of scleraxis expression

scleraxis as a histidine (His) fusion protepossible binding to a series of labeled prodifferent E-box sequences. The His-sclshowed little specific DNA-binding activthe presence of a GST-E12 fusion proteito a sequence corresponding to the left Eenhancer was detected (Fig. 6A). DN

Fig. 3. Expressionsclerotome of dayembryos. Transcrdetected by in situtransverse (A) ansections of day 12embryos. In A, sc

be seen throughout the sclerotome and limb buds, as well as in the body expression of scleraxis in the somites can also be seen in B. drg, dorsal rbud; h, heart; hl, hindlimb bud; n, neural tube; s, sclerotome; t, trachea.

in and looked for itsbes corresponding to

eraxis fusion proteinity alone, whereas inn, significant binding-box from the MCKA binding by His-

of scleraxis in the 12.5 p.c. mouseipts for scleraxis were hybridization to

d tangential (B).5 p.c. mouseleraxis transcripts can wall and trachea. Theoot ganglia; fl, forelimb

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1105Scleraxis and skeletal formation in mouse

Fig. 4. Detection of scleraxis transcripts on sequential days of mouse development by in situ hybridization. Transcripts for scleraxis weredetected by in situ hybridization to parasagittal sections of (A) day 11 p.c., (B) day 11.5 p.c., and (C) day 12.5 p.c. mouse embryos. Senseprobes for scleraxis showed no detectable hybridization above background. (D) A transverse section through the pelvis and hindlimb of a day15.5 p.c. embryo. Scleraxis transcripts are present in the intervertebral discs (id), prevertebrae (pv), pre-ribs (r), condensing cartilage of thenose and face, and in connective tissues. c, cartilage; cw, chest wall; d, diaphragm; fb, forebrain; h, heart; hp, hindpaw; hv, heart valves; l, liver;lb, limb bud; mn, mandible; n, nose; p, pelvis; tn, tendon; t, tongue.

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1106 P. Cserjesi and others

Fig

. 5. D

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Page 9: during mouse embryogenesis - Development · mouse embryo cDNA library (Novagen) at high stringency with a 400-bp fragment from the 5 ′ end of the scleraxis cDNA obtained from the

1107mouse

scleraxcognatesequenE-box others E12 shpresumformati

WheMr ~30the sclthe pre

To tscriptioreportesite wepromottransfecin the pwas strwas covector tivator.sites fasclerax

ChromThe chwas dprogen× C57Bhas beamong(CopelDNAs SouthefragmeThe 6.

raxis protein. the left E-box a probe in gelnd E12d in vitro (B).bsence ofce of a 100-folde (S) or an MEF2 siterobe is

tom in A and B.diograph of

Scleraxis and skeletal formation in

Fig. 6. DNA-binding activity of scleAn oligonucleotide corresponding tofrom the MCK enhancer was used asmobility shift assays with scleraxis aexpressed in bacteria (A) or translateIn A, assays were performed in the acompetitor DNA (−) or in the presenexcess of the cognate oligonucleotidoligonucleotide corresponding to thefrom the MCK enhancer (NS). Free pindicated by an arrowhead at the botThe lane on the left in B is an autora

is plus GST-E12 was specific and was competed by the DNA sequence but not by nonspecific DNA

ces. Among 10 E-box sequences tested, the MCK leftwas bound most avidly by scleraxis plus E12, whereasshowed weak or no binding. Since neither scleraxis norowed appreciable binding to this sequence alone, wee that the binding of the two together represents theon of a scleraxis/E12 heterodimer. n translated in a reticulocyte lysate, scleraxis showed an,000 (Fig. 6B). Like the bacterially expressed protein,

eraxis in vitro translation product bound DNA only insence of E12 (Fig. 6B).est whether scleraxis had the potential to activate tran-n through its DNA-binding site, we created a CATr gene in which 4 tandem copies of the scleraxis-bindingre inserted upstream of the thymidine kinase basaler. This reporter was not expressed above background inted COS cells, which do not express scleraxis. However,resence of a scleraxis expression vector, the reporter geneongly transactivated (Fig. 7). The level of transactivationmparable to that seen in the presence of an expressionfor MyoD (Davis et al., 1987), which is a potent transac- A tk-CAT reporter gene lacking the scleraxis-bindingiled to be transactivated by scleraxis. We conclude thatis can function as a strong transactivator.

osomal mapping of the mouse scleraxis generomosomal location of scleraxis (locus designation Scx)etermined by interspecific backcross analysis usingy derived from matings of [C57BL/6J×Mus spretus)F1L/6J] mice. This interspecific backcross mapping panel

en typed for over 1600 loci that are well distributed all the autosomes as well as the X chromosomeand and Jenkins, 1991). C57BL/6J and M. spretuswere digested with several enzymes and analyzed byrn blot hybridization for informative restrictionnt length polymorphisms (RFLPs) using a cDNA probe.0 kb M. spretus EcoRI RFLP (see Materials and

Methods) was used to f the Scx locusin backcross mice. Th ted that Scx islocated in the distal reg e 15 linked toTgn, Pdgfb and Wnt1. re analyzed forevery marker and are analysis (Fig.8), up to 171 mice wer markers. Eachlocus was analyzed in for recombina-tion frequencies using e ratios of the

[35S]met tained fromin vitro t markers are tothe left.

Fig. 7. Transcriptional actransiently transfected wµg of scleraxis or MyoDand Methods. An expresstransfected to determine cells were harvested and extract containing equiva

follow the segregation oe mapping results indicaion of mouse chromosom

Although 142 mice weshown in the segregatione typed for some pairs of pairwise combinations the additional data. Th

hionine-labeled scleraxis obranscription/translation. Mr

tivity of scleraxis. COS-1 cells wereith 5 µg of the indicated reporter gene and 5 expression vector, as described in Materialsion vector lacking a cDNA insert was

background. 48 hours after transfection,CAT activity was determined in aliquots oflent quantities of protein.

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1108

total numthe totalmost lik155–Pdg[expressstandardWnt1. Nin 155 aare with

We hwith a locationM. T. DDoolittle

maintained at the Jackson Laboratory, Bar Harbor, ME). Scxmapped in a region of the composite map that lacks mousemutations with a phenotype that might be expected to have analteration in this locus (data not shown).

The distal region of mouse chromosome 15 shares a regionof homology with human chromosomes 22q and 12q (summa-rized in Fig. 8). In particular, Pdgfb has been placed on human22q12.3-q13.1. The tight linkage between Pdgfb and Scx inmouse suggests that Scx will also reside on 22q.

DISCUSSION

Because ubiquitous Class A bHLH proteins heterodimerizepreferentially with cell-type-specific bHLH proteins (Murre etal., 1989b), we were able to use the yeast two-hybrid system,which detects protein-protein interactions, to screen for novel

P

Fig. 8. Scwas placanalysis.backcrosthe figuretyped (sein the bacspretus) C57BL/6spretus achromosochromosoto linkeddistanceschromosoknown, apositions(Genomeinformatiof The Jo

. Cserjesi and others

e

e oniac

dimerization partners for E12 from a mouse embryo cDNA

e Ts

e

FJl

g

r oh

expression library. From this screen, we cloned a novel bHLHprotein, scleraxis, which was expressed in mesenchymal pre-cursors of cartilage and in connective tissue.

Skeleton formation occurs as a result of an ordered series ofevents in which mesenchymal cells aggregate into regions ofhigh cell density that foreshadow the future skeletal elements.These cells then acquire a chondrogenic phenotype and secretelarge amounts of extracellular matrix. Finally, these chondro-genic cells undergo ossification. Scleraxis is expressed in mes-enchymal and chondrogenic cells that form the skeleton and itbecomes downregulated when ossification is initiated.

Scleraxis transcripts were first detected within the sclero-tomal region of the rostral-most somites between day 9.5 and10.5 p.c. of embryogenesis. The somites form in a rostrocau-dal progression by segmentation of the paraxial mesoderm,beginning at day 8 p.c. of mouse embryogenesis. Newlyformed somites appear as an epithelial sphere, which becomescompartmentalized beginning at day 9.0 p.c. to form the scle-rotome (which forms the axial skeleton), the myotome (theorigin of skeletal muscle) and the dermatome (the origin ofdermis). Scleraxis transcripts were not detected in the somitesuntil after the sclerotome had formed and their expressionappeared to be restricted to the sclerotomal compartment.

Cells from the sclerotome give rise to the ribs and vertebrae

x maps in the distal region of mouse chromosome 15. Scxd on mouse chromosome 15 by interspecific backcrosshe segregation patterns of Scx and flanking genes in 142

animals that were typed for all loci are shown at the top of. For individual pairs of loci, more than 142 animals were text). Each column represents the chromosome identifiedkcross progeny that was inherited from the (C57BL/6J × M.1 parent. The shaded boxes represent the presence of a allele and white boxes represent the presence of the M.

lele. The number of offspring inheriting each type ofme is listed at the bottom of each column. A partialme 15 linkage map showing the location of Scx in relationenes is shown at the bottom of the figure. Recombination

between loci in centiMorgans are shown to the left of theme, and the position of loci in human chromosomes, wheree shown to the right. References for the human mapof loci cited in this study can be obtained from GDB

ber of mice exhibiting recombinant chromosomes tonumber of mice analyzed for each pair of loci and thely gene order were centromere –Tgn–12/145–Scx–0/fb–23/171–Wnt1. The recombination frequenciesd as genetic distances in centiMorgans (cM) ± theerror] were –Tgn–8.3±2.3–[Scx, Pdgfb]–13.5±2.6– recombinants were detected between Scx and Pdgfbimals typed in common, suggesting that the two loci

n 1.9 cM of each other (upper 95% confidence limit).ve compared our interspecific map of chromosome 15omposite mouse linkage map that reports the mapof many uncloned mouse mutations (compiled by avisson, T. H. Roderick, A. L. Hillyard and D. P., and provided from GBASE, a computerized database

(Christ and Wilting, 1992). Scleraxis transcripts wereexpressed at high levels in the prevertebrae and in the ribprimordia by day 10.5 p.c., which is well before the onset ofchondrogenesis, which is normally initiated between 12.5 and14.0 days p.c. (Rugh, 1990). Scleraxis was also expressed inchondrogenic precursors of the long bones of the limbs at atime when there are no recognizable cartilaginous structures inthese regions. Within the bone primorida, scleraxis expressionwas confined to the periphery, which corresponds to the peri-chondrium and periosteum, which are a source of stem cellsfor appositional growth of bone and cartilage. Upon osteoge-nesis scleraxis expression declined to low levels. Scleraxis wasnot detected in cranial bones or their progenitors. These bonesdevelop without a cartilaginous intermediate, suggesting thatscleraxis is involved in chondrogenesis but not osteogenesis.

Scleraxis was also expressed in mesenchyme cells within thefrontonasal region that give rise to facial structures and in mes-enchymal cells in the mandibular region. These cells arederived from cranial neural crest cells (Noden 1988; Le

Data Base), a computerized database of human linkagen maintained by The William H. Welch Medical Libraryns Hopkins University (Baltimore, MD).

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Douarineural undergthey b1993).migratneural region

In adwas exdiaphrahave inmatrix,Whethextrace

Pax-1988; Kthe venthe prexpresare oveventromnotochscleraxlateral scleraxCells fand giwherealaminaPax-1of the derivatlevel in

The itors o(Zhao drocyticartilag1 or CAto be d

The to possvitro asites o1994).direct wherebticularlBMP5genesisfunctio

The erodimability bHLH formattarget 1989b)of the cannot

1109Scleraxis and skeletal formation in mouse

n et al., 1993), which begin emigrating from the dorsaltube at the time of closure (about day 8.0 p.c.) ando an epithelial-to-mesenchymal transition at the timeegin to migrate (Lumsden et al., 1991; Sechrist et al., Scleraxis was not detected in neural crest cells prior toion. Thus, it must be expressed after the mesenchymalcrest cells reach their destinations in the frontonasaland first branchial arch.dition to its expression in cartilage progenitors, scleraxis

pressed at high levels in tendons in the limbs, tongue andgm, and in cartilage of bronchi. All of these cell types common the secretion of large amounts of extracellular such as types I and II collagens (Cheah et al., 1991).er scleraxis activates the expression of these types ofllular matrix molecules remains to be determined.1 is also expressed in the sclerotome (Deutsch et al.,oseki et al., 1993) and is required for the formation of

tral parts of the vertebrae, the intervertebral discs and

family are not expressed in differentiating somites (Wang etal., 1992; Evans and O’Brien, 1993), suggesting that het-erodimeric interactions between lineage-specific bHLHproteins and E-proteins can occur as the somites give rise todifferent cell lineages.

Like other cell-type-specific bHLH proteins, scleraxis bindsthe E-box consensus sequence as a heterodimer with E12.Scleraxis/E12 heterodimers do not bind to the majority of E-boxes that we tested, including the right E-box from the MCKenhancer, which binds members of the MyoD family with highaffinity. Our results also demonstrate that scleraxis is a potenttransactivator through its multimerized DNA-binding site.Although we do not yet know the target genes for scleraxis,based on its expression pattern, DNA-binding properties andtranscriptional activity, scleraxis may regulate genes involvedin establishing chondrogenic and dense connective tissue celltypes.

It is particularly intriguing that compartmentalization of the

osir

oirirovseesoiv e

f ece

ebenf Hcyy (.n

e

pios,I b

ximal parts of the ribs (Wallin et al., 1994). Theon patterns of scleraxis and Pax-1 in the sclerotomelapping, but clearly distinct. Pax-1 is localized to theedial region of the sclerotome adjacent to therd and is expressed earlier than scleraxis. Whens first appears in the sclerotome, it is localized to theegion. Later, as the sclerotome begins to differentiate,s expression extends into the ventromedial region.m the medial sclerotome migrate toward the notochord

e rise to the vertebral bodies and intervertebral discs, cells from the lateral sclerotome form the pedicles and of neural arches and ribs (Christ and Wilting, 1992).xpression is also restricted to chondrocytic derivativesmites, whereas scleraxis is expressed in chondrocytices throughout the embryo, as well as at a low basalmany nonchondrocytic cell types.xpression pattern of scleraxis in chondrocytic progen-bones is similar to that of the homeobox gene CART1t al., 1993), which is expressed in condensed prechon- mesenchymal cells and in early chondrocytes of primordia. Whether scleraxis collaborates with Pax-

RT1 to regulate chondrocytic gene expression remainstermined.one morphogenetic proteins (BMPs) have been shownss bone- and cartilage-inducing activity in vivo and in

somite is accompanied by the expression of myogenic bHLHproteins in the myotome and scleraxis in the sclerotome. Inaddition, we have recently identified a bHLH protein calledparaxis, which is closely related to scleraxis and is expressed innewly formed epithelial somites and undifferentiated sclerotome(Burgess et al., in press). Another bHLH protein called Dermo-1 is expressed specifically in the dermamyotome and dermatome(L. Li and E. Olson, unpublished). Thus, the generation ofmyogenic, chondrogenic and dermal cell lineages from somiticmesodermal cells may involve independent pathways involvinglineage-specific bHLH proteins. The expression of scleraxis inchondrogenic progenitors of the ribs is also interesting in lightof the phenotype of myf5-null mice, which lack ribs (Braun etal., 1992). Whether this rib defect reflects cross-talk betweenmyotomal and sclerotomal bHLH proteins or arises as a resultof a disruption of the expression or function of scleraxis in thesclerotome is an interesting question for the future.

By comparison with other cell-type-specific bHLH genessuch as the MyoD and achaete scute gene families, whichregulate muscle and neural cell fates, respectively, it is temptingto speculate that scleraxis may play a role in the formation ofchondrogenic cell types. In this regard, we have attempted todetermine whether scleraxis is sufficient to activate theexpression of chondrogenic or osteogenic markers in trans-fected fibroblasts, but have thus far not observed this type of

d certain members of the BMP family are expressed atbone formation in the embryo (reviewed in Reddi,owever, little is known of the genetic pathways that

artilage and bone formation or of the mechanisms the BMPs stimulate these processes. There is a par- striking correlation between the expression patterns ofKing et al., 1994) and scleraxis during mouse embryo- It will be of interest to determine whether these factors in the same developmental pathway.activities of bHLH proteins are controlled by het-ric interactions and are influenced by partner avail-(Kadesch, 1993). Dimerization of cell-type-specificroteins with E-proteins such as E12 results in then of heterodimers that are able to bind their specificequences and activate transcription (Murre et al., whereas dimerization with the inhibitory HLH proteinsd family results in the formation of heterodimers thatind DNA (Benezra et al., 1990). Members of the Id

activity (unpublished data). Gene inactivation through homol-ogous recombination in transgenic mice should shed light onthe possible functions of scleraxis in chondrogenic cell lineages.

Supported by grants from the NIH, The Muscular Dystrophy Asso-ciation, and The Robert A. Welch Foundation to E. N. O., from theNIH and Muscular Dystrophy Association to G. E. L., the NationalCancer Institute, DHHS, under contract N01-CO-74101 with ABL toN. A. J. We thank A. Tizenor for assistance with graphics andK. Tucker for editorial assistance. We thank D. Barnhart for excellenttechnical assistance (N. A. J.).

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(Accepted 6 January 1994)