SWiP-1: novel SOCS box containing WD-protein regulated · PDF fileSWiP-1: novel SOCS box containing WD-protein regulated by signalling centres and by Shh during development Daniel

SWiP-1: novel SOCS box containing WD-protein regulated by signallingcentres and by Shh during development

Daniel Vasiliauskas, Sarah Hancock, Claudio D. Stern*

Department of Genetics and Development, College of Physicians and Surgeons of Columbia University,701 West 168th Street, New York, NY 10032, USA

Received 18 November 1998; received in revised form 6 January 1999; accepted 6 January 1999

Abstract

We describe a novel chick WD-protein, cSWiP-1, expressed in somitic mesoderm and developing limb buds as well as in otherembryonic structures where Hedgehog signalling has been shown to play a role. Using embryonic manipulations we show that in somitescSWiP-1expression integrates two signals originating from structures adjacent to the segmental mesoderm: a positive signal from thenotochord and a negative signal from intermediate and/or lateral mesoderm. In explant cultures of somitic mesoderm, Shh protein inducescSWiP-1, while a blocking antibody to Shh inhibits the induction ofcSWiP-1by the notochord. These results show that the positive signalfrom the notochord is mediated by Shh. We also show that in limb budscSWiP-1is upregulated by ectopic Shh. This occurs in about thesame time period as upregulation ofBMP2, placingcSWiP-1among the earliest markers for the change of limb pattern caused by ectopicShh. We also describe a human homologue ofcSWiP-1and a mouse gene,mSWiP-2, that is more distantly related toSWiP-1, suggestingthat SWiP-1belongs to a novel subfamily of WD-proteins. 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:SOCS box; WD proteins; SwiP; Sonic hedgehog; Somites; Limb buds; Feather buds; Gut; Chick embryo; Notochord

1. Introduction

The Hedgehog proteins are involved in a number ofimportant tissue interactions during embryonic develop-ment. Three Hedgehog proteins have been described inhigher vertebrates: Sonic Hedgehog (Shh), Indian Hedge-hog (Ihh) and Desert Hedgehog (Dhh) (Echelard et al.,1993; Riddle et al., 1993; Chang et al., 1994; Roelink etal., 1994; Bitgood and McMahon, 1995; Marigo et al., 1995;Vortkamp et al., 1996). Of these, Shh has been studied mostintensively because of its involvement at early stages ofdevelopment. Its roles include regulation of left-right asym-metry (Levin et al., 1997; Pagań-Westphal and Tabin,1998), ventral patterning of the neural tube (Echelard etal., 1993; Roelink et al., 1994; Ericson et al. 1996; Chianget al., 1996), subdivision of the somite into dermomyotomeand sclerotome (Fan and Tessier-Lavigne, 1994; Johnson etal., 1994; Bumcrot and McMahon, 1995; Fan et al., 1995;

Chiang et al., 1996; Borycki et al., 1998), induction of themyotome (Johnson et al., 1994; Mu¨nsterberg et al., 1995;Borycki et al., 1998), mediation of the activity of the Zoneof Polarising Activity (ZPA) in the limb bud (Riddle et al.,1993; Chang et al., 1994; Lo´pez-Martınez et al., 1995; Yanget al., 1997), tissue interactions in the gut (Roberts et al.,1995; Apelqvist et al., 1997; Kim et al., 1997; Hebrok et al.,1998; Roberts et al., 1998) and feather patterning (Nohno etal., 1995; Noveen et al., 1996; Ting-Berreth and Chuong,1996a; Ting-Berreth and Chuong, 1996b; Jung et al., 1998).

Several components of the hedgehog signal transductionpathway have been identified in Drosophila. Two types oftransmembrane proteins are involved in signal reception:patched and smoothened (reviewed in Perrimon, 1996; Ing-ham, 1998a,b). Downstream of these, Protein Kinase A(PKA), fused, Suppressor of fused, costal-2, slimb and thetranscription factor Cubitus interuptus (Ci) have been impli-cated in transducing the signal to the nucleus, resulting intranscriptional upregulation of a number of genes, amongthem Patched(Ingham, 1995; Perrimon, 1995; Kalderon,1997; Ruiz i Altaba, 1997; reviewed in Ingham, 1998a,b;Jiang and Struhl, 1998). At least parts of this pathway have

Mechanisms of Development 82 (1999) 79–94

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* Corresponding author. Tel.: +1-212-305-7915; fax: +1-212-923-2090;e-mail: [email protected]

been conserved in vertebrates. Vertebrate homologues ofpatched, smoothened, PKA, slimb (b-TRCP) and Ci(Gli1,2,3) are known (reviewed in Ruiz i Altaba, 1997;Ingham, 1998a,b; Jiang and Struhl, 1998). Patched proteinhas been shown to interact directly with Shh, confirming itsrole as a Hedgehog receptor (Marigo et al., 1996a; Stone etal., 1996; reviewed in Ingham, 1998a,b). PKA, as in Dro-sophila, antagonizes Hedgehog signalling (reviewed in Per-rimon, 1995; Ingham, 1995) and at least one of three knownCi homologues,Gli-1, is a target and a mediator of Hedge-hog signalling (Marigo et al., 1996b; Lee et al., 1997;reviewed in Ruiz i Altaba, 1997). Furthermore, in responseto a Hedgehog signal, transcription ofPatchedis upregu-lated (Concordet et al., 1996; Hahn et al., 1996; Marigo andTabin, 1996; Marigo et al., 1996c; Lee et al., 1997; Boryckiet al., 1998; reviewed in Ingham, 1998a,b), which allows theuse ofPatchedas a marker for cells responding to Hedge-hog signals.

Interestingly, of a handful of genes that are known to betranscriptionally regulated in different structures inresponse to Shh signal, onlyPtc and Gli1 are universallyupregulated in all systems tested to date. All other markersare expressed in only some of the developing structures thatare patterned by Shh. For example, the responses to Shhsignalling in the limb bud and somites share no knownmarkers aside fromPtc andGli1.

Here, we describe the cloning, expression pattern andregulation of a novel chick gene,cSWiP-1 (for SOCSbox and WD-repeats in Protein). Its expression patternsuggests participation in many of the inductive processesknown to involve hedgehog. We demonstrate thatcSWiP-1is regulated by Shh in two different developing sys-tems, the somites and the limb bud. In the somites,cSWiP-1transcription is regulated by Shh emitted by thenotochord and repressed by signals from the lateral plate. Inthe limb bud cSWiP-1 is induced by ectopic Shh. Wealso describe the cloning of the human homologue ofhSWiP-1and of a related mouse gene,mSWiP-2, suggest-ing that these are members of a novel WD-protein sub-family.

2. Results

2.1. Expression of cSWiP-1 in the developing embryo

We studied the expression ofcSWiP-1(a gene identifiedin a screen unrelated to this project) by whole-mount in situhybridization using antisense riboprobe generated from acDNA clone containing most of the transcript of the gene(see below).

2.1.1. General featuresAt stage 4 (Hamburger and Hamilton, 1951),cSWiP-1is

expressed in almost the entire epiblast layer of the areapellucida, but is excluded from Hensen’s node (Fig. 1A),

but by stage 6 expression has become restricted to the neuralplate (Fig. 1B). As the neural tube develops,cSWiP-1expression evolves and becomes patterned along both thedorsoventral (DV) and anteroposterior (AP) axes (Fig. 1B–D). At stage 10,cSWiP-1starts to be expressed in thenotochord and somite mesoderm and somewhat later inthe intermediate mesoderm. Expression ofcSWiP-1 isquite dynamic during maturation of the somites (Fig. 1D–I) and is described below in greater detail. The strongest siteof expression ofcSWiP-1during development is the bran-chial arches: from stage 20,cSWiP-1is expressed in the firstbranchial arch (along the posterior edge of the maxillaryprocess and the anterior edge of the mandibular arch) aswell as along the posterior edge of the second (hyoid)arch (Fig. 2A,B). From this stage until at least stage 29,cSWiP-1is also expressed in the gut (particularly in thegizzard, duodenum/pancreas and caecal primordia; Fig.2C). Expression ofcSWiP-1in the developing limb budsis dynamic (Fig. 1J–L) and is described below in greaterdetail. In older embryos (stage 34)cSWiP-1expression ismost prominent in the forming feather buds and in thescleral papillae of the eye (Fig. 2D–G). It is interestingthat many of these sites ofcSWiP-1expression either over-lap or lie close to regions where members of theHedgehoggene family are expressed (for example, compare Fig. 2D–G with H–K, or Fig. 2A–B with Wall and Hogan, 1995 andHelms et al., 1997).

2.1.2. Expression in somitesExpression ofcSWiP-1 in somites becomes apparent

around stage 10. After stage 11, expression in somites dis-plays a constant pattern along the axis (Fig. 1D–I): a lowlevel of cSWiP-1transcript is detectable in the segmentalplate. This expression is upregulated in somite I (numberingafter Christ and Ordahl, 1995), in the ventromedial region ofthe prospective sclerotome. As somites mature this expres-sion domain shifts to the medial side of the somite andthen into the dorso-medial lip, the region of the formingmyotome. Expression then shifts ventrolaterally with theforming myotome. At this point the dermomyotome alsoexpressescSWiP-1but at lower level, while the sclerotomedoes not express at all.

2.1.3. Expression in limb budscSWiP-1expression in the limb buds is initially diffuse

(stage 18), but resolves to an area of stronger expression atthe posterior margin by stage 21 (Fig. 1J). This domainoverlaps with, but is not identical to, the expression ofShh: the latter extends more distally (not shown). At thesame time there is a weaker domain of expression in theproximal-central region of the limb bud. The posteriordomain of expression resolves to a thin stripe along theposterior margin at stage 25 (Fig. 1K) and disappears com-pletely by stage 28 (Fig. 1L). Meanwhile, at around stage23, cSWiP-1is upregulated in the central domain along theAP axis both dorsally and ventrally. By stage 28, the distal

80 D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94

Fig. 1. Expression ofcSWiP-1in the early embryo, somites and limbs. (A) At stage 4,cSWiP-1is expressed in the prospective neural plate and is particularlystrong in the presumptive cephalic region. (B) At stage 6,cSWiP-1expression is restricted to the neural plate and is strongest in the forebrain and hindbrain.(C) Head region of stage 11 embryo.cSWiP-1expression in the neural ectoderm has started to disappear from specific axial levels, such as the isthmus andmost of the hindbrain. (D–I)cSWiP-1expression in somitic mesoderm shifts medially and dorsally during somite maturation (arrows in E–I). (D) Dorsal viewof 23-somite embryo. (E–I) Transverse sections of the embryo shown in D at the levels of somites II (I), IV (H), V (G), X (F) and XV (E).cSWiP-1isexpressed at a low level in segmental plate. The expression becomes localized ventro-medially in somites I and II. As the somite matures,cSWiP-1expression becomes restricted medially and then shifts dorsally. Finally,cSWiP-1becomes expressed at high levels in the myotome-forming region(E,F). At this stagecSWiP-1is also expressed in the notochord and Wolffian duct. In the neural tubecSWiP-1is expressed dorsally or ventrally dependingon the axial level. Note the expression in endoderm and associated splanchnic mesoderm (E). (J–L)cSWiP-1is expressed in two domains during limb buddevelopment. (J) Dorsal view of stage 21 wing bud.cSWiP-1is strongly expressed at the posterior margin of the limb bud. Weaker expression is also apparentin the proximal central portion of the limb bud. (K) At stage 25,cSWiP-1remains expressed posteriorly and in the centre of the wing bud. (L) Ventral view ofstage 28 leg bud. By this stage the central domain in the toe plate has resolved into an interdigital pattern of expression. The posterior domain has virtuallydisappeared.

81D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94

Fig. 2. Expression ofcSWiP-1in branchial arches, gut, feather buds and scleral papillae. (A,B)cSWIP-1is strongly expressed in the branchial arches. (A)Stage 20. Expression is detected along the anterior edge of the mandibular arch (top arrow) and along the posterior edge of the second arch (bottom arrow).(B) Stage 24. In addition to this pattern (bottom two arrows),cSWiP-1is also expressed along the posterior edge of the maxillary process (top arrow). (C)cSWiP-1is expressed in the gut in stage 29 embryo, particularly in the duodenum/pancreas (top arrow) and caecal primordia (bottom arrow). In day 8embryos,cSWiP-1(A–G) andShh(H–K) are expressed in the forming feather buds and in the scleral papillae in the eye. (E,I) Dorsal feather buds of embryosin (D) and (H) shown at a higher magnification. Anterior is to the top. (F,J) Sagittal sections of feather buds shown in (E) and (I); anterior is to the left. Notethat there are two domains of expression ofcSWiP-1in each feather bud (E,F). At this stage bothcSWiP-1andShhare also expressed in the scleral papillae(arrows) in the developing eye (G,K).


portion of this domain is refined to interdigital regions (Fig.1L). Later still (by stage 31), expression is also detected inthe approximate region of tendon formation (not shown).Histological analysis revealed that at all stages, transcriptsare confined to the mesoderm underlying the surface ecto-derm (not shown).

2.2. Regulation of cSWiP-1 in somites

The somite mesoderm is flanked by structures that havebeen shown to be involved in its patterning along dorso-ventral (DV) and medio-lateral (ML) axes (reviewed inCossu et al., 1996; Lassar and Mu¨nsterberg, 1996; Yama-guchi, 1997). Medially, it is bordered by the notochord andneural tube, and laterally by intermediate and lateralmesoderm.cSWiP-1 expression in somites is strikinglyreminiscent of the expression ofPatched(Marigo et al.,1996c; Marigo and Tabin, 1996; Borycki et al., 1998), amarker for cells that are responding to Shh signalling. Shhsecreted by notochord and floor plate cells has been shownto play a role in DV patterning of somites (Fan and Tessier-Lavigne, 1994; Johnson et al., 1994; Bumcrot and McMa-hon, 1995; Fan et al., 1995; Mu¨nsterberg et al., 1995;Chiang et al., 1996; Borycki et al., 1998), which raises thepossibility that axial structures may be involved in regula-tion of cSWiP-1expression in the somites. In addition,cSWiP-1expression is restricted medially as the somitematures, a hint that structures lateral to the segmental meso-derm may downregulatecSWiP-1expression in the somites.To address these questions we performed a series of micro-surgical experiments on stage 10–12 embryos to isolate thesomitic mesoderm from neighbouring structures.

2.2.1. Axial structures are necessary for the induction ofcSWiP-1 in somites

To assess the role of the neural tube and notochord inregulatingcSWiP-1we made a cut through all three germlayers of the embryo just medial to the anterior segmentalplate (Fig. 3A). 10–16 h after the operation, we assessed theexpression ofcSWiP-1in the newly formed somites.cSWiP-1 was not expressed in the somites that had developed adja-cent to the cut (13/13 embryos), suggesting that axial struc-tures are necessary for the induction ofcSWiP-1in somitemesoderm.

2.2.2. The neural tube is not necessary for the expression ofcSWiP-1 in somites

To determine which of the axial structures provide signalsnecessary for the induction ofcSWiP-1, we removed theneural tube at the level of the segmental plate (Fig. 3B).This operation often leads to the pairwise fusion of rightand left somites. Despite the absence of the neural tubeand the new midline location of the somites,cSWiP-1isexpressed in the ventral aspect of the fused somites, adja-cent to the notochord (3/3 embryos). The neural tube istherefore not necessary for expression ofcSWiP-1.

2.2.3. Notochord can induce cSWiP-1 in somitesThe above experiments suggest that the notochord emits

signals that regulatecSWiP-1expression. To confirm this,we grafted an ectopic notochord medial to the segmentalplate, and then made a cut to separate the segmental platetogether with the attached notochord graft from the axialstructures of the host embryo (Fig. 3C).cSWiP-1 wasinduced in the somites that developed close to the graft(3/3 embryos). As expected from our other experiments,the somites that developed adjacent to the cut but furtheraway from the graft did not expresscSWiP-1. Therefore, theloss of cSWiP-1expression due to separation of somitemesoderm from axial structures can be rescued by the noto-chord, suggesting that the notochord emits signals thatinducecSWiP-1in the medial portion of the somites duringnormal development.

2.2.4. Shh mediates the signal from the notochord thatupregulates cSWiP-1

Shh, a secreted molecule expressed in the notochord andthe floor plate, has been shown to be involved in somitepatterning (Fan and Tessier-Lavigne, 1994; Johnson et al.,1994; Bumcrot and McMahon, 1995; Fan et al., 1995;Munsterberg et al., 1995; Chiang et al., 1996; Borycki etal., 1998). The finding that the notochord emits a signal thatupregulatescSWiP-1and the similarity between the expres-sion patterns ofcSWiP-1and Patchedin somites suggestthat Shh may be the signal that inducescSWiP-1. We usedan explant culture system to investigate this.

First, we examinedcSWiP-1(andPatched, as a positivecontrol) expression in cultures of stage 11–12 segmentalplates with or without added Shh protein. The results wereidentical regardless of the inclusion of the ectoderm in theexplant: bothPatchedandcSWiP-1were induced in treatedexplants (Patched: 8/8; cSWiP-1: 13/13) (Fig. 4B,D,F).Neither gene was expressed in explants cultured withoutShh protein (Patched: 0/7, cSWiP-1: 0/11) (Fig. 4A,C,E).Shh protein can therefore substitute for the notochord ininducingcSWiP-1transcription in somites.

To test whether thecSWiP-1inducing signal produced bythe notochord requires Shh, we placed notochords and seg-mental plates in contact with each other and cultured themin the presence or absence of the Shh-blocking antibody5E1 (Ericson et al., 1996). In the absence of antibody,Patchedexpression is detected in the notochord and inpart of segmental plate explant (4/4) (Fig. 4G). In the pre-sence of antibody,Patchedexpression is detectable onlyin the notochord (0/4 explants expressedPatchedin seg-mental mesoderm) (Fig. 4H). The antibody also greatlyreduced both the intensity of expression ofcSWiP-1(com-pare Fig. 4I and 4J) and the proportion of explants thatexpressed the gene: 4/6 untreated control explants expressedcSWiP-1and treatment with antibody reduced this to 1/4.Together, these experiments indicate that Shh produced bythe notochord accounts for induction ofcSWiP-1expressionin somites.


2.2.5. Structures lateral to the somite mesoderm inhibitcSWiP-1 expression in somites

To assess the role of lateral structures in regulatingcSWiP-1expression we made a cut through all three germlayers of the embryo just lateral to the anterior segmentalplate (Fig. 3D). The resulting gap isolates paraxial fromintermediate and lateral mesoderm. In the somites thatdevelop adjacent to the cut, the level ofcSWiP-1increasesas compared to the somite on the contralateral side and thedomain of cSWiP-1 expression extends laterally (10/11embryos). In the youngest affected somites the ectopicexpression on the lateral side is not restricted to the ventralsomite (as it is on the medial side) (Fig. 3Db). In olderaffected somites, ectopic expression is confined to the dor-

solateral lip of the dermomyotome (Fig. 3Da). Because theisolation of the paraxial mesoderm from lateral tissues leadsto upregulation ofcSWiP-1in somites, we conclude thatthese tissues produce signals that normally inhibitcSWiP-1 expression in somites.

3. Regulation of cSWiP-1 expression by Shh indeveloping limb buds

3.1. Sonic hedgehog induces cSWiP-1 in the limb bud

The ZPA is largely responsible for patterning the limbalong the AP axis (Saunders and Gasseling, 1968; MacCabe

Fig. 3. Regulation ofcSWiP-1by axial and lateral tissues. The panels at the top show diagrammatic representations of the operations performed at the anteriorsegmental plate level in stage 11–12 embryos. The central panels show whole-mount views and the bottom panels show a transverse section at the levelindicated by the horizontal lines in the whole mount. (A) ‘Medial cut’ experiment. The segmental plate was separated from the notochord and the neuraltubeby a cut through all three layers of the embryo. No expression ofcSWiP-1is detected in the somites that developed adjacent to the cut. (B) The neural tubewas removed at the level of the anterior segmental plate. The operation resulted in fusion of somites across the midline. Despite the absence of neuraltube,expression ofcSWiP-1is maintained adjacent to the notochord in the fused somites (arrow in lower panel). (C) A rescue of the ‘medial cut’ experimentshown in A. A piece of notochord (red in the top panel, red arrow in the bottom panel) was grafted between the neural tube and the segmental plate. Thesegmental plate and notochord graft were then separated from the axial structures of the host embryo.cSWiP-1expression is downregulated in somites thatdevelop adjacent to the cut except in the immediate vicinity of the graft. Note that the expression (black arrow in lower panel) rescued by the notochord graftis stronger in the medial domain of the somite than laterally. (D) ‘Lateral cut’ experiment. The segmental plate was separated from the intermediate andlateral mesoderm by a cut through all three layers of the embryo. On the operated side, expression ofcSWiP-1is expanded laterally. In somite VI (a),expression ofcSWiP-1is seen at both the medial (normal) and the lateral (ectopic, arrow) margin of the dermomyotome. In somite III (b), the domain ofcSWiP-1is expanded dorsally and laterally (arrow).


Fig. 4. Expression ofcSWiP-1in somites is regulated by Shh. Segmental plates with (E,F) or without (A–D) overlying ectoderm were cultured in collagen inthe presence (B,D,F) or absence (A,C,E) of Shh protein. BothPatched(A,B) and cSWiP-1(C–F) are induced by Shh. Segmental plate and notochordconjugates (G–J) cultured in the presence (H,J) or absence (G,I) of Shh-blocking antibody. The notochord can induce expression ofPatchedin segmentalplate (arrow in G) andcSWiP-1(I). Shh-blocking antibody prevents the induction of bothPatched(H) andcSWiP-1(J). The remaining expression ofPatchedin H is in the notochord (‘not’ in G and H).


et al., 1973; Tickle et al., 1975; Summerbell, 1979). Sonichedgehog (Shh) is expressed in posterior limb buds in aregion that has been equated with the ZPA and appears tomediate ZPA activity: misexpression of Shh in anterior limbbuds leads to mirror-image duplication of the limb along itsAP axis (Riddle et al., 1993). As part of this process, Shhinduces ectopic expression of a number of genes normallyexpressed in the posterior limb bud (e.g.Patched, BMP2,Hoxd-13) (Izpisua-Belmonte et al., 1991; Nohno et al.,1991; Francis et al., 1994; Laufer et al., 1994; Marigo etal., 1996c; Nelson et al., 1996).

Because the posterior domain ofcSWiP-1expression inthe limb buds overlaps with the proximal aspect of the ZPAand with the domain ofShhexpression, and becausecSWiP-1 is induced in segmental mesoderm by Shh, we investi-gated whethercSWiP-1can also be induced in the anteriorlimb bud by ectopic expression of Shh. We used a replica-tion-competent retrovirus-encoding chickShh(Riddle et al.,1993), which was injected into anterior mesodermal tissueof stage 19–20 limb buds (both wing and leg). Approxi-mately 16 h after infection we assessedcSWiP-1expressionby in situ hybridization (Fig. 5).cSWiP-1was upregulatedin the infected region as compared to the uninfected con-tralateral limb bud (22/22 limb buds). Similar infection oflimb buds with a control virus encodingalkaline phospha-tase (Fekete and Cepko, 1993) did not affectcSWiP-1expression.

3.2. Ectopic Shh induces cSWiP-1 and BMP2 at the sametime

Patchedand BMP2 are two early markers commonlyused to identify cells responding to Shh signalling in verte-brate limb buds (Francis et al., 1994; Laufer et al., 1994;Marigo et al., 1996c). This relationship is conserved in limbdevelopment between vertebrates andDrosophila, whereHedgehog upregulatesPatchedand inducesdpp (a Droso-phila homologue ofBMP2) (reviewed in Ingham, 1998a,b)at the transcriptional level. In normal stage 22 limb budsPatchedis upregulated in the mesenchyme in and aroundthe ZPA whileBMP2 is expressed in an overlapping but

larger domain that includes the posterior two-thirds of theAER.

To assess the timing of induction ofSWiP-1by Shh rela-tive to other genes in the Shh signalling pathway, we com-pared the time necessary for upregulation ofPatched, BMP2andcSWiP-1following ectopic expression ofShh(Table 1).While ectopicPatchedexpression was upregulated within8–9 h after virus injection, neitherBMP2norcSWiP-1wereinduced at this time. However, bothBMP2 and cSWiP-1were induced just over 1 h later (Table 1). AlthoughcSWiP-1does not appear to be an immediate target ofShh, these results suggest that its regulation by Shh is asrapid as that ofBMP2.

4. Molecular nature of SWiP-1

A 2.3 kbcSWiP-1clone was isolated from a stage 12–15chick embryo cDNA library (Nieto et al., 1994). Sequenceanalysis of the clone revealed an open reading frame encod-ing a putative polypeptide of 421 amino acids (Fig. 6A).

4.1. SWiP-1 contains WD repeats and a SOCS box

A database search for sequences similar to the predictedamino acid sequence of cSWiP-1 revealed significanthomology with a number of different WD proteins (Neeret al., 1994). Detailed analysis of the cSWiP-1 amino acidsequence shows that it contains eight WD-repeats (Fig.6A,B), spanning most of the protein. Six of the WD-repeatshave four or fewer mismatches from the consensus sequenceproposed by Neer et al. (1994). The two N-terminal repeatsare more divergent from the consensus and can be recog-nized only by their position adjacent to the well-definedrepeats, which is not unusual (Neer et al., 1994).

To identify homologies to non-WD-proteins, databasesearches were performed using the cSWiP-1 sequences out-side the WD-repeat region of the protein. The C-terminus ofcSWiP-1 was found to contain homology to members of theCIS/SOCS/JAB/SSI family of cytokine inducible genes(Yoshimura et al., 1995; Endo et al., 1997; Matsumoto et

Fig. 5. Ectopic expression of Shh upregulatescSWiP-1, BMP2 andPatchedin anterior wing buds. The right wing bud was injected withShh-virus in theanterior margin at stage 19–20. After a 16-h incubation, the embryos were fixed and hybridized withcSWiP-1(A); BMP2 (B); andPatched(C); probes. Allthree genes were ectopically expressed in the anterior region of the injected wing buds (arrows).BMP2 is induced in the anterior AER as well as inmesoderm.


al., 1997; Minamoto et al., 1997; Starr et al., 1997). Thishomology is restricted to a region known as the SOCS box(Fig. 6A,C), a motif of unknown function common to thesecytokine inducible proteins (Starr et al., 1997). In addition,three other as yet uncharacterized sequences contain this

region: two different human genes that are similar to Rasand aC. elegansankyrin-repeat containing protein (Fig.6C). In all of these genes the SOCS box is located at ornear the C-terminus of the peptide. cSWiP-1 also containsa potential tyrosine phosphorylation site in the SOCS box(Fig. 6A).

4.2. Homologues and related genes in human and mouse

The similarity between cSWiP-1 and other known WD-proteins is limited to the WD repeats themselves and doesnot extend to the regions between or outside the repeats.This suggests that no true homologue of this gene has yetbeen characterized. Therefore we searched the expressed

Table 1

Time-course of induction ofPatched, BMP2 andcSWiP-1after infectionof limb buds with Shh-virus

8–9 h 10.3–11.3 h 15–17 h

Patched 5/5 7/7 6/6BMP2 0/17 16/24 21/21cSWiP-1 0/19 17/21 22/22

Fig. 6. Molecular nature ofcSWiP-1. (A) Comparison of the amino-acid sequences of chick and human SWiP-1 and mouse SWiP-2. The shaded residues areeither identical (black) or similar (grey) in at least two sequences. WD-repeats are underlined. Solid lines indicate cSWiP-1 WD-repeats with fewerthan fivemismatches from the consensus (Neer et al., 1994). Dashed lines indicate the more divergent repeats. The SOCS box is boxed. The asterisk indicates apotential tyrosine phosphorylation site. (B) Alignment of the SOCS box sequences from SWiP family proteins, SOCS family proteins (which includes CIS)and three as yet uncharacterized amino-acid sequences:C. elegansankyrin-repeat containing protein and two human sequences similar to Ras GTPase. Theshaded residues are either identical (black) or similar (grey) in at least five sequences. Asterisks indicate the C-terminal residues.


sequence tag (EST) database for similar sequences andidentified a large number of human and mouse ESTs withhigh homology to chickcSWiP-1. These ESTs could beassembled into contiguous sequences and fell into twogroups: one representing an apparent homologue ofcSWiP-1and the other representing acSWiP-related gene,which we namedSWiP-2. Both human and mouse ESTswere available for each gene, however only the humanESTs spanned the entire open reading frame of thecSWiP-1homologue, and only the mouse ESTs spannedan entire open reading frame ofSWiP-2.

Sequence analysis of the humanhSWiP-1clone revealedan open reading frame encoding a putative 421 amino acidpeptide (Fig. 6A). hSWiP-1 shares 88% identity to chickcSWiP-1, suggesting that these are true orthologues. Atthe DNA level, the region of high similarity between thegenes coincides with the open reading frame, supporting ourassignment of the translation initiation site (as in chickcSWiP-1, the human gene has a poor match to the Kozaktranslation initiation consensus (Kozak, 1987)). In addition,the 3′ UTRs of chick and humanSWiP-1contain an approxi-mately 200 bp long region of similarity, which begins about200 bases 3′ of the stop codon (data not shown). This regioncould be involved in the regulation of expression of the twohomologues at either the DNA or RNA level.

Sequence analysis of mousemSWiP-2revealed an openreading frame encoding a putative peptide of 404 aminoacids (Fig. 6A), which shares 49.8% identity with chickcSWiP-1 and 49.5% with human hSWiP-1. It also has asimilar structure of eight WD-repeats comprising most ofthe protein and a SOCS box at its C-terminal end. Therefore,the two peptides are related and define a new subfamily ofWD-proteins.

5. Discussion

5.1. SWiP-1 and -2, novel SOCS box-containing WD-proteins

We isolated chickSWiP-1 from a stage 12–15 cDNAlibrary. Its sequence revealed a putative peptide that belongsto a large family of WD-proteins. The members of thisfamily all contain at least four WD repeats, a repeatedmotif of approximately 40 amino acids that often ends intryptophan and asparagine. A consensus sequence for theWD repeats has been proposed by Neer et al. (1994).Recently, the crystal structure of Gb, a WD-protein, hasbeen solved (Wall et al., 1995; Lambright et al., 1996; Son-dek et al., 1996), demonstrating that WD repeats formb-sheet ‘blades’ of ab-propeller structure. The non-conservedstretches of amino acids located betweenb-strands form thesurface of the propeller and have been shown to be involvedin, and to provide specificity for, interactions with otherproteins (Whiteway et al., 1994; Wall et al., 1995; Lamb-right et al., 1996; Komachi and Johnson, 1997; Ng et al.,

1997; Li et al., 1998). Since the WD repeat is a structuralmotif, it is therefore not surprising that WD-proteins shareno discernible functional similarity: proteins containing thismotif have been identified whose functions range from sig-nal transduction to RNA splicing (Neer et al., 1994).

Many WD-proteins also contain other motifs; SWiP-1contains a SOCS box at its C-terminus. The SOCS box isa domain of unknown function that has been found in afamily of cytokine inducible repressors of cytokine signal-ling (CIS/SOCS/JAB/SSI proteins) (Yoshimura et al., 1995;Endo et al., 1997; Matsumoto et al., 1997; Minamoto et al.,1997; Starr et al., 1997). Homology searches with SOCSbox sequences revealed a number of as yet uncharacterizedputative proteins. All identified SOCS box-containing pro-teins also contain domains known to be involved in protein-protein interactions, such as an SH2 domain (in CIS/SOCS/JAB/SSI proteins), ankyrin repeats, Ras-like domains andWD repeats (in SWiPs). Because all of these domains areknown to be present in proteins involved in signal transduc-tion and because SOCS proteins have been shown to beinvolved in signal transduction we are tempted to speculatethat SWiP-1 might also be involved in signal transduction.Elucidation of the function of the SOCS box motif in otherproteins might suggest possible functions for SWiP-1, andallow the design of a rational study of a truncated form ofSWiP-1 lacking the SOCS box. Such a protein might beconstitutively active or act as a dominant negative form ofSWiP-1 and will be useful for learning about the mechanismof SWiP-1 function during embryogenesis.

Homology searches of the EST database led us to identifya human homolog ofSWiP-1and a mouse gene,mSWiP-2,related toSWiP-1. Other ESTs in the database indicate thatmouseSWiP-1and humanSWiP-2also exist. The generalstructure of SWiP-1 is conserved in SWiP-2. Therefore, theSWiPs define a new subfamily of WD-proteins.

5.2. cSWiP-1 is regulated in somites by Shh secreted by thenotochord and by signals from the lateral plate

The dynamic expression ofcSWiP-1during somite devel-opment led us to investigate the regulation of this gene bysignals from neighbouring tissues. We show thatcSWiP-1ispositively regulated by signals from notochord and nega-tively regulated by signals from the intermediate and/orlateral mesoderm.

The notochord is a source of ventralising signals forsomites (Brand-Saberi et al., 1993; Dietrich et al., 1993,1997; Koseki et al., 1993; Pourquie´ et al., 1993; Fan andTessier-Lavigne, 1994; Goulding et al., 1994). One of thesesignals is mediated by Shh (Fan and Tessier-Lavigne, 1994;Johnson et al., 1994; Bumcrot and McMahon, 1995; Fan etal., 1995; Chiang et al., 1996; Borycki et al., 1998), asecreted molecule produced by the notochord and thefloor plate (Echelard et al., 1993; Krauss et al., 1993; Riddleet al., 1993; Roelink et al., 1994). In response to Shh, theventral region of the somite differentiates into sclerotome


and expresses sclerotomal markers such asPax1 (Fan andTessier-Lavigne, 1994; Johnson et al., 1994; Fan et al.,1995; Borycki et al., 1998). Some cells at the medial andcranial edges of the dermomyotome, the dorsolateral portionof the developing somite, later give rise to the myotome(Christ and Ordahl, 1995; Cossu et al., 1996; Denetclaw etal., 1997). Shh is also involved in this process (Johnson etal., 1994; Munsterberg et al., 1995; Borycki et al., 1998), butit must act in conjunction with other signals from the dorsalneural tube (Gallera, 1966; Vivarelli and Cossu, 1986;Kenny-Mobbs and Thorogood, 1987; Mu¨nsterberg andLassar, 1995; Stern et al., 1995; Spence et al., 1996; Dietrichet al., 1997), perhaps Wnt-1 or a related molecule(Munsterberg et al., 1995; Stern et al., 1995; Hirsinger etal., 1997; Marcelle et al., 1997; Capdevila et al., 1998).Interestingly, cSWiP-1 expression seems to correlate intime and space with regions being patterned by Shh. Inthe youngest somites,cSWiP-1is expressed in the ventraldomain. As the somite matures, expression shifts mediallyand dorsally and eventually is maintained in the myotome.Therefore,cSWiP-1expression is unlikely to be associatedwith a particular differentiated cell type, but rather withsome intermediate step(s) in somite patterning.

The expression pattern ofcSWiP-1in somites suggeststhat it is positively regulated by signals produced by thenotochord. The similarity ofcSWiP-1expression patternto that ofPatched(Borycki et al., 1998) is consistent withthis hypothesis. To test this directly, we first showed thataxial structures (neural tube and notochord) are necessaryfor upregulation ofcSWiP-1in somite mesoderm, by separ-ating segmental mesoderm from axial structures (the ‘med-ial cut’ experiment). Then we showed that the notochord is asource of signal(s) that can inducecSWiP-1. We did this firstby removing the neural tube, and observed thatcSWiP-1expression was maintained adjacent to the notochord;then, we showed that the ‘medial cut’ experiment can berescued with a graft of notochord to the ventro-medial sideof the segmental plate. The advantage of this type ofexperiment is that the geometry and the context of the inter-acting structures is largely preserved, in contrast to experi-ments in which signalling structures are grafted to ectopiclocations.

BecausecSWiP-1is positively regulated by signals fromthe notochord and because its expression resembles that ofPatched, we used explant cultures to test whethercSWiP-1is upregulated in the somite by Shh signalling from the mid-line. We show that Shh protein can substitute for the noto-chord in inducingSWiP-1 expression in segmental plateexplants. Induction by the notochord is suppressed by aShh-blocking antibody, suggesting that the regulation ofSWiP-1by the notochord is mediated by Shh.

The medial restriction ofcSWiP-1 expression in thedeveloping somites led us to consider the possibility thatcSWiP-1is also negatively regulated by structures lateralto the somitic mesoderm. Our hypothesis was correct:separation of segmental mesoderm from lateral structures

by a cut through the embryo (the ‘lateral cut’ experiment)results in expansion ofcSWiP-1expression laterally. Lateralplate mesoderm has been shown to be important in medio-lateral patterning of somites (Pourquie´ et al., 1995); it isnecessary for the expression of the lateral dermomyotomemarkerSim1(Pourquieet al., 1996) and for the suppressionof myotome differentiation in the lateral dermomyotome.BMP4 is a secreted molecule likely to mediate this signal;it is expressed in the intermediate and lateral plate meso-derm and when introduced ectopically near the medialsomite it inducesSim1 and inhibits myotome formation(Pourquieet al., 1995, 1996). This lateralization of thesomite has been shown to occur at relatively low concentra-tions of BMP4 protein (Tonegawa et al., 1997). At higherconcentrations, BMP4 specifies intermediate and lateralmesoderm. Because of this evidence, we hypothesized thatBMP4 is the signal from lateral structures that inhibitscSWiP-1expression. We attempted to rescue the ‘lateralcut’ experiment by grafting pellets of BMP4 expressingCOS cells to the lateral side of the segmental plate. Thisdid result in downregulation ofcSWiP-1adjacent to thepellet (data not shown). However, somite segmentation aswell as the expression ofparaxis(a general somite marker)were also suppressed, whileSim1was induced not adjacentto the pellet but at some distance from it (data not shown).From these data we conclude that under our experimentalconditions the level of BMP4 was high enough to changesegmental mesoderm to a presumably more lateral fate.Therefore the role of BMP4 in the downregulation ofcSWiP-1within the somite, independently of its effect onsomite character, remains hypothetical.

Taken together, our results show thatcSWiP-1expressionin somites integrates two signals originating from structuresadjacent to the segmental mesoderm: a positive signal fromthe notochord, mediated by Shh, and a negative signal fromintermediate and/or lateral mesoderm (possibly BMP4).Additional signals may also influencecSWiP-1expression.For example, since signals from the dorsal neural tube areinvolved in the formation of myotome in the dorso-medialsomite (Gallera, 1966; Vivarelli and Cossu, 1986; Kenny-Mobbs and Thorogood, 1987; Mu¨nsterberg and Lassar,1995; Stern et al., 1995; Spence et al., 1996; Dietrich etal., 1997), they might also play a role in the regulation ofcSWiP-1expression.

5.3. Regulation of cSWiP-1 by Shh in limb buds

Having shown that Shh upregulatescSWiP-1in somites,we were curious whether the relationship between the twomolecules holds in systems other than segmental mesoderm.We investigated the regulation ofcSWiP-1expression in thelimb bud, where Shh has been implicated as a major pattern-ing signal (Riddle et al., 1993; Chang et al., 1994; Lo´pez-Martınez et al., 1995; Yang et al., 1997).

The ZPA, located at the posterior margin of the early limbbud, has long been recognized as an organising centre that


patterns the limb along the anterior-posterior axis (Saundersand Gasseling, 1968; MacCabe et al., 1973; Tickle et al.,1975; Summerbell, 1979). When grafted into the anteriorregion of another limb bud, the ZPA induces a mirror imageduplication of the limb. One molecule likely to mediate thisactivity is Shh (Riddle et al., 1993; Chang et al., 1994;Lopez-Martınez et al., 1995; Yang et al., 1997). Its expres-sion domain coincides with the region defined embryologi-cally as the ZPA and misexpression of Shh in the anteriorlimb bud also induces mirror image duplication. This effect,as with the ZPA (Tickle, 1981), depends on both the dose ofShh and the duration of exposure of the limb to the signal(Yang et al., 1997). There is a minimum time window ofShh signalling necessary for an irreversible change to occurin the digit pattern. The same time period is necessarybefore changes are observed in the earliest known limbbud patterning markers,BMP-2 andHoxd-13(Yang et al.,1997).

cSWiP-1 is expressed at a high level in the posteriorregion during early limb development. Later it is also upre-gulated in the central domain. The posterior domain appearsto overlap the ZPA, which prompted us to test whetherectopic expression of Shh in the anterior limb bud can upre-gulatecSWiP-1. We found this to be the case, and that thisoccurs as quickly as the upregulation ofBMP2 in responseto Shh. By contrast,Patched, which marks the cellsresponding to hedgehog signalling, is induced by ectopicShh a few hours beforecSWiP-1andBMP2.

In an attempt to obtain more direct information about theroles ofcSWiP-1in limb patterning, we constructed a retro-viral vector encoding this gene. The virus was injected intoeither the anterior or the posterior domains of developinglimb buds, but we observed no morphological phenotype(data not shown). Anterior misexpression ofcSWiP-1hadno effect on the expression ofBMP2 or FGF4 (data notshown), which are normally expressed in the posteriorlimb bud (BMP2 in posterior mesoderm and AER, andFGF4 in posterior AER) (Francis et al., 1994; Laufer etal., 1994; Niswander et al., 1994). Therefore expression ofcSWiP-1 alone is not sufficient to affect the patterning of thedeveloping limb.

In summary, we have identified a novel WD-protein,expressed in somitic mesoderm and developing limb budsas well as other embryonic structures in which hedgehogsignalling has been shown to play a role (such as ventralneural tube, branchial arches, gut and feather buds). InsomitescSWiP-1 is upregulated by Shh produced in thenotochord and downregulated by signals from the lateralplate mesoderm. In limb budscSWiP-1is induced by ecto-pic expression of Shh at a time when irreversible cell fatechanges are taking place. The structural features of thenovel SWiP proteins are consistent with a possible role insignal transduction. Finally,SWiP-1is only the third gene(afterPtcandGli1, both of which are involved in Shh signaltransduction) known to be upregulated in both developinglimb buds and somites in response to Shh signal, and which

is also expressed close to other sources of Hedgehog duringdevelopment. Together, these findings make it tempting tospeculate that SWiP-1 may be part of a signal transductioncascade, downstream of Hedgehog.

6. Materials and methods

Fertile hens’ eggs (White Leghorn; Spafas, Mass.) andquails’ eggs (Karasoulas, CA) were incubated at 38°C for 14h–8 days to produce embryos between stages 4 and 34(Hamburger and Hamilton, 1951).

6.1. Whole-mount in situ hybridization

In situ hybridization of chick embryos using DIG-labelled RNA probes and sectioning were performed asdescribed previously (The´ry et al., 1995; Streit et al.,1997). For all probes, hybridization and post-hybridizationwashes were done at 68–70°C. The probes used were:cSWiP-1, 2.3 kb, chick Patched, 3.8 kb (Marigo et al.,1996c; Marigo and Tabin, 1996) (kind gift of Dr. CliffTabin), chickSonic hedgehog, 1.4 kb (Riddle et al., 1993)(kind gift of Dr. Cliff Tabin), chickBMP2, 0.8 kb (Francis etal., 1994) (kind gift of Drs. Karel Liem and Tom Jessell),chick sim-1, 0.6 kb (Pourquie´ et al., 1996) (kind gift of Dr.Olivier Pourquie), chickparaxis, 1.4 kb (Barnes et al., 1997;Sosic et al., 1997) (kind gift of Dr. Eric Olson) andFGF4,0.6 kb (Laufer et al., 1994; Niswander et al., 1994) (kind giftof Dr. Lee Niswander).

6.2. Dissection and grafting techniques

Somite, neural tube and notochord surgery was per-formed in ovo, with stage 10–12 embryos, using techniquesdescribed by Stern (1993) in a standing drop of calcium-,magnesium-free Tyrode’s solution (CMF) with 0.11%trypsin (1:250, Difco Laboratories) using a micro knife(15° blade, Xomed Surgical Products). A small hole wasmade in the vitelline membrane over the segmental plateregion. Cuts medial or lateral to the segmental plate wereperformed in three steps. First, the surface ectoderm was cutat the level of the segmental plate, overlying the neural tubeor intermediate mesoderm. Then, the anterior two-thirds ofthe segmental plate were completely separated from eitherthe neural tube and notochord or from the intermediatemesoderm and lateral plate. Finally, the endoderm was cut.

For neural tube ablation, the surface ectoderm was cutadjacent to both sides of the neural tube at the level of thesegmental plate. The posterior region of the neural tube wasgently separated from the flanking segmental plates andnotochord, and removed. For notochord grafts, a piece ofposterior notochord (about 4–6 somites in length, from thelevel of the segmental plate) was isolated from stage 10–12quail donor embryos. The surface ectoderm adjacent to theneural tube of a chick host embryo was cut and the segmen-


tal plate gently separated from the neural tube. The noto-chord graft was placed between the neural tube and thesegmental plate, about 4 somite-lengths posterior to the tipof the segmental plate. The egg was sealed and incubated at38°C for 1–2 h to allow the graft to attach to surroundingtissues. The egg was then reopened and a cut (involvingall three layers) made to separate the segmental plate andthe grafted notochord from the axial structures of thehost.

After surgery, embryos were washed in CMF, lowered byremoval of a few ml of thin egg albumen and the window inthe eggshell sealed with electrical tape. The eggs were incu-bated at 38°C for a further 10–16 h, after which the operatedregions were adjacent to the newly formed somites I-XIII(nomenclature after Christ and Ordahl, 1995: the mostrecently formed somite is designated I, then somites arecounted in the caudal to rostral direction). In notochordgrafting experiments, after a 16-h incubation, the graftswere adjacent to somites IV-V. Embryos were then fixedand processed for in situ hybridization.

6.3. Explant cultures

Segmental plates (excluding the first presumptive somiteand the most posterior quarter) and the notochord adjacentto this region were dissected from stage 11–12 embryos, insome cases using 1 mg/ml Dispase (Boehringer Manheim).Segmental plates (with or without attached surface ecto-derm in separate experiments) were cultured in the presenceor absence of 0.75mg/ml Shh protein (kind gift of Drs. JohanEricson and Tom Jessell). Juxtaposed segmental plate withnotochord explants were cultured in the presence or absenceof 20 mg/ml 5E1 Shh-blocking antibody (kind gift of Drs.Johan Ericson and Tom Jessell) (Ericson et al., 1996). Theexplants were cultured in collagen as described previously(Stern et al., 1986) with or without added Shh or 5E1 anti-body for 16 h at 37°C in 5% CO2.

Explants were fixed in 4% paraformaldehyde with 20 mMEGTA for about 8 h at 4°C and processed for in situ hybri-dization using the protocol described above for embryos.

6.4. Retroviral constructs and infection

A 1.3 kb fragment ofcSWiP-1containing the open read-ing frame (see below) was inserted into RCAS-BP(A) retro-viral vector (with an intermediate cloning step in pSlax13vector). Both untagged and haemagglutinin epitope-taggedversions were made.

Replication-competent RCAS-BP(A) retrovirus encodingSonic hedgehog(Shh-virus) (Riddle et al., 1993),alkalinephophatase(AP-virus) (Fekete and Cepko, 1993) (both kindgifts of Drs. Cliff Tabin and Connie Cepko) orcSWiP-1-virus were grown in chick embryo fibroblasts and concen-trated as described by Morgan et al. (1992) and Fekete andCepko (1993). Infection of chick limb buds was performedin ovo. Shh-virus was injected under the apical ectodermal

ridge (AER) into the anterior margin of stage 19–20 wingand leg buds. Embryos were incubated for 8–9, 10.3–11.3or 15–17 h, then fixed and processed for in situ hybridiza-tion. Infections withAP-virus were used as negative con-trols.

6.5. Molecular biology and sequence analysis

Standard molecular biology methods (Ausubel et al.,1997) were used.cSWiP-1was identified in a stage 12–15chick cDNA library kindly provided by Drs. A. Nieto and D.Wilkinson (Nieto et al., 1994). The 2276 bp fragment wassubcloned into pBlusecript KS (Stratagene) and used as atemplate for sequencing and in situ hybridization. Sequenceanalysis of the clone revealed a 1263 bp open reading frameflanked by 79 bp of 5′ UTR and 934 bp of 3′ UTR. Northernanalysis identified two transcripts of approximate sizes 2.6kb and 1.5 kb (data not shown), suggesting that the clone isonly a few hundred bases shorter than the longer transcript.Since the clone does not terminate in a polyA tail or containa polyadenylation site it is likely to be missing some 3′untranslated sequence. The predicted open reading framebegins with an ATG at position 80, which is immediatelypreceded by an in-frame stop codon. The homologybetween chick cSWiP-1 and its human homologue(described below) begins at this site. Therefore we assignbase 80 as the putative translation initiation site, eventhough it is not contained within a good match to theKozak sequence (Kozak, 1987).

A human EST clone (GenBank accession numberN57397/N32303) was sequenced to obtain humanSWiP-1sequence. Two overlapping mouse EST clones (accessionnumbers AA030104 and AA119135) were sequenced toobtain mouseSWiP-2sequence. Other human EST clones(e.g. accession number W47621) were identified thatencode fragments of a human homologue ofSWiP-2andother mouse EST clones (e.g. accession number W85275)were identified that encode fragments of a mouse homolo-gue ofSWiP-1. Sequencing was performed on both strandsby a strategy of subcloning and specific oligonucleotidepriming, using AmpiTaq DNA polymerase and an ABI373 DNA sequencer.

Database searches were performed using BLAST pro-grams (Altschul et al., 1990; Gish and States, 1993), toaccess sequences in GenBank, EMBL, DDBJ and PDB data-bases. Alignments were made using ALIGN (Pearson andLipman, 1988; Pearson, 1990) and ClustalW (Thompson etal., 1994) programs. Chick and humanSWiP-1and mouseSWiP-2sequences were submitted to GenBank and assignedaccession numbers: chickSWiP-1, AF072879; humanSWiP-1, AF072880; mouseSWiP-2, AF072881. The acces-sion numbers of other unpublished amino acid sequencesare as follows: human Rar protein, 466271; human Raslike GTPase, 2117166; open reading frame inC. elegansgenomic sequence (Wilson et al., 1994) containing ankyrin-like repeats, 1055113.


Acknowledgements

This project was funded by a grant from NIH (HD31942).We are indebted to Drs. Connie Cepko, Johan Ericson, TomJessell, Karel Liem, Lee Niswander, Eric Olson, OlivierPourquieand Cliff Tabin for generous gifts of probes andreagents; to Alyson Berliner, James Briscoe, Artur Kaniaand Ed Laufer for help with the manuscript; to Andrea Streitfor discussions; and to Dr. Ellie Larsen for inspiration.

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