Mechanisms of Development 82 (1999) 79–94
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 other embryonic structures where Hedgehog signalling has been shown to play a role. Using embryonic manipulations we show that in somites
cSWiP-1 expression integrates two signals originating from structures adjacent to the segmental mesoderm: a positive signal from the notochord and a negative signal from intermediate and/or lateral mesoderm. In explant cultures of somitic mesoderm, Shh protein induces
cSWiP-1, while a blocking antibody to Shh inhibits the induction of cSWiP-1 by the notochord. These results show that the positive signal from the notochord is mediated by Shh. We also show that in limb buds cSWiP-1 is upregulated by ectopic Shh. This occurs in about the same time period as upregulation of BMP2, placing cSWiP-1 among the earliest markers for the change of limb pattern caused by ectopic
Shh. We also describe a human homologue of cSWiP-1 and a mouse gene, mSWiP-2, that is more distantly related to SWiP-1, suggesting that SWiP-1 belongs to a novel subfamily of WD-proteins.
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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 of important tissue interactions during embryonic development. Three Hedgehog proteins have been described in higher vertebrates: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) (Echelard et al.,
1993; Riddle et al., 1993; Chang et al., 1994; Roelink et al., 1994; Bitgood and McMahon, 1995; Marigo et al., 1995;
Vortkamp et al., 1996). Of these, Shh has been studied most intensively because of its involvement at early stages of development. Its roles include regulation of left-right asymmetry (Levin et al., 1997; Paga´n-Westphal and Tabin,
1998), ventral patterning of the neural tube (Echelard et al., 1993; Roelink et al., 1994; Ericson et al. 1996; Chiang et al., 1996), subdivision of the somite into dermomyotome and sclerotome (Fan and Tessier-Lavigne, 1994; Johnson et al., 1994; Bumcrot and McMahon, 1995; Fan et al., 1995;
* Corresponding author. Tel.: +1-212-305-7915; fax: +1-212-923-2090; e-mail: cds20@columbia.edu
Chiang et al., 1996; Borycki et al., 1998), induction of the myotome (Johnson et al., 1994; Mu¨nsterberg et al., 1995;
Borycki et al., 1998), mediation of the activity of the Zone of Polarising Activity (ZPA) in the limb bud (Riddle et al.,
1993; Chang et al., 1994; Lo´pez-Martı´nez et al., 1995; Yang et 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 et al., 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 transduction pathway have been identified in Drosophila. Two types of transmembrane proteins are involved in signal reception: patched and smoothened (reviewed in Perrimon, 1996; Ingham, 1998a,b). Downstream of these, Protein Kinase A
(PKA), fused, Suppressor of fused, costal-2, slimb and the transcription factor Cubitus interuptus (Ci) have been implicated in transducing the signal to the nucleus, resulting in transcriptional upregulation of a number of genes, among them 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
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1999 Elsevier Science Ireland Ltd. All rights reserved.
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80 D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 been conserved in vertebrates. Vertebrate homologues of patched, 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 protein has been shown to interact directly with Shh, confirming its role as a Hedgehog receptor (Marigo et al., 1996a; Stone et al., 1996; reviewed in Ingham, 1998a,b). PKA, as in Drosophila, antagonizes Hedgehog signalling (reviewed in Perrimon, 1995; Ingham, 1995) and at least one of three known
Ci homologues, Gli-1, is a target and a mediator of Hedgehog signalling (Marigo et al., 1996b; Lee et al., 1997; reviewed in Ruiz i Altaba, 1997). Furthermore, in response to a Hedgehog signal, transcription of Patched is upregulated (Concordet et al., 1996; Hahn et al., 1996; Marigo and
Tabin, 1996; Marigo et al., 1996c; Lee et al., 1997; Borycki et al., 1998; reviewed in Ingham, 1998a,b), which allows the use of Patched as a marker for cells responding to Hedgehog signals.
Interestingly, of a handful of genes that are known to be transcriptionally regulated in different structures in response to Shh signal, only Ptc and Gli1 are universally upregulated in all systems tested to date. All other markers are expressed in only some of the developing structures that are patterned by Shh. For example, the responses to Shh signalling in the limb bud and somites share no known markers aside from Ptc and Gli1.
Here, we describe the cloning, expression pattern and regulation of a novel chick gene, cSWiP-1 (for SOCS box and WD-repeats in Protein). Its expression pattern suggests participation in many of the inductive processes known to involve hedgehog. We demonstrate that cSWiP-1 is regulated by Shh in two different developing systems, the somites and the limb bud. In the somites,
cSWiP-1 transcription is regulated by Shh emitted by the notochord and repressed by signals from the lateral plate. In the limb bud cSWiP-1 is induced by ectopic Shh. We also describe the cloning of the human homologue of
hSWiP-1 and of a related mouse gene, mSWiP-2, suggesting that these are members of a novel WD-protein subfamily.
but by stage 6 expression has become restricted to the neural plate (Fig. 1B). As the neural tube develops, cSWiP-1 expression evolves and becomes patterned along both the dorsoventral (DV) and anteroposterior (AP) axes (Fig. 1B–
D). At stage 10, cSWiP-1 starts to be expressed in the notochord and somite mesoderm and somewhat later in the intermediate mesoderm. Expression of cSWiP-1 is quite dynamic during maturation of the somites (Fig. 1D–
I) and is described below in greater detail. The strongest site of expression of cSWiP-1 during development is the branchial arches: from stage 20, cSWiP-1 is expressed in the first branchial arch (along the posterior edge of the maxillary process and the anterior edge of the mandibular arch) as well as along the posterior edge of the second (hyoid) arch (Fig. 2A,B). From this stage until at least stage 29,
cSWiP-1 is also expressed in the gut (particularly in the gizzard, duodenum/pancreas and caecal primordia; Fig.
2C). Expression of cSWiP-1 in the developing limb buds is dynamic (Fig. 1J–L) and is described below in greater detail. In older embryos (stage 34) cSWiP-1 expression is most prominent in the forming feather buds and in the scleral papillae of the eye (Fig. 2D–G). It is interesting that many of these sites of cSWiP-1 expression either overlap or lie close to regions where members of the Hedgehog gene family are expressed (for example, compare Fig. 2D–
G with H–K, or Fig. 2A–B with Wall and Hogan, 1995 and
Helms et al., 1997).
2.1.2. Expression in somites
Expression of cSWiP-1 in somites becomes apparent around stage 10. After stage 11, expression in somites displays a constant pattern along the axis (Fig. 1D–I): a low level of cSWiP-1 transcript is detectable in the segmental plate. This expression is upregulated in somite I (numbering after Christ and Ordahl, 1995), in the ventromedial region of the prospective sclerotome. As somites mature this expression domain shifts to the medial side of the somite and then into the dorso-medial lip, the region of the forming myotome. Expression then shifts ventrolaterally with the forming myotome. At this point the dermomyotome also expresses cSWiP-1 but at lower level, while the sclerotome does not express at all.
2. Results
2.1. Expression of cSWiP-1 in the developing embryo
We studied the expression of cSWiP-1 (a gene identified in a screen unrelated to this project) by whole-mount in situ hybridization using antisense riboprobe generated from a cDNA clone containing most of the transcript of the gene
(see below).
2.1.1. General features
At stage 4 (Hamburger and Hamilton, 1951), cSWiP-1 is expressed in almost the entire epiblast layer of the area pellucida, but is excluded from Hensen’s node (Fig. 1A),
2.1.3. Expression in limb buds
cSWiP-1 expression in the limb buds is initially diffuse
(stage 18), but resolves to an area of stronger expression at the posterior margin by stage 21 (Fig. 1J). This domain overlaps with, but is not identical to, the expression of
Shh: the latter extends more distally (not shown). At the same time there is a weaker domain of expression in the proximal-central region of the limb bud. The posterior domain of expression resolves to a thin stripe along the posterior margin at stage 25 (Fig. 1K) and disappears completely by stage 28 (Fig. 1L). Meanwhile, at around stage
23, cSWiP-1 is upregulated in the central domain along the
AP axis both dorsally and ventrally. By stage 28, the distal

D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 81
Fig. 1. Expression of cSWiP-1 in the early embryo, somites and limbs. (A) At stage 4, cSWiP-1 is expressed in the prospective neural plate and is particularly strong in the presumptive cephalic region. (B) At stage 6, cSWiP-1 expression is restricted to the neural plate and is strongest in the forebrain and hindbrain.
(C) Head region of stage 11 embryo. cSWiP-1 expression in the neural ectoderm has started to disappear from specific axial levels, such as the isthmus and most of the hindbrain. (D–I) cSWiP-1 expression in somitic mesoderm shifts medially and dorsally during somite maturation (arrows in E–I). (D) Dorsal view of 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-1 is expressed at a low level in segmental plate. The expression becomes localized ventro-medially in somites I and II. As the somite matures, cSWiP-1 expression becomes restricted medially and then shifts dorsally. Finally, cSWiP-1 becomes expressed at high levels in the myotome-forming region
(E,F). At this stage cSWiP-1 is also expressed in the notochord and Wolffian duct. In the neural tube cSWiP-1 is expressed dorsally or ventrally depending on the axial level. Note the expression in endoderm and associated splanchnic mesoderm (E). (J–L) cSWiP-1 is expressed in two domains during limb bud development. (J) Dorsal view of stage 21 wing bud. cSWiP-1 is strongly expressed at the posterior margin of the limb bud. Weaker expression is also apparent in the proximal central portion of the limb bud. (K) At stage 25, cSWiP-1 remains expressed posteriorly and in the centre of the wing bud. (L) Ventral view of stage 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 virtually disappeared.

82 D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94
Fig. 2. Expression of cSWiP-1 in branchial arches, gut, feather buds and scleral papillae. (A,B) cSWIP-1 is 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-1 is also expressed along the posterior edge of the maxillary process (top arrow). (C)
cSWiP-1 is expressed in the gut in stage 29 embryo, particularly in the duodenum/pancreas (top arrow) and caecal primordia (bottom arrow). In day 8 embryos, cSWiP-1 (A–G) and Shh (H–K) are expressed in the forming feather buds and in the scleral papillae in the eye. (E,I) Dorsal feather buds of embryos in (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. Note that there are two domains of expression of cSWiP-1 in each feather bud (E,F). At this stage both cSWiP-1 and Shh are also expressed in the scleral papillae
(arrows) in the developing eye (G,K).

2.2.1. Axial structures are necessary for the induction of cSWiP-1 in somites
To assess the role of the neural tube and notochord in regulating cSWiP-1 we made a cut through all three germ layers of the embryo just medial to the anterior segmental plate (Fig. 3A). 10–16 h after the operation, we assessed the expression of cSWiP-1 in the newly formed somites. cSWiP-
1 was not expressed in the somites that had developed adjacent to the cut (13/13 embryos), suggesting that axial structures are necessary for the induction of cSWiP-1 in somite mesoderm.
D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 portion of this domain is refined to interdigital regions (Fig.
1L). Later still (by stage 31), expression is also detected in the approximate region of tendon formation (not shown).
Histological analysis revealed that at all stages, transcripts are confined to the mesoderm underlying the surface ectoderm (not shown).
2.2. Regulation of cSWiP-1 in somites
The somite mesoderm is flanked by structures that have been shown to be involved in its patterning along dorsoventral (DV) and medio-lateral (ML) axes (reviewed in
Cossu et al., 1996; Lassar and Mu¨nsterberg, 1996; Yamaguchi, 1997). Medially, it is bordered by the notochord and neural tube, and laterally by intermediate and lateral mesoderm. cSWiP-1 expression in somites is strikingly reminiscent of the expression of Patched (Marigo et al.,
1996c; Marigo and Tabin, 1996; Borycki et al., 1998), a marker for cells that are responding to Shh signalling. Shh secreted by notochord and floor plate cells has been shown to play a role in DV patterning of somites (Fan and Tessier-
Lavigne, 1994; Johnson et al., 1994; Bumcrot and McMahon, 1995; Fan et al., 1995; Mu¨nsterberg et al., 1995;
Chiang et al., 1996; Borycki et al., 1998), which raises the possibility that axial structures may be involved in regulation of cSWiP-1 expression in the somites. In addition,
cSWiP-1 expression is restricted medially as the somite matures, a hint that structures lateral to the segmental mesoderm may downregulate cSWiP-1 expression in the somites.
To address these questions we performed a series of microsurgical experiments on stage 10–12 embryos to isolate the somitic mesoderm from neighbouring structures.
2.2.2. The neural tube is not necessary for the expression of cSWiP-1 in somites
To determine which of the axial structures provide signals necessary for the induction of cSWiP-1, we removed the neural tube at the level of the segmental plate (Fig. 3B).
This operation often leads to the pairwise fusion of right and left somites. Despite the absence of the neural tube and the new midline location of the somites, cSWiP-1 is expressed in the ventral aspect of the fused somites, adjacent to the notochord (3/3 embryos). The neural tube is therefore not necessary for expression of cSWiP-1.
2.2.3. Notochord can induce cSWiP-1 in somites
The above experiments suggest that the notochord emits signals that regulate cSWiP-1 expression. To confirm this, we grafted an ectopic notochord medial to the segmental plate, and then made a cut to separate the segmental plate together with the attached notochord graft from the axial structures of the host embryo (Fig. 3C). cSWiP-1 was induced 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 further away from the graft did not express cSWiP-1. Therefore, the loss of cSWiP-1 expression due to separation of somite mesoderm from axial structures can be rescued by the notochord, suggesting that the notochord emits signals that induce cSWiP-1 in the medial portion of the somites during normal development.
2.2.4. Shh mediates the signal from the notochord that upregulates cSWiP-1
83
Shh, a secreted molecule expressed in the notochord and the floor plate, has been shown to be involved in somite patterning (Fan and Tessier-Lavigne, 1994; Johnson et al.,
1994; Bumcrot and McMahon, 1995; Fan et al., 1995;
Mu¨nsterberg et al., 1995; Chiang et al., 1996; Borycki et al., 1998). The finding that the notochord emits a signal that upregulates cSWiP-1 and the similarity between the expression patterns of cSWiP-1 and Patched in somites suggest that Shh may be the signal that induces cSWiP-1. We used an explant culture system to investigate this.
First, we examined cSWiP-1 (and Patched, as a positive control) expression in cultures of stage 11–12 segmental plates with or without added Shh protein. The results were identical regardless of the inclusion of the ectoderm in the explant: both Patched and cSWiP-1 were induced in treated explants (Patched: 8/8; cSWiP-1: 13/13) (Fig. 4B,D,F).
Neither gene was expressed in explants cultured without
Shh protein (Patched: 0/7, cSWiP-1: 0/11) (Fig. 4A,C,E).
Shh protein can therefore substitute for the notochord in inducing cSWiP-1 transcription in somites.
To test whether the cSWiP-1 inducing signal produced by the notochord requires Shh, we placed notochords and segmental plates in contact with each other and cultured them in the presence or absence of the Shh-blocking antibody
5E1 (Ericson et al., 1996). In the absence of antibody,
Patched expression is detected in the notochord and in part of segmental plate explant (4/4) (Fig. 4G). In the presence of antibody, Patched expression is detectable only in the notochord (0/4 explants expressed Patched in segmental mesoderm) (Fig. 4H). The antibody also greatly reduced both the intensity of expression of cSWiP-1 (compare Fig. 4I and 4J) and the proportion of explants that expressed the gene: 4/6 untreated control explants expressed
cSWiP-1 and treatment with antibody reduced this to 1/4.
Together, these experiments indicate that Shh produced by the notochord accounts for induction of cSWiP-1 expression in somites.

84 D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94
2.2.5. Structures lateral to the somite mesoderm inhibit cSWiP-1 expression in somites
To assess the role of lateral structures in regulating
cSWiP-1 expression we made a cut through all three germ layers of the embryo just lateral to the anterior segmental plate (Fig. 3D). The resulting gap isolates paraxial from intermediate and lateral mesoderm. In the somites that develop adjacent to the cut, the level of cSWiP-1 increases as compared to the somite on the contralateral side and the domain of cSWiP-1 expression extends laterally (10/11 embryos). In the youngest affected somites the ectopic expression on the lateral side is not restricted to the ventral somite (as it is on the medial side) (Fig. 3Db). In older affected somites, ectopic expression is confined to the dorsolateral lip of the dermomyotome (Fig. 3Da). Because the isolation of the paraxial mesoderm from lateral tissues leads to upregulation of cSWiP-1 in somites, we conclude that these tissues produce signals that normally inhibit cSWiP-
1 expression in somites.
3. Regulation of cSWiP-1 expression by Shh in developing limb buds
3.1. Sonic hedgehog induces cSWiP-1 in the limb bud
The ZPA is largely responsible for patterning the limb along the AP axis (Saunders and Gasseling, 1968; MacCabe
Fig. 3. Regulation of cSWiP-1 by axial and lateral tissues. The panels at the top show diagrammatic representations of the operations performed at the anterior segmental plate level in stage 11–12 embryos. The central panels show whole-mount views and the bottom panels show a transverse section at the level indicated by the horizontal lines in the whole mount. (A) ‘Medial cut’ experiment. The segmental plate was separated from the notochord and the neural tube by a cut through all three layers of the embryo. No expression of cSWiP-1 is detected in the somites that developed adjacent to the cut. (B) The neural tube was removed at the level of the anterior segmental plate. The operation resulted in fusion of somites across the midline. Despite the absence of neural tube, expression of cSWiP-1 is maintained adjacent to the notochord in the fused somites (arrow in lower panel). (C) A rescue of the ‘medial cut’ experiment shown 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. The segmental plate and notochord graft were then separated from the axial structures of the host embryo. cSWiP-1 expression is downregulated in somites that develop 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 graft is stronger in the medial domain of the somite than laterally. (D) ‘Lateral cut’ experiment. The segmental plate was separated from the intermediate and lateral mesoderm by a cut through all three layers of the embryo. On the operated side, expression of cSWiP-1 is expanded laterally. In somite VI (a), expression of cSWiP-1 is seen at both the medial (normal) and the lateral (ectopic, arrow) margin of the dermomyotome. In somite III (b), the domain of
cSWiP-1 is expanded dorsally and laterally (arrow).

D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 85
Fig. 4. Expression of cSWiP-1 in somites is regulated by Shh. Segmental plates with (E,F) or without (A–D) overlying ectoderm were cultured in collagen in the presence (B,D,F) or absence (A,C,E) of Shh protein. Both Patched (A,B) and cSWiP-1 (C–F) are induced by Shh. Segmental plate and notochord conjugates (G–J) cultured in the presence (H,J) or absence (G,I) of Shh-blocking antibody. The notochord can induce expression of Patched in segmental plate (arrow in G) and cSWiP-1 (I). Shh-blocking antibody prevents the induction of both Patched (H) and cSWiP-1 (J). The remaining expression of Patched in H is in the notochord (‘not’ in G and H).

86 D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 et al., 1973; Tickle et al., 1975; Summerbell, 1979). Sonic hedgehog (Shh) is expressed in posterior limb buds in a region that has been equated with the ZPA and appears to mediate ZPA activity: misexpression of Shh in anterior limb buds leads to mirror-image duplication of the limb along its
AP axis (Riddle et al., 1993). As part of this process, Shh induces ectopic expression of a number of genes normally expressed in the posterior limb bud (e.g. Patched, BMP2,
Hoxd-13) (Izpisu´a-Belmonte et al., 1991; Nohno et al.,
1991; Francis et al., 1994; Laufer et al., 1994; Marigo et al., 1996c; Nelson et al., 1996).
Because the posterior domain of cSWiP-1 expression in the limb buds overlaps with the proximal aspect of the ZPA and with the domain of Shh expression, and because cSWiP-
1 is induced in segmental mesoderm by Shh, we investigated whether cSWiP-1 can also be induced in the anterior limb bud by ectopic expression of Shh. We used a replication-competent retrovirus-encoding chick Shh (Riddle et al.,
1993), which was injected into anterior mesodermal tissue of stage 19–20 limb buds (both wing and leg). Approximately 16 h after infection we assessed cSWiP-1 expression by in situ hybridization (Fig. 5). cSWiP-1 was upregulated in the infected region as compared to the uninfected contralateral limb bud (22/22 limb buds). Similar infection of limb buds with a control virus encoding alkaline phospha-
tase (Fekete and Cepko, 1993) did not affect cSWiP-1 expression.
larger domain that includes the posterior two-thirds of the
AER.
To assess the timing of induction of SWiP-1 by Shh relative to other genes in the Shh signalling pathway, we compared the time necessary for upregulation of Patched, BMP2 and cSWiP-1 following ectopic expression of Shh (Table 1).
While ectopic Patched expression was upregulated within
8–9 h after virus injection, neither BMP2 nor cSWiP-1 were induced at this time. However, both BMP2 and cSWiP-1 were induced just over 1 h later (Table 1). Although
cSWiP-1 does not appear to be an immediate target of
Shh, these results suggest that its regulation by Shh is as rapid as that of BMP2.
4. Molecular nature of SWiP-1
A 2.3 kb cSWiP-1 clone was isolated from a stage 12–15 chick embryo cDNA library (Nieto et al., 1994). Sequence analysis of the clone revealed an open reading frame encoding a putative polypeptide of 421 amino acids (Fig. 6A).
4.1. SWiP-1 contains WD repeats and a SOCS box
3.2. Ectopic Shh induces cSWiP-1 and BMP2 at the same time
Patched and BMP2 are two early markers commonly used to identify cells responding to Shh signalling in vertebrate limb buds (Francis et al., 1994; Laufer et al., 1994;
Marigo et al., 1996c). This relationship is conserved in limb development between vertebrates and Drosophila, where
Hedgehog upregulates Patched and induces dpp (a Droso-
phila homologue of BMP2) (reviewed in Ingham, 1998a,b) at the transcriptional level. In normal stage 22 limb buds
Patched is upregulated in the mesenchyme in and around the ZPA while BMP2 is expressed in an overlapping but
A database search for sequences similar to the predicted amino acid sequence of cSWiP-1 revealed significant homology with a number of different WD proteins (Neer et al., 1994). Detailed analysis of the cSWiP-1 amino acid sequence shows that it contains eight WD-repeats (Fig.
6A,B), spanning most of the protein. Six of the WD-repeats have four or fewer mismatches from the consensus sequence proposed by Neer et al. (1994). The two N-terminal repeats are more divergent from the consensus and can be recognized only by their position adjacent to the well-defined repeats, which is not unusual (Neer et al., 1994).
To identify homologies to non-WD-proteins, database searches were performed using the cSWiP-1 sequences outside the WD-repeat region of the protein. The C-terminus of cSWiP-1 was found to contain homology to members of the
CIS/SOCS/JAB/SSI family of cytokine inducible genes
(Yoshimura et al., 1995; Endo et al., 1997; Matsumoto et
Fig. 5. Ectopic expression of Shh upregulates cSWiP-1, BMP2 and Patched in anterior wing buds. The right wing bud was injected with Shh-virus in the anterior margin at stage 19–20. After a 16-h incubation, the embryos were fixed and hybridized with cSWiP-1 (A); BMP2 (B); and Patched (C); probes. All three genes were ectopically expressed in the anterior region of the injected wing buds (arrows). BMP2 is induced in the anterior AER as well as in mesoderm.

D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94
Table 1
Time-course of induction of Patched, BMP2 and cSWiP-1 after infection of limb buds with Shh-virus
8–9 h 10.3–11.3 h 15–17 h
Patched
BMP2 cSWiP-1
5/5
0/17
0/19
7/7
16/24
17/21
6/6
21/21
22/22 4.2. Homologues and related genes in human and mouse
87 region: two different human genes that are similar to Ras and a C. elegans ankyrin-repeat containing protein (Fig.
6C). In all of these genes the SOCS box is located at or near the C-terminus of the peptide. cSWiP-1 also contains a potential tyrosine phosphorylation site in the SOCS box
(Fig. 6A).
al., 1997; Minamoto et al., 1997; Starr et al., 1997). This homology is restricted to a region known as the SOCS box
(Fig. 6A,C), a motif of unknown function common to these cytokine inducible proteins (Starr et al., 1997). In addition, three other as yet uncharacterized sequences contain this
The similarity between cSWiP-1 and other known WDproteins is limited to the WD repeats themselves and does not extend to the regions between or outside the repeats.
This suggests that no true homologue of this gene has yet been characterized. Therefore we searched the expressed
Fig. 6. Molecular nature of cSWiP-1. (A) Comparison of the amino-acid sequences of chick and human SWiP-1 and mouse SWiP-2. The shaded residues are either identical (black) or similar (grey) in at least two sequences. WD-repeats are underlined. Solid lines indicate cSWiP-1 WD-repeats with fewer than five mismatches from the consensus (Neer et al., 1994). Dashed lines indicate the more divergent repeats. The SOCS box is boxed. The asterisk indicates a potential 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. elegans ankyrin-repeat containing protein and two human sequences similar to Ras GTPase. The shaded residues are either identical (black) or similar (grey) in at least five sequences. Asterisks indicate the C-terminal residues.

88 D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 sequence tag (EST) database for similar sequences and identified a large number of human and mouse ESTs with high homology to chick cSWiP-1. These ESTs could be assembled into contiguous sequences and fell into two groups: one representing an apparent homologue of
cSWiP-1 and the other representing a cSWiP-related gene, which we named SWiP-2. Both human and mouse ESTs were available for each gene, however only the human
ESTs spanned the entire open reading frame of the
cSWiP-1 homologue, and only the mouse ESTs spanned an entire open reading frame of SWiP-2.
Sequence analysis of the human hSWiP-1 clone revealed an open reading frame encoding a putative 421 amino acid peptide (Fig. 6A). hSWiP-1 shares 88% identity to chick cSWiP-1, suggesting that these are true orthologues. At the DNA level, the region of high similarity between the genes coincides with the open reading frame, supporting our assignment of the translation initiation site (as in chick
cSWiP-1, the human gene has a poor match to the Kozak translation initiation consensus (Kozak, 1987)). In addition, the 3
′
UTRs of chick and human SWiP-1 contain an approximately 200 bp long region of similarity, which begins about
200 bases 3
′ of the stop codon (data not shown). This region could be involved in the regulation of expression of the two homologues at either the DNA or RNA level.
Sequence analysis of mouse mSWiP-2 revealed an open reading frame encoding a putative peptide of 404 amino acids (Fig. 6A), which shares 49.8% identity with chick cSWiP-1 and 49.5% with human hSWiP-1. It also has a similar structure of eight WD-repeats comprising most of the protein and a SOCS box at its C-terminal end. Therefore, the two peptides are related and define a new subfamily of
WD-proteins.
1997; Li et al., 1998). Since the WD repeat is a structural motif, it is therefore not surprising that WD-proteins share no discernible functional similarity: proteins containing this motif have been identified whose functions range from signal transduction to RNA splicing (Neer et al., 1994).
Many WD-proteins also contain other motifs; SWiP-1 contains a SOCS box at its C-terminus. The SOCS box is a domain of unknown function that has been found in a family of cytokine inducible repressors of cytokine signalling (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 SOCS box sequences revealed a number of as yet uncharacterized putative proteins. All identified SOCS box-containing proteins also contain domains known to be involved in proteinprotein interactions, such as an SH2 domain (in CIS/SOCS/
JAB/SSI proteins), ankyrin repeats, Ras-like domains and
WD repeats (in SWiPs). Because all of these domains are known to be present in proteins involved in signal transduction and because SOCS proteins have been shown to be involved in signal transduction we are tempted to speculate that SWiP-1 might also be involved in signal transduction.
Elucidation of the function of the SOCS box motif in other proteins might suggest possible functions for SWiP-1, and allow the design of a rational study of a truncated form of
SWiP-1 lacking the SOCS box. Such a protein might be constitutively active or act as a dominant negative form of
SWiP-1 and will be useful for learning about the mechanism of SWiP-1 function during embryogenesis.
Homology searches of the EST database led us to identify a human homolog of SWiP-1 and a mouse gene, mSWiP-2, related to SWiP-1. Other ESTs in the database indicate that mouse SWiP-1 and human SWiP-2 also exist. The general structure of SWiP-1 is conserved in SWiP-2. Therefore, the
SWiPs define a new subfamily of WD-proteins.
5. Discussion
5.1. SWiP-1 and -2, novel SOCS box-containing WDproteins
5.2. cSWiP-1 is regulated in somites by Shh secreted by the notochord and by signals from the lateral plate
We isolated chick SWiP-1 from a stage 12–15 cDNA library. Its sequence revealed a putative peptide that belongs to a large family of WD-proteins. The members of this family all contain at least four WD repeats, a repeated motif of approximately 40 amino acids that often ends in tryptophan and asparagine. A consensus sequence for the
WD repeats has been proposed by Neer et al. (1994).
Recently, the crystal structure of G b , a WD-protein, has been solved (Wall et al., 1995; Lambright et al., 1996; Sondek et al., 1996), demonstrating that WD repeats form b sheet ‘blades’ of a b -propeller structure. The non-conserved stretches of amino acids located between b -strands form the surface of the propeller and have been shown to be involved in, and to provide specificity for, interactions with other proteins (Whiteway et al., 1994; Wall et al., 1995; Lambright et al., 1996; Komachi and Johnson, 1997; Ng et al.,
The dynamic expression of cSWiP-1 during somite development led us to investigate the regulation of this gene by signals from neighbouring tissues. We show that cSWiP-1 is positively regulated by signals from notochord and negatively regulated by signals from the intermediate and/or lateral mesoderm.
The notochord is a source of ventralising signals for somites (Brand-Saberi et al., 1993; Dietrich et al., 1993,
1997; Koseki et al., 1993; Pourquie´ et al., 1993; Fan and
Tessier-Lavigne, 1994; Goulding et al., 1994). One of these signals is mediated by Shh (Fan and Tessier-Lavigne, 1994;
Johnson et al., 1994; Bumcrot and McMahon, 1995; Fan et al., 1995; Chiang et al., 1996; Borycki et al., 1998), a secreted molecule produced by the notochord and the floor plate (Echelard et al., 1993; Krauss et al., 1993; Riddle et al., 1993; Roelink et al., 1994). In response to Shh, the ventral region of the somite differentiates into sclerotome

D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 and expresses sclerotomal markers such as Pax1 (Fan and
Tessier-Lavigne, 1994; Johnson et al., 1994; Fan et al.,
1995; Borycki et al., 1998). Some cells at the medial and cranial edges of the dermomyotome, the dorsolateral portion of the developing somite, later give rise to the myotome
(Christ and Ordahl, 1995; Cossu et al., 1996; Denetclaw et al., 1997). Shh is also involved in this process (Johnson et al., 1994; Mu¨nsterberg et al., 1995; Borycki et al., 1998), but it must act in conjunction with other signals from the dorsal neural tube (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 et al., 1997), perhaps Wnt-1 or a related molecule
(Mu¨nsterberg et al., 1995; Stern et al., 1995; Hirsinger et al., 1997; Marcelle et al., 1997; Capdevila et al., 1998).
Interestingly, cSWiP-1 expression seems to correlate in time and space with regions being patterned by Shh. In the youngest somites, cSWiP-1 is expressed in the ventral domain. As the somite matures, expression shifts medially and dorsally and eventually is maintained in the myotome.
Therefore, cSWiP-1 expression is unlikely to be associated with a particular differentiated cell type, but rather with some intermediate step(s) in somite patterning.
The expression pattern of cSWiP-1 in somites suggests that it is positively regulated by signals produced by the notochord. The similarity of cSWiP-1 expression pattern to that of Patched (Borycki et al., 1998) is consistent with this hypothesis. To test this directly, we first showed that axial structures (neural tube and notochord) are necessary for upregulation of cSWiP-1 in somite mesoderm, by separating segmental mesoderm from axial structures (the ‘medial cut’ experiment). Then we showed that the notochord is a source of signal(s) that can induce cSWiP-1. We did this first by removing the neural tube, and observed that cSWiP-1 expression was maintained adjacent to the notochord; then, we showed that the ‘medial cut’ experiment can be rescued with a graft of notochord to the ventro-medial side of the segmental plate. The advantage of this type of experiment is that the geometry and the context of the interacting structures is largely preserved, in contrast to experiments in which signalling structures are grafted to ectopic locations.
Because cSWiP-1 is positively regulated by signals from the notochord and because its expression resembles that of
Patched, we used explant cultures to test whether cSWiP-1 is upregulated in the somite by Shh signalling from the midline. We show that Shh protein can substitute for the notochord in inducing SWiP-1 expression in segmental plate explants. Induction by the notochord is suppressed by a
Shh-blocking antibody, suggesting that the regulation of
SWiP-1 by the notochord is mediated by Shh.
The medial restriction of cSWiP-1 expression in the developing somites led us to consider the possibility that
cSWiP-1 is also negatively regulated by structures lateral to 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 of cSWiP-1 expression laterally. Lateral plate mesoderm has been shown to be important in mediolateral patterning of somites (Pourquie´ et al., 1995); it is necessary for the expression of the lateral dermomyotome marker Sim1 (Pourquie´ et al., 1996) and for the suppression of 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 mesoderm and when introduced ectopically near the medial somite it induces Sim1 and inhibits myotome formation
(Pourquie´ et al., 1995, 1996). This lateralization of the somite has been shown to occur at relatively low concentrations of BMP4 protein (Tonegawa et al., 1997). At higher concentrations, BMP4 specifies intermediate and lateral mesoderm. Because of this evidence, we hypothesized that
BMP4 is the signal from lateral structures that inhibits
cSWiP-1 expression. We attempted to rescue the ‘lateral cut’ experiment by grafting pellets of BMP4 expressing
COS cells to the lateral side of the segmental plate. This did result in downregulation of cSWiP-1 adjacent to the pellet (data not shown). However, somite segmentation as well as the expression of paraxis (a general somite marker) were also suppressed, while Sim1 was induced not adjacent to the pellet but at some distance from it (data not shown).
From these data we conclude that under our experimental conditions the level of BMP4 was high enough to change segmental mesoderm to a presumably more lateral fate.
Therefore the role of BMP4 in the downregulation of
cSWiP-1 within the somite, independently of its effect on somite character, remains hypothetical.
Taken together, our results show that cSWiP-1 expression in somites integrates two signals originating from structures adjacent to the segmental mesoderm: a positive signal from the notochord, mediated by Shh, and a negative signal from intermediate and/or lateral mesoderm (possibly BMP4).
Additional signals may also influence cSWiP-1 expression.
For example, since signals from the dorsal neural tube are involved in the formation of myotome in the dorso-medial somite (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 et al., 1997), they might also play a role in the regulation of
cSWiP-1 expression.
5.3. Regulation of cSWiP-1 by Shh in limb buds
89
Having shown that Shh upregulates cSWiP-1 in somites, we were curious whether the relationship between the two molecules holds in systems other than segmental mesoderm.
We investigated the regulation of cSWiP-1 expression in the limb bud, where Shh has been implicated as a major patterning signal (Riddle et al., 1993; Chang et al., 1994; Lo´pez-
Martı´nez et al., 1995; Yang et al., 1997).
The ZPA, located at the posterior margin of the early limb bud, has long been recognized as an organising centre that

90 D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 patterns the limb along the anterior-posterior axis (Saunders and Gasseling, 1968; MacCabe et al., 1973; Tickle et al.,
1975; Summerbell, 1979). When grafted into the anterior region of another limb bud, the ZPA induces a mirror image duplication of the limb. One molecule likely to mediate this activity is Shh (Riddle et al., 1993; Chang et al., 1994;
Lo´pez-Martı´nez et al., 1995; Yang et al., 1997). Its expression domain coincides with the region defined embryologically as the ZPA and misexpression of Shh in the anterior limb bud also induces mirror image duplication. This effect, as with the ZPA (Tickle, 1981), depends on both the dose of
Shh and the duration of exposure of the limb to the signal
(Yang et al., 1997). There is a minimum time window of
Shh signalling necessary for an irreversible change to occur in the digit pattern. The same time period is necessary before changes are observed in the earliest known limb bud patterning markers, BMP-2 and Hoxd-13 (Yang et al.,
1997).
cSWiP-1 is expressed at a high level in the posterior region during early limb development. Later it is also upregulated in the central domain. The posterior domain appears to overlap the ZPA, which prompted us to test whether ectopic expression of Shh in the anterior limb bud can upregulate cSWiP-1. We found this to be the case, and that this occurs as quickly as the upregulation of BMP2 in response to Shh. By contrast, Patched, which marks the cells responding to hedgehog signalling, is induced by ectopic
Shh a few hours before cSWiP-1 and BMP2.
In an attempt to obtain more direct information about the roles of cSWiP-1 in limb patterning, we constructed a retroviral vector encoding this gene. The virus was injected into either the anterior or the posterior domains of developing limb buds, but we observed no morphological phenotype
(data not shown). Anterior misexpression of cSWiP-1 had no effect on the expression of BMP2 or FGF4 (data not shown), which are normally expressed in the posterior limb bud (BMP2 in posterior mesoderm and AER, and
FGF4 in posterior AER) (Francis et al., 1994; Laufer et al., 1994; Niswander et al., 1994). Therefore expression of cSWiP-1 alone is not sufficient to affect the patterning of the developing limb.
In summary, we have identified a novel WD-protein, expressed in somitic mesoderm and developing limb buds as well as other embryonic structures in which hedgehog signalling has been shown to play a role (such as ventral neural tube, branchial arches, gut and feather buds). In somites cSWiP-1 is upregulated by Shh produced in the notochord and downregulated by signals from the lateral plate mesoderm. In limb buds cSWiP-1 is induced by ectopic expression of Shh at a time when irreversible cell fate changes are taking place. The structural features of the novel SWiP proteins are consistent with a possible role in signal transduction. Finally, SWiP-1 is only the third gene
(after Ptc and Gli1, both of which are involved in Shh signal transduction) known to be upregulated in both developing limb buds and somites in response to Shh signal, and which is also expressed close to other sources of Hedgehog during development. Together, these findings make it tempting to speculate that SWiP-1 may be part of a signal transduction cascade, downstream of Hedgehog.
6. Materials and methods
Fertile hens’ eggs (White Leghorn; Spafas, Mass.) and quails’ eggs (Karasoulas, CA) were incubated at 38
°
C for 14 h–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 DIGlabelled RNA probes and sectioning were performed as described previously (The´ry et al., 1995; Streit et al.,
1997). For all probes, hybridization and post-hybridization washes 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. Cliff
Tabin), chick Sonic hedgehog, 1.4 kb (Riddle et al., 1993)
(kind gift of Dr. Cliff Tabin), chick BMP2, 0.8 kb (Francis et al., 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´), chick paraxis, 1.4 kb (Barnes et al., 1997;
Sosic et al., 1997) (kind gift of Dr. Eric Olson) and FGF4,
0.6 kb (Laufer et al., 1994; Niswander et al., 1994) (kind gift of Dr. Lee Niswander).
6.2. Dissection and grafting techniques
Somite, neural tube and notochord surgery was performed in ovo, with stage 10–12 embryos, using techniques described 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 was made in the vitelline membrane over the segmental plate region. Cuts medial or lateral to the segmental plate were performed in three steps. First, the surface ectoderm was cut at the level of the segmental plate, overlying the neural tube or intermediate mesoderm. Then, the anterior two-thirds of the segmental plate were completely separated from either the neural tube and notochord or from the intermediate mesoderm and lateral plate. Finally, the endoderm was cut.
For neural tube ablation, the surface ectoderm was cut adjacent to both sides of the neural tube at the level of the segmental plate. The posterior region of the neural tube was gently separated from the flanking segmental plates and notochord, and removed. For notochord grafts, a piece of posterior notochord (about 4–6 somites in length, from the level of the segmental plate) was isolated from stage 10–12 quail donor embryos. The surface ectoderm adjacent to the neural tube of a chick host embryo was cut and the segmen-

6.3. Explant cultures
D. Vasiliauskas et al. / Mechanisms of Development 82 (1999) 79–94 tal plate gently separated from the neural tube. The notochord graft was placed between the neural tube and the segmental plate, about 4 somite-lengths posterior to the tip of the segmental plate. The egg was sealed and incubated at
38
°
C for 1–2 h to allow the graft to attach to surrounding tissues. The egg was then reopened and a cut (involving all three layers) made to separate the segmental plate and the grafted notochord from the axial structures of the host.
After surgery, embryos were washed in CMF, lowered by removal of a few ml of thin egg albumen and the window in the eggshell sealed with electrical tape. The eggs were incubated at 38
°
C for a further 10–16 h, after which the operated regions were adjacent to the newly formed somites I-XIII
(nomenclature after Christ and Ordahl, 1995: the most recently formed somite is designated I, then somites are counted in the caudal to rostral direction). In notochord grafting experiments, after a 16-h incubation, the grafts were adjacent to somites IV-V. Embryos were then fixed and processed for in situ hybridization.
Segmental plates (excluding the first presumptive somite and the most posterior quarter) and the notochord adjacent to this region were dissected from stage 11–12 embryos, in some cases using 1 mg/ml Dispase (Boehringer Manheim).
Segmental plates (with or without attached surface ectoderm in separate experiments) were cultured in the presence or absence of 0.75
m g/ml Shh protein (kind gift of Drs. Johan
Ericson and Tom Jessell). Juxtaposed segmental plate with notochord explants were cultured in the presence or absence of 20 m g/ml 5E1 Shh-blocking antibody (kind gift of Drs.
Johan Ericson and Tom Jessell) (Ericson et al., 1996). The explants were cultured in collagen as described previously
(Stern et al., 1986) with or without added Shh or 5E1 antibody for 16 h at 37
°
C in 5% CO
2
.
Explants were fixed in 4% paraformaldehyde with 20 mM
EGTA for about 8 h at 4
°
C and processed for in situ hybridization using the protocol described above for embryos.
6.4. Retroviral constructs and infection
A 1.3 kb fragment of cSWiP-1 containing the open reading frame (see below) was inserted into RCAS-BP(A) retroviral vector (with an intermediate cloning step in pSlax13 vector). Both untagged and haemagglutinin epitope-tagged versions were made.
Replication-competent RCAS-BP(A) retrovirus encoding
Sonic hedgehog (Shh-virus) (Riddle et al., 1993), alkaline
phophatase (AP-virus) (Fekete and Cepko, 1993) (both kind gifts of Drs. Cliff Tabin and Connie Cepko) or cSWiP-1virus were grown in chick embryo fibroblasts and concentrated as described by Morgan et al. (1992) and Fekete and
Cepko (1993). Infection of chick limb buds was performed in ovo. Shh-virus was injected under the apical ectodermal ridge (AER) into the anterior margin of stage 19–20 wing and leg buds. Embryos were incubated for 8–9, 10.3–11.3
or 15–17 h, then fixed and processed for in situ hybridization. Infections with AP-virus were used as negative controls.
6.5. Molecular biology and sequence analysis
91
Standard molecular biology methods (Ausubel et al.,
1997) were used. cSWiP-1 was identified in a stage 12–15 chick cDNA library kindly provided by Drs. A. Nieto and D.
Wilkinson (Nieto et al., 1994). The 2276 bp fragment was subcloned into pBlusecript KS (Stratagene) and used as a template for sequencing and in situ hybridization. Sequence analysis of the clone revealed a 1263 bp open reading frame flanked by 79 bp of 5
′
UTR and 934 bp of 3
′
UTR. Northern analysis identified two transcripts of approximate sizes 2.6
kb and 1.5 kb (data not shown), suggesting that the clone is only a few hundred bases shorter than the longer transcript.
Since the clone does not terminate in a polyA tail or contain a polyadenylation site it is likely to be missing some 3
′ untranslated sequence. The predicted open reading frame begins with an ATG at position 80, which is immediately preceded by an in-frame stop codon. The homology between chick cSWiP-1 and its human homologue
(described below) begins at this site. Therefore we assign base 80 as the putative translation initiation site, even though it is not contained within a good match to the
Kozak sequence (Kozak, 1987).
A human EST clone (GenBank accession number
N57397/N32303) was sequenced to obtain human SWiP-1 sequence. Two overlapping mouse EST clones (accession numbers AA030104 and AA119135) were sequenced to obtain mouse SWiP-2 sequence. Other human EST clones
(e.g. accession number W47621) were identified that encode fragments of a human homologue of SWiP-2 and other mouse EST clones (e.g. accession number W85275) were identified that encode fragments of a mouse homologue of SWiP-1. Sequencing was performed on both strands by a strategy of subcloning and specific oligonucleotide priming, using AmpiTaq DNA polymerase and an ABI
373 DNA sequencer.
Database searches were performed using BLAST programs (Altschul et al., 1990; Gish and States, 1993), to access sequences in GenBank, EMBL, DDBJ and PDB databases. Alignments were made using ALIGN (Pearson and
Lipman, 1988; Pearson, 1990) and ClustalW (Thompson et al., 1994) programs. Chick and human SWiP-1 and mouse
SWiP-2 sequences were submitted to GenBank and assigned accession numbers: chick SWiP-1, AF072879; human
SWiP-1, AF072880; mouse SWiP-2, AF072881. The accession numbers of other unpublished amino acid sequences are as follows: human Rar protein, 466271; human Ras like GTPase, 2117166; open reading frame in C. elegans genomic sequence (Wilson et al., 1994) containing ankyrinlike repeats, 1055113.

92
Acknowledgements
This project was funded by a grant from NIH (HD31942).
We are indebted to Drs. Connie Cepko, Johan Ericson, Tom
Jessell, Karel Liem, Lee Niswander, Eric Olson, Olivier
Pourquie´ and Cliff Tabin for generous gifts of probes and reagents; to Alyson Berliner, James Briscoe, Artur Kania and Ed Laufer for help with the manuscript; to Andrea Streit for discussions; and to Dr. Ellie Larsen for inspiration.
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