Development 105, 541-548 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

541

Molecular differences between the rostral and caudal halves of the

sclerotome in the chick embryo

WENDIE E. NORRIS1, CLAUDIO D. STERN1 and ROGER J. KEYNES2

1 Department of Human Anatomy, South Parks Road, Oxford 0X1 3QX, UK2 Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK

Summary

It is known that both neural crest cell migration andmotor axon outgrowth in most vertebrate embryos aresegmented because of restrictions imposed upon theirdistribution by the neighbouring sclerotomes, each ofwhich is divided into a rostral and a caudal half. Thecaudal half does not allow crest migration or axonoutgrowth, while the rostral half does. In this paper, weinvestigate the expression of proteins and glycoproteinsin the two halves of the sclerotome of the chick embryo atstages between 20 and 32 pairs of somites bytwo-dimensional SDS-polyacrylamide gel electrophor-esis. We find that the patterns of expression are complex,

and that polypeptides and glycoproteins vary bothspatially and temporally: of those that are expresseddifferentially by the sclerotome, some differ quantitat-ively and others qualitatively. Some macromoleculeschange their spatial distribution with developmentalage, and some appear or disappear as the embryosbecome older.

Key words: segmentation, 2-D gel electrophoresis,glycoproteins, chick embryo, somite, sclerotome, peripheralnervous system.

Introduction

A striking feature of higher vertebrate embryos is theirsegmented body plan, which is first obvious duringdevelopment in the arrangement of the somites, lyingadjacent to the neural tube. Segmentation is alsoreflected in the pattern of the peripheral nerves as theyemerge from the spinal cord, and indeed there is muchevidence that it is the segmentation of the somiticmesoderm that determines the segmented pattern ofthe peripheral nervous system (reviewed by Keynes &Stern, 1988). Lewis et al. (1981) showed that in theabsence of somite tissue, motor nerves grow out of theneural tube in an unsegmented fashion. Both themigration of neural crest cells and the outgrowth ofmotor axons occurs only through the rostral half of eachsclerotome (Keynes & Stern, 1984; Rickmann et al.1985); this pattern is determined by differences betweenrostral and caudal half-sclerotome cells, since rostro-caudal rotation of somitic tissue by 180° at an appropri-ate stage of development results in a reversed pattern ofmotor nerves (Keynes & Stern, 1984) and neural crestmigration (Stern & Bronner-Fraser, in preparation).The rostral-caudal subdivision of the sclerotome alsoappears to play a role in modulating the interactionsbetween the cells of the two halves of the sclerotome(Stern & Keynes, 1987) and results in the formation of

an intrasclerotomal boundary, now known as vonEbner's fissure (von Ebner, 1888; Keynes & Stern,1984).

The molecular nature of the differences betweenrostral and caudal half-sclerotome cells is not under-stood. However, the cells of the rostral half appear toexpress cytotactin (Tan et al. 1987), tenascin (Mackie etal. 1988) and butyrylcholinesterase activity (Layer et al.1988), while the cells of the caudal half bind peanutlectin (Stern et al. 1986) and antibodies directed againsta cytotactin-binding proteoglycan ('CTB-proteogly-can'; Tan et al. 1987). However, no differences in thedistribution of these molecules can be detected untilsome time after the onset of neural crest migration intothe rostral half-sclerotome. Nevertheless, it is possiblethat one or more of these molecules might influencemotor nerve outgrowth. Other molecules known toinfluence neural crest migration in vitro, such as lam-inin, fibronectin, integrin and N-CAM show no differ-ence in their distribution within the sclerotome (Rick-mann et al. 1985; Krotoski et al. 1986; Tosney et al.1986). In this study, using two-dimensional polyacryl-amide gel electrophoresis and Western blotting, wereport the existence of novel differences in macromol-ecules present in the two halves of the sclerotome ofembryos at much earlier stages than those used inprevious investigations.

542 W. E. Norris, C. D. Stern and R. J. Keynes

Materials and methods

All solutions were prepared using deionized distilled water.

DissectionHens' eggs were incubated at 38°C to stages 13-17 (Ham-burger & Hamilton, 1951) to obtain embryos with 20-32 pairsof somites. The embryos were explanted and pinned out,ventral side uppermost, on Sylgard dishes in Ca2+- and Mg2+-free Tyrode's solution (CMF) at 30°C. The embryos werewashed twice with CMF, immersed in double-strength CMFcontaining 0-1% EDTA (2xCMF/EDTA) at 30°C and thesclerotome halves dissected out from both left and right sidesof the embryo. These half-sclerotomes were collected intorostral and caudal pools, and deposited on the embryo;collections from six somites at a time were transferred toEppendorf tubes containing 200^1 CMF at 4°C. Half-sclero-tomes were not collected from the most-rostral five pairs ofsomites (occipital) or the most-caudal five to six (epithelial)pairs of somites. The sclerotome halves were then washedwith cold CMF (4°C) by centrifugation for 5 min at the 'low'speed in an MSE Micro Centaur centrifuge. The cell pelletswere then resuspended in cold CMF containing 1 mM-PMSFand centrifuged at 'high' speed (11600g) for 5 min. The finalpellets were fast frozen on solid CO2 in 10 /A of CMFcontaining 1 mM-PMSF and 1 mM-EDTA and stored at —20°Cuntil required.

2D-gel electrophoresisApart from Trizma base (Sigma), reagents used for preparingthe gels were of electrophoretic (BioRad) or isoelectrophor-etic grade (NP-40, ampholines, agarose: LKB).

Sample preparationTo each cell pellet was added, at room temperature: 60^1 of3 mM-EDTA containing 1-5 mM-PMSF, 100^1 of lysis buffer(9-5M-urea, 10% NP-40, 1-6% ampholines pH5-7, 0-4%ampholines pH 3-5-10, 5 % mercaptoethanol) and the equiv-alent of 100^1 of lyophilized buffer (9-5M-urea, 1% SDS).The cell pellet was dispersed in this solubilization buffer bythree passages through a 100 1̂ Hamilton microsyringe. Eachsample was then airfuged at 100000 # (30 p.s.i.) for lh atroom temperature. The supernatants were then assayed forprotein content using a modification of the Bramhall et al.(1969) assay. This involved drying protein samples onto filterpaper squares, staining with Coomassie blue R-250, elutioninto 66% methanol/1% ammonia and reading the opticaldensity (OD) at 560 nm. A standard curve was constructedfrom known amounts of BSA. Supernatants were storedfrozen at —20°C for not longer than one week prior to use.

For gels of neural crest cells, neural tubes were cultured ontissue culture plastic as described previously (Stern et al.1986). After 24 h, the cultures were washed with CMF, theneural tube removed and the emigrated crest cells scraped offtheir substrate in the presence of 1 mM-PMSF. Eight neuraltube cultures were used per gel.

ElectrophoresisIsofocusing gels were prepared and run as described byO'Farrell (1975) with modifications. 5 ml of 4% w/v poly-acrylamide gel solution containing 2% ampholines (1-6%pH5-7; 0-4% pH 3-5-10) were degassed in vacuo for lmin,and then 10^1 of 10% ammonium persulphate and 5/ul ofTEMED added. Using a lml disposable syringe fitted withsurgical tubing, the solution was used to fill glass tubes 130 mmlong and lmm internal diameter sealed at their base with

Nescofilm. The tubes were filled up to an 80mm mark andthen overlaid with either deionized water or overlay buffer[9M-urea, 2% NP-40, 2% ampholines (0-8% pH5-7: 0-2%pH 3-5-10)]. After sealing the top of each tube with Nesco-film, they were allowed to gel overnight at room temperature.

Prior to use, the overlay solution was removed and the baseof the gel sealed with dialysis membrane secured by latexrings. The gels were overlaid with overlay buffer and degassed50mM-NaOH, and prefocused at 200V for lh, with 50mM-NaOH in the upper (—) reservoir and 10mM-H3PO4 in thelower (+) reservoir. The overlay buffer and NaOH were thenremoved and up to 40/d of sample applied to each gel,followed by fresh overlay buffer and NaOH. The samplecontained 5 jig of cell protein plus 2/d of lx stock carbonicanhydrase (Carbamylyte) pi reference peptides. The upperreservoir was refilled with fresh degassed NaOH and the gelsfocused for 17h with 400 V at room temp. On one gel per run,sample was substituted with solubilization buffer containingno protein. This gel was later divided into lcm lengths andeach length eluted into 0-5 ml degassed deionized water for30 min at room temperature. The pH of each elution was usedto construct a pH profile.

Following removal of the gels by extrusion onto Nescofilm,the bottom (+) of each gel was marked by insertion of aninsect dissection pin. All gels, except for the one for pHprofile, were then dehydrated sequentially by 5 min incu-bations in 25% ethanol, 50% ethanol and 100% ethanol.This procedure both removed the ampholines and fixed theproteins. The dehydrated gels were stored in 100% ethanol inscrew-top jars at -20°C.

The second dimension SDS-PAGE was performed on4-15 % acrylamide 0-5 mm thick minigels (9cm x 9cm), usingthe Laemmli (1970) buffer system in apparatus of the Matsu-daira & Burgess (1978) design. Using a perspex trough andcomb, agarose (1% IEF agarose, 0-125M-Tris-HCl, pH6-8)wells for the isofocused gel and molecular weight standardswere made atop the minigels. The isofocused gel was equilib-rated for 10 min in 10 ml of sample buffer (0-0625 M-TrispH6-8, 10% SDS, 10% glycerol, 0-25 ing ml"1 DTT andSO^gml"1 bromophenol blue). The equilibrated gel wasplaced in the agarose well so that the top (—) was adjacent tothe molecular weight standard well(s) and fixed in place withmore agarose (pH6-8). 1-2/il of standards (14C rainbowmarkers, diluted 1:3; Amersham) were placed in the smallerwell(s). Electrophoresis was performed at 150 V for 90 min, oruntil the tracking dye was 1-5 cm above the bottom of the gel.

Silver stainingThis was performed according to the method of Morrissey(1981) except that 15-20 min incubations were used.

ImmunoblottingWestern blots were prepared from the 2D-gels by transfer for90 min to Immobilon membrane (Millipore) in the continuousbuffer system (39mM-glycine, 48mM-Tris, 0-0375% [w/v]SDS, 20% methanol) with the Novablot semidry apparatus(LKB). Transfer was checked by staining with the reversibleprotein stain Ponceau S (BDH). The blots were then blockedby incubation for 30 min in 0-5% Tween 20/10 mM-Tris/150mM-NaCl (pH7-4), washed with buffer A (50mM-Tris/150mM-NaCl, pH7-4, containing 0-5mM-CaCl2 and 0-5 mM-MnSO,») and then incubated in 20/igml"1 of Concanavalin A(ConA, Miles) in buffer A containing 005 % Tween 20 andSmgmr1 haemoglobin (crystalline, BDH) for 2h at roomtemperature. After three washes with buffer A/Tween 20, theblots were incubated for 2h with 20/igml"1 of horseradishperoxidase (Sigma), diluted as for ConA. After three further

Molecular differences between somite halves 543

200

1vr ^ 45

Tv

200

45

5-9 5-4 7 0 6-8 6-6 6-4Pi

6-2 5-9 5-4

Fig. 1. 2-D gel analysis of rostral and caudal half-sclerotomes from chick embryos with 20 pairs of somites. Topphotographs, rostral; Bottom photographs, caudal. Left photographs, silver staining; Right photographs, ConA-probedblots. The large white arrowhead marks the position of actin, and the large white arrow shows the train of pi referenceproteins at 31Y.V& M,. Black arrows, polypeptides present in both the rostral and caudal halves of the sclerotome. Two-tonearrows, polypeptides showing differences between the two halves (see Table 1).

washes with buffer A/Tween 20, the blots were washed oncein buffer A and then incubated in 0-05 % diaminobenzidine(DAB) (Aldrich) in OlM-Tris-HCl (pH7-4) for about15min. Cobalt enhancement was not usually necessary. Theuse of Tween 20 as a blocking agent rather than haemoglobindid not alter the binding pattern of ConA. All ConA bindingusing this system was confirmed by comparison with n^ConA overlay on gels according to the method of Burridge(1978), using lectins iodinated using the Iodogen methoddescribed by Fraker & Speck (1978).

Although the mannose-specificity of ConA was not testedby competition with the appropriate carbohydrate, the speci-ficity was confirmed by its binding to the molecular weightstandard that contained mannose (ovalbumin; Mr = 46xl(r).Non-carbohydrate-containing protein standards did not bindConA.

Results

The use of a miniaturized format for 2D-gel electro-phoresis has enabled us to examine, on a single gel, theprotein content of half-sclerotomes from as few as two

embryos. To eliminate differences between rostral andcaudal halves due to the peculiarities of individuals, wepooled dissections from six to eight embryos. Theseembryos had been carefully selected so that theypossessed the same number of somite pairs and were atthe same stage of development.

2D-gel analysisRostral and caudal half-sclerotomes from embryospossessing 20 pairs (Fig. 1), 24 pairs (Fig. 2) and 32pairs (Fig. 3) of somites were analysed in 2D-minigels.Each figure shows rostral (top) and caudal (bottom)halves examined for total polypeptide content by silver-staining of gels (left) and for glycoprotein content byprobing of Western blots with the mannose-specificlectin ConA (right). On the silver-stained gels, a trainof pi reference proteins at 31xlO3(Mr) can also beseen. This train consists of the protein carbonic anhyd-rase and its carbamylated derivatives, and was added toeach sample prior to running the first dimension. Itprovides an internal standard for the pH gradient, each

544 W. E. Norris, C. D. Stern and R. J. Keynes

16171

V

200

L45

V

-200

L45

7-0 6-8 6-6 6-4 6-2 5-9 5-4 6-8 6 6 6-4 6-2 5-9 5-4Pi pl

Fig. 2. 2-D gel analysis of rostral and caudal half-sclerotomes from chick embryos with 24 pairs of somites. Details as inFig. 1.

spot having a unique and reproducible pi, enablingprecise location and comparison of spot positions ondifferent gels. At the basic ( - ) end of each gel,molecular weight markers were run in order to stan-dardize the second dimension (not shown in Figures).

Polypeptides showing differences were numbered notwith respect to Mr and pi, but rather in order of theirappearance from the earliest stage of developmentexamined.

Polypeptide differencesMany differences between rostral and caudal half-sclerotomes could be seen in silver-stained gels. Boththe number of polypeptides seen in such gels to beexpressed differently in the two halves of the somite andthe complexity of their distribution appeared to in-crease with the stage of development. A few of thevariants were identified as glycoproteins on the basis ofConA binding. The changes in the polypeptide patternare summarized in Table 1, which shows their MT, pivalues, rostral/caudal distribution and whether they areConA-binding glycoproteins (*). Three glycoproteins(doublet la / lb and 7) that are common to both halves

and are not differentially expressed at any of the stagesexamined are not included.

Qualitative differencesA number of polypeptides were expressed exclusivelyin one half of the sclerotome (e.g. spots 2, 11,12, 23, 24in the rostral portion; spots 3, 4, 5, 6, 19, 20, 21, 22 inthe caudal portion). All of these appear to be expressedin a stage-specific manner. In particular, the glyco-protein train 15, 16, 17, 18 showed a complex pattern ofexpression, being absent from 20-somite embryos, ex-pressed exclusively in the rostral half-sclerotome at 24somites and present in both halves at the 32-pair stage.Expression of spots 3,4,5,6 was seen only in 20-somiteembryos, in which it was restricted to the caudal half ofthe sclerotome.

Quantitative differencesOther polypeptides were found to differ in amountbetween the two halves (e.g. 8, 9, 10, 13, 14 and 25).Again, some of these were stage specific (e.g. spot 25)but others were expressed differentially at more thanone stage (e.g. 8, 9, 10, 13, 14). It may be significantthat these quantitative differences occurred as reduced

Molecular differences between somite halves 545

r200

7-0 6-9 6-7 6-5 6-0 5-5 70 6 9 6-7 6-5 60 5 5 5-0

-200

Fig. 3. 2-D gel analysis of rostral and caudal half-sclerotomes from chick embryos with 32 pairs of somites. Details as inFig. 1.

amounts in the rostral half compared to the caudal half;since neural crest cells are present only in the rostralhalf-sclerotome (Rickmann et al. 1985), their presencemay dilute proteins expressed by rostral half-sclero-tome cells. In addition, none of these differencescorresponds with spots seen in 2D gels of neural crestcells (Fig. 4). A feature of the 32-somite stage was thepresence of spot 19, which was associated with the train8, 9, 10. However, it is not clear whether this is a stage-specific quantitative change, since, in gels of earlierstage sclerotomes, the sample did not penetrate as farinto the basic end of the gel. This is demonstrated bythe presence of an extra spot in the carbamylyte train atthe basic ( - ) end of the gel in Fig. 3.

Discussion

Our results suggest that the differences in polypeptideexpression between rostral and caudal half-sclerotomesare complex and that they change with the stage ofdevelopment. In general, the differences between thetwo halves become more numerous with age betweenthe 20- and the 32-somite stage. A large number of

polypeptides were focused in the pH range 5-7 and yetonly a few of these showed differences in expressionbetween the two halves. When a polypeptide snowed aquali\&i\\z difference in expression between the twohalves of the sclerotome, the differential expression wasonly seen at a single stage of development: some of thepolypeptides localized in one half of the sclerotome atan early stage were later found in both halves or in theopposite half. For example, the 150xlO3 glycoproteintrain (15, 16, 17, 18) is not detectable in 20-somiteembryos, is expressed in the rostral half at the 24-somitestage and appears in both halves at later stages (in the32-somite embryo, the presence of this glycoproteintrain in the rostral half-sclerotome was clearly demon-strable by its binding to ConA, but, for an unknownreason, it was not seen in the silver-stained gel shown inFig. 3).

One possibility, suggested by its rostral location, isthat this 150xlO3 train is neural crest derived. How-ever, two observations argue against this: first, it is notdetectable in gels of proteins extracted from culturedcrest cells (Fig. 4), and second, it is subsequentlyexpressed in the caudal half, where neural crest cells arenot found (Rickmann etal. 1985; Teillet etal. 1987). It is

546 W. E. Norris, C. D. Stern and R. J. Keynes

Table 1. Summary of polypeptides showing region-specific expression in the sclerotome of chick embryos

of between 20 and 32 pairs of somites

Spot

2345689

10111213141516171819202122232425

Pi

5-86-86-786-756-76-946-96-856-36-35-75-56-56-46-36-286-986-725-885-66-846-865-7

MT

(xlO3)

44*7272727270707037396266

150*150*150*150*70926866584560

Rostral/caudal

Rostral onlyCaudal onlyCaudal onlyCaudal onlyCaudal onlyRostral < caudalRostral < caudalRostral < caudalRostral onlyRostral onlyRostral < caudalRostral < caudalRostral, then bothRostral, then bothRostral, then bothRostral, then bothCaudal onlyCaudal onlyCaudal onlyCaudal onlyRostral onlyRostral onlyRostral < caudal

iiii

Stage of difference

20 pair only20 pair only20 pair only20 pair only20 pair only24-32 pair24-32 pair24-32 pair24 pair only24 pair only24 (and 32?) pair24 (and 32?) pairR at 24, both at 32R at 24, both at 32R at 24, both at 32R at 24, both at 3232 pair only32 pair only32 pair only32 pair only32 pair only32 pair only24-32 pair

In the molecular weight column, an asterisk marks ConA-bindingglycoproteins. In the fourth column, the regional characteristics ofexpression of each is summarized. The last column indicates thestage at which differential expression is seen. Polypeptides la, lband 7, although glycoproteins pointed out in the Figures, are notincluded in this table because they do not show tissue-specificity.

Mrx10"3

116-

92-5-

66-

45-

31- -

70 6-8

- W

6-6 6-4Pi

t 1 *

60 5-6 5-3

Fig. 4. Silver stained, 2-D gel of cultured neural crest cells.

also clear that the 150xlO3 glycoprotein train is notcytotactin or tenascin (Tan et al. 1987; Mackie et at.1988; Hoffman et al. 1988; Faissner et al. 1988), forseveral reasons: its MT is too low, it changes in locationfrom the rostral half to both halves (cytotactin/tenascin/Jl-related molecules change in the reverse

direction; Stern, Norris, Bronner-Fraser, Fraser, Carl-son, Faissner, Schachner & Keynes, in preparation) andimmunostaining with antibodies to these proteins oncryostat sections show that they are not present withinthe rostral half-sclerotome at these early stages (Tan etal. 1987). The expession of cytotactin/tenascin/Jl-related molecules will be described in more detailelsewhere (Stern, Norris, Bronner-Fraser, Fraser, Carl-son, Faissner, Schachner & Keynes, in preparation).Another macromolecule, CTB-proteoglycan, has beenreported to change location within the sclerotome (Tanet al. 1987). However, not only is this proteoglycanconsiderably larger than the 150X103 polypeptide train(280X103) but it was reported to have a homogeneousdistribution within the sclerotome, subsequently be-coming localized to the caudal half of the sclerotome ata later stage than those examined here. The change inthe distribution of the 150X103 train occurs in theopposite manner at a much earlier stage. It is possible,nevertheless, that the homogeneous distribution of the150x 103 train in the 32-somite embryo is related to thatof CTB-proteoglycan at the 34-somite stage.

In contrast with the findings made on moleculesexpressed differentially in a qualitative way, some ofthe quantitative differences observed do occur at morethan one of the stages studied (e.g. 8, 9, 10). All ofthese show reduced expression in the rostral half andnone is present in gels obtained from cultured neuralcrest. It is therefore unlikely that they are due to thepresence of neural crest cells in the rostral half-sclero-tome, although the possibility that neural crest cellsexpress different polypeptides in vitro and in vivocannot be excluded.

Stage-specific differences might be explained by con-sidering that the somites included in the samples fromeach stage include different regions of the embryo. Inthe fowl, there are 15 cervical, 5 thoracic, 9-10 lumbar/.sacral, and 12 caudal/coccygeal vertebrae, followed bya fused structure, the pygostyle (Robinson, 1970). In20- and 24-somite embryos, all the somites dissectedwere presumptive cervical vertebrae, while the 32-somite stage included all of the cervical and thoracicregions as well as some presumptive lumbar vertebrae.Transplantation of prospective thoracic segmental plateto the cervical region gives rise to ribs (Kieny et al.1972) and to a thoracic-like plumage pattern (Mauger,1972) in the neck. These findings have been interpretedto mean that presumptive sclerotome and dermatomecells are regionally determined for their ultimate ver-tebral and dermal fate many hours in advance of theformation of the sclerotome, while they still reside inthe segmental plate. It is therefore possible that some ofthe polypeptides seen as stage-specific in this study arereally region-specific. A further possibility is that thosepolypeptides that change their pattern of expressionbetween the different stages studied here reflectchanges in the maturation of a somite and its deriva-tives. We are presently involved in raising monoclonaland polyclonal antibodies to some of these peptides.

The polypeptide train 3, 4, 5, 6 is probably the mostinteresting with regard to rostral-caudal specificity. It is

Molecular differences between somite halves 547

caudal-half specific, and therefore not neural crestderived, and is seen to be present at a very early stage.Preliminary data (not shown) indicate that this train isalso present in the caudal halves of epithelial somites atstage 13. These polypeptides satisfy some of the criteriaexpected of molecules that may inhibit neural crest cellsfrom entering into the caudal half of the sclerotome, asthey are the earliest ones found to be expressed in thishalf prior to neural crest migration. If this is the case,however, it is puzzling that they are only seen inembryos with 20 pairs of somites. One reason for thismight be that in half-sclerotomes dissected from olderembryos more somites are included in the sample,which may dilute any polypeptides specific to one halfbut expressed only at the stage at which neural crestcells are starting to migrate segmentally through thesclerotome. One way to resolve this question is to carryout direct functional studies to establish their involve-ment in the decision of neural crest cells to migrate onlythrough the rostral half of the sclerotome. Such studiesare currently in progress. It will also be interesting tosee if cells of the segmental plate also express any ofthese polypeptides, because this tissue has been foundto inhibit neural crest migration (Stern & Bronner-Fraser, in preparation).

Conclusions

Our results show that the two halves of the sclerotomeof chick embryos between stage 13 and 17 expressdifferent proteins and glycoproteins, that their patternsof expression are complex, and that they vary in a stage-and region-specific manner from early stages in thedevelopment of somites. Studies using either a singleexisting antibody or a variety of antibodies directedagainst a single identified macromolecule should bear inmind this complexity before claims can be made aboutthe involvement of such molecules in the guidance ofneural crest cells or motor axons. Since all previousmarkers described to be specific to one or the other halfof the sclerotome appear too late to be involved in theguidance of neural crest cells, we conclude that none ofthe sclerotome-half-specific markers described pre-viously (peanut lectin receptors, butyrylcholinesteraseactivity, cytotactin, tenascin, CTB-proteoglycan) arelikely to be responsible for guiding neural crest cellsthrough the rostral half of the sclerotome. Futurestudies to identify suitable candidate molecules shouldbe directed, instead, at those found to be expressed atappropriate stages of development.

The research reported in this paper was funded by a grantfrom Action Research for the Crippled Child to C.D.S. Weare grateful to Mr Geoffrey Carlson for technical assistance,and to Mr Brian Archer for help with photography.

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