Development 111, 15-22 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

15

T-cadherin expression alternates with migrating neural crest cells in the

trunk of the avian embryo

BARBARA RANSCHT1* and MARIANNE BRONNER-FRASER2

xLa Jolla Cancer Research Foundation, La Jolla, CA 92037, USA2Developmental Biology Center, University of California, Irvine, CA 92717, USA

* To whom correspondence should be addressed

Summary

Trunk neural crest cells and motor axons move in asegmental fashion through the rostral (anterior) half ofeach somitic sclerotome, avoiding the caudal (posterior)half. This metameric migration pattern is thought to becaused by molecular differences between the rostral andcaudal portions of the somite. Here, we describe thedistribution of T-cadherin (truncated-cadherin) duringtrunk neural crest cell migration. T-cadherin, a novelmember of the cadherin family of cell adhesionmolecules was selectively expressed in the caudal half ofeach sclerotome at all times examined. T-cadherinimmunostaining appeared graded along the rostrocau-dal axis, with increasing levels of reactivity in the caudalhalves of progressively more mature (rostral) somites.The earliest T-cadherin expression was detected in asmall population of cells in the caudal portion of the

somite three segments rostral to last-formed somite. Thisinitial T-cadherin expression was observed concomitantwith the invasion of the first neural crest cells into therostral portion of the same somite in stage 16 embryos.When neural crest cells were ablated surgically prior totheir emigration from the neural tube, the pattern ofT-cadherin immunoreactivity was unchanged comparedto unoperated embryos, suggesting that the metamericT-cadherin distribution occurs independent of neuralcrest cell signals. This expression pattern is consistentwith the possibility that T-cadherin plays a role ininfluencing the pattern of neural crest cell migration andin maintaining somite polarity.

Key words: trunk neural crest cell migration, cell adhesionmolecules, chick embryos, somite, sclerotome.

Introduction

Neural crest cells initiate their migration from theneural tube shortly after fusion of the neural folds andneural tube closure. These cells migrate along severalwell-defined pathways and give rise to numerousderivatives, including pigment cells, Schwann cells,adrenal chromafnn cells and cartilagenous elements ofthe face (ref. LeDouarin, 1982). In addition, neuralcrest cells contribute the precursors for most peripheralneurons and glia. In the trunk region, the neural crest-derived sensory and sympathetic ganglia with theirassociated nerve roots are organized in a segmentalpattern along the vertebral column. This segmentalorganization results from the metameric migratorypattern of neural crest cells that selectively movethrough the rostral (anterior) half of each somiticsclerotome during early phases in development (Rick-mann et al. 1985; Bronner-Fraser, 1986; Teillet et al.1987; Lallier and Bronner-Fraser, 1988).

The majority of trunk neural crest cells migrate in aventral direction away from the neural tube andthrough the adjacent somites. As the epithelial somites

undergo a transition to form the dermatome (presump-tive dermis), myotome (presumptive muscle), andsclerotome (presumptive vertebrae), most ventrallymigrating neural crest cells invade the rostral half ofeach sclerotome (see Fig. 1); in contrast, the caudal(posterior) half of each sclerotome lacks migratingneural crest cells (Rickmann et al. 1985; Bronner-Fraser, 1986; Teillet et al. 1987; Serbedzija et al. 1989).Similarly, motor axons, emerging from the ventralneural tube several hours after neural crest cells, extendtheir growth cones through the rostral half of thesomitic sclerotome (Keynes and Stern, 1984). For bothpopulations, the signals responsible for the metamericpattern appear to reside within the somite itself. Afterexperimental rotation of the segmental plate to reverseits rostrocaudal polarity, neural crest cells and motoraxons recognize the appropriate rostral portion of thesomite even when presented in a caudal position(Keynes and Stern, 1984; Bronner-Fraser and Stern,1991). Furthermore, in the absence of the somiticsclerotome, neural crest cells and motor axons migratein a non-segmental pattern (Lewis et al. 1981; Tosney,1988; Stern et al. 1986; Kalcheim and Teillet, 1989).

16 B. Ranscht and M. Bronner-Fraser

Molecular differences between the cells of the rostraland caudal halves of the somitic sclerotome are likely toaccount for the selective migration pattern of neuralcrest cells and motor axons through the rostral half ofthe somites. Cells in the rostral half of each somite mayselectively expose or secrete molecules that attractneural crest cells and/or axons, whereas cells in thecaudal half of each somite may contain molecules thatare repulsive or inhibitory to cell and/or axonmovements. Furthermore, it may be a combination ofboth attractant molecules in the rostral and repulsivemolecules in the caudal half of each sclerotome thatregulate the metameric pattern of neural crest celland/or motor axon locomotion through somite tissues.Some molecular differences between rostral and caudalsomites have been noted. Peanut lectin bindingglycoproteins of 48 and 55 x 103 MT have been localizedspecifically to the caudal half of the somitic sclerotome(Stern et al. 1986; Davies et al. 1990). In the rostral halfof the sclerotome, a 70xl03Mr protein (Tanaka et al.1989), butyrylcholinesterase activity (Layer et al. 1988),and cytotactin/tenascin/Jl (Tan et al. 1987; Mackie etal. 1988; Stern et al. 1989) have been detected. Inaddition, differences in the polypeptide compositionbetween rostral and caudal somite portions have beenrevealed by two-dimensional gel electrophoresis (Nor-ris et al. 1989).

Cadherins are a class of cell adhesion molecules thathave been suggested to regulate morphogenesis by acalcium-dependent adhesion mechanism (Takeichi,1988). A novel member of this family, T-cadherin(truncated-cadherin), was recently identified by mol-ecular cloning and sequencing of corresponding cDNAs(Ranscht and Dours, 1989; Ranscht and Dours-Zimmerman, 1991). In the present study, we haveexamined the onset and distribution of T-cadherin inrelation to neural crest cell migration. T-cadherin waslocalized selectively in the caudal half of the somites atboth initial and advanced stages of neural crest cellmigration. Furthermore, T-cadherin expression in thecaudal half of each somite coincided with the initialpenetration of neural crest cells into the rostral half ofeach somite. This distribution is consistent with apossible role for T-cadherin in influencing the pattern oftrunk neural crest cell migration.

Materials and methods

EmbryosWhite Leghorn chick embryos ranging from stages 11 to 19(according to the criteria of Hamburger and Hamilton, 1951)were used for this study. Eggs were incubated in a forced airincubator at 37 °C until they reached the desired stage ofdevelopment.

Fixation and tissue processingCryostat sections

Embryos were removed from the eggs and washed inHoward's Ringers solution. The embryos were straightened inwax dishes by placing insect pins or cactus needles into theirsurrounding membranes. They were then fixed in 4%

paraformaldehyde in phosphate-buffered saline (PBS) at 4CCovernight, washed in PBS, transferred to 5 % sucrose in PBSwith azide for 4-24 h, and immersed in 15% sucrose in PBSwith azide overnight at 4°C. Embryos were placed in 7%gelatin (Sigma; 300 Bloom) in 15 % sucrose/PBS for 3 h at37°C, and embedded in fresh gelatin. Storage of the embryoswas up to two weeks in the refrigerator until the time ofsectioning when they were rapidly frozen in liquid nitrogen.14 /an sections were cut on a Reichardt cryostat and mountedon gelatin-subbed slides.

Paraffin sectionsEmbryos were fixed in Zenkers fixative for 1.25 h, rinsed inrunning water for 15min, and placed in 70% ethanol.Embryos were dehydrated through a series of alcohols,followed by three changes of histosol, and three changes ofparaffin before embedding in fresh paraffin. Sections were cuton a Leitz microtome at a thickness of 5/an.

ImmunocytochemistryCryostat sections were air dried for 15 min prior to applicationof antibody. A polyclonal antiserum against T-cadherin(Ranscht and Dours-Zimmermann, 1991) was applied diluted1:150 in PBS containing 0.1 % BSA and 0.05 % Triton X-100at room temperature for two hours or overnight. Sectionswere washed in phosphate buffer and incubated for one hourwith FITC-goat anti-rabbit IgG (Zymed) diluted 1:30 in 0.1 MPBS, pH7.4, with 1 % BSA (PBS/BSA). Slides treated withpreimmune instead of immune antiserum gave no immuno-reactivity. In some experiments, sections were labelledsimultaneously with anti-T-cadherin and the HNK-1 antibodywhich recognizes migrating neural crest cells (Tucker et al.1984). The HNK-1 antibody is a mouse IgM and was detectedusing an RITC-rabbit anti-mouse IgM (Axell) for 1 h.

Paraffin sections were rehydrated and stained with theHNK-1 antibody as described above and elsewhere (Bronner-Fraser, 1986). After antibody staining, the cell nuclei in thesame sections were stained by placing them in 0.2/tgml"1 of4-6-diamidino-2-phenylindole (DAPI) solution in 0.1M phos-phate buffer for 3 min.

Neural crest ablationsChicken eggs were incubated until they reached stages 11-13(according to the criteria of Hamburger and Hamilton, 1951).A small hole was made at one end of the egg and 1 ml ofalbumin was removed to lower the embryo away from theshell. A hole slightly larger than the blastoderm was cut in theshell overlying the embryo. India Ink (Pelikan Fount; diluted1:4 in Eagle's Minimum Essential Medium containing 15 %horse serum and 10% 11-day chick embryo extract) wasinjected underneath the blastoderm to aid in visualizing theembryo. The vitelline membrane was removed with anelectrolytically sharpened tungsten needle.

In order to remove the presumptive neural crest cells, thedorsal portion of the neural tube was excised using glassneedles and Dumont no. 5 forceps. The rostral limit of theablation was typically three to four somites above the mostrecently formed somite. A length of dorsal neural tubecorresponding to six or more somite lengths was removed.Following the operation, embryos were sealed with adhesivetape and returned to the incubator for 24h prior to fixation.Embryos were prepared for cryostat sectioning and immuno-cytochemistry as described above.

Analysis of cell densityLongitudinal paraffin sections of 5 /an thickness were stained

T-cadherin expressed in caudal somite 17

with HNK-1 antibody and DAPI as described above. Thenumbers of somites were counted using the most recentlyformed somite as a landmark. The sections were viewedthrough a SIT camera onto a video screen. On randomlyselected sections, somite borders were traced and the cellnuclei in a given somite were counted. The line demarcatingthe rostral and caudal halves of the sclerotome wasdetermined by the distribution of neural crest cells, whichselectively migrate through the rostral half only. To determinethe number of sclerotomal cells, the number of HNK-1-positive cells was substracted from the total number of cells inthe rostral half of the sclerotome. A Student's two-sided Mestwas used to determine the statistical significance of differ-ences in the number of cells per unit area.

Immunoblotting techniquesSomites were dissected from stage 16-17 chick embryos atlevels between the 8th rostral- and 6th caudal-most somites.The tissue was homogenized immediately in cold 10 mMTris-HCl, pH7.6, 2HIM CaCl2> 2% Nonidet P40, lmMdithiothreiol, 1 mM phenylmethylsulfonyl fluoride, 50/iMleupeptin, 5/IM pepstatin and 4ngml~' aprotinin. Proteinswere separated by SDS-PAGE (Laemmli, 1970) and trans-ferred to polyvinylidene difluoride transfer membranes(Immobilon P, Millipore) for several hours or overnight(Towbin et al. 1979). Non-specific binding was blocked with5% nonfat dry milk in TBST (10 mM Tris-HCl, pH8.0,150 mM NaCl, 0.05% Tween 20) for lh. T-cadherin wasprobed with rabbit anti-T-cadherin antiserum (1:500, Ranschtand Dours-Zimmermann, 1991) followed by 125I-goat anti-rabbit immunoglobulin (lxlCrctsmin"1 ml"1) diluted inTBST. Each incubation was for one hour and followed by fivewashes in TBST. Reacted blots were exposed for 1-5 h toKodak XAR5-film with an intensifying screen.

Results

Neural crest cells begin to emigrate from the neuraltube shortly after neural tube closure. The majority ofneural crest cells move ventrally through the rostral halfof each somite (Fig. 1), first at rostral axial levels of theembryo and subsequently at more caudal levels.Therefore, several stages of neural crest cell migrationexist simultaneously within a single embryo. Neuralcrest migration is well advanced in the rostral (older)portion of the embryo, when it is just beginning in morecaudal (younger) regions. We have performed most ofour analyses on stage 16-18 chick embryos becauseearly and advanced stages of neural crest cell migrationexist simultaneously in the embryo at these times.

Anti-T-cadherin antiserum primarily detects a9Ox26^Mr protein in somitesSince T-cadherin is expressed in a variety of neuronaland non-neuronal tissues (Ranscht and Dours-Zimmer-mann, 1991), immunoblots were used to determine ifthe antiserum specifically identifies T-cadherin insomite tissue. Somites dissected from stage 16-17chicken embryos were homogenized in the presence ofCa2+ and protease inhibitors, separated by SDS-PAGE and transferred to polyvinylidene difluoridemembranes. After reaction with anti-T-cadherin anti-serum and 125I-goat anti-rabbit IgG, a single polypep-tide of approximately 90X103 MT was detected (Fig. 2).This reactivity compares well with results reportedelsewhere (Ranscht and Dours-Zimmerman, 1991) andindicates that the immunoreactivity described belowreflects genuine T-cadherin.

Fig. 1. Schematic diagramillustrating the early pathways oftrunk neural crest cell migration.Neural crest cells emerge fromthe neural tube (NT) and proceedeither ventrally (indicated bylarge curved arrow) through thesomitic sclerotome (Scl) ordorsolaterally (indicated by smallcurved arrow) between theectoderm (Ec) anddermomyotome (DM). Theventrally migrating cells onlymove through the rostral (R) halfof each sclerotome and are notobserved in the caudal (C) half(Rickmann et al. 1985; Bronner-Fraser, 1986). Neural crest cellsalso avoid the region around thenotochord (No) (Pettway et al.1990) Ao=dorsal aorta.

18 B. Ranscht and M. Bronner-Fraser

- 3x10

2 0 0 -

1 1 6 -9 7 -

6 6 -

Fig. 2. Reactivity of T-cadherinantiserum on immunoblots. Insomites isolated from stage 16-17chicken embryos, the anti-T-cadherin antiserum used in thisstudy recognizes a singlepolypeptide band of approximately

Appearance of T-cadherin in stage 16-18 chickembryosT-cadherin immunoreactivity within somite tissue wasobserved in the caudal half of the sclerotome at all timesexamined. A gradation of staining intensity wasobserved along the rostrocaudal axis with most intenseimmunoreactivity at rostral levels (Fig.3A,D). Usingthe last-formed somites as reference, the caudal-mostregion containing detectable T-cadherin immunoreac-tivity was about three somites rostral to the last-formedsomite; T-cadherin appeared on a small population ofcells in the caudal portion of this somite (Fig. 3C). Thelevel of immunoreactivity increased with distance fromthe last somite, with rostral portions of the embryoexpressing higher levels of T-cadherin than caudal ones(Fig. 3A,D).As the somites completed the epithelial-mesenchymaltransition to form the dermomyotome and sclerotome,T-cadherin expression became restricted to the sclero-tome. In newly formed sclerotomes, T-cadherin immu-noreactivity appeared as a diffuse signal. Although theimmunoreactivity was most prominent in the caudalhalf of the somite, there was no sharp discontinuity instaining intensity distinguishing the rostral from caudalhalves of the sclerotome. Rather, the transitionbetween the caudal and rostral halves was diffuse andgradual. Approximately six somites rostral to the lastformed somite, the level of immunoreactivity in thecaudal somite region intensified markedly. In additionto the prominent immunoreactivity in the caudal halfofthe somite, a few faint fibrils of T-cadherin immuno-reactive material appeared to extend into rostralportions of the sclerotome.

Eleven to twelve somites rostral to the last formed

somite, both the morphology of the somites and theT-cadherin immunoreactivity underwent an apparenttransition. T-cadherin expression appeared more dis-tinct, with a sharp discontinuity in immunostainingseparating the caudal from the rostral halves of thesclerotome. At this stage and beyond, T-cadherin wasclearly associated with the surfaces of caudal sclero-tomal cells (Fig. 4), and absent from the rostralsclerotome cells. Morphologically, the somites ap-peared more compact and shorter in rostral-caudalextent than somites in less mature regions of theembryo.

In transverse sections at the level of the wingbud,T-cadherin immunoreactivity in the sclerotome ap-peared on the surface of all cells in the caudal half of thesclerotome including the dorsal portion of the sclero-tome, the region between the dermomyotome andneural tube, and the ventral portion of the sclerotomesurrounding the notochord (Fig. 5A). In contrast,immunoreactivity was absent from all regions of therostral half of the sclerotome. In more caudal (younger)regions of the embryo, T-cadherin immunoreactivitywas observed in the dorsal sclerotome, but appeared tobe absent from the ventral sclerotome surrounding thenotochord (Fig. 5B). The dermomyotome and subepi-dermal region appeared to lack T-cadherin immuno-reactivity at all times examined. Migrating neural crestcells were not seen to express T-cadherin, though motoraxons that also move through the rostral half of thesclerotome, expressed T-cadherin at all stages exam-ined.

T-cadherin expression in stage 16-18 embryosinversely correlates with neural crest cell migrationThe first neural crest cells to emigrate from the neuraltube in stage 16 —18 chicken embryos can be observedthree somite lengths rostral to the last-formed somite(Bronner-Fraser, 1986). Although HNK-1-positiveneural crest cells emerge in an unsegmented patternalong the entire dorsal surface of the neural tube, theyonly enter the rostral half of each somite. The firstneural crest cells enter the somites at the time ofsclerotome formation. In embryos double-labelled withanti-T-cadherin and FfNK-1 antibodies, we observedneural crest cells entering the rostralmost portion of thesomite slightly after or concomitant with the appear-ance of T-cadherin in the caudalmost portion of thesame somite.

The number of neural crest cells progressivelyincreased with distance from the last somite. Relativelyfew neural crest cells were detected in the tencaudalmost somites. Those were widely dispersedthroughout the rostral half of each sclerotome and hadno clear orientation. The distribution of the HNK-1neural crest cells was complementary to the diffusestaining for T-cadherin in the caudal halves of the samesomites (Fig. 3B).

In more mature regions of the embryo (11 or moresegments above the most recently formed somite), thesomites appeared more compact. Larger numbers ofneural crest cells with a clear orientation in the

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T-cadherin expressed in caudal somite 19

mediolateral direction were migrating through therostral half of each sclerotome. At these more matureaxial levels, the expression of T-cadherin was morepronounced than in more caudal regions of the embryo.The boundary between the T-cadherin-positive caudalhalves and T-cadherin-negative rostral halves of thesclerotomes was sharp, with T-cadherin expressionalternating with the HNK-1 -positive neural crest cells(Fig. 4).

T-cadherin expression in stage 19 embryosBy stage 19, neural crest cells along the ventral pathway

Fig. 5. Fluorescence photomicrographs of transversesections through the rostral (A) and caudal (B) portions ofstage 18 embryos after staining with T-cadherin. (A) Aglancing section with the left-hand side through the rostral(R) sclerotome and the right-hand side through the caudal(C) sclerotome. T-cadherin immunoreactivity wasprominent on the motoraxons (indicated by curved arrow).Although T-cadherin immunoreactivity was absent from therostral sclerotome, it was noted throughout the caudalsclerotome, including the region around the notochord (N).(B) In more caudal regions of the embryo, T-cadherinimmunore activity appeared fibril l ar within the caudalsclerotome and was faint or absent in the neural tube.Immunoreactivity was absent from the notochordal region.Intense staining was observed on primordial germ cells(indicated by straight arrows). Bar=47^m.

are in their final stages of migration and are beginningto aggregate to form dorsal root and sympatheticganglia. At this time (Fig. 6), T-cadherin immunoreac-tivity remained prominent in the caudal half of thesclerotome, but now was also observed throughout themyotome, though it was absent from the dermomyo-tome at earlier stages. It contrast to the sclerotome,staining of the myotome was non-segmented.T-cadherin was detected on motor axons that, likeneural crest cells, move through the rostral half of eachsclerotome.

Cell density in the somitesOne possible explanation for the absence of neural crestcells from the caudal half of the sclerotome is that closecell packing in this region may mechanically excludeneural crest cells. To investigate if T-cadherin ex-pression correlates with a possible compaction of thecaudal-half sclerotomal cells, we compared the lengthof the somites and the density of sclerotomal cells in therostral versus caudal halves of the sclerotome. Thenumber of nuclei in a 1200 ̂ m2 area was determined inrandomly chosen sections through the rostral andcaudal halves of the sclerotome at 5, 7, 9, 13, and 15somites above the last-formed somite in stage 16-17embryos. Nuclei of HNK-1-positive neural crest cellswere substracted prior to quantitation. The presence orabsence of HNK-1 immunoreactivity also distinguishedthe rostral half (neural crest-containing) from thecaudal half (neural crest-free) of the sclerotome. Thecell density in the caudal half of the sclerotome was43-67% higher than that in the rostral half at all somitelevels examined (P=s0.001; Table 1). Therefore, the celldensity in the caudal half of the sclerotome increases

Fig. 6. Fluorescent photomicrographs of longitudinalsections through a stage 19 embryo. (A) At the level of theventral neural tube, neural crest-derived Schwann cells andmotoraxons stain with the HNK-1 antibody. (B) In thesame section, T-cadherin immunoreactivity is observed onthe motoraxons in the rostral (R) half of the sclerotome aswell as in the caudal (C) half sclerotomal cells, myotomal(M) cells and cells in the ventral neural tube (NT).

Fig. 7. Fluorescent photomicrographs of two embryos fixedat stage 17 from which the dorsal neural tube was removedby surgical ablation at stage 11-12. Longitudinal sections areillustrated at the level of the neural tube (NT). Even in theabsence of neural crest cells assessed by the lack of HNK-1immunoreactivity in the rostral half of the sclerotome,T-cadherin immunoreactivity is observed in the caudal half ofthe sclerotome (B), where it appears on schedule. R=rostral;C=caudal; DM=dermomyotome. Bar=40/an.

20 B. Ranscht and M. Bronner-Fraser

Table 1. Somite length and sclerotomal cell density instage 16-17 embryos

SomiteSomiteSomiteSomiteSomite

5 (*=19)7 (n=18)9 (n=20)13 (n=8)15 (n=14)

Rostrocaudalextent(/an)

218 ±25218±20221±34198±23205±34

Number ofcells±s.D./

1200 ftm2

Rostral

9.4±2.8*10.5±3.7*10.3±2.5*10.4±2.2*11.5±2.6*

Caudal

14.6±2.716.3+2.815.8±4.117.4±2.416.4±3.7

Ratiocaudal/rostral

1.551.551.531.671.43

Somites are numbered with respect to the most recently formedsomite.

* Significant differences between cell density in rostral andcaudal halves of the sclerotome; P=£0.001.

shortly after the initial expression of T-cadherin, andthis increased density persists with somitic maturation.

T-cadherin expression is independent of signals fromneural crest cellsSurgical removal of the dorsal portion of the neuraltube in the trunk region produces embryos lacking theneural crest in the operated region (Yntema andHammond, 1945). To determine if T-cadherin ex-pression depends on the presence of neural crest cells,the neural crest was ablated by surgically removingeither the whole neural tube (n=3) or the dorsal portionof the neural tube (n=13). Fifteen embryos completelylacked neural crest cells over several somite lengthseither unilaterally (n=3) or bilaterally (n=12), with oneadditional embryo having a greatly reduced neural crestcell population in the ablated region. In most operatedanimals, neural crest cells were present rostral andcaudal to the ablated region.

In the absence of HNK-1-positive neural crest cells,T-cadherin immunoreactivity persisted in the caudalhalf of the sclerotome. The staining pattern appearedidentical to that observed in unoperated embryos(Fig. 7). The caudalmost extent of immunostaining wasdetected about three segments rostral to the last-formed somite and immunoreactivity was confined tothe caudal half of each sclerotome, increasing inintensity in progressively more mature somites.T-cadherin staining was similar to that of normalanimals in longitudinal sections both at the level of theneural tube (Fig. 7A,B) and the notochord (notshown). These observations indicate that the segmentalexpression of T-cadherin is independent of the presenceof neural crest cells.

Discussion

T-cadherin is a novel member of the cadherin celladhesion family that shares significant similarities withknown cadherins in its biochemical characteristics andextracellular structure (Ranscht and Dours-Zimmer-mann, 1991)..T-cadherin, however, differs from knowncadherins in that it lacks cytoplasmic sequences that are

highly conserved in other cadherins. The polyclonalanti-T-cadherin antiserum used in this study recognizesa predominant polypeptide of 90xl(fiMT on immuno-blots of somites. This pattern compares well with thatobserved in other tissues (Ranscht and Dours-Zimmer-man, 1991) and suggests that genuine T-cadherin isdetected in somites.

Here, we report the selective distribution ofT-cadherin in the caudal half of each sclerotome duringneural crest cell migration as examined by immunohis-tochemistry. The segmental expression of T-cadherinwas inversely correlated with the pattern of migratingneural crest cells at all times examined. T-cadherinimmunoreactivity appeared graded along the rostrocau-dal axis, with the staining intensity in the caudal half ofthe sclerotome increasing with distance from the mostrecently formed somite. T-cadherin immunoreactivityfirst appeared on a small population of cells in thecaudal portion of the somite approximately threesomites rostral to the most recently formed somite. Itsexpression was concurrent with the initial entry ofneural crest cells into the rostralmost portion of thesame somite. Several cell diameters separate the entrypoint of neural crest cells into the rostral half of thesomite from cells expressing T-cadherin in the caudalhalf of the somite. This would argue against a directcontact-mediated repulsion or mechanical exclusionmechanism of T-cadherin in the initial selection of therostral half of the sclerotome by neural crest cells. It ispossible, however, that T-cadherin is expressed in acaudal-to-rostral gradient along the entire newlyformed somite, despite the fact that such a subtlegradient cannot be detected by immunocytochemistry.The possibility that T-cadherin acts as a diffusiblemolecule cannot be ruled out since it lacks amino acidsconserved in the cytoplasmic domains of other cadher-ins (Ranscht and Dours-Zimmerman, 1991).

As somites undergo the transition to form thedermomyotome and sclerotome, T-cadherin was ex-pressed preferentially in the caudal half of eachsclerotome. Staining became increasingly pronouncedwith sclerotomal maturation and, eleven or moresomites rostral to the last-formed somite, a sharpdiscontinuity demarcated the T-cadherin immunostain-ing in the caudal half of each sclerotome from non-stained rostral-half sclerotomal tissue. Above this level,the somitic sclerotome appeared shortened in rostro-caudal extent, with a significantly greater cell density inthe caudal half of the sclerotome than in the rostral halfof the sclerotome.

While a number of different mechanisms couldexplain the segmental organization of the peripheralnervous system in vertebrates (Keynes and Stern,1988), convincing evidence has been presented that themechanisms responsible for segmentation reside withinthe somites themselves (Keynes and Stern, 1984; Sternand Keynes, 1987; Oakley and Tosney, 1989; Bronner-Fraser and Stern, 1991). Molecules expressed in eitherhalf of the somitic sclerotome are possible candidatesfor influencing or determining segmentation in ver-tebrates. Neural crest cells may preferentially interact

T-cadherin expressed in caudal somite 21

with molecules in the rostral somite region, whilemolecules expressed in the caudal somites may be lesspermissive or inhibitory for neural crest cell migration.The selective interaction of neural crest cells with celladhesion molecules in either half of the somiticsclerotome is a compelling possibility to explain theirmetameric migration pattern. An alternative or ad-ditional possibility is that cell adhesion moleculesselectively expressed by caudal sclerotomal cells causecell compaction and mechanical exclusion of cellsand/or axons from this area. Consistent with the latterpossibility, we observed significantly higher cell den-sities, apparently correlating with increased levels ofT-cadherin expression, in caudal-half sclerotomes com-pared to their rostral counterparts. This observation,however, does not rule out the possibility thatT-cadherin also may act as a less permissive orinhibitory substrate for neural crest cells than moleculesexpressed in the rostral sclerotome.

The expression of T-cadherin during somite develop-ment is distinct in several aspects from the stainingpattern described by others for N-cadherin (Hatta et al.1987; Duband et al. 1988). Unlike T-cadherin, whichwas not detected in the segmental plate or in the mostrecently formed epithelial somites, N-cadherin isdetected in the segmental plate and the epithelialsomite, predominantly on the apical cell surfaces Liningthe somitic lumen (Duband et al. 1988). At the time ofsclerotome formation, T-cadherin was detected on asmall population of cells in the caudal portion of thesomite. In more posterior regions of the embryo, theremay be some overlap between T-cadherin andN-cadherin in the caudal portion of the somite. As theepithelial somites form the dermomyotome and sclero-tome, T-cadherin expression persisted specifically in thecaudal sclerotome, while N-cadherin seems to disap-pear completely from sclerotomal cells. The transitionfrom N-cadherin to T-cadherin expression duringsclerotome formation may reflect the epithelial-mesenchymal transition, consistent with the proposedrole of cadherins in tissue morphogenesis (Takeichi,1988).

To date, only one group of molecules other than T-cadherin has been localized specifically to the caudalhalf of the sclerotome during neural crest migration andmotor axon growth. Stern et al. (1986) and Davies et al.(1990) described peanut agelutinin (PNA) bindingglycoproteins of 48 and 55 x l(r MT that are expressed inthe caudal half of the sclerotome. These molecule(s)differ from T-cadherin not only in their apparentmolecular mass, but also in their glycosylation andpattern of expression. T-cadherin has an apparentrelative molecular mass of 90xl03Mr and exhibits noapparent peanut lectin binding activity in stage 16-17somite homogenates (Ranscht, unpublished obser-vation). In addition, T-cadherin is expressed on motoraxons whereas the PNA-binding molecules are not. ThePNA-binding proteins cause growth cones to collapsesuggesting that inhibitory mechanisms may participatein establishing segmentation in vertebrate embryos(Davies et al. 1990). It remains to be established if

T-cadherin has a similar function. The anti-T-cadherinantiserum used in this study appears to recognizeT-cadherin mainly in a denatured or fixed configurationand was not effective in preliminary functional studies.Because multiple molecules are present in the rostraland caudal halves of the sclerotome (Stern et al. 1986;Norris et al. 1989; Layer et al. 1988; Tanaka and Obata,1989; Davies et al. 1990), it may be a mistake to arguefor a causal role for only one molecule; instead, acombination of cell adhesion molecules, extracellularmatrix components, and soluble factors may berequired for the establishment and maintenance ofsomite polarity.

For several reasons, T-cadherin is a logical candidateto play a central role in maintaining rostrocaudalpolarity in the somites and to contribute to themetameric migration of neural crest cells. First,T-cadherin expression is confined primarily to thecaudal half of the sclerotome as early as the sclerotomeis formed and is, therefore, one of the first obviousmanifestations of somite polarity. Second, T-cadherin isexpressed before or concomitant with neural crest cellimmigration into the sclerotome. Third, T-cadherinlocalization is maintained in a polarized fashion bothduring neural crest migration and motor axon growth.Fourth, high levels of T-cadherin expression correlatewith close packing of cells in the caudal half of thesclerotome. One possible mode of operation is thatT-cadherin causes dense packing of caudal-half sclero-tomal cells, thereby mechanically excluding neural crestcells and possibly axons from these regions. Alterna-tively, and perhaps in addition, T-cadherin mayfunction similarly to the PNA-binding proteins (Davieset al. 1990) by acting as an inhibitory or repulsivesubstrate for neural crest cell and/or growth conelocomotion. Although functional tests will be requiredto establish the role of T-cadherin in development, itsspatial and temporal distribution and homology tocadherins make it an attractive candidate for influenc-ing the pattern of spinal segmentation in vertebrateembryos.

We thank Drs Andrew Lumsden and Scott Fraser forcritical reading of the manuscript, and Kristin Bruk and MaryFlowers for excellent technical assistance. This work wassupported by HD15527 and a Grant from the MuscularDystrophy Association to M.B.-F. and by HD 25938 and BasilO'Connor Starter Scholar Research Award 5-752 from theMarch of Dimes Birth Defects Foundation to B.R. M.B.-F. isa Sloan Foundation Fellow and B.R. is a McKnight Scholar.

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