Cytotactin expression in somites after dorsal neural ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6398-6402, August 1991 Developmental Biology

Cytotactin expression in somites after dorsal neural tube and neural crest ablation in chicken embryos (substrate adhesion/extraceilular matrix/in situ hybridization/wounding)

SEONG-SENG TAN*, ANNE L. PRIETOt, DONALD F. NEWGREEN*, KATHRYN L. CROSSINt, AND GERALD M. EDELMANt tThe Rockefeller University, 1230 York Avenue, New York, NY 10021; *Department of Anatomy, The University of Melbourne, Victoria, Australia; and tDepartment of Pediatrics, Westmead Hospital, New South Wales, Australia Contributed by Gerald M. Edelman, May 3, 1991

ABSTRACT The spatiotemporal expression of the extracellular matrix protein cytotactin/tenascin during somitogenesis suggests that it plays a role in the morphogenetic events that give rise to the pattern of neural crest (NC) development. In the present study, the spatial distribution and molecular forms of cytotactin in somites were examined using in situ hybridization, Western blotting, and immunohistochemistry during normal development and after injury. In situ hybridization showed that prior to NC cell invasion cytotactin mRNA was restricted to the caudal half of the newly formed epithelial somites. As each epithelial somite matured, giving rise to a sclerotome and dermamyotome, the mRNA was first restricted to the dermamyotome and later restricted to the rostral portion of the sclerotome, consistent with the previously reported protein distribution. Immunocytochemical analysis of the distribution of cytotactin and NC cells in embryos with ablations that removed NC cells, or with simple wounds that left NC cells in place, demonstrated that the presence of NC cells is neither necessary nor sufficient for the correct positioning of cytotactin. Immunoblotting analysis showed that cytotactin synthesized by sclerotomes in the absence of NC cells was of similar molecular mass to that produced in their presence. These fmdings are in accord with the notion that the abnormalities of cytotactin distribution are related to the wounding process. We conclude that, contrary to the suggestion of Stern et al. [Stern, C. D., Norris, W. E., Bronner-Fraser, M., Carlson, G. J., Faissner, A., Keynes, R. J. & Schachner, M. (1989) Development 107, 309-319], there is no causal link between the presence of NC cells and the distribution and molecular mass of sclerotomal cytotactin.

rostral half of the sclerotome concurrent with NC invasion (8, 9, 17); somewhat later one of its ligands, cytotactin-binding proteoglycan (18), becomes localized to the caudal half of the sclerotome (8). Cell migration assays in vitro suggest that both of these molecules affect cell shape and inhibit migration (8, 19, 20); their effects were mitigated by fibronectin, suggesting that the roles of cytotactin and the proteoglycan in affecting cell movement may depend upon interaction with other substrate adhesion molecules. A recent study (21) has reexamined the question of molecular patterning within the sclerotome. Based on their results, Stern et al. (21) proposed that NC cells are necessary for the rostral expression of cytotactin. They also suggest that somite cells are capable of producing only low molecular weight forms of tenascin/J1220/200 that require complementation by NC or other cells for assembly into high molecular mass forms. The present study was conducted to examine the spatial distribution and molecular forms of cytotactin in somites using in situ hybridization, Western blotting, and immunohistochemistry, in normal development and after ablation of the dorsal neural tube and NC. The results confirm our original observations (8) and indicate that the presence of NC cells is neither necessary nor sufficient for the patterned expression of cytotactin or for the expression of its high molecular mass components.

MATERIALS AND METHODS Tissues and in Situ Hybridization. White Leghorn chicken embryos (Hamburger and Hamilton stages 11-20) (22) were prepared for in situ hybridization and parallel immunocytochemistry as described (23). The protocol for in situ hybridization using cytotactin RNA probes was previously used and described in detail (24). Dorsal Neural Tube and NC Ablations. Stage 12-14 (16-22 somite) embryos were prepared for ablation by standard methods. The dorsal one-half to one-third of the neural tube extending from about the third to last somite caudally for 8-12 somite lengths was separated from adjacent tissues using tungsten needles. This region includes the premigratory NC cells (3-5). In experimental specimens (n = 61) this region was removed with a micropipette, and in control specimens (n = 24) it was left in place. After 48 hr, the embryos (stages 19-23) that did not survive the operation (experimental, n = 11; control, n = 1) or that showed gross axial distortions (experimental, n = 6; control, n = 2) were excluded from further study. Embryos (experimental, n = 10; control, n = 3) were randomly selected for immunohistology. Serial paraffin sections (8) in the horizontal plane were stained with HNK-1 monoclonal antibody [to visualize NC cells (25)] and rabbit

The migration of neural crest (NC) cells is one of the most accessible models for studying morphogenetic events in vertebrates (1). At the trunk level in chicken embryos, crest cells normally invade only the rostral half of each somite unit (2-5). This pattern is controlled by the somites (6), but their ability to accept continued immigration is transient (7). The nature of the mechanisms controlling spatiotemporal patterning in the somites has been eagerly sought because it is likely to provide important clues regarding the rostral-caudal asymmetry of NC cell migration into the somites. Several molecules have now been identified that show a polarized distribution in the somite and that may therefore affect NC cell and motor neuron patterning (8-12). One of these is cytotactin, a large hexameric glycoprotein, related or identical to tenascin (13) and J1220/200 antigen (14); the chicken monomer has two major isoforms of 200 and 220 kDa arising from differential splicing of a single gene (15, 16). Cytotactin is of particular interest since it is localized in the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: NC, neural crest.

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FIG. 1. Posterior expression of cytotactin mRNA in epithelial somites and dorsal expression in the dermamyotome. Parasagittal sections of 15-somite (A and B) and 19-somite (C) embryos were hybridized with a cytotactin RNA probe. (A) Twelve most caudal somites. (B) Somites 9 and 10 from a different 15-somite embryo. (C) Somites 6-12 of a 19-somite embryo. Rostral is to the right in all panels. (Bar = 240 Am for A and C and 70 Aum for B.)

antiserum to cytotactin (8) and examined under epifluorescence with a Nikon Optiphot microscope. Biochemistry. Experimental (n = 34) and control (n = 18) embryos were collected in calcium-, magnesium-free phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 1 mg of aprotinin per ml, and 1 mM EDTA (Sigma). A trunk section three somites long was obtained by microdissection from the center of the ablated region, and the neural tube, notochord, somatopleure, and tissue ventral to the dorsal aorta were discarded. The remainder, consisting mainly of somites but also including epidermal ectoderm, peripheral ganglia, and blood vessels, was solubilized in 5% NaDodSO4/5% 2-mercaptoethanol/10o glycerol in 50 mM Tris buffer (pH 6.8) (2 1.l per specimen). Younger somites were obtained by microdissection from the 12th to 8th segment of stage 14-15 embryos (n = 15) and were pooled in 10 ul of solubilization buffer. Aliquots (1 ILI; equivalent to three somites) were applied to 1 x 5 mm strips of cellulose acetate paper and loaded on 100-pym-thick 7.5% acrylamide slab gels (26). The gels were passively blotted for 2 hr onto nitrocellulose (Bio-Rad) or charged nylon membranes (Hybond N+; Amersham) and silver stained as described (27)

(except that the AgNO3 concentration was doubled) or blotted with the indicated antibodies.

RESULTS Segmental Distribution of Cytotactin mRNA in the Posterior Half of the Epithelial Somite and in the Dermamyotome Is Independent of the Presence of NC Cells. To identify the tissues capable of synthesizing cytotactin, we analyzed the distribution of cytotactin mRNA by in situ hybridization. Each somite from the 4th through the 15th somite of a 15-somite embryo (Fig. LA) contained cytotactin mRNA in a different pattern that was related to the maturity of the somite. The mRNA was restricted to the posterior half of the epithelial somites (Fig. 1 A and B), even in the most newly formed somite in which little to no protein staining was observed (5, 8, 21, 28). The epithelial somite reorganizes to give rise to the sclerotome and dermamyotome (29). In the 15-somite embryo, this transition occurs approximately at the level of the 9th somite, where the cytotactin mRNA became dorsal as well as posterior (Fig. LA). In the more anterior somites, only dorsal (i.e., dermamyotomal) hybridization

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FIG. 2. Immunofluorescence and in situ hybridization of embryos of stages 18 and 20. Parasagittal sections of stages 18 (A-C) and 20 (D-F) were double-labeled with HNK-1 monoclonal antibodies (fluorescein) (B and E) or cytotactin polyclonal antibodies (rhodamine) (A and D). In situ hybridization for cytotactin is shown in parallel sections (C and F). Rostral is to the right in all panels. r, Rostral; c, caudal; dm, dermamyotome; drg, dorsal root ganglion; sp, sympathetic plexi. (Bar = 240 Am.)

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FIG. 3. Double-labeled immunofluorescence of operated regions with HNK-1 (rhodamine) and anti-cytotactin (fluorescein) antibodies. In all micrographs, the anterior end of the embryo lies to the right. (A) In the rostral aspect of the ablation field. HNK-1 immunoreactivity is seen in neural tube and in two somites at the anterior end of the field (top of the picture) where the excision was performed. NC cells are absent

Developmental Biology: Tan et al. was observed; dermamyotome staining was also evident in the 19-somite embryo (Fig. 1C). No NC cells were present at this stage, as evidenced by the lack of HNK-1 antibody staining (not shown). These results indicate that, at this stage of development, the segmental expression of cytotactin mRNA is independent of NC influence. Cytotactin mRNA Expression in the Anterior Portion of the Sclerotome. During sclerotomal maturation, cytotactin protein first appeared in the rostral half-sclerotome at stage 14. Although weak protein immunostaining in both halves was detected (5, 9, 21, 38), no mRNA above background levels was detected in the sclerotome at stage 13 (Fig. 1C). The protein present in the young sclerotome in the absence of mRNA either is remnant from the epithelial stage (when it is caudally expressed) or secreted by the dermamyotome. Cytotactin mRNA colocalized with HNK-1 immunostaining, in the sclerotome and in the presumptive dorsal root ganglion and sympathetic plexus in the ventral region (compare Fig. 2B with C and E with F, respectively). Although mRNA and protein were found in the rostral aspect of the sclerotome, the message was more discretely localized than the protein as seen in other regions (24) (compare Fig. 2 C and F with A and D, respectively). The hybridization signal associated with the presumptive dorsal root ganglia probably corresponds to Schwann cell precursors, inasmuch as Schwann cells have been shown to secrete cytotactin during development and regeneration (30). Cytotactin Expression in Sclerotomes of Operated Embryos. Staining for cytotactin in the somites opposite the ablated dorsal neural tube and NC showed variable patterns of immunoreactivity from one operated embryo to another and, within a single embryo, from one region to another. This is in direct contrast to cytotactin distribution in normal embryos (8), in which immunoreactivity in the rostral sclerotome was invariant in every specimen. Ingression of NC cells from more anterior or posterior levels into the ablated region was not a frequent occurrence. In most cases, there was an abrupt decrease of HNK-1 staining in the somites opposite the limits of the ablation zone, with further diminished numbers of HNK-1-positive cells in the next somite in the ablation zone (Fig. 3 A and E). Somites found lying further into the operated region did not show HNK-1 immunoreactivity, as judged by examination of every horizontal section. After ablation, rostral staining of cytotactin was seen in four specimens (40%) (Fig. 3B); the staining was evident in the first four somites in the anterior-most (and therefore older) part of the ablated field. Crest cells were present in only the first two of these somites (Fig. 3A). In the remaining more caudal somites, the predominant pattern of cytotactin staining was in both rostral and caudal halves of the sclerotome, especially in somites found at the middle region of the ablated field (Fig. 3 C and D); this pattern was found in the central region of every ablated specimen (100%). Predominant staining in the caudal half of the sclerotome was seen in five embryos (50%) and usually involved the somites found in the posterior-most part of the ablated field (Fig. 3 Eand F); in the posterior ablation region, rostral staining was never seen. Interestingly, this pattern of caudal staining did not terminate at the posterior border of surgical excision. In one instance,

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predominant caudal half staining was seen in 4 or 5 somites of the operated area, continuing into the posterior flanking region of undisturbed somites containing a normal complement of NC cells for at least eight somite lengths (not shown). In sham-operated embryos, patterns of cytotactin staining were also variable, although the NC cell distribution was unperturbed. Two control embryos showed clear rostral half staining of cytotactin similar to previously described patterns (8), but in one case (Fig. 3 G and H) (which also showed abnormal neural tube closure), cytotactin staining was also seen in the caudal halves of the sclerotome in somites opposite to operated and unoperated areas. Immunoblotting of Somites of Control and Dorsal Neural Tube-Ablated Embryos and of Young Somites. Immunoblotting of somites from sham-operated controls and from embryos with dorsal neural tube ablation showed distinct bands for fibronectin, laminin, and cytotactin that could not be distinguished between control and ablated tissues (Fig. 4). The results of all four dorsal neural tube ablation series were closely comparable (not shown). Two- sharp bands of 72 and 116 kDa bound to the labeled streptavidin in the absence of either primary or secondary antibodies (not shown). Fibronectin immunoreactivity was seen in the region of 200 kDa, with less clear bands of slightly higher and lower molecular sizes. Cytotactin showed major bands of 220, 200, and 190 kDa and weaker bands of about 230, 180, and 165 (diffused) kDa. In younger somites, into which NC cells had not migrated, cytotactin polypeptides of 220, 200, and 190 kDa were observed (Fig. 4). In no case was cytotactin immunoreactivity seen in the range below 150 kDa.

DISCUSSION Cell and substrate adhesion molecules have been proposed to play a key role in somitogenesis (31). The present study showed that cytotactin mRNA was expressed in a distinct sequential pattern during development. The mRNA was already polarized in the caudal half of the youngest somite as it detached from the segmental plate. As the somite reorganized, cytotactin expression was restricted to the dorsomedial aspect, as also is the case for N-cadherin (31), and it remained so until stage 14, when cells in the rostral aspect of the sclerotome expressed cytotactin. This raises the question of how the change in the expression pattern of cytotactin mRNA is correlated with cell rearrangements that give rise to an epithelial dermamyotome and mesenchymal sclerotome. This could be due to changes in number or position of cells in the epithelial somite, analogous to that occurring in amphibian somitogenesis (32, 33), or to an induction of expression of the message in the cells of the dorsal aspect of the somite that subsequently give rise to the dermamyotome. Experiments using lineage tracers could distinguish between these possibilities. Immunocytochemical analysis of axial regions of chicken embryos deprived of NC cells showed variable alterations in the pattern of cytotactin expression. All three modes of cytotactin distribution in the sclerotome may be found after ablation of the NC and dorsal neural tube depending on their proximity to the anterior or posterior parts of the ablation

from the sclerotome of the remaining two somites at the posterior end. (B) Cytotactin staining is predominant in the rostral halves of the sclerotome with and without NC cells. Faint but detectable levels of cytotactin are also visible in the caudal halves. (C and D) View across five somite lengths near the middle aspect of the ablation field. Cytotactin (D) is expressed in rostral and caudal halves of the sclerotome in somites devoid of NC cells (C). The most posterior somite in this field (left side of the picture) shows slightly increased cytotactin expression in the caudal half compared to the rostral half. (E) View of five somite lengths in the posterior end of the ablation field where the neural tube is still intact near the site of excision. NC cells are seen in the posterior three somites, but further inward toward the ablated site (right side of picture) the somites do not show HNK-1 staining. (F) Cytotactin is visible in rostral and caudal halves of the sclerotome but predominates in the caudal half of somites with or without NC cells. (G and H) HNK-1 staining (G) and cytotactin staining (H) in sham-operated embryos. Although NC cells are properly positioned in the rostral portion of the sclerotome (G), cytotactin is preferentially expressed in the caudal half somite. r, Rostral; c, caudal. (Bar = 300 ,um.)

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FIG. 4. Biochemical analysis of cytotactin in somites. Somite tissues from sham-operated control embryos at embryonic day 4 (C), embryonic day 4 embryos that had dorsal neural tube ablation at embryonic day 2 (0), or young somites at embryonic day 2 (y) were separated on NaDodSO4/PAGE. Gels were stained with silver (Ag) or blotted and labeled for fibronectin (FN), laminin (LN), and cytotactin (CT) using biotin and 35S-labeled streptavidin and autoradiographed. Ablated and control embryos showed identical patterns, and cytotactin immunoreactivity was exclusively in high molecular mass components. Markers (arrowheads) are 205, 116, 97, and 68 kDa. Bands at 116 kDa and 72 kDa in autoradiographs are endogenous streptavidin-binding proteins.

field. Distribution of cytotactin in both somite halves was seen for some somites in all cases; such somites coexisted with others that showed predominantly rostral or caudal staining. In one instance, all three modes of cytotactin distribution were found in series along the ablated zone. The closer approach to normality at the anterior (and developmentally older) end of the ablation field suggests that the older somites are more committed to their normal differentiation program. The spatial misexpression of cytotactin around the caudal limits of the ablation and in one sham-operated embryo, despite the presence of correctly localized NC cells, indicates that the presence of crest cells is not sufficient for the normal pattern of cytotactin expression. Furthermore, the normal pattern of cytotactin expression in the sclerotomes at the rostral extent of ablation, despite the absence of crest cells, indicates that crest cells are not necessary for the generation of the cytotactin pattern. The abnormalities in cytotactin distribution are likely to be due to generalized tissue disorganization following a wound. Such changes have been observed in other wounding paradigms (34, 35). These changes in the pattern of cytotactin expression may be the result of growth factors or other molecules generated at the wound site that affect expression of cytotactin through one of its many upstream motifs (36). Alternatively, the distribution of inducing factors arising from the neural tube itself may be altered by the surgical intervention. Given the homologies to the DNA-binding sequences for homeotic gene products found upstream of the cytotactin gene (36), it is plausible that hox genes (37) may be among those that regulate cytotactin expression. It would be of particular interest to determine the expression patterns of such hox genes after NC ablation. It was reported that numerous unusual low molecular mass components of cytotactin were isolated from somites by immunoaffinity chromatography (21). Using SDS/PAGE followed by immunoblotting, conditions where the opportunity for degradation or for molecular interactions are minimized, we found that young somites and those from control and ablated specimens gave identical profiles of high molecular mass immunoreactivity, with no molecular mass species smaller than 150 kDa. Other matrix molecules (fibronectin, laminin) were likewise unaltered by dorsal neural tube ablation. Thus somite cells themselves, independent of the presence of NC cells, synthesize high molecular mass forms of

cytotactin. This interpretation is consistent with previous results (9) as well as with the localization of cytotactin mRNA independent of NC cells shown here. The present results strengthen our previous observations (8, 28) that cytotactin is normally codistributed with the final crest cell pattern, although its distribution is not positively correlated with early routes of NC cell migration. An interaction with crest cells, therefore, is neither necessary nor sufficient for the synthesis in the sclerotome of correctly positioned and sized forms of cytotactin. We thank Frank Weissenborn for excellent technical assistance. This work was supported by U.S. Public Health Service Grant DK-04256 (G.M.E.), a grant from The Irma T. Hirschl Trust (K.L.C.), Medical Research Council Grant 900344 (S.-S.T.), and grants from the George Hicks and Ian Potter Foundations (S.-S.T.). A.L.P. is supported by National Research Service Award Training Grant GM 07524-14. K.L.C. is a Becton Dickinson Young Faculty Fellow. 1. Newgreen, D. & Thiery, J.-P. (1980) Cell Tissue Res. 211, 269-291. 2. Rickman, M., Fawcett, J. W. & Keynes, R. J. (1985) J. Embryol. Exp. Morphol. 90, 437-455. 3. Bronner-Fraser, M. (1986) Dev. Biol. 115, 44-55. 4. Loring, J. F. & Erickson, C. A. (1987) Dev. Biol. 121, 220-236. 5. Newgreen, D. F., Powell, M. E. & Moser, B. (1990) Dev. Biol. 139, 100-120. 6. Keynes, R. J. & Stem, C. (1984) Nature (London) 310, 786-789. 7. Weston, J. A. & Butler, S. L. (1966) Dev. Biol. 14, 246-266. 8. Tan, S.-S., Crossin, K. L., Hoffman, S. & Edelman, G. M. (1987) Proc. Natl. Acad. Sci. USA 84, 7977-7981. 9. Mackie, E. J., Tucker, R. P., Halfter, W., Chiquet-Ehrismann, R. & Epperlein, H. H. (1988) Development 102, 237-250. 10. Davies, J. A., Cook, G. M. W., Stem, C. D. & Keynes, R. J. (1990) Neuron 2, 11-20. 11. Ranscht, B. & Bronner-Fraser, M. (1991) Development 111, 15-22. 12. Perris, R., Krotoski, D., Lallier, T., Domingo, C., Sorrel, J. M. & Bronner-Fraser, M. (1991) Development 111, 583-599. 13. Chiquet-Ehrismann, R., Mackie, E. J., Pearson, C. A. & Sakakura, T. (1986) Cell 47, 131-139. 14. Faissner, A., Kruse, J., Chiquet-Ehrismann, R. & Mackie, E. (1988) Differentiation 37, 104-114. 15. Jones, F. S., Hoffman, S., Cunningham, B. A. & Edelman, G. M. (1989) Proc. Natl. Acad. Sci. USA 86, 1905-1909. 16. Spring, J., Beck, K. & Chiquet-Ehrismann, R. (1989) Cell 59, 325-334. 17. Bronner-Fraser, M. (1988) J. Neurosci. Res. 21, 135-147. 18. Hoffman, S. & Edelman, G. M. (1987) Proc. Natd. Acad. Sci. USA 84, 2523-2527. 19. Friedlander, D. R., Hoffman, S. & Edelman, G. M. (1988) J. Cell Biol. 107, 2329-2340. 20. Halfter, W., Chiquet-Ehrismann, R. & Tucker, R. P. (1989) Dev. Biol. 132, 14-25. 21. Stem, C. D., Norris, W. E., Bronner-Fraser, M., Carlson, G. J., Faissner, A., Keynes, R. J. & Schachner, M. (1989) Development 107, 309-319. 22. Hamburger, V. & Hamilton, H. L. (1951) J. Morphol. 88, 49-92. 23. Prieto, A. L., Crossin, K. L., Cunningham, B. A. & Edelman, G. M. (1989) Proc. Natl. Acad. Sci. USA 86, 9579-9583. 24. Prieto, A. L., Jones, F. S., Cunningham, B. A., Crossin, K. L. & Edelman, G. M. (1990) J. Cell Biol. 111, 685-698. 25. Tucker, G. C., Aoyama, H., Lipinski, M., Tursz, T. & Thiery, J.-P. (1984) Cell Djff. 14, 223-230. 26. Neukirchen, R. O., Schlosshauer, B., Baars, S., Jackel, H. & Schwarz, U. (1982) J. Biol. Chem. 257, 15229-15234. 27. vonBoxberg, Y. (1988) Anal. Biochem. 169, 372-375. 28. Crossin, K. L., Hoffman, S., Grumet, M., Thiery, J.-P. & Edelman, G. M. (1986) J. Cell Biol. 102, 1917-1930. 29. Bellairs, R. (1979) J. Embryol. Exp. Morphol. 51, 227-243. 30. Daniloff, J. K., Crossin, K. L., Pingon-Raymond, M., Murawsky, M., Rieger, F. & Edelman, G. M. (1989) J. Cell Biol. 108, 625-635. 31. Duband, J.-L., Dufour, S., Hatta, K., Takeichi, M., Edelman, G. M. & Thiery, J.-P. (1987) J. Cell Biol. 104, 1361-1374. 32. Youn, B. W. & Malacinski, G. M. (1981) J. Embryol. Exp. Morphol. 64, 23-43. 33. Neff, A. W., Malacinski, G. M. & Chung, H.-M. (1989) Dev. Biol. 132, 529-543. 34. Mackie, E. J., Halfter, W. & Liverani, D. (1988) J. Cell Biol. 107, 2757-2767. 35. Lightner, V. A., Gumkowski, F., Bigner, D. D. & Erickson, H. P. (1989) J. Cell Biol. 108, 2483-2493. 36. Jones, F. S., Crossin, K. L., Cunningham, B. A. & Edelman, G. M. (1990) Proc. Natl. Acad. Sci. USA 87, 6497-6501. 37. Kessel, M. & Gruss, P. (1990) Science 249, 374-379. 38. Wehrle, B. & Chiquet, M. (1990) Development 110, 401-415.