Institute of Histology and Embryology, School of Medicine at Split, University of Zagreb, Split, Croatia. Accepted in revised form: 28 March 1995. Abstract. Indirect ...
Differentiation (1995) 59:4349
Differentiation Ontogeny, Neoplasia and Differentiation Therapy
0 Springer-Verlag 1995
Changes in the expression of intermediate filaments and desmoplakins during development of human notochord Eero Lehtonen', Vedran Stefanovic'.2 , Mirna Saraga-Babic3 I
Department of Pathology, University of Helsinki, P.O.B. 21 (Haartmaninkatu 3), FIN-00014, Helsinki, Finland of Obstetrics and Gynecology, University of Helsinki, FIN-00014 Helsinki, Finland Institute of Histology and Embryology, School of Medicine at Split, University of Zagreb, Split, Croatia
2 Department
Accepted in revised form: 28 March 1995
Abstract. Indirect immunofluorescence was used to study the expression of desmosomal and intermediate filament (IF) proteins in the human notochord between the 4th and 12th weeks of embryonic development. Towards the end of this period, the development of the notochord is characterized by its gradual physiological atrophy and disappearance inside the vertebral bodies. In all of our embryos, the notochord cells expressed cytokeratin and vimentin but not desmin, neurofilament protein or glial fibrillary acidic protein. Throughout the stages studied, the expression of cytokeratin was strong. Vimentin expression, on the other hand, changed during the stages studied. In our youngest embryos, vimentin could be detected only in the peripheral cells of the notochord. During development, a distinct increase occurred in vimentin expression, and in the oldest embryos, all notochord cells showed bright vimentin-specific fluorescence. Simultaneously with this modification, a change occured in the expression of desmosomal proteins: The notochord cells expressed desmoplakins abundantly during early stages, but weakly or not at all during later stages. Correspondingly, electron microscopy of the same stages showed a striking decrease in the number of desmosomes between notochord cells. Our results confirm that, during early development, the notochord displays features specific for epithelial cells. This accords with the view that notochord is of epithelial origin. The modifications observed in the expression of IF and desmosomal proteins were temporally correlated with developmentally regulated atrophy of the notochord. The programmed regression of the notochord cells is thus associated with a switch from a predominantly epithelial phenotype to a more mesenchymal one.
the development of the neural plate and chondrogenesis in the surrounding mesenchyme [3, 39, 471. During embryonic development it progressively atrophies and disappears except in the central parts of the intervertebral discs [28, 481. The origin of the notochord is controversial [l, 11, 27, 381. During embryogenesis, cytodifferentiation is accompanied by expression of cell- and tissue-specific subtypes of IFs (e.g. [37]). Of the IF subtypes, notochord cells have been reported to express cytokeratin in mouse [13] and Xenopus embryos [7]. In human embryos, the nucleus pulposus cells seem to express both cytokeratin and vimentin [14, 381. The latter study, using 4-week and 6-week embryos as well as late foetal and postnatal stages reported expression of cytokeratin and vimentin in all notochord cells. Ultrastructurally, IF-type filaments are found in the notochord cells of human [6, 35, 40, 42, 431, chick [lo], and mouse embryos [l 11 as well as in the cells of the juvenile human nucleus pulposus [22]. During early development, notochord cells are characteristically connected by desmosomes [6]. In the present investigation we studied human embryos at several successive stages, during which the notochord develops and undergoes a programmed, almost complete atrophy. We show distinct changes in the expression of IFs and desmosomal proteins during this morphogenetically important period. Methods Tissues. Human embryos and foetuses were obtained from induced abortions according to all legal and ethical processes in conformity with the Helsinki Declaration (Table 1). The age was estimated from menstrual data and correlated with crown-rump length (CRL), crown-heel length (CHL) and Carnegie stages [28].
Introduction Human notochord is a transitory organ which plays an important role during early embryogenesis. It induces Correspondence to: E. Lehtonen
Immunofuorescence microscopy. Eleven young embryos (4-8 weeks of development, CRL 5-31 mm, Carnegie stages 13-23) were used in this study; they were fixed in toto. In the case of the five older stages studied (9-12 weeks of development, CHL 31-100 mm), only the cranial and caudal ends were dissected
44 gen, Federal Republic of Germany), and the antibodies to keratin No. 19 by Dr. James Rheinwald (Dana-Farber Cancer Institute, Boston, MA, USA). The antibodies LE65, LLOO1, and LP2K were kindly provided by Dr. Birgitte Lane (Department of Biochemistry, University of Dundee, Dundee, UK). The antibodies 3B4 and those to desmoplakins I+II were from Boehringer, Mannheim, FRG.
from the embryos; this was necessary to ascertain good preservation. The samples were fixed in 1% acetic acid in 96% ethanol at 4 "C for 20-24 h [33], dehydrated in ethanol and xylol, embedded in paraffin, and sectioned sagittally. For indirect immunofluorescence, the deparaffinized sections were incubated with the specific antibodies for 45 min at room temperature followed by appropriate FITC- or TRITC-coupled second antibodies for 45 min at room temperature. The conjugates were from Cappel Laboratories (Cochranville, PA, USA). The samples were mounted in Elvanol and studied in a Zeiss Axiophot microscope equipped with filters for FITC and TRITC fluorescence.
Electron microscopy. Tissue pieces from three human conceptuses between the 5th and 9th developmental weeks were fixed in a formaldehyde solution, made by dissolving 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2), for 24 h at 4 "C, postfixed in 2% osmium tetroxide, and embedded in LX 112. Sagittal ultrathin sections, selected on the basis of serial semi-thin sections, were studied in a Jeol 1200 EX electron microscope.
Antibodies. The antibodies used and their specificities are summarized in Table 2. The antibodies PKK1, PKK3, 65EE3, FV24BA6, NFI3AA8, NF14BA8, U251Mg, and 37EH11 were kindly provided by Dr. Ismo Virtanen (Department of Anatomy, University of Helsinki, Helsinki, Finland). The TROMA 1 antibodies were kindly provided by Dr. Rolf Kemler (Max-Planck-Institut, Tiibin-
Results Electron microscopy
Table 1. Developmental stage and number of embryos Age (weeks)
Carnegie stage
No.
4 5 5-6 6 6-7 8 8-9 9 10-1 1 12
13 15 16 17 19 23
1 3 3 2 3 2 1 2 1 1
From the 4th to the 6th week of development, the notochord runs like a continuous cylinder of cells through the developing vertebral column. The closely packed notochord cells contained abundant glycogen deposits and cytofilaments. The cells were connected by numerous desmosomes (Fig. 1). From the 6th to the 12th week of development, the notochord degenerates within the vertebral bodies. This stage was characterized by progressive formation of intercellular spaces and accumulation of glycogen and cytoplasmic filaments within the notochord cells (Fig. 2). Occasional desmosomes were observed only at the few
Table 2. Expression of intermediate filaments and desmoplakins in developing human notochord Antigen
Antibody
Ref.
Age of embryos (weeks) 4-6 C.C.
Cytokeratins of simple epithelium 8 TROMA 1 18 PKK3 18 LE65 19 Rheinwald 19 LP2K 8, 18, 19 PKKl Cytokeratins of epidermis/mucosa 14 LLOOl Vimentin 65EE3 3B4 FV24BA6 Desmin 37EH11 NF protein NF13AA8 NF14BA8 GFAP U25 1Mg Anti-desmoDesmoplakin I+II plakin I+II
p.c.
8-12 C.C.
++
++ ++ ++ ++
++ ++ ++ ++ ++ ++
++ ++ ++ ++ ++ ++
++ + ++ ++ ++
n.d.
-
-
-
-
-
+ + +
+ + +
++ ++ +
++ ++ ++
++ ++ ++
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
++
++
+
13 46 16 49 17,36 46
++ ++ ++ ++ n.d. ++
32 45
-
n.d. -
46 44 41 41 30 24
p.c.
6-7 C.C.
++
++
++ ++
++
n.d.
++
+
-
p.c.
++
-
++, strong positivity; +, weak positivity; -, negative reaction. Cytokeratin polypeptide numbers refer to the classification in [25]. GFAP, glial fibrillary acidic protein. NF, neurofilament; c.c., central cells; P.c., peripheral cells
45
Fig. 1. Human notochord at the beginning of the 6th embryonic week. Closely packed notochord cell are connected by frequent desmosomes (arrows). The cells have euchromatic nuclei and abundant cytoplasmic glycogen (G), and a continuous basal lamina (BL) covers their surface; insert,a higher magnification of two desmosomes connecting notochord cells. Note the bundles of intermediate filaments; bar; 1bm; bar in insert,200 nm
Fig. 2. Notochord cells (N) of a 10-week foetus are separated by numerous intercellular spaces ( I S ) of different sizes. Note the discontinuities in the basal lamina (BL) lining the surface of the notochord and the intercellular spaces; G, glycogen; bur; 1 pm
46
Fig. 3a, b. Cytokeratin in human notochord. a A section through the lumbar level of a 4-week embryo shows bright cytokeratin 18 positivity in the notochord (N) and skin epithelium (S). The spinal cord (Soand the surrounding mesenchyme (M) are negative. b Lumbar level of an 8-week embryo. The notochord cells (N) show strong expression of cytokeratin 18. The nonfluorescent areas (arrow) represent the intercellular vacuoles present in the notochord at this stage. The chondrocytes in the surrounding cartilage (C) are negative; bar; 100 km Fig. 4a-c. Vimentin in human notochord. a Lumbar level of a 5week embryo. Antibodies to vimentin have strongly stained the mesenchymal cells (M)of the future vertebral column. In the notochord (N), only a few cells on the surface were stained. b Lumbar level of a 6-week embryo. Vimentin is strongly expressed in all peripheral cells of the notochord, but weakly in the cells in the
interior of the structure; M, vertebral column mesenchyme. C. Lumbar level of an 8-week embryo. All cells of the notochord (fl have reacted with antibodies to vimentin. The chondrocytes (C) in the surrounding vertebral column have been stained weakly; bar; 100 pm
Fig. 5a-c. Desmoplakin I+II in human notochord. a Lumbar level of a 5-week embryo. The notochord (N) strongly expresses desmoplakin I+II, while the surrounding mesenchyme (M) is negative. b Tail area of a 7-week embryo. The notochord (N) has still weakly reacted with antibodies to desmoplakin 1+1I, while the chondrocytes (C) of the vertebral column are negative. c An 8-week embryo. Desmoplakin I+II is expressed by the skin epithelium (S), but not by the underlying connective tissue (C); bar; 100 pm
47
longer areas of contact which at this stage could be found between the notochord cells.
Immunof uorescence microscopy When stained with antibodies to simple epithelial keratin polypeptides (Table 2), all the notochord cells showed strong reactivity (Fig. 3a), which remained practically unchanged during the period from the 4th to the 6th week of development. During further development, the presence of large intercellular spaces within the notochord tissue sometimes suggested a decrease in keratin expression in the notochord (Fig. 3b). No expression of the epidermal/mucosal keratin 14 was found in the notochord cells, while its presence could be observed in appropriate tissues in the same sections. During the 4th and 5th developmental weeks, antibodies to vimentin (Table 2) revealed only a weak positive staining in the peripheral cells of the notochord (Fig. 4a). On the 6th week, the reactivity became more pronounced and gradually spread to the cells inside the notochord (Fig. 4b). During further development, the expression of vimentin became stronger and spread to all cells both in the periphery and the interior of the notochord (Fig. 4c). During all stages investigated, we found no expression of desmin, neurofilament protein, or glial fibrillary acidic protein in the notochord cells. Expression of these proteins could be observed in appropriate tissues in the same sections. Notochord cells reacted strongly with antibodies to desmoplakin I+II (Table 2) at early stages (Fig. 5a). The reactivity with the desmoplakin I+II antibodies gradually weakened during the 7th week (Fig. 5b) and disappeared by the end of the 8th week of development, while it remained positive in various epithelial tissues in the same sections (Fig. 5c).
Discussion The present study is the first one to demonstrate that spatiotemporal changes occur in the expression of IF and desmosomal proteins during the development of the human notochord. During early development, keratin was strongly expressed by all notochord cells, while vimentin was expressed weakly by the peripheral cells and not at all by the central ones. During further development, the expression of keratin remained at a high level. The expression of vimentin clearly increased and spread from the peripheral notochord cells to all of them. Simultaneously, modifications occured in notochordal desmosomes: Desmoplakins were strongly expressed in the early stages, whereafter the expression gradually decreased and disappeared. Electron microscopy revealed a simultaneous dramatic decrease in the number of desmosomes between notochord cells. The above findings fit with the epithelial-type morphology of the notochord cells during the early stages, and the atrophy and acqui-
sition of a more mesenchymal phenotype during further development [6, 351. During mammalian embryogenesis, the first type of IF proteins detected is cytokeratin (e.g. [2, 20, 311). In mouse embryos only cytokeratin-type IFs are expressed before the primitive streak stage. During further development cytokeratins are replaced by vimentin in primary mesenchyme cells [4] and in mesenchyme-derived cells [25]. Other types of IFs then gradually become expressed in the differentiating tissues, and eventually each cell type expresses its specific class of IFs. In addition to the notochord, there are several other tissues which coexpress two types of IFs during development (e.g. [9, 18, 191) or in mature tissues [12]. In the present study we found both vimentin and keratin in peripheral cells of the notochord throughout development. The central cells, on the other hand, showed stage-specific modulation in their IFs: During early development, the cells expressed keratin only. Later, both keratin and vimentin were seen. Cells of chordomas, tumours derived from notochord, have similarly been shown to coexpress vimentin and keratin [8, 141. Interestingly, modulation of IF expression may also occur in chordoma tumour cells [21, 231. Keratin polypeptides are expressed in different combinations during epithelial differentiation and in mature epithelial cells [15, 251. Our present work shows that the notochord expresses simple epithelial keratins but not the epidermal/mucosal keratin 14. Consistent with this, the expression of the latter type of keratins seems to be exceptional in chordomas [8]. The development-associated molecular changes we observed in the notochord are accompanied by the appearance of numerous intercellular spaces and accumulation of extracellular matrix and intracellular cytofilaments [6, 401. In later stages, the widely separated notochord cells were found to communicate mostly by long cytoplasmic extensions and very rarely by cytoplasmic areas bound by desmosomes. This accords with the progressive decrease and final disappearance of desmoplakins we observed in the notochord cells. In the second half of intrauterine life [34] and in the early postnatal, period, the notochord is gradually replaced by fibrocartilage, originating from the surrounding mesenchyme. Finally, only scattered notochord cells are found in the postnatal nucleus pulposus [22]. It has been suggested that the notochord induces chondrogenesis in the sclerotomal cells of the surrounding vertebral column [3, 5, 111. The changes we observed in the IFs and desmosomes of the notochord cells were spatially and temporally correlated with these developmental changes in the vertebral column. Our data thus corroborate the view that developmentally important tissue interactions take place between the two organ structures, the vertebral column and the notochord. Acknowledgements. We thank Ms. Ulla Kiiski and Ms. Asja Miletic for skillful technical assistance and Ms. Marja-Leena Rissanen for skilled secretarial help. M.S.-B. was a recipient of a short-term fellowship from the Programme on Developmental Biology of the European Science Foundation. This work was supported by the Medical Research Council of the Academy of Finland, the Uni-
48 versity of Helsinki, the Sigrid Justlius Foundation, Finland, and the Ministry of Science, Technology and Informatics of the Republic of Croatia.
References 1. Bancroft M, Bellairs R (1976) The development of the notochord in the chick embryo, studied by scanning and transmission electron microscopy. J Embryol Exp Morphol 35:38340 1 2. Capco DG, Gallicano GI, McGaughey RW, Downing KH, Larabell CA (1993) Cytoskeletal sheets of mammalian eggs and embryos: a lattice-like network of intermediate filaments. Cell Motil Cytoskel24:85-99 3. Carlson EC (1973) Intercellular connective tissue fibrils in the notochord epithelium of the early chick embryo. Am J Anat 136:77-90 4. Franke WW, Grund C, Kuhn C, Jackson BW, Illmensee K (1982) Formation of cytoskeletal elements during mouse embryogenesis. 111. Primary mesenchymal cell and the first appearance of vimentin filaments. Differentiation 23:43-59 5. Frederickson RG, Law FN (1971) The fine structure of prinotochordal microfibrils in control and enzyme treated chick embryos. Am J Anat 130:347-376 6. Galic M, Saraga-Babic M, Svajger A (1986) Electron microscopic observations on the notochord of human embryos and fetuses. Proc Tu Acad Sci 424:239-273 7. Godsave SF, Anderton BH, Wylie CC (1986) The appearance and distribution of IFs during differentiation of the central nervous system, skin and notochord of Xenopus luevis. J Embryol Exp Morphol97:201-223 8. Heikinheimo K, Persson S, Kindblom L-G, Morgan PR, Virtanen I (1991) Expression of different cytokeratin subclasses in human chordoma. J Pathol 164:145-150 9. Holthofer H, Miettinen A, Lehto V-P, Lehtonen E, Virtanen I (1984) Expression of vimentin and cytokeratin types of intermediate filament proteins in developing and adult human kidneys. Lab Invest 5032-559 10. Jurand A (1962) The development of the notochord in chick embryos. J Embryol Exp Morphol 10:612-621 1 I . Jurand A (1974) Some aspects of the development of the notochord in mouse embryos. J Embryol Exp Morphol32: 1-33 12. Kasper M, Stosiek P (1990) The expression of vimentin in epithelial cells from human nasal mucosa. Eur Arch Otorhinolaryngol248:53-56 13. Kemler R, Brulet P, Schnebelen M-T, Gailard J, Jacob F (1981) Reactivity of monoclonal antibodies against IFs during embryonic development. J Embryol Exp Morphol 64:4660 14. Krech R, Loy V, Iglesia JR, Gerdes J, Stein H (1987) Immunohistologische Characterisierung von Chordomen. Patologe 8:207-212 15. LaFlamme SE, Dawid IB (1990) XK endo B is preferentially expressed in several induced embryonic tissues during the development of Xenopus laevis. Differentiation 43: 1-9 16. Lane EB (1982) Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J Cell Biol92:665-672 17. Lane EB, Bartek J, Purkis PE, Leigh M (1985) Keratin antigens in differentiating skin. Ann NY Med Sci 455:241-258 18. Lane EB, Hogan BLM, Kurkinen M, Carrels JI (1983) Coexpression of vimentin and cytokeratins in parietal endoderm cells of the early mouse embryo. Nature 303:701-704 19. Lehtonen E, Lehto V-P, Paasivuo R, Virtanen I (1983) Parietal and visceral endoderm differ in their expression of intermediate filaments. EMBO J 2: 1023-1 028 20. Lehtonen E, Ordonez G, Reima I (1988) Cytoskeleton in preimplantation mouse development. Cell Differ 24: 165-178
21. Maiorano E, Renzulli G, Favia G, Ricco R (1992) Expression of intermediate filaments in chordomas. An immunohistochemical study of five cases. Pathol Res Pract 188:901-907 22. Meachim G, Cornah S (1970) Fine structure of juvenile human nucleus pulposus. J Anat 107:337-350 23. Miettinen M, Lehto V-P, Virtanen I (1984) Malignant fibrous histiocytoma within a recurrent chordoma. A light microscopic, electron microscopic and immunohistochemical study. Am J Clin Pathol 82:738-743 24. Moll R, Cowin P, Kapprell H-P, Franke WW (1986) Biology of disease. Desmosomal proteins: new markers for identification and classification of tumors. Lab Invest 54:4-25 25. Moll R, Franke WW, Schiller D, Geiger B, Krepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 3 I : 1 1-24 26. van Muijen GNP, Ruiter DJ, Franke WW, Achtstatter T, Haasnoot WHB, Ponec M, Warnaar SO (1986) Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas as demonstrated by monoclonal antibodies specific for cytokeratins Nos. 4 and 13. Exp Cell Res 162:97-113 27. Nakamura J, Becker LE, Marks A (1983) SlOO protein in human chordoma and human and rabbit notochord. Arch Pathol Lab Med 107:118-120 28. O’Rahilly R, Gardner R (1971) The timing and sequence of events in the development of the human nervous system during the embryonic period proper. Z Anat Entwickl-Gesch 134:1-12 29. O’Rahilly R, Meyer DB (1979) The timing and sequence of events in the development of the human vertebral column during the embryonic period proper. Anat Embryol 157:167176 30. Paetau A, Virtanen I (1986) Cytoskeletal properties and endogenous degradation of glial fibrillar acidic protein and vimentin in cultured human glioma cells. Acta Neuropathol 69~73-80 31. Plancha CE, Carmo-Fonseca M, David-Ferreira JF (1989). Cytokeratin filaments are present in golden hamster oocytes and early embryos. Differentiation 42: 1-19 32. Purkis PE, Steel JB, Mackenzie IC, Nathrath WBJ, Leigh IM, Lane EB (1990) Antibody markers of basal cells in complex epithelia. J Cell Sci 97:39-50 33. Sainte-Marie G (1962) A paraffin embedding technique for studies employing immunofluorescence. J Histochem Cytochem 10:250-256 34. Saraga-Babic M, Svajger A, Galic M (1984) Light and electron microscopic investigation on developmental changes in the human notochord. Acta Facultatis Medicae Zagrebiensis 25:111-120 25. Shinohara H, Tanaka 0 (1988) Development of the notochord in human embryos: ultrastructural, histochemical and immunohistochemical studies. Anat Rec 220: 171-1 78 36. Stasiak PC, Purkis PE, Leigh IM, Lane EB (1989) Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins. J Invest Dermato1 92:707-716 37. Steinert PM, Roop DR (1988) Molecular and cellular biology of intermediate filaments. Annu Rev Biochem 57:593-625 38. Stosiek P, Kasper M, Karsten U (1988) Expression of cytokeratin and vimentin in nucleus pulposus cells. Differentiation 39:78-8 1 39. Strudel G (1976) The primary connective tissue matrix of the bird embryo. Front Biol 3:77-100 40. Svajger A, Galic M, Saraga-Babic M (1984) The role of the cell death in human notochord in the formation of nucleus pulposus of intervertebral disc. Acta Orthop Jugosl 15:7376 41. Tienari J, Virtanen I, Soinila S, Lehtonen E (1987) Neuronlike derivatives of cultured F9 embryonal carcinoma cells express characteristics of parietal endoderm cells. Dev Biol 123~566-573
49 42. Trout JJ, Buckwalter JA, Moore KC (1982a) Ultrastructure of the human intervertebral disc. 11. Cells of the nucleus pulposus. Anat Rec 204:307-314 43. Trout JJ, Buckwalter JJ, Moore KC, Landas SK (1982b) Ultrastructure of the human intervertebral disc. I. Changes in notochordal cells with age. Tissue Cell 14:359-369 44. Virtanen I, Kallajoki M, Narvanen 0, Paranko J, Thornell LE, Miettinen M, Lehto V-P (1986) Peritubular myoid cells of human and rat testis are smooth muscle cells that contain desmin-type intermediate filaments. Anat Rec 21 5 : 10-20 45. Virtanen I, Kivela T, Bugnoli M. Mencarelli C, Pallini V, Albert DM, Tarkkanen A (1988) Expression of intermediate filaments and synaptophysin show neuronal properties and lack
46. 47. 48. 49.
of glial characteristics in Y79 retinoblastoma cells. Lab Invest 59:649-656 Virtanen I, Miettinen M, Lehto V-P, Kariniemi A-L, Paasivuo R (1985) Diagnostic application of monoclonal antibodies to intermediate filaments. Ann NY Acad Sci 455:635-648 Willis RA (1962) The first two months in human development. In: The Borderland of Embryology and Pathology. Butterworths, London, pp. 19-26 Williams LW (1908) The later development of the notochord in mammals. Am J Anat 8:251-284 Wu Y-J, Rheinwald JG (1981) A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas. Cell 25:627-635
