0736-5748/92 $5.00+0.00 Pergamon Press Ltd. © 1992 ISDN
Int. J. Devl. Neuroscience, Vol. 10, No. 6, pp. 473-480, 1992
Printed in Great Britain.
BRAIN FILAMENT PROTEINS IN PRIMARY CULTURES DERIVED FROM CHICK EMBRYOS EARLY IN DEVELOPMENT D. DAHL,* L. MAGGINIand V. H. GILAD Spinal Cord Injury Research Laboratory, Brockton/West Roxbury VA Medical Center; and tDepartment of Pathology, Harvard Medical School, Boston, MA, U.S.A.
(Received 31 July 1992; in revisedform 29 September 1992; accepted 29 September 1992)
Abstract--Brain filament expression and neurofilament post-translational modifications (phosphorylations) were studied in primary cultures derived from whole 3-4 day chick embryos. After 2-3 days in culture, neurofilament-positivecells formed neuronal aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. Aggregates and neuritic bundles were stained with several monoclonal antibodies reacting with phosphorylated neurofilament epitopes. With two monoclonal antibodies reacting with phosphorylated forms of the high molecular weight neurofilament subunit, staining was only observed after 8 and 10 days in vitro. There was a major difference between rat and chicken with respect to astrocyte differentiation in culture. In chicken, the fiat cells surrounding the neuronal aggregates remained constantly GFAP-negative throughout the whole experimental period (10 days). GFAP-positive cells were first observed within the neuronal aggregates on day 8 in vitro. Key words: neurofilament, glial fibrillary acidic protein, phosphorylation.
In this laboratory, in vitro studies on the expression of brain intermediate filaments have been conducted in primary cultures of murine CNS. 5,7'11'18'22 In the present study we report similar studies conducted in a different system, i.e. primary cultures derived from chick embryos early in development. 21'28 The purpose of the work was to provide for the first time a close comparison between two systems used in several studies of brain development, i.e. primary cultures derived from chicken and rat brain. A n o t h e r purpose was to find out whether brain filament expression and post-translational modification remain basically unchanged in phylogeny. EXPERIMENTAL PROCEDURES White Leghorn e m b r y o n a t e d eggs were obtained from Connecticut Valley Biological Supply (Southampton, MA). Cultures were prepared from 3- or 4-day chick embryos according to Vernadakis et al. 28 After removing the m e m b r a n e s , embryos were mechanically dissociated and plated at a density of 200/mm z on glass coverslips coated with poly-L-lysine (100 p~g/ml, Sigma, St Louis, M O ) in D M E M containing 10% (v/v) fetal bovine serum, antibiotic-antimycotic mixture (both from G I B C O Laboratories, G r a n d Island, NY) and 2 m M glutamine. Tissue culture glass coverslips were fixed in cold acetone and stored at - 2 0 ° C before use. Antibodies
Neurofilament polyclonal antibody R96 raised in rabbit against bovine antigen 1° was used in conjunction with monoclonal antibodies derived from mice for double staining experiments. A m o n g the neurofilament monoclonal antibodies previously characterized in this laboratory, those selected for this study (Table 1) stained cryostat sections of chick spinal cord and dorsal root ganglia as previously reported in the rat. 16 Monoclonals NE1424 and SMI3126 were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN) and from Sternberger-Meyer Immunocytochemicals (Jarretsville, M D ) , respectively. Monoclonal RT9719 was a gift from D r B. H. Anderton. All other neurofilament antibodies were produced in this laboratory. 12.14,15 For the staining of GFAP-positive astrocytes, we used monoclonal antibody A 2 D 4 raised against chicken antigen. 13 Conditions for immunolabeling with monoclonal and polyclonal antibodies were as reported in previous studies. 5,11 *Author to whom correspondence should be addressed at: Spinal Cord Injury Research Laboratory (151), Brockton/ West Roxbury VA Medical Center, 1400 VFW Parkway, West Roxbury, MA 02132, U.S.A. 473
474
D. Dahl et al. Table 1. Characterization of neurofilament monoclonat antibodies Monoclonals A9 C21 I120 J J8 J J28 J J29 J J32 J J36 J J47 J J51 NEI4 RT97 SMI31
Neurofilament immunoreactivity
lmrnunoreactivity after phosphatase
NF-H NF-M NF-H NF-H, NF-M NF-H, NF-M NF-H,NF-M NF-H, NF-M NF-H, NF-M NF-H, NF-M NF-H, NF-M NF-H NF-H NF-H, NF-M
Abolished Unchanged Reduced Reduced Reduced Reduced Reduced Reduced Reduced Reduced Abolished Abolished Abolished
NF-M: middle molecular weight neurofilament subunit. NF-H: high molecular weight neurofilament subunit.
RESULTS Two-three days after plating, neurons had formed large aggregates which persisted for the whole experimental period, i.e. 10 days in culture (Fig. 1). The aggregates were surrounded by an extensive neurite outgrowth. Some of these neurites formed rectilinear bundles connecting the aggregates. Cultures derived from 3- and 4-day chick embryo had a similar appearance. Aggregates and neurites, as well as a few isolated neurons, were stained by polyclonal and monoclonal neurofilament antibodies (Fig. 2). Among the antibodies listed in Table 1, only two (monoclonals 1120 and A9) did not stain the cultures at this stage of development. With these antibodies, staining was first observed on day 8 and 10 in vitro and appeared to be confined to a few neurites in the culture (Fig. 3). As previously reported, 25"29several neurofilament monoclonal antibodies reacting with phosphorylated epitopes also stained cell nuclei (Fig. 3). According to Schilling et al. ,23 the staining is due to crossreactivity with hitherto unidentified nuclear proteins and not, as suggested before,17 to the presence in histones of the repetitive sequences (Lys-Ser-Pro) which are the major phosphorylation sites of the middle and high molecular weight neurofilament subunits. Individual neurons within the aggregates could only be identified with monoclonal C21 (Table 1). This antibody selectively stained dorsal root ganglion neurons in primary cultures derived from rat embryos. 11 The large neurons decorated by monoclonal C21 in chicken cultures are illustrated in Fig. 4. As reported before,el the flat or stellate cells forming a monolayer between the aggregates were GFAP-negative, and it was not possible to tell whether they were fibroblasts or immature glia. GFAP immunoreactivity was first observed within the aggregates 8 days after plating. As shown in Fig. 5, it was in the form of a dense mesh of fibers. Individual cells were difficult to identify. DISCUSSION As indicated by neurofilament expression and post-translational modification, neuronal differentiation in chick and rat primary cultures appeared to follow a similar course. In both species, early neurofilament expression and phosphorylation were followed by phosphorylation events occurring with considerable delay (see this paper and Ref. 11). It is important to note that in the two studies the cultures were derived from embryos at comparable stages of neuronal development. Specifically, neurofilament proteins were first detected in rat spinal cord on embryonal day 12 and in dorsal root ganglia on day 13. 22 The corresponding ages for the chick embryo were day 3 and 4, respectively. 6 Similar findings have been reported by other investigators both in murine and chick embryos, e'3,8,9"z7Interestingly, if chick and rat embryonal development is measured in Carnegie stages, 3-day chick embryos and 13-day rat embryos are at Carnegie stage 14 (Fedoroff, personal communication).
Brain intermediate filament proteins in chick primary cultures
475
Fig. 1. Primarydissociatedcultures derived from 4-daychick embryosat day 4 (A) and day 8 (B) in vitro. Neurons form aggregates interconnected by neuritic bundles. Note that on day 8 (B) flat cells surrounding the aggregates form a monolayer. Phase contrast × 136. The demonstration of early and late neurofilament phosphorylation events in such different species as rat and chicken strongly suggests that the heterogeneity of phosphorylated neurofilament epitopes is functionally significant. Little is known of the function of neurofilament phosphorylation except that phosphorylation of cdc2 kinase dissociated the binding of dephosphorylated N F - H to microtubules. 2°
476
D. D a h l et al.
Fig. 2. A primary dissociated culture of 4-day chick embryos at day 4 in vitro is stained with two ncur~filament monoclonal antibodies reacting with phosphorylated epitopes. Note that both antibodies also stain nuclear chromatin. A. Decoration of neuronal aggregate and surrounding neuritic outgrowth with monoclonal SMI31 reacting with phosphorylated forms of the middle and high molecular weight neurofilament subunits. B. Decoration of isolated neurons between the aggregates with monoclonal NE14 reacting with phosphorylated form~ of the high molecular weight neurofilament subunit. The stained neurons probably derive from dor:,al root ganglia. Most neurofilament antibodies reacting with phosphorylated epitopes are axon specific except in dorsal root ganglia where they decorate a subpopulation of sensory neuron. 16 Indirect immunofluorescence, × 136 (A) and × 340 (B).
B r a i n i n t e r m e d i a t e filament p r o t e i n s in chick p r i m a r y cultures
Fig. 3. In a culture derived from 4-day chick embryos immunoreactivity with monoclonal A9 recognizing a phosphorylated form of the high molecular weight neurofilament subunit is confined to few neurites when it first appears on day 10 in vitro. A9-positive neurites in bundles originating from a neuronal aggregate (ag) or apparently not connected with aggregates are shown in A and B, respectively. Arrows in A point to the border of the A9-negative aggregate faintly visible in the background. Indirect immunofluorescence, x 340.
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Fig. 4. In an 8-day primary dissociated culture derived from 4-day chick embryos, neurofilament monoclonal antibody C21 selectively stains large neurons within a neuronal aggregate and between aggregate~ (insert). Indirect immunofluorescence, x 340.
Fig. 5. In a culture derived from 4-day chick embryos, immunoreactivity with G F A P monoclonal antibody A 2 D 4 is confined to a neuronal aggregate on its first appearance at day 8 in vitro. A small aggregate on the left of the figure faintly visible in the background (arrow) is GFAP-negative. Indirect immunofluorescence, × 136.
Brain intermediate filament proteins in chick primary cultures
479
Neuronal differentiation followed a similar course in chick and rat primary cultures, but we observed a major difference between the two species with respect to astrocyte development. As previously reported in rat, 1'22 GFAP-positive astrocytes appeared approximately 'on time' in chick cultures, that is after 8 days in vitro. In chick spinal cord, GFAP immunoreactivity was first observed on embryonal day 12 in vivo. 4 However, the site of astrocyte differentiation was markedly different in the two species. In rat, GFAP-positive cells on their first appearance in culture were located in the monolayer of non-neuronal cells, leading to the suggestion that the neuronal aggregates were colonized by astrocytes migrating from the surrounding monolayer.5 As reported before, 21 the cells forming the monolayer remained GFAP-negative in chick cultures. GFAP immunoreactivity first appeared within neuronal aggregates in 8-day cultures. The findings thus suggest that the conditions allowing the development of GFAP-positive cells in monolayers of non-neural cells still remain to be determined in chick. This is perhaps the reason why GFAP has not become a standard astrocyte marker in glial cultures derived from chick embryo. Acknowledgements--This work was supported by USPHS grant NS 13034 and by the Department of Veterans Affairs.
REFERENCES 1. Abney E. R., Bartlett P. P. and Raft M. C. (1981) Astrocytes, ependymal cells, and oligodendrocytes develop on schedule in dissociated cell cultures of embryonic rat brain. Devl Biol. 83, 291-300. 2. Bennett G. S. and DiLullo C. (1985) Expression of a neurofilament protein by the precursors of a subpopulation of ventral spinal cord neurons. Devl Biol. 107, 94-106. 3. Bennett G. S. and DiLullo C. (1985) Transient expression of a neurofilament protein by replicating neuroepithelial cells of the embryonic chick brain. Devl Biol. 107, 107-127. 4. Bignami A. and Dahl D. (1975) Astroglial protein in the developing spinal cord of the chick embryo. Devl Biol. 44, 204-209. 5. Bignami A. and Dahl D. (1989) Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord. Int. J. Devl Neurosci. 7, 343-357. 6. Bignami A., Dahl D. and Seiler M. W. (1980) Neurofilaments in the chick embryo during early development. 1. Immunofluorescent study with antisera to neurofilament protein. Devl Neurosci. 3, 151-161. 7. Bignami A., Raju T. R. and Dahl D. (1982) Localization of vimentin, the nonspecific intermediate filament protein, in embryonal glia and in early differentiating neurons. Devl Biol. 91,286-295. 8. Carden M. J., Trojanowski J. Q., Schlaepfer W. W. and Lee V. M.-Y. (1987) Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7, 3489-3504. 9. Cochard P. and Paulin D. (1984) Initial expression of neurofilaments and vimentin in the central and peripheral system of the mouse embryo in vivo. J. Neurosci. 4, 2080-2094. 10. Dahl D. (1983) Immunohistochemical differences between neurofilaments in perikarya, dendrites and axons. Immunofluorescence study with antisera raised to neurofilament polypeptides (200k, 150k, 70k) isolated by anion exchange chromatography. Expl Cell Res. 149, 397-408. 11. Dahl D. (1988) Early and late appearance of neurofilament phosphorylated epitopes in rat nervous system development. In vivo and in vitro study with monoclonal antibodies. J. Neurosci. Res. 20, 431-441. 12. Dahl D., Crosby C. J., Gardner E. E. and Bignami A. (1986) Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development. J. Neurosci. Res. 15, 513-519. 13. Dahl D., Crosby C. J., Seithi J. S. and Bignami A. (1985) Glial fibrillary acidic (GFA) protein in vertebrates: immunofluorescence and immunoblotting study with monoclonal and polyclonal antibodies. J. comp. Neurol. 239, 75-88. 14. Dahl D., Gardner E. E. and Crosby C. J. (1987) Axonal maturation in development - - I. Characterization of monoclonal antibodies reacting with axon-specific neurofilament epitopes. Int. J. Devl Neurosci. 5, 17-27. 15. Dahl D., Grossi M. and Bignami A. (1984) Masking of epitopes in tissue sections. A study of glial fibrillary acidic (GFA) protein with antisera and monoclonal antibodies. Histochemistry 81,525-531. 16. Dahl D., Labkovsky B. and Bignami A. (1988) Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies, J. comp. Neurol. 271, 445-450. 17. Geisler N.,Vandekerckhove J. andWeberK. (1987) Location and sequence characterization of the major phosphorylation sites of the high molecular mass neurofilament proteins M and H. Fedn Eur. Biochem. Soc. Lett. 221,403--407. 18. Gilad G. M., Gilad V. H. and Dahl D. (1989) Expression of neurofilament immunoreactivity in developing rat cerebellum in vitro and in vivo. Neurosci. Lett. 96, 7-12. 19. Haugh M. C., Probst A., Ulrich J., Kahn J. and Anderton B. H. (1986) Alzheimer neurofibrillary tangles contain phosphorylated and hidden neurofilament epitopes. J. Neurol. Neurosurg. Psych. 49, 1213-1220. 20. Hisanaga S., Kusubata M., Okumura E. and Kishimoto T. (1991) Phosphorylation of neurofilament H subunit at the tail domain by cdc2 kinase dissociates the association to microtubules. J. biol. Chem. 266, 21798-21803. 21. Mangoura D., Sakellaridis N. and Vernadakis A. (1988) Factors influencing neuronal growth in primary cultures derived from 3-day-old chick embryos. Int. J. Devl Neurosci. 6, 89-102. 22. Raju T., Bignami A. and Dahl D. (1981) In vivo and in vitro differentiation of neurons and astrocytes for the rat embryo. Immunofluorescence study with neurofilament and glial filament antisera. Devl Biol. 85, 344-357. 23. Schilling K., Duvernoy C., Keck S. and Pilgrim C. (1989) Detection and partial characterization of a developmentally regulated nuclear antigen in neural cells in vitro and in vivo. J. Histochem. Cytochem. 37, 241-247.
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24. Shaw G., Osborn M. and Weber K. (1986) Reactivity of a panel of neurofilament antibodies on phosphorylated and dephosphorylated neurofilaments. Eur. J. Cell Biol. 42, 1-9. 25. Sternberger L. A. (1986) Immunocytochemistry, 3rd ed. John Wiley, New York, Chichester, Brisbane, Torontt~. 26. Sternberger L. A. and Sternberger N. H. (1983) Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc. natn. Acad. Sci. U.S.A, 80, 6126-6130. 27. Tapscott S. J., Bennett G. S., Toyoma Y., Kleinbart F. and Holtzer H. (1981) Intermediate filament proteins in lhc developing chick spinal cord. Devl Biol. 86, 40-54. 28. Vernadakis A., Sakellaridis N. and Mangoura D. (1986) Growth patterns of primary cultures dissociated from 3-dayold chick embryos: morphological and biochemical comparisons. J. Neurosci. Res. 16, 397-407. 29. Wood J. N., Lathangue N. B., McLachlan D. R., Smith B. J., Anderton B. H. and Dowding A. J. (1985) Chromatin proteins share antigenic determinants with neurofilaments. J. Neurochem. 44, 149-154.
Int. J. Devl. Neuroscience, Vol. 10, No. 6, pp. 473-480, 1992
Printed in Great Britain.
BRAIN FILAMENT PROTEINS IN PRIMARY CULTURES DERIVED FROM CHICK EMBRYOS EARLY IN DEVELOPMENT D. DAHL,* L. MAGGINIand V. H. GILAD Spinal Cord Injury Research Laboratory, Brockton/West Roxbury VA Medical Center; and tDepartment of Pathology, Harvard Medical School, Boston, MA, U.S.A.
(Received 31 July 1992; in revisedform 29 September 1992; accepted 29 September 1992)
Abstract--Brain filament expression and neurofilament post-translational modifications (phosphorylations) were studied in primary cultures derived from whole 3-4 day chick embryos. After 2-3 days in culture, neurofilament-positivecells formed neuronal aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. Aggregates and neuritic bundles were stained with several monoclonal antibodies reacting with phosphorylated neurofilament epitopes. With two monoclonal antibodies reacting with phosphorylated forms of the high molecular weight neurofilament subunit, staining was only observed after 8 and 10 days in vitro. There was a major difference between rat and chicken with respect to astrocyte differentiation in culture. In chicken, the fiat cells surrounding the neuronal aggregates remained constantly GFAP-negative throughout the whole experimental period (10 days). GFAP-positive cells were first observed within the neuronal aggregates on day 8 in vitro. Key words: neurofilament, glial fibrillary acidic protein, phosphorylation.
In this laboratory, in vitro studies on the expression of brain intermediate filaments have been conducted in primary cultures of murine CNS. 5,7'11'18'22 In the present study we report similar studies conducted in a different system, i.e. primary cultures derived from chick embryos early in development. 21'28 The purpose of the work was to provide for the first time a close comparison between two systems used in several studies of brain development, i.e. primary cultures derived from chicken and rat brain. A n o t h e r purpose was to find out whether brain filament expression and post-translational modification remain basically unchanged in phylogeny. EXPERIMENTAL PROCEDURES White Leghorn e m b r y o n a t e d eggs were obtained from Connecticut Valley Biological Supply (Southampton, MA). Cultures were prepared from 3- or 4-day chick embryos according to Vernadakis et al. 28 After removing the m e m b r a n e s , embryos were mechanically dissociated and plated at a density of 200/mm z on glass coverslips coated with poly-L-lysine (100 p~g/ml, Sigma, St Louis, M O ) in D M E M containing 10% (v/v) fetal bovine serum, antibiotic-antimycotic mixture (both from G I B C O Laboratories, G r a n d Island, NY) and 2 m M glutamine. Tissue culture glass coverslips were fixed in cold acetone and stored at - 2 0 ° C before use. Antibodies
Neurofilament polyclonal antibody R96 raised in rabbit against bovine antigen 1° was used in conjunction with monoclonal antibodies derived from mice for double staining experiments. A m o n g the neurofilament monoclonal antibodies previously characterized in this laboratory, those selected for this study (Table 1) stained cryostat sections of chick spinal cord and dorsal root ganglia as previously reported in the rat. 16 Monoclonals NE1424 and SMI3126 were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN) and from Sternberger-Meyer Immunocytochemicals (Jarretsville, M D ) , respectively. Monoclonal RT9719 was a gift from D r B. H. Anderton. All other neurofilament antibodies were produced in this laboratory. 12.14,15 For the staining of GFAP-positive astrocytes, we used monoclonal antibody A 2 D 4 raised against chicken antigen. 13 Conditions for immunolabeling with monoclonal and polyclonal antibodies were as reported in previous studies. 5,11 *Author to whom correspondence should be addressed at: Spinal Cord Injury Research Laboratory (151), Brockton/ West Roxbury VA Medical Center, 1400 VFW Parkway, West Roxbury, MA 02132, U.S.A. 473
474
D. Dahl et al. Table 1. Characterization of neurofilament monoclonat antibodies Monoclonals A9 C21 I120 J J8 J J28 J J29 J J32 J J36 J J47 J J51 NEI4 RT97 SMI31
Neurofilament immunoreactivity
lmrnunoreactivity after phosphatase
NF-H NF-M NF-H NF-H, NF-M NF-H, NF-M NF-H,NF-M NF-H, NF-M NF-H, NF-M NF-H, NF-M NF-H, NF-M NF-H NF-H NF-H, NF-M
Abolished Unchanged Reduced Reduced Reduced Reduced Reduced Reduced Reduced Reduced Abolished Abolished Abolished
NF-M: middle molecular weight neurofilament subunit. NF-H: high molecular weight neurofilament subunit.
RESULTS Two-three days after plating, neurons had formed large aggregates which persisted for the whole experimental period, i.e. 10 days in culture (Fig. 1). The aggregates were surrounded by an extensive neurite outgrowth. Some of these neurites formed rectilinear bundles connecting the aggregates. Cultures derived from 3- and 4-day chick embryo had a similar appearance. Aggregates and neurites, as well as a few isolated neurons, were stained by polyclonal and monoclonal neurofilament antibodies (Fig. 2). Among the antibodies listed in Table 1, only two (monoclonals 1120 and A9) did not stain the cultures at this stage of development. With these antibodies, staining was first observed on day 8 and 10 in vitro and appeared to be confined to a few neurites in the culture (Fig. 3). As previously reported, 25"29several neurofilament monoclonal antibodies reacting with phosphorylated epitopes also stained cell nuclei (Fig. 3). According to Schilling et al. ,23 the staining is due to crossreactivity with hitherto unidentified nuclear proteins and not, as suggested before,17 to the presence in histones of the repetitive sequences (Lys-Ser-Pro) which are the major phosphorylation sites of the middle and high molecular weight neurofilament subunits. Individual neurons within the aggregates could only be identified with monoclonal C21 (Table 1). This antibody selectively stained dorsal root ganglion neurons in primary cultures derived from rat embryos. 11 The large neurons decorated by monoclonal C21 in chicken cultures are illustrated in Fig. 4. As reported before,el the flat or stellate cells forming a monolayer between the aggregates were GFAP-negative, and it was not possible to tell whether they were fibroblasts or immature glia. GFAP immunoreactivity was first observed within the aggregates 8 days after plating. As shown in Fig. 5, it was in the form of a dense mesh of fibers. Individual cells were difficult to identify. DISCUSSION As indicated by neurofilament expression and post-translational modification, neuronal differentiation in chick and rat primary cultures appeared to follow a similar course. In both species, early neurofilament expression and phosphorylation were followed by phosphorylation events occurring with considerable delay (see this paper and Ref. 11). It is important to note that in the two studies the cultures were derived from embryos at comparable stages of neuronal development. Specifically, neurofilament proteins were first detected in rat spinal cord on embryonal day 12 and in dorsal root ganglia on day 13. 22 The corresponding ages for the chick embryo were day 3 and 4, respectively. 6 Similar findings have been reported by other investigators both in murine and chick embryos, e'3,8,9"z7Interestingly, if chick and rat embryonal development is measured in Carnegie stages, 3-day chick embryos and 13-day rat embryos are at Carnegie stage 14 (Fedoroff, personal communication).
Brain intermediate filament proteins in chick primary cultures
475
Fig. 1. Primarydissociatedcultures derived from 4-daychick embryosat day 4 (A) and day 8 (B) in vitro. Neurons form aggregates interconnected by neuritic bundles. Note that on day 8 (B) flat cells surrounding the aggregates form a monolayer. Phase contrast × 136. The demonstration of early and late neurofilament phosphorylation events in such different species as rat and chicken strongly suggests that the heterogeneity of phosphorylated neurofilament epitopes is functionally significant. Little is known of the function of neurofilament phosphorylation except that phosphorylation of cdc2 kinase dissociated the binding of dephosphorylated N F - H to microtubules. 2°
476
D. D a h l et al.
Fig. 2. A primary dissociated culture of 4-day chick embryos at day 4 in vitro is stained with two ncur~filament monoclonal antibodies reacting with phosphorylated epitopes. Note that both antibodies also stain nuclear chromatin. A. Decoration of neuronal aggregate and surrounding neuritic outgrowth with monoclonal SMI31 reacting with phosphorylated forms of the middle and high molecular weight neurofilament subunits. B. Decoration of isolated neurons between the aggregates with monoclonal NE14 reacting with phosphorylated form~ of the high molecular weight neurofilament subunit. The stained neurons probably derive from dor:,al root ganglia. Most neurofilament antibodies reacting with phosphorylated epitopes are axon specific except in dorsal root ganglia where they decorate a subpopulation of sensory neuron. 16 Indirect immunofluorescence, × 136 (A) and × 340 (B).
B r a i n i n t e r m e d i a t e filament p r o t e i n s in chick p r i m a r y cultures
Fig. 3. In a culture derived from 4-day chick embryos immunoreactivity with monoclonal A9 recognizing a phosphorylated form of the high molecular weight neurofilament subunit is confined to few neurites when it first appears on day 10 in vitro. A9-positive neurites in bundles originating from a neuronal aggregate (ag) or apparently not connected with aggregates are shown in A and B, respectively. Arrows in A point to the border of the A9-negative aggregate faintly visible in the background. Indirect immunofluorescence, x 340.
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Fig. 4. In an 8-day primary dissociated culture derived from 4-day chick embryos, neurofilament monoclonal antibody C21 selectively stains large neurons within a neuronal aggregate and between aggregate~ (insert). Indirect immunofluorescence, x 340.
Fig. 5. In a culture derived from 4-day chick embryos, immunoreactivity with G F A P monoclonal antibody A 2 D 4 is confined to a neuronal aggregate on its first appearance at day 8 in vitro. A small aggregate on the left of the figure faintly visible in the background (arrow) is GFAP-negative. Indirect immunofluorescence, × 136.
Brain intermediate filament proteins in chick primary cultures
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Neuronal differentiation followed a similar course in chick and rat primary cultures, but we observed a major difference between the two species with respect to astrocyte development. As previously reported in rat, 1'22 GFAP-positive astrocytes appeared approximately 'on time' in chick cultures, that is after 8 days in vitro. In chick spinal cord, GFAP immunoreactivity was first observed on embryonal day 12 in vivo. 4 However, the site of astrocyte differentiation was markedly different in the two species. In rat, GFAP-positive cells on their first appearance in culture were located in the monolayer of non-neuronal cells, leading to the suggestion that the neuronal aggregates were colonized by astrocytes migrating from the surrounding monolayer.5 As reported before, 21 the cells forming the monolayer remained GFAP-negative in chick cultures. GFAP immunoreactivity first appeared within neuronal aggregates in 8-day cultures. The findings thus suggest that the conditions allowing the development of GFAP-positive cells in monolayers of non-neural cells still remain to be determined in chick. This is perhaps the reason why GFAP has not become a standard astrocyte marker in glial cultures derived from chick embryo. Acknowledgements--This work was supported by USPHS grant NS 13034 and by the Department of Veterans Affairs.
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