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Fibronectin-regulated methionine enkephalin-like and somatostatin-like immunoreactivity in quail neural crest cell cultures
Neuropeptides
4:
457-466,
1984
FIBRONECTIN-REGULATED METHIONINE ENKEPHALIN-LIKE AND SOMATOSTATIN-LIKE IMMUNOREACTMTY IN QUAIL NEURAL CREST CELL CULTURES Maya Sieber-Blum Department of Cell Biology and Anatomy, The Johns Hopkins University, Medicine, Baltimore, Maryland 21205 U.S.A.
School of
ABSTRACT The presence of cells with peptidergic immunoreactivity in neural crest cell cultures and the influence of fibronectin on their in vitro differentjation were evaluated by indirect immunostaining. Met-enkephaxn-E immunoreactive and somatostatin-like immunoreactive cells were observed. Met-enkephalin-like immunoreactive cells resembled endocrine cells. A variety of morphologies was observed in the cells with somatostatin-like immunoreactivity: cells without processes which looked like endocrine cells, multipolar cells with long varicose processes resembling sympathetic neurons, and bipolar cells which were similar to sensory neuroblasts. Differentiation of both types of peptidergic cells was promoted by adding fibronectin to the culture medium. The results suggest that neural crest cell cultures may become a valuable experimental system with which to study the early development of peripheral peptidergic neurons and endocrine cells. INTRODUCTION The neural crest is a transitory tissue of the vertebrate embryo. In avian embryos, as well as in the mouse, neural crest cells of the trunk region disseminate from the neural tube shortly after closure of the neural folds and assume a midline position on the dorsal neural tube (1, 2). Shortly thereafter, the neural crest cells leave the dorsal neural tube bilaterally and migrate into the embryo, localize in different areas, and give rise to a variety of cell types and tissues: they form sensory and autonomic neurons, nerve supporting cells, melanocytes, endocrine cells, and contribute to the head mesenchyme (3-5). Many crest-derived neurons and endocrine cells contain neuropeptides in addition to their primary transmitters. Contrary to the common view, the so-called APUD (Amine Precursor Uptake and Decarboxylation) cells of the gut and pancreas, which also contain these peptides, are not from the neural crest but are of endodermal origin (6, 7). The variety of progeny makes the neural crest an attractive experimental system with which to study the mechanisms that regulate cell differentiation. The most direct way to address this issue is to follow differentiation in cell culture under controlled conditions. Quail neural crest cells of the trunk region differentiate in vitro into melanocytes, adrenergic neurons (81, cholinergic neurons (91, and serotonergicurons IAbbreviations
used are:
PFN - plasma fibronectin; saline. 457
PBS - phosphate-buffered
(10). By _in vitro clonal analysis, we had shown previously that there are at least three distinct types of progenitor cells at the onset of neural crest cell migration: a) pluripotent Cells able to give rise to melanocytes as well as to unpigmented cells, b) cells committed to the pigmented lineage, and c) cells committed to the unpigmented pathway. Some cells in unpigmented and mixed clones have been identified as adrenergic neurons (11, 12). There are several ways of influencing the expression of the different phenotypes. The expression of the adrenergic phenotype is promoted by fibronectin, as judged by the increase in the number of colonies containing adrenergic neurons and the initiation of process formation by these cells in unpigmented and mixed colonies (13). Since the glycoprotein does not influence melanogenesis, fibronectin most likely generates an environment that is conducive to the expression of the adrenergic phenotype. Conversely, tumor-promoting phorbol esters direct prospective unpigmented cells to differentiate preferentially into melanocytes (14). Heart cell conditioned medium promotes the cholinergic phenotype and suppresses expression of the adrenergic phenotype (15). The present study was designed to extend our previous investigation of the formation of neural crest cell lineages and of signals that influence neural crest cell differentiation by seeking additional, crest-derived phenotypes in culture. I report here that, in primary cultures, crest cells differentiate into peptidergic cells that have somatostatin-like and met-enkephalin-like immunoreactivity. Expression of both peptidergic phenotypes is enhanced in the presence of fibronectin. MATERIALS AND METHODS Materials Embryonated quail (Coturnix coturnix japonica) and chick (White Leghorn) eggs: Truslow Farms, Inc., Chestertown, Maryland. Culture medium: alpha-modified MEM (75%; GIBCO, Grand Island, New York); horse serum (15%; GIBCO); extract from U-day-old chick embryo (10%; 11); gentamycin (50 ug/ml; Schering Corp., Kenilworth, pepsin-solubilized bovine dermal collagen (type I, Flow Collagen: New Jersey). Tousimis Research Corp., Rockville, Laboratories, McLean, Virginia). Glutaraldehyde: Sigma Chemical Co., St. Louis, Missouri. Glycine and glyoxylic acid: Maryland. Rabbit antibodies (1:lOO) made against synthetic somatostatin and met-enkephalin: PITC-labeled goat anti-rabbit IgG: Immuno Nuclear Corp., Stillwater, Minnesota. Antibodies Inc., Davis, California. Culture dishes (35 mm): Corning Glass Works, Bethesda Research Laboratories, Inc., Corning, New York. Human plasma fibronectin: Gaithersburg, Maryland. Primary
Neural Crest Cell Cultures
Cultures were prepared as previously described (11): The last six segments of The neural tubes were stage 14 (16) quail embryos were excised and trypsinized. released by vigorous pipetting and placed into collagen-coated 35 mm Culture dishes. The neural crest cells left the neural tubes and migrated onto the collagen substratum. Eighteen hours after explantation, the neural tubes and contaminating mesenchymal cells were removed with a sharpened tungsten needle. The cultures were kept in a humidified atmosphere with 5% CO2 at 36.5OC. The culture medium was replaced every other day. Where indicated, PPNl was added to the culture medium 1 hour and 24 hours after explantation of the neural tubes at a COnCentdiOn of 50 I.% per CuhnC dish.
458
Indirect Immunofluorescence The cultures were rinsed with PBS at pH 7.5, fixed on ice with 4% paraformaldehyde in PBS, and then rinsed again with PBS. The fixative was quenched with 0.15 M glycine in PBS for 30 min on ice. After an additional three rinsing cycles, the cultures were exposed to the primary antibody diluted 1:lOO in PBS containing 0.1% Triton X-100 for 18 hours at 10°C. The cultures were rinsed again and then incubated with the secondary antibody for 1 hour at room temperature. After several rinses with PBS, the cultures were post-fixed with 2% glutaraldehyde for 10 min at room temperature and subsequently mounted with 50% glycerol and a coverslip. For control experiments, either PBS was substituted for the primary antibody, or 50 ul of the primary antibody were preincubated with 50 ul of a 200 pg/ml solution of the antigen for 18 hours at 10°C. Fluorescein fluorescence was observed with a Zeiss Universal microscope (Zeiss, Oberkochen, Federal Republic of Germany) with a 100 W Halogen lamp (Osram, Federal Republic of Germany) at the following filter settings: short pass excitation filter KP 500, dichroic mirror 500 nm, barrier filter 530 nm. RESULTS Cells with Somatostatin-like
Immunoreactivity
In primary cultures, faint somatostatin-like immunoreactivity was observed as early as day 5 and some cultures contained intensely stained cells by day 6. Many The number of immunoreactive cells did not increase cultures were still negative. noticeably between days 6 and 10. By day 12, however, all cultures were positive and contained up to several hundred fluorescent cells per culture. Another slight increase was observed between days 12 and 20 (Table I). When early cultures were treated with Table I Number of Somatostatin-immunoreactive Cells per Culture
Culture
Day
6 6 (colchicinel 8 10 12 20
Number of Immunoreactive Cells per CulSEM ture 8.67 2.50 11.00 12.83 235.00 330.00
+ 5.70 T 1.63 7 9.29 T 8.20 T 41.59 T 83.51
Some cultures were first treated with 10 ug/ml of colchicine for 24 hours to enhance the intensity of staining. Six cultures were tested on each culture day indicated, except on day 20, where the numbers of positive cells in 4 cultures were averaged. Data are expressed as mean counts + SEM.
colchicine in order to increase the level of transmitter within the cells, and thus the intensity of fluorescence (Fig. lE1, the number of positive cells was not noticeably increased (Table I).
of day-12 neural crest cell cultures with Indirect immunostaining Cells with different morphology were observed: multipolar %$%&tin antibodies. cells with long processes that had multiple ramifications and varicosities (A, B); dipolar cells (Cl; and intensely fluorescent cells lacking processes (D). When the cultures were pretreated with colchicine, processes disappeared, cells were rounded, and fluorescence was increased (El. Bar, 10 urn. 460
As judged by their morphology, several different types of somatostatin-like immunoreactive cells were observed: Multipolar cells that had long, varicose processes (Fig. lA, B), bipolar cells (Fig. lC), and very intensely fluorescent cells lacking processes (Fig. 1D). Fluorescence was granular in all of these cells and absent from the nuclear area (Fig. 11. In another series of experiments, neural crest cell cultures were grown in the presence and absence of exogenously added fibronectin. Whereas there was always some cell death by day 4 in normal culture medium, many fewer floating cells were observed at that time if fibronectin had previously been added to the cultures. Moreover, in the presence of fibronectin, there was an increase of positive cultures and these cultures showed significantly more immunoreactive cells than untreated cultures (Table II). Table II Number of Somatostatin-immunoreactive Cells 5 m the Presence and Absence of Added Plasma Fibronectin at Day 6 of Culture
Number of Immunoreactive Cells per Culture
Conditions Without added PFN
1.40 +
With added PFN
0.79
53.70 + 19.75
Plasma fibronectin was added at a concentration of 50 ug/plate 1 hour after plating, and again at 24 hours in culture. Ten cultures were tested for each condition. In an attempt to test the specificity of the somatostatin antibodies, they were treated with somatostatin before being incubated with the cells. This prior treatment inhibited staining of somatostatin-like lmmunoreactive cells completely; no immunoreactive cells were observed in nine day-14 cultures. Likewise, when the primary antibody was replaced by buffer, no fluorescence was observed. Methionine
Enkephalin-like
Immunoreactivity
Cells with met-enkephalin-like immunoreactivity were first observed on culture day 5. They were much less numerous than somatostatin-like immunoreactive cells and had a completely different morphology. Fluorescence was usually limited to the cell body (Fig. 2). Occasionally, some fluorescent granulation was observed within very short (5-10 urn) coarse processes (Fig. 2C, Dl, but these processes did not have As in cells with varicosities which are characteristic of autonomic neurons. somatostatin immunoreactivity, fluorescence was granular. However, the granules in met-enkephalin-immunoreactive cells were somewhat larger (Figs. 2A-Dl than in somatostatin-like immunoreactive cells. Occasionally, cells were observed where the 461
fluorescence had a finer granulation (Fig. 2EL When the cultures were first treated with colchicine, the immunoreactive cells were rounded and intensely fluorescent (Fig. 2FL
Indirect immunostaining of day-14 neural crest cell cultures with Fluorescence was granular, restricted to the cell met iomne-enkephalin antibodies. TV body and absent in the nuclear area. Occasionally, short cell processes were observed (C, D). Most granules (El Pretreatment increased the
cells contained large fluorescent granules (A, B, D). Cells with small or both types of fluorescence (C, F) were observed more seldom. of the cultures with colchicine caused the cells to become round and fluorescence (F). Bar, 10 urn.
Unlike somatostatin immunoreactivity, met-enkephalin immunoreactivity was somewhat increased after colchicine treatment (Table III). A still larger increase in the number of immunoreactive cells was obtained when the cells were grown in the presence of fibronectin (Table III). 462
Table III Number of Met-Enkephalin Immunoreactive Cells in Day 8 Cultures
Number of Immunoreactive Ceils per Culture
Conditions Collagen
2.00 2 2.00
Collagen, colchicine
4.75 + 3.45 11.50 + 2.63
Collagen, PFN
All culture dishes were coated with collagen. Some cultures were treated with 10 ug/ml of colchicine during the 24 hours before antibody adsorption. Other cultures were grown in the presence of PFN (50 ug/dish) that was added at day 0 and again at day 1. Four cultures were tested for each condition. When the met-enkephalin antibodies were pretreated with the antigen to serve as controls, no immunoreactive cells were observed (four day-14 cultures). In addition, the pretreatment inhibited staining in met-enkephalin immunoreactive central nervous system neurons present in neural tube explants (data not shown). In another experiment, the met-enkephalin antibodies were preincubated with leucine-enkephalin and again no immunoreactive neurons were observed (four day-14 cultures). DISCUSSION The following cells differentiate me t-enkephalin-like when the cells were types of cells were
are the main observations reported in this study: 1) Neural crest in vitro into peptidergic neurons with somatostatin-like and 15 m=ac tivity. 2) Expression of both phenotypes was enhanced grown in the presence of fibronectin. 3) Morphologically different observed with both phenotypes.
In addition to the different locations in the central nervous system, somatostatin-like immunoreactivity was observed in the following neurons of the rodent peripheral nervous system and in endocrine cells: primary sensory neurons, sympathetic ganglia, adrenal medulla, and enteric (parasympathetic) ganglia of the gut. Enkephalin immunoreactivity was observed in rodent sympathetic and parasympathetic ganglia and in the adrenal medulla (17-22). In the avian gut, somatostatin-like immunoreactivity was first observed in neurofilament-positive cells on embryonic days 4 (gizzard), 7 (duodenum), and in newly hatched chicks (proventriculus). Met-enkephalin immunoreactivity was first seen on embryonic day 5 (gizzard, duodenum) and day 9 (proventriculus; 231. Thus, the appearance of peptidergic cells in neural crest cell cultures was delayed by 2 to 3 days. This may indicate either that differentiation in culture is delayed (possibly due to prolonged ceII proliferation) or that the antisera used 463
in the different laboratories have different sensitivities. The time course in our cultures coincides with the one observed in culture by Maxwell -et al (24), who, however, obtained the antibodies from the same source as we did. The main function of peptide transmitters seems to be the modulation of primary transmitter action. They may modify the sensitivity to a primary transmitter either of their own nerve endings, or of the postsynaptic neurons, or they may also have a larger spatial range of action. Thus, there are probably no neurons that contain only peptide transmitters but no classical transmitters. Rather, peptide transmitters coexist with classical transmitters. Enkephalins were observed to coexist with catecholamines in neurons of the superior cervical ganglion, in small intensely fluorescent cells, and in cells of the adrenal medulla. Somatostatin was observed to coexist with catecholamines in sympathetic ganglia, in the adrenal medulla (reviewed in 21, 25), and in cultured neural crest cells (24). Judged by the different morphologies of the immunoreactive cells, it is conceivable that there are autonomic neurons (Fig. lA, B), young sensory neuroblasts (Fig. lC), and endocrine cells (Fig. 1D) with somatostatin-like immunoreactivity present Most met-enkephalin immunoreactive cells had large fluorescent in our cultures. granules (Fig. 2A, B, D), while a few cells had more even, small, granular fluorescence (Fig. 2E) or mixed large and small granulation (Fig. 2D). The cells with large granules may contain large dense cored storage granules that may reflect an early stage of vesicle processing (22). The fact that somatostatin immunoreactivity was abolished when the antibody was pretreated with somatostatin suggests that the neural crest cells synthesize either In the case of enkephalin, it is not clear somatostatin or a closely related peptide. whether the antibody can distinguish between met-enkephalin and leu-enkephalin, since prior incubation of the antibody with both peptides abolished staining of the neural crest cells. The enhancement of phenotypic expression in the presence of fibronectin most likely reflects an increased rate of survival and is consistent with our earlier findings that fibronectin promotes adrenergic differentiation (Sieber-Blum -et al, 1981). The data presented here suggest that neural crest cell cultures will be useful in future studies on the formation of neural crest-derived cell lineages and on the regulation of peptidergic neuron expression. ACKNOWLEDGMENTS I thank Mrs. Bella Lee for expert technical assistance, Dr. Pamela Talalay for critically reading the manuscript, Mr. Chester F. Reather for his help with the photomicrographs, and Mrs. Alverta Fields for typing the manuscript. This project was supported by USPHS Grant HD15311. Some of the results of this study have been published in abstract form [M. Sieber-Blum (1983) In vitro differentiation of quail met-enkephalin-immunoreactive and somatoneural crest cells into serotoninergic, statin-immunoreactive neurons, Neuroscience Abstr. 9: 8971.
464
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Noden DM. (1980) The migration and cytodifferentiation of cranial neural crest cells. In Current Research Trends in Prenatal Craniofacial Development (eds RM Pratt, RL Christiansen), pp 3-25. Elsevier/North Holland, New York.
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Andrew A, Kramer B, Rawdon BB. (1983) Gut and pancreatic amine precursor uptake and decarboxylation cells are not neural crest derivatives. Gastroenterology 84, 429-431.
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Cohen AM. (1977) Independent expression of the adrenergic phenotype by neural crest cells -in vitro. Proceedings of the National Academy of Science USA 74, 2899-2903.
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Sieber-Blum M, Sieber F. (1984) Membrane heterogeneity among early quail neural crest cells. Developmental Brain Research, in press.
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Sieber-Blum M, Sieber F, Yamada KM. (1981) Cellular fibronectin promotes Experimental adrenergic differentiation of quail neural crest cells -in vitro. Cell Research 133, 285-295.
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Sieber-Blum M, Sieber F. (1981) Tumor-promoting phorbol esters melanogenesis and prevent expression of the adrenergic phenotype 20, 117-123. neural crest cells. Differentiation
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Sieber-Blum M, Kahn CR. (1982) Suppression of catecholamine and melanin synthesis and promotion of cholinergic differentiation of quail neural CreSt CeUS by heart cell conditioned medium. Stem Cells 2, 344-353.
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Hamburger V, Hamilton of the chick embryo.
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Del Fiacco M, Cuello AC. (1980) Substance P- and enkephalin-containing Neuroscience 5, 803-815. in the rat trigeminal system.
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H. (1951) A series of normal stages in the development Journal of Morphology 88, 49-92.
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Kimura S, Lewis RV, Stern AS, Rossier J, Stein S, Udenfriend S. (1980) Probable precursors of [leulenkephalin and [metlenkephalin in adrenal medulla: peptides of 3-5 kilodaltons. Proceedings of the National Academy of Sciences USA 77, 1681-1685.
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Schultzberg M, Dreyfus CF, Gershon M, Hhkfelt T, Elde RP, Nilsson G, Said S, Goldstein M. (1978) VIP-enkephalin-, substance P- and somatostatin-like immunoreactivity in neurons intrinsic to the intestine: immunohistochemical evidence from organotypic tissue cultures. Brain Research 155, 239-248.
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Schultzberg M, Hiikfelt T, Nilsson G, Terenius L, Rehfels JF, Brown M, Elde R, Goldstein M, Said S. (1980) Distribution of peptide- and catecholamine-containing neurons in the gastro-intestinal tract of rat and guinea pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin and dopamine- @hydroxylase. Neuroscience 5, 689-744.
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Burnstock C, Hhkfelt T, Gershon MD, Iversen LL, Kosterlitz HW, Szurszewski JH (eds). (1979) Non-adrenergic, non-cholinergic autonomic neurotransmission Neurosciences Research Program Bulletin, Vol. 17, MIT Press, mechanisms. Boston.
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Wilson SP, Cubeddu LX, Chang K-J, Viveros OH. (19811 Met-enkephalin, leu-enkephalin and other opiate-like peptides in human pheochromocytoma tumors. Neuropeptides 1, 273-281.
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Epstein ML, Hudis J, Dahl JL. (1983) The development the foregut of the chick. Journal of Neurosciences
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Maxwell AD, Sietz PD, Jean S. (1984) Somatostatin-like immunoreactivity Developmental Biology 101, 357-366. expressed in neural crest cultures.
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Hikfelt T, Johansson, Peptidergic neurons.
26.
Thureson-Klein A. (1983) Exocytosis from large and small dense cored vesicles in noradrenergic nerve terminals. Neuroscience 10, 245-252.
Accepted
0, Ljungdahl A, Lundberg Nature 284, 515-521.
14/g/ 84
466
of peptidergic 3, 2431-2447.
JM, Schultzberg
neurons in is
M. (19801
4:
457-466,
1984
FIBRONECTIN-REGULATED METHIONINE ENKEPHALIN-LIKE AND SOMATOSTATIN-LIKE IMMUNOREACTMTY IN QUAIL NEURAL CREST CELL CULTURES Maya Sieber-Blum Department of Cell Biology and Anatomy, The Johns Hopkins University, Medicine, Baltimore, Maryland 21205 U.S.A.
School of
ABSTRACT The presence of cells with peptidergic immunoreactivity in neural crest cell cultures and the influence of fibronectin on their in vitro differentjation were evaluated by indirect immunostaining. Met-enkephaxn-E immunoreactive and somatostatin-like immunoreactive cells were observed. Met-enkephalin-like immunoreactive cells resembled endocrine cells. A variety of morphologies was observed in the cells with somatostatin-like immunoreactivity: cells without processes which looked like endocrine cells, multipolar cells with long varicose processes resembling sympathetic neurons, and bipolar cells which were similar to sensory neuroblasts. Differentiation of both types of peptidergic cells was promoted by adding fibronectin to the culture medium. The results suggest that neural crest cell cultures may become a valuable experimental system with which to study the early development of peripheral peptidergic neurons and endocrine cells. INTRODUCTION The neural crest is a transitory tissue of the vertebrate embryo. In avian embryos, as well as in the mouse, neural crest cells of the trunk region disseminate from the neural tube shortly after closure of the neural folds and assume a midline position on the dorsal neural tube (1, 2). Shortly thereafter, the neural crest cells leave the dorsal neural tube bilaterally and migrate into the embryo, localize in different areas, and give rise to a variety of cell types and tissues: they form sensory and autonomic neurons, nerve supporting cells, melanocytes, endocrine cells, and contribute to the head mesenchyme (3-5). Many crest-derived neurons and endocrine cells contain neuropeptides in addition to their primary transmitters. Contrary to the common view, the so-called APUD (Amine Precursor Uptake and Decarboxylation) cells of the gut and pancreas, which also contain these peptides, are not from the neural crest but are of endodermal origin (6, 7). The variety of progeny makes the neural crest an attractive experimental system with which to study the mechanisms that regulate cell differentiation. The most direct way to address this issue is to follow differentiation in cell culture under controlled conditions. Quail neural crest cells of the trunk region differentiate in vitro into melanocytes, adrenergic neurons (81, cholinergic neurons (91, and serotonergicurons IAbbreviations
used are:
PFN - plasma fibronectin; saline. 457
PBS - phosphate-buffered
(10). By _in vitro clonal analysis, we had shown previously that there are at least three distinct types of progenitor cells at the onset of neural crest cell migration: a) pluripotent Cells able to give rise to melanocytes as well as to unpigmented cells, b) cells committed to the pigmented lineage, and c) cells committed to the unpigmented pathway. Some cells in unpigmented and mixed clones have been identified as adrenergic neurons (11, 12). There are several ways of influencing the expression of the different phenotypes. The expression of the adrenergic phenotype is promoted by fibronectin, as judged by the increase in the number of colonies containing adrenergic neurons and the initiation of process formation by these cells in unpigmented and mixed colonies (13). Since the glycoprotein does not influence melanogenesis, fibronectin most likely generates an environment that is conducive to the expression of the adrenergic phenotype. Conversely, tumor-promoting phorbol esters direct prospective unpigmented cells to differentiate preferentially into melanocytes (14). Heart cell conditioned medium promotes the cholinergic phenotype and suppresses expression of the adrenergic phenotype (15). The present study was designed to extend our previous investigation of the formation of neural crest cell lineages and of signals that influence neural crest cell differentiation by seeking additional, crest-derived phenotypes in culture. I report here that, in primary cultures, crest cells differentiate into peptidergic cells that have somatostatin-like and met-enkephalin-like immunoreactivity. Expression of both peptidergic phenotypes is enhanced in the presence of fibronectin. MATERIALS AND METHODS Materials Embryonated quail (Coturnix coturnix japonica) and chick (White Leghorn) eggs: Truslow Farms, Inc., Chestertown, Maryland. Culture medium: alpha-modified MEM (75%; GIBCO, Grand Island, New York); horse serum (15%; GIBCO); extract from U-day-old chick embryo (10%; 11); gentamycin (50 ug/ml; Schering Corp., Kenilworth, pepsin-solubilized bovine dermal collagen (type I, Flow Collagen: New Jersey). Tousimis Research Corp., Rockville, Laboratories, McLean, Virginia). Glutaraldehyde: Sigma Chemical Co., St. Louis, Missouri. Glycine and glyoxylic acid: Maryland. Rabbit antibodies (1:lOO) made against synthetic somatostatin and met-enkephalin: PITC-labeled goat anti-rabbit IgG: Immuno Nuclear Corp., Stillwater, Minnesota. Antibodies Inc., Davis, California. Culture dishes (35 mm): Corning Glass Works, Bethesda Research Laboratories, Inc., Corning, New York. Human plasma fibronectin: Gaithersburg, Maryland. Primary
Neural Crest Cell Cultures
Cultures were prepared as previously described (11): The last six segments of The neural tubes were stage 14 (16) quail embryos were excised and trypsinized. released by vigorous pipetting and placed into collagen-coated 35 mm Culture dishes. The neural crest cells left the neural tubes and migrated onto the collagen substratum. Eighteen hours after explantation, the neural tubes and contaminating mesenchymal cells were removed with a sharpened tungsten needle. The cultures were kept in a humidified atmosphere with 5% CO2 at 36.5OC. The culture medium was replaced every other day. Where indicated, PPNl was added to the culture medium 1 hour and 24 hours after explantation of the neural tubes at a COnCentdiOn of 50 I.% per CuhnC dish.
458
Indirect Immunofluorescence The cultures were rinsed with PBS at pH 7.5, fixed on ice with 4% paraformaldehyde in PBS, and then rinsed again with PBS. The fixative was quenched with 0.15 M glycine in PBS for 30 min on ice. After an additional three rinsing cycles, the cultures were exposed to the primary antibody diluted 1:lOO in PBS containing 0.1% Triton X-100 for 18 hours at 10°C. The cultures were rinsed again and then incubated with the secondary antibody for 1 hour at room temperature. After several rinses with PBS, the cultures were post-fixed with 2% glutaraldehyde for 10 min at room temperature and subsequently mounted with 50% glycerol and a coverslip. For control experiments, either PBS was substituted for the primary antibody, or 50 ul of the primary antibody were preincubated with 50 ul of a 200 pg/ml solution of the antigen for 18 hours at 10°C. Fluorescein fluorescence was observed with a Zeiss Universal microscope (Zeiss, Oberkochen, Federal Republic of Germany) with a 100 W Halogen lamp (Osram, Federal Republic of Germany) at the following filter settings: short pass excitation filter KP 500, dichroic mirror 500 nm, barrier filter 530 nm. RESULTS Cells with Somatostatin-like
Immunoreactivity
In primary cultures, faint somatostatin-like immunoreactivity was observed as early as day 5 and some cultures contained intensely stained cells by day 6. Many The number of immunoreactive cells did not increase cultures were still negative. noticeably between days 6 and 10. By day 12, however, all cultures were positive and contained up to several hundred fluorescent cells per culture. Another slight increase was observed between days 12 and 20 (Table I). When early cultures were treated with Table I Number of Somatostatin-immunoreactive Cells per Culture
Culture
Day
6 6 (colchicinel 8 10 12 20
Number of Immunoreactive Cells per CulSEM ture 8.67 2.50 11.00 12.83 235.00 330.00
+ 5.70 T 1.63 7 9.29 T 8.20 T 41.59 T 83.51
Some cultures were first treated with 10 ug/ml of colchicine for 24 hours to enhance the intensity of staining. Six cultures were tested on each culture day indicated, except on day 20, where the numbers of positive cells in 4 cultures were averaged. Data are expressed as mean counts + SEM.
colchicine in order to increase the level of transmitter within the cells, and thus the intensity of fluorescence (Fig. lE1, the number of positive cells was not noticeably increased (Table I).
of day-12 neural crest cell cultures with Indirect immunostaining Cells with different morphology were observed: multipolar %$%&tin antibodies. cells with long processes that had multiple ramifications and varicosities (A, B); dipolar cells (Cl; and intensely fluorescent cells lacking processes (D). When the cultures were pretreated with colchicine, processes disappeared, cells were rounded, and fluorescence was increased (El. Bar, 10 urn. 460
As judged by their morphology, several different types of somatostatin-like immunoreactive cells were observed: Multipolar cells that had long, varicose processes (Fig. lA, B), bipolar cells (Fig. lC), and very intensely fluorescent cells lacking processes (Fig. 1D). Fluorescence was granular in all of these cells and absent from the nuclear area (Fig. 11. In another series of experiments, neural crest cell cultures were grown in the presence and absence of exogenously added fibronectin. Whereas there was always some cell death by day 4 in normal culture medium, many fewer floating cells were observed at that time if fibronectin had previously been added to the cultures. Moreover, in the presence of fibronectin, there was an increase of positive cultures and these cultures showed significantly more immunoreactive cells than untreated cultures (Table II). Table II Number of Somatostatin-immunoreactive Cells 5 m the Presence and Absence of Added Plasma Fibronectin at Day 6 of Culture
Number of Immunoreactive Cells per Culture
Conditions Without added PFN
1.40 +
With added PFN
0.79
53.70 + 19.75
Plasma fibronectin was added at a concentration of 50 ug/plate 1 hour after plating, and again at 24 hours in culture. Ten cultures were tested for each condition. In an attempt to test the specificity of the somatostatin antibodies, they were treated with somatostatin before being incubated with the cells. This prior treatment inhibited staining of somatostatin-like lmmunoreactive cells completely; no immunoreactive cells were observed in nine day-14 cultures. Likewise, when the primary antibody was replaced by buffer, no fluorescence was observed. Methionine
Enkephalin-like
Immunoreactivity
Cells with met-enkephalin-like immunoreactivity were first observed on culture day 5. They were much less numerous than somatostatin-like immunoreactive cells and had a completely different morphology. Fluorescence was usually limited to the cell body (Fig. 2). Occasionally, some fluorescent granulation was observed within very short (5-10 urn) coarse processes (Fig. 2C, Dl, but these processes did not have As in cells with varicosities which are characteristic of autonomic neurons. somatostatin immunoreactivity, fluorescence was granular. However, the granules in met-enkephalin-immunoreactive cells were somewhat larger (Figs. 2A-Dl than in somatostatin-like immunoreactive cells. Occasionally, cells were observed where the 461
fluorescence had a finer granulation (Fig. 2EL When the cultures were first treated with colchicine, the immunoreactive cells were rounded and intensely fluorescent (Fig. 2FL
Indirect immunostaining of day-14 neural crest cell cultures with Fluorescence was granular, restricted to the cell met iomne-enkephalin antibodies. TV body and absent in the nuclear area. Occasionally, short cell processes were observed (C, D). Most granules (El Pretreatment increased the
cells contained large fluorescent granules (A, B, D). Cells with small or both types of fluorescence (C, F) were observed more seldom. of the cultures with colchicine caused the cells to become round and fluorescence (F). Bar, 10 urn.
Unlike somatostatin immunoreactivity, met-enkephalin immunoreactivity was somewhat increased after colchicine treatment (Table III). A still larger increase in the number of immunoreactive cells was obtained when the cells were grown in the presence of fibronectin (Table III). 462
Table III Number of Met-Enkephalin Immunoreactive Cells in Day 8 Cultures
Number of Immunoreactive Ceils per Culture
Conditions Collagen
2.00 2 2.00
Collagen, colchicine
4.75 + 3.45 11.50 + 2.63
Collagen, PFN
All culture dishes were coated with collagen. Some cultures were treated with 10 ug/ml of colchicine during the 24 hours before antibody adsorption. Other cultures were grown in the presence of PFN (50 ug/dish) that was added at day 0 and again at day 1. Four cultures were tested for each condition. When the met-enkephalin antibodies were pretreated with the antigen to serve as controls, no immunoreactive cells were observed (four day-14 cultures). In addition, the pretreatment inhibited staining in met-enkephalin immunoreactive central nervous system neurons present in neural tube explants (data not shown). In another experiment, the met-enkephalin antibodies were preincubated with leucine-enkephalin and again no immunoreactive neurons were observed (four day-14 cultures). DISCUSSION The following cells differentiate me t-enkephalin-like when the cells were types of cells were
are the main observations reported in this study: 1) Neural crest in vitro into peptidergic neurons with somatostatin-like and 15 m=ac tivity. 2) Expression of both phenotypes was enhanced grown in the presence of fibronectin. 3) Morphologically different observed with both phenotypes.
In addition to the different locations in the central nervous system, somatostatin-like immunoreactivity was observed in the following neurons of the rodent peripheral nervous system and in endocrine cells: primary sensory neurons, sympathetic ganglia, adrenal medulla, and enteric (parasympathetic) ganglia of the gut. Enkephalin immunoreactivity was observed in rodent sympathetic and parasympathetic ganglia and in the adrenal medulla (17-22). In the avian gut, somatostatin-like immunoreactivity was first observed in neurofilament-positive cells on embryonic days 4 (gizzard), 7 (duodenum), and in newly hatched chicks (proventriculus). Met-enkephalin immunoreactivity was first seen on embryonic day 5 (gizzard, duodenum) and day 9 (proventriculus; 231. Thus, the appearance of peptidergic cells in neural crest cell cultures was delayed by 2 to 3 days. This may indicate either that differentiation in culture is delayed (possibly due to prolonged ceII proliferation) or that the antisera used 463
in the different laboratories have different sensitivities. The time course in our cultures coincides with the one observed in culture by Maxwell -et al (24), who, however, obtained the antibodies from the same source as we did. The main function of peptide transmitters seems to be the modulation of primary transmitter action. They may modify the sensitivity to a primary transmitter either of their own nerve endings, or of the postsynaptic neurons, or they may also have a larger spatial range of action. Thus, there are probably no neurons that contain only peptide transmitters but no classical transmitters. Rather, peptide transmitters coexist with classical transmitters. Enkephalins were observed to coexist with catecholamines in neurons of the superior cervical ganglion, in small intensely fluorescent cells, and in cells of the adrenal medulla. Somatostatin was observed to coexist with catecholamines in sympathetic ganglia, in the adrenal medulla (reviewed in 21, 25), and in cultured neural crest cells (24). Judged by the different morphologies of the immunoreactive cells, it is conceivable that there are autonomic neurons (Fig. lA, B), young sensory neuroblasts (Fig. lC), and endocrine cells (Fig. 1D) with somatostatin-like immunoreactivity present Most met-enkephalin immunoreactive cells had large fluorescent in our cultures. granules (Fig. 2A, B, D), while a few cells had more even, small, granular fluorescence (Fig. 2E) or mixed large and small granulation (Fig. 2D). The cells with large granules may contain large dense cored storage granules that may reflect an early stage of vesicle processing (22). The fact that somatostatin immunoreactivity was abolished when the antibody was pretreated with somatostatin suggests that the neural crest cells synthesize either In the case of enkephalin, it is not clear somatostatin or a closely related peptide. whether the antibody can distinguish between met-enkephalin and leu-enkephalin, since prior incubation of the antibody with both peptides abolished staining of the neural crest cells. The enhancement of phenotypic expression in the presence of fibronectin most likely reflects an increased rate of survival and is consistent with our earlier findings that fibronectin promotes adrenergic differentiation (Sieber-Blum -et al, 1981). The data presented here suggest that neural crest cell cultures will be useful in future studies on the formation of neural crest-derived cell lineages and on the regulation of peptidergic neuron expression. ACKNOWLEDGMENTS I thank Mrs. Bella Lee for expert technical assistance, Dr. Pamela Talalay for critically reading the manuscript, Mr. Chester F. Reather for his help with the photomicrographs, and Mrs. Alverta Fields for typing the manuscript. This project was supported by USPHS Grant HD15311. Some of the results of this study have been published in abstract form [M. Sieber-Blum (1983) In vitro differentiation of quail met-enkephalin-immunoreactive and somatoneural crest cells into serotoninergic, statin-immunoreactive neurons, Neuroscience Abstr. 9: 8971.
464
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