Cyclic AMP Signaling Functions as a Bimodal Switch in ...

MOLECULAR AND CELLULAR BIOLOGY, May 2000, p. 3004–3014 0270-7306/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 20, No. 9

Cyclic AMP Signaling Functions as a Bimodal Switch in Sympathoadrenal Cell Development in Cultured Primary Neural Crest Cells MATTHEW L. BILODEAU, THERESA BOULINEAU, RONALD L. HULLINGER, AND OURANIA M. ANDRISANI* Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907 Received 3 August 1999/Returned for modification 28 September 1999/Accepted 17 February 2000

Cells of the vertebrate neural crest (crest cells) are an invaluable model system to address cell fate specification. Crest cells are amenable to tissue culture, and they differentiate to a variety of neuronal and nonneuronal cell types. Earlier studies have determined that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that elevate intracellular cyclic AMP (cAMP) stimulate the development of the sympathoadrenal (SA, adrenergic) lineage in neural crest cultures. To investigate whether interactive mechanisms between signaling pathways influence crest cell differentiation, we characterized the combinatorial effects of BMP-2 and cAMP-elevating agents on the development of quail trunk neural crest cells in primary culture. We report that the cAMP signaling pathway modulates both positive and negative signals influencing the development of SA cells. Specifically, we show that moderate activation of cAMP signaling promotes, in synergy with BMP-2, SA cell development and the expression of the SA lineage-determining gene Phox2a. By contrast, robust activation of cAMP signaling opposes, even in the presence of BMP-2, SA cell development and the expression of the SA lineage-determining ASH-1 and Phox2 genes. We conclude that cAMP signaling acts as a bimodal regulator of SA cell development in neural crest cultures. regulated by cAMP and is dependent on a CRE (27, 65). In neural crest cultures, CA and elevated cAMP promote the development of the SA lineage (9, 74). The developmental relevance of CA is supported in vivo by the presence of CA (26), ␤-adrenergic receptors, and CA biosynthetic enzymes (14) in early-stage embryos, prior to the differentiation of neurons. As crest cells form the sympathetic trunks, the notochord mesenchyme can synthesize CA from l-DOPA (2, 34), and both notochord and ventral neural tube accumulate and store CA (2, 34, 38, 50). Furthermore, the induction of SA cell differentiation in vivo is dependent on the presence of either notochord or neural tube (18, 64, 67). Bone morphogenetic protein 2 (BMP-2), BMP-4, or BMP7/OP-1 also promotes development of the SA lineage in neural crest cultures (55, 68, 70). BMP-2, -4, and -7 are produced by endothelial cells of the aorta as crest cells form the sympathetic trunks and adrenal gland primordia (55, 58), suggesting that these factors are relevant to SA cell development in vivo. Moreover, the causal link of BMP-2 to SA cell development has been demonstrated by expression of a constitutively active mutant of the BMP type I receptor in cultured crest cells (69). Although the mechanism of regulation has not been elucidated, BMPs induce the expression of genes essential to SA lineage determination. For example, BMP-2 induces expression of the basic helix-loop-helix transcription factor ASH-1 (58), a homolog of the Drosophila proneural achaete-scute complex (29) with multiple roles in vertebrate autonomic neurogenesis (reviewed in reference 17). Targeted deletion of the ASH-1 gene in the mouse has demonstrated that ASH-1 is essential for development of the SA lineage (7, 19, 25, 36). In primary cultures of neural crest stem cells (63), ASH-1 maintains competence for neurogenesis (43) and promotes autonomic differentiation of committed neuronal precursor cells (61). In addition, the closely related paired-like homeodomain transcription factors Phox2a (49) and Phox2b (52, 53) are essential for development of the SA

The neural crest is a stem cell-like population of multipotent embryonic cells which separates from the neuroepithelium as the neural tube forms, migrates to numerous and diverse locations, and differentiates to diverse cell types (reviewed in reference 35). Neural crest cells from the trunk region of the embryo produce sensory and sympathetic neurons, peripheral glial cells, adrenergic (chromaffin) cells of the adrenal medulla, and melanocytes (reviewed in reference 40). It is accepted that the developmental fate of most crest cells is determined by the interplay of signals from the microenvironments encountered during and after migration (reviewed in reference 3). Consistent with this model, sympathetic neurons and adrenergic cells differentiate from fate-restricted sympathoadrenal (SA) progenitor cells (5). These SA progenitor cells arise from trunkderived crest cells migrating to dorsal-lateral aspects of the aorta, forming the sympathetic trunks (strands), and to the adrenal gland primordia (2, 33). At other axial positions, SA progenitor cells are precursors for vagal-derived crest cells differentiating to subsets of enteric neurons and thyroid parafollicular (calcitonin) cells and for sacral-derived crest cells differentiating to parasympathetic neurons (reviewed in reference 4). Those cells committed to the SA lineage are distinguished by their at least transient expression of catecholamines (CA) and enzymes for CA biosynthesis, i.e., tyrosine-3-hydroxylase (TH), the rate-limiting enzyme in CA biosynthesis, and dopamine-␤-hydroxylase (DBH), the enzyme converting dopamine to norepinephrine. Expression of TH is tissue specific and dependent, in part, on a cyclic AMP (cAMP)-responsive element (CRE) (11, 32, 39) which is activated by CA and ␤-adrenergic receptors in immortalized crest cells (9). Similarly, expression of DBH is * Corresponding author. Mailing address: Department of Basic Medical Sciences, 1246 Lynn Hall, Purdue University, West Lafayette, IN 47907-1246. Phone: (765) 494-8131. Fax: (765) 494-0781. E-mail: [email protected]. 3004

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EFFECT OF cAMP AND BMP-2 ON CULTURED NEURAL CREST CELLS

lineage. Phox2 proteins function synergistically with the cAMP signaling pathway, via neighboring homeodomain and CRE cis-acting elements, to directly regulate transcription of both the TH and DBH genes (15, 31, 66, 71, 72). The Phox2 proteins, in addition to being necessary (42) and sufficient (62) for SA cell development, likely promote survival of SA progenitors by regulating the expression of the glial-derived neurotrophic factor receptor c-Ret (10, 44, 49, 53). Although expression of the Phox2a gene, but not the Phox2b gene, is regulated by ASH-1 (25, 44), these genes display cross-regulation (52, 53, 62); i.e., the expression of one induces expression of the other. Moreover, Phox2b is necessary for the maintenance, but not the induction, of ASH-1 (53). The molecular mechanisms that regulate the expression of these SA lineage-determining genes remain to be elucidated. Likewise, the molecular mechanisms that integrate the function of the SA lineage-determining transcription factors with other transcription factors in directing the neurogenic program, the specification of neurotransmitter identity, and the survival of the progenitor cell have yet to be deciphered. Accordingly, the combinatorial effect of the BMP-2 and cAMP signaling pathways on SA cell development and, specifically, on the expression and function of the SA lineage-determining factors remains to be explored. Using primary cultures of avian neural crest cells, we explored interactive mechanisms between the BMP-2 and cAMP signaling pathways. Herein, we report that the cAMP signaling pathway is a dynamic regulator of SA cell development. Moderate levels of cAMP signaling exert an SA-promoting influence which functions in synergy with BMP-2 to dramatically stimulate SA cell development. By contrast, high levels of cAMP signaling modulate a dominant antagonism that, even in the presence of BMP-2, blocks SA cell development. Furthermore, we show that the SA-promoting effect of cAMP signaling involves the induction of the Phox2a gene and that the SA-antagonizing effect involves inhibition of the expression of the SA lineage-determining genes ASH-1, Phox2a, and Phox2b. MATERIALS AND METHODS Neural crest cultures. Primary cultures of trunk neural crest cells were prepared from Japanese quail (Coturnix coturnix) embryos stage 12 to 13 (23) essentially as described by Maxwell et al. (47). Eggs were incubated for 47.5 h at 37.5°C with 58% humidity. Embryos were separated from the yolk and rinsed in Hanks’ balanced salt solution buffered with 15 mM HEPES (pH 7.4). The neural tubes and associated neural crests were dissected from embryos as explants of the trunk region including the last five somites and extending through the unsegmented mesoderm to the chordal neural hinge. Explants were incubated in pancreatin (6.25 mg/ml) until the neural tubes were freed from adjacent somites, notochord, and surface ectoderm. Neural tubes were washed in growth medium and plated in dishes coated with Vitrogen 100 (Collagen Corporation). In primary culture, numerous crest cells emigrate from the neural tubes onto the surface of the culture dish. After 42 h, neural tubes were dissected free using tungsten needles and removed by washing from the plate with calcium- and magnesium-free phosphate-buffered saline, pH 7.4 (CMF-PBS). Crest cells were harvested in growth medium following a brief treatment with trypsin-EDTA. Mass cultures. Cells were suspended in growth medium and seeded at a density of 320 cells/mm2 in tissue culture dishes treated with bovine fibronectin (40 ␮g/ml; Sigma). Cells were allowed to attach for 2 h, and then the seeding medium was replaced with 2 ml of growth medium. Cells were fed by an exchange of 1 ml of growth medium on day 3 after passage into secondary culture and every other day thereafter. Clonal cultures. Cells were suspended in growth medium at a density of 100 cells/ml, and 1-ml aliquots were seeded into 35-mm-diameter tissue culture dishes treated with both Vitrogen 100 and bovine fibronectin (20 ␮g/ml). After allowing 2 h for cells to attach, the clonal cultures were maintained as described for mass cultures. Growth medium and other reagents. All cultures were grown at 37°C in a 5% CO2, humidified incubator. Growth medium contained 75 ml of Dulbecco modified Eagle medium—Ham’s F-12 (Life Technologies), 15 ml of heat-inactivated horse serum (HyClone), 10 ml of day 9 chick embryo extract (8), 10 mg of gentamicin sulfate, 10 mg of kanamycin sulfate, 1 ml of 7.5% sodium bicarbon-

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ate, 1 ml of 0.2 M L-glutamine, and 1 ml of 100⫻ stock vitamin mix (47). Stock vitamin mix contained 1 mg of 6,7-dimethyl-5,6,7,8-tetrahydropterine, 100 mg of L-ascorbic acid, and 5 mg of oxidized glutathione in 20 ml of distilled water, pH 6.0 (45, 47). BMP-2 was generously provided by Genetics Institute, Inc., and was reconstituted as recommended by the provider. 8-Bromo-cAMP (8-Br-cAMP) and norepinephrine were obtained from Sigma and reconstituted in water. Forskolin, 3-isobutyl-1-methylxanthine (IBMX; Sigma), vinpocetine, Ro 20-1724, and MY5445 were reconstituted in dimethyl sulfoxide. The presence of 0.1% dimethyl sulfoxide had no observable effect on crest cell growth or differentiation (data not shown). Stock solutions (1,000⫻) for all reagents were maintained at ⫺80°C. Unless otherwise noted, all experimental reagents were obtained from Biomol (Plymouth Meeting, Pa.) and added to the cultures at the designated feeding times. Western blot analysis. Cells from primary neural crest cultures were subcultured in 24-well dishes and maintained as described above. Cultures were harvested by triturating in 120 ␮l of radioimmunoprecipitation buffer (150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 10 ␮g of leupeptin and 25 ␮g of aprotinin per ml, 1.0% Triton X-100, 50 mM Tris [pH 7.6]) and sonicating on ice for 20 s. Total protein concentration was determined by the Bio-Rad protein assay. Cell extract (20 ␮g) was boiled with a 1:5 dilution of sample buffer (10% sodium dodecyl sulfate [SDS], 50% glycerol, 5% 2-mercaptoethanol, 0.4 M Tris [pH 6.8]), electrophoresed on an SDS–10% polyacrylamide gel, and transferred to nitrocellulose. Immunoblot analysis used a 1:5 dilution of supernatant containing the monoclonal anti-TH antibody (13) (Developmental Studies Hybridoma Bank, University of Iowa) and a 1:300 dilution of the rabbit antiactin antibody (Sigma). Detection was with 1:10,000 and 1:2,000 dilutions of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (Jackson Laboratory) and anti-rabbit IgG (Vector) antibodies, respectively, using the Amersham ECL (enhanced chemiluminescence) detection system. Immunochemical and histochemical fluorescence. TH-expressing cells were visualized using indirect immunofluorescence. Neural crest cultures were fixed for 20 min in 4% paraformaldehyde and permeabilized for 30 min with PBT (0.2% bovine serum albumin and 0.1% Triton X-100 in CMF-PBS [pH 7.4]). The monoclonal anti-TH antibody (see “Western blot analysis” above) was applied as undiluted supernatant for 40 min followed by three washes with PBT. A 1:64 dilution of the fluorescein isothiocyanate-conjugated donkey anti-mouse IgG antibody (Sigma) was applied in the dark for 40 min followed by washing once with PBT and twice with CMF-PBS. Cultures were rinsed with distilled water and mounted using FluorSave reagent (Calbiochem) and a coverslip. CA-containing cells were visualized by the procedure of Furness et al. (16), which produces a water-stable fluorophore. Neural crest cultures were washed twice with CMF-PBS followed by incubation in 4% paraformaldehyde and 0.1% glutaraldehyde for 2 h at room temperature. Cultures were washed once with CMF-PBS and mounted using glycerol and a coverslip. PKA activity. Cells from primary neural crest cultures were subcultured as described above except that a 6 ⫻ 105 cells were seeded in a 35-mm-diameter dish. Following 2 h for cell attachment, seeding medium was replaced with 2 ml of growth medium and the indicated cAMP-elevating agents. After 2 h, the cells were harvested by scraping in extraction buffer (5 mM EDTA, 50 mM Tris [pH 7.5]) and lysed by briefly sonicating on ice. Protein kinase A (PKA) activity was analyzed using a radiometric PKA assay system (Life Technologies) as described by the manufacturer. RNA and RT-PCR. Total RNA was isolated from neural crest cells subcultured in 35-mm-diameter plates using 1 ml of TRIZOL reagent (Life Technologies) as described by the manufacturer. Reverse transcription-PCR (RT-PCR) was performed with total RNA (1 ␮g) and gene-specific primers using the Titan One Tube RT-PCR system (Roche Biomolecular). Typically 30 cycles of amplification were conducted using the following conditions: denaturation at 94°C, annealing at 55°C, and extension at 68°C, with 1 min at each step. Primers for ASH-1 (5⬘ sense oligonucleotide, 5⬘-AACCGAGTCAAGCTGGTGAA-3⬘; 3⬘ antisense oligonucleotide, 5⬘-TCAGAACCAGCTGGTGAA-3⬘), Phox2a (5⬘ sense oligonucleotide, 5⬘-CGTCCGCCTACGATTTCAACC-3⬘; 3⬘ antisense oligonucleotide, 5⬘-TGATGGCCGATGGGTCCGAA-3⬘), Phox2b (5⬘ sense oligonucleotide, 5⬘TCGAGCCTGGCTTCAGCGTAT-3⬘; 3⬘ antisense oligonucleotide, 5⬘-TCAAA CCGCCGTGGTCGGTG-3⬘), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5⬘ sense oligonucleotide, 5⬘-GTGAAAGTCGGAGTCAAC-3⬘; 3⬘ antisense oligonucleotide, 5⬘-TGGTGCACGATGCATTGC-3⬘) were derived from the sequenced chicken genes (J.-F. Brunet, personal communication; 28, 51). Primers for TH (5⬘ sense oligonucleotide, 5⬘-CTGGAAGGAGGTGTACA GTA-3⬘; 3⬘ antisense oligonucleotide, 5⬘-AGCAGCGTCAGGATCAAAGT-3⬘) were derived from the sequenced quail gene (12). To enhance photocopy reproducibility, negative digital images were produced from photographs of ethidium bromide-stained agarose gels by using Adobe Photoshop software.

RESULTS Effects of BMP-2 and cAMP on SA cell development. To investigate whether the BMP-2 and cAMP signaling pathways exert a combinatorial influence on SA cell development, we used primary cultures of quail neural crest cells and assessed

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FIG. 1. Western blot analysis of TH protein expression in neural crest cultures treated with cAMP-elevating agents and BMP-2. Cellular extracts were prepared from cultures grown 7 days in the presence of BMP-2 (10 ng/ml) and the cAMP-elevating agents IBMX (100 ␮M), forskolin (10 ␮M), and 8-Br-cAMP (100 ␮M) as indicated. The 63-kDa TH protein and 42-kDa actin protein, serving as an internal control, are indicated by arrows. The histogram was derived by densitometric scanning of blots from three independent cellular extract preparations. The effects of IBMX and forskolin compared to the control, the combination of BMP-2 with IBMX compared to treatment with either agent alone, and the combination of BMP-2 with forskolin or 8-Br-cAMP are significant at P ⬍ 0.05 (analysis of variance).

the expression of phenotypic markers of the SA lineage, namely, TH and CA. We activated the cAMP signaling pathway by using three distinct types of cAMP-elevating agents: IBMX, a nonselective inhibitor of cyclic nucleotide phosphodiesterases (PDE) (6); forskolin, an activator of adenylate cyclase (37, 57); and 8-Br-cAMP, an analog of cAMP with specificity for activating PKA and reduced hydrolysis by PDE (24, 48, 56). These cAMP-elevating agents, applied alone or in combination with BMP-2, resulted in complex effects on the development of SA cells (Fig. 1 and 2) and on the overall growth of the neural crest cultures (Table 1). Reported herein, we measured SA cell development by quantifying the relative expression levels of TH detected in Western blot assays (Fig. 1). In a related study, results from this method of analysis were shown to closely correlate with the proportion of TH-positive crest cells detected by indirect immunofluorescence (data not shown). Additionally, we qualitatively confirmed the effects on SA cell development by monitoring the generation of THimmunoreactive (TH-positive) and CA-histofluorescent (CApositive) cells (Fig. 2). Consistent with results reported by Varley and Maxwell (68), treatment of the cultures with BMP-2 (10 ng/ml) dramatically increased both the expression of TH protein (Fig. 1, compare lanes 1 and 2) and the development of TH-positive and CApositive SA cells (Fig. 2). Treatment of the cultures with either 10 ␮M forskolin or 100 ␮M IBMX significantly increased the

FIG. 2. Differentiation of TH- and CA-positive cells in neural crest cultures. Crest cells were grown for 6 days in the presence of 10 ng of BMP-2 per ml and 100 ␮M IBMX as indicated. (A) Indirect immunofluorescence to visualize THexpressing cells; (B) formaldehyde-induced fluorescence to visualize CA-containing cells (bar ⫽ 100 ␮m).

expression of TH protein (Fig. 1, lanes 3 and 5 compared to lane 1); however, treatment with 100 ␮M 8-Br-cAMP produced no detectable increase in TH protein expression (lane 7). Interestingly, the combination of BMP-2 and IBMX resulted in a synergistic 2.7-fold increase in the expression of TH protein (Fig. 1, lane 4 compared to lanes 2 and 3). In contrast to the synergistic effect of BMP-2 and IBMX, the combination of BMP-2 with forskolin or 8-Br-cAMP decreased the expres-

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TABLE 1. Effects of BMP-2 and cAMP-elevating agents on overall growth of crest cell cultures Total cell no.a Agent

Control BMP-2

Control

IBMX (100 ␮M)

Forskolin (10 ␮M)

8-Br-cAMP (100 ␮M)

339,000 ⫾ 10,800 431,000 ⫾ 15,200

357,000 ⫾ 2,100 310,000 ⫾ 3,500

322,000 ⫾ 7,900 237,000 ⫾ 7,100

59,500 ⫾ 2,300 27,700 ⫾ 1,800

a Cultures were grown in the presence of BMP-2 (10 ng/ml) and cAMP-elevating agents as indicated and analyzed for total cell number. Values are means ⫾ standard errors of the means from at least three independent cultures analyzed per condition.

sion of TH protein by 37% and greater than 93%, respectively (Fig. 1, lanes 6 and 8 compared to lane 2). The differential effects of the cAMP-elevating agents on neural crest cultures were confirmed by microscopy using immunochemical and histochemical fluorescence assays (Fig. 2). The combination of BMP-2 and IBMX stimulated an obvious increase in the development of TH-positive and CA-positive SA cells (Fig. 2). However, the multilayer nature of the cultures and the large number of TH-positive and CA-positive cells precluded our ability to accurately measure these effects. In contrast, 8-Br-cAMP had a profound negative impact on the development of SA cells, abolishing the generation of THpositive and CA-positive cells, both in the absence and in the presence of BMP-2 (data not shown). It is possible that the negative effect of 8-Br-cAMP on SA cell development is related to its profound effect on the overall growth of the neural crest cultures (Table 1). By comparison, treatment with IBMX or forskolin, alone or in combination with BMP-2, had a less pronounced effect on overall growth (Table 1). These data indicate that cAMP-elevating agents differentially influence SA cell development (Fig. 1) and crest cell growth (Table 1). To gain further insight into the developmental role of BMP-2 and IBMX, we studied crest cell survival and development in cultures initiated at clonal density (Table 2). The total number of colonies generated in the assay was similar in all treatment groups, suggesting that BMP-2 and IBMX, alone or in combination, do not affect the survival of colony founder cells (Table 2). In agreement with results reported by Varley and Maxwell (68), the development of CA-positive colonies was stimulated by treatment with BMP-2. IBMX alone generated a slight 0.3-fold increase relative to BMP-2 in the proportion of CA-positive colonies. Interestingly, the combination of BMP-2 and IBMX resulted in a dramatic increase in the proportion of CA-positive colonies, i.e., a 2.2-fold increase relative to BMP-2 and a 27-fold increase relative to IBMX. These data provide further evidence that BMP-2 and IBMX function in synergy to promote the development of SA cells.

IBMX is a nonselective PDE inhibitor and could act by inhibiting a PDE other than the cAMP-specific (type III) PDE (6). Therefore, we analyzed the effects of several selective PDE inhibitors to confirm that the cAMP signaling pathway was specifically promoting SA cell development (Fig. 3). The agents included vinpocetine, an inhibitor of the Ca2⫹/calmodulin-dependent (type I) PDE (1, 21); Ro 20-1724, an inhibitor of cAMP-specific (type III) PDE (30, 54, 59); and MY-5445, an inhibitor of the cGMP-specific (type V) PDE (22). In the absence and presence of BMP-2, neither 100 ␮M vinpocetine nor 30 ␮M MY-5445 had a significant effect on TH protein expression (Fig. 3A, lanes 5 and 9 compared to lane 1; lanes 6 and 10 compared to lane 2). By contrast, the combination of BMP-2 and Ro 20-1724 produced a synergistic 2.4-fold increase in the expression of TH protein (Fig. 3A, lane 8 compared to lanes 2 and 7) and stimulated an obvious increase in the development of SA cells (data not shown). These increases were comparable to the effects of BMP-2 and IBMX (Fig. 2 and 3A, lane 4) and support the conclusion that IBMX, like Ro 20-1724, promotes SA cell development by activating the cAMP signaling pathway. Furthermore, the combination of Ro 20-1724 and forskolin decreased the expression of TH protein (Fig. 3B, lanes 4 and 6 compared to lanes 3 and 5, respectively). Cotreatment with Ro 20-1724 and forskolin undoubtedly results in higher intracellular cAMP levels than treatment with either agent alone. Accordingly, these data suggest that the intracellular level of cAMP, which reflects the level of activation of the cAMP signaling pathway, is a criterion for selecting the pathway’s function either to promote or to antagonize SA cell development. Dose-dependent modulation of SA cell development by cAMP-elevating agents. To investigate the model that the specific function of the cAMP pathway, i.e., as an SA-promoting or SA-antagonizing mechanism, is determined by the level of cAMP signaling, we tested the ability of the various cAMPelevating agents to activate the cAMP signaling pathway. Initially we used a radiometric PKA assay to test the ability of

TABLE 2. BMP-2 and IBMX stimulate the generation of CA-positive crest cell coloniesa Expt 1 Treatment

Control BMP-2 IBMX IBMX ⫹ BMP-2

No. of

Expt 2

CA-positive colonies

Total colonies

% CA-positive colonies

0 4.7 ⫾ 0.5 0.3 ⫾ 0.2 11.2 ⫾ 0.8

32.1 ⫾ 0.8 29.5 ⫾ 0.7 31.5 ⫾ 0.9 31.8 ⫾ 1.6

0 15.8 ⫾ 1.4 0.9 ⫾ 0.5 35.0 ⫾ 1.2

No. of: CA-positive colonies

0.1 ⫾ 0.1 5.8 ⫾ 0.3 0.7 ⫾ 0.2 13.1 ⫾ 4.1

Avg

Total colonies

% CA-positive colonies

24.5 ⫾ 0.7 25.1 ⫾ 0.8 24.7 ⫾ 0.8 26.2 ⫾ 0.8

0.4 ⫾ 0.4 23.2 ⫾ 1.2 2.9 ⫾ 0.8 50.1 ⫾ 1.2

0.2 19.5 1.9 42.6

a Clonal cultures were prepared as described in Materials and Methods. Cells grown 12 days in the presence of 10 ng of BMP-2 per ml and 100 ␮M IBMX as indicated and processed to reveal CA-containing cells. Data are presented as the mean colony counts ⫾ standard error of the mean from 10 replicate plates each for control and experimental conditions. The data for two independent experiments are presented. In both experiments, the effects of BMP-2 and IBMX, added alone or in combination, on the percentage of CA-positive colonies are significantly different from the control (P ⬍ 0.05, analysis of variance).

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MOL. CELL. BIOL. TABLE 3. Differential activation of PKA by cAMP-elevating agents in neural crest cultures Agent

Control IBMX (100 ␮M) Ro 20-1724 (20 ␮M) Forskolin (10 ␮M) 8-Br-cAMP (100 ␮M)

% of total endogenous PKA activateda Expt 1

Expt 2

Expt 3

Avg ⫾ SEM

2.0 4.4 0.4 8.5 56.1

2.4 0.0 6.5 6.0 58.3

0.2 1.8 4.7 13.4 30.0

1.5 ⫾ 0.7 2.0 ⫾ 1.3 3.9 ⫾ 1.8 9.3 ⫾ 2.2 48.1 ⫾ 9.1

a Cultures for PKA assays were prepared as described in Materials and Methods. cAMP-elevating agents were applied as indicated to crest cells for 2 h and processed to measure the percentage of total endogenous PKA activated. Data represent the average of duplicate radiometric values and three independent experiments.

FIG. 3. Effects of PDE inhibitors on TH protein expression in neural crest cultures. (A) Western blot analysis of TH protein expression in cultures grown for 7 days in the presence of BMP-2 (10 ng/ml) and PDE inhibitor IBMX (100 ␮M), vinpocetine (100 ␮M), Ro 20-1724 (20 ␮M), or MY-5445 (30 ␮M) as indicated. The histogram was derived by densitometric scanning of blots from three independent cellular extract preparations. The effects of BMP-2 and Ro 20-1724 compared to treatment with either agent alone are significant at P ⬍ 0.05 (analysis of variance). The effects of BMP-2 and either vinpocetine or MY-5445 are not significantly different from treatment with only BMP-2. (B) Western blot analysis of TH protein expression in cultures grown for 7 days in the presence of 10 ng of BMP-2 per ml, 20 ␮M Ro 20-1724, 10 ␮M forskolin, and 100 ␮M 8-Br-cAMP as indicated.

cAMP-elevating agents to activate the endogenous PKA enzyme in neural crest cultures (Table 3). Both forskolin and 8-Br-cAMP stimulated marked activation, 6- and 32-fold, respectively, of endogenous PKA. By contrast, IBMX or Ro 20-1724 stimulated only small increases in PKA activity. In related studies, we used a nonradioactive assay to measure cAMP levels (Amersham Pharmacia) in cultures treated with combinations of BMP-2 and either IBMX or forskolin. We observed that only forskolin, independent of the presence of BMP-2, stimulated a detectable increase in intracellular cAMP (data not shown). Additionally, we used a phospho-specific

CREB (Ser133) antibody (New England Biolabs) and Western blot assays to monitor the activation of endogenous CREB following treatment of the cultures with combinations of BMP-2 and either IBMX or forskolin. Only forskolin, independent of the presence of BMP-2, induced a detectable increase in CREB phosphorylation (data not shown). Taken together, these observations indicate that the distinct effects of IBMX, forskolin, and 8-Br-cAMP on SA cell development (Fig. 1) are linked to their ability to activate the cAMP signaling pathway to different degrees. To further confirm this model, we measured the dose-dependent effects of cAMP-elevating agents on BMP-2-induced TH protein expression. As shown in Fig. 4, low doses (0.1 to 1.0 ␮M) of forskolin or 8-Br-cAMP dramatically enhanced BMP2-induced TH protein expression (Fig. 4, lanes 6, 7, 10, and 11 compared to lane 1) and produced an obvious increase in the development of SA cells (data not shown). Moreover, the combinatorial effect of BMP-2 and low dose of forskolin or 8-Br-cAMP on the expression of TH protein was similar to the synergistic influence of BMP-2 and IBMX (Fig. 4, lanes 4 and 5). Conversely, a high dose (100 ␮M) of forskolin or 8-BrcAMP largely blocked the stimulatory effect of BMP-2 on TH protein expression (Fig. 4, lanes 9 and 13 compared to lane 1). These data are consistent with the model that moderate levels of cAMP signaling promote SA cell development, whereas high levels of cAMP signaling antagonize SA cell development. Early requirement of cAMP-elevating agents in antagonizing SA cell development. To further understand the mechanism by which the cAMP signaling pathway antagonizes SA cell development, we tested the temporal requirements for the inhibitory effect of high dose of forskolin on the BMP-2-induced expression of TH protein. In parallel assays, we also examined the inhibitory effect of high dose of norepinephrine, a natural effector of the cAMP signaling pathway. Importantly, 100 ␮M forskolin or 100 ␮M norepinephrine had a significantly reduced impact on overall crest cell growth (P ⬍ 0.05) (Table 4) compared to 100 ␮M 8-Br-cAMP (Table 1), suggesting that the antagonism of SA cell development is not solely dependent on an inhibition of crest cell growth. As shown in Fig. 5, continuous treatment with either 100 ␮M forskolin or 100 ␮M norepinephrine for the entire culturing period greatly reduced the ability of BMP-2 to stimulate TH protein expression (compare lanes 1 and 2). Exposure of the cultures to a high dose of forskolin or norepinephrine for at least the initial 24 h of culturing was crucial for either agent to reduce the expression of TH protein (Fig. 5, lane 7 compared to lane 1). Treatment with forskolin beyond the first 24 h of culturing reduced TH protein expression to levels comparable to those obtained by treatment for the entire culturing period (Fig. 5A,

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FIG. 4. Low dose of forskolin or 8-Br-cAMP acts cooperatively with BMP-2 to stimulate development of SA cells in neural crest cell cultures. Shown is Western blot analysis of TH protein expression in cultures grown 5 days in the presence of 10 ng of BMP-2 per ml and 0.1, 1.0, 10, or 100 ␮M cAMP-elevating agent as indicated. The histogram was derived by densitometric scanning of blots from three independent cellular extract preparations. The effects of BMP-2 and either low (0.1 to 1.0 ␮M) or high (100 ␮M) doses of forskolin or 8-Br-cAMP are significant compared to treatment with BMP-2 alone (P ⬍ 0.05, analysis of variance). The effects of BMP-2 and low-dose forskolin or 8-Br-cAMP are not significantly different from treatment with BMP-2 and 1.0 to 100 ␮M IBMX.

lanes 4 to 6 compared to lane 2). Norepinephrine required continuous exposure for a minimum of 72 h to reduce TH protein expression (Fig. 5B, lanes 5 and 6 compared to lane 1). Surprisingly, the addition of norepinephrine for only the initial 48 h of culturing reproducibly increased TH protein (n ⫽ 2; Fig. 5B, lane 4 compared to lane 1). The mechanism of this effect has not been further examined. Synergy of BMP-2 and cAMP signaling regulates the SA lineage-determining gene Phox2a. The SA-promoting and SAantagonizing influences of cAMP signaling (Fig. 4) suggest that the cAMP signaling pathway regulates the expression of SA lineage-determining genes, such as ASH-1 (7, 19, 25, 36), Phox2a (49), and Phox2b (52, 53). To test this hypothesis, we used RT-PCR to monitor the expression of the ASH-1, Phox2a, and Phox2b genes under cAMP signaling conditions that either promote or antagonize SA cell development (as discussed above). The TH and GAPDH genes were monitored as controls for SA cell differentiation and RNA loading, respectively. TABLE 4. Effect of high-dose forskolin or norepinephrine on the overall growth of crest cell cultures Total cell no.a Agent

Forskolin (100 ␮M)

Norepinephrine (100 ␮M)

Control BMP-2

312,000 ⫾ 6,900 240,000 ⫾ 5,500

353,000 ⫾ 9,200 334,000 ⫾ 1,700

a

Cultures were grown in the presence of BMP-2 (10 ng/ml) and cAMPelevating agents as indicated and analyzed for total cell number. Values are means ⫾ standard errors of the means of three independent cultures per condition.

In agreement with previous reports (42, 58), BMP-2 increased the expression of ASH-1, Phox2b, Phox2a, and TH (Fig. 6B and D, lane 2 compared to lane 1). Treatment of the cultures with IBMX or 0.1 ␮M forskolin had no detectable effect on the expression of the SA lineage-determining genes (Fig. 6B and D, lanes 3 and 5 compared to lane 1); however, IBMX did produce an increase in TH gene expression (Fig. 6D, lane 3). Interestingly, the combination of BMP-2 with IBMX or low dose of forskolin, conditions that synergistically promote SA cell development, increased the expression of Phox2a and TH (Fig. 6D, lanes 4 and 6 compared to lane 2), but these conditions had no detectable effect on ASH-1 or Phox2b (Fig. 6B, lanes 4 and 6 compared to lane 2). On the contrary, 100 ␮M forskolin, a condition that antagonizes SA cell development, blocked the expression of SA lineage-determining genes, inhibiting the BMP-2-induced expression of ASH-1, Phox2b, Phox2a, and TH (Fig. 6B and D, compare lanes 2 and 8). Consistent with work by others (17), these results support a conclusion that regulation of ASH-1, Phox2a, and Phox2b gene expression is central to the development of SA cells. Moreover, these results identify possible transcriptional targets of SA-promoting and SA-antagonizing cAMP signals. Thus, moderate levels of cAMP signaling may stimulate SA cell differentiation by inducing the expression of Phox2a. High levels of cAMP signaling may oppose SA cell differentiation by blocking the expression of the ASH-1 and Phox2 genes. DISCUSSION The ability of neural crest cells in primary culture to give rise to an array of cell types provides a powerful model system to explore molecular mechanisms that determine cell fate. Using

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FIG. 5. Temporal requirements of high doses of cAMP-elevating agents to antagonize SA cell development in neural crest cultures, determined by Western blot analysis of TH protein expression in 6-day cultures treated with 100 ␮M forskolin (A) and 100 ␮M norepinephrine (B).

primary cultures of quail neural crest cells, we investigated the combinatorial effects of the BMP-2 and cAMP signaling pathways on the development of the SA lineage. Our findings indicate that the cAMP signaling pathway acts as a bimodal switch on SA cell development, governing both positive and negative signals which influence crest cell fate. We have shown that moderate activation of the cAMP signaling pathway stimulates the development of SA cells, whereas robust activation of the pathway opposes SA cell development. Furthermore, our data indicate that the cAMP signaling pathway influences the ability of BMP-2 to induce SA cell development. Moderate levels of cAMP signaling act synergistically with BMP-2 to further promote the development of SA cells. By contrast, high levels of cAMP signaling oppose BMP-2 signaling and SA cell development. We demonstrate that the combinatorial effect of BMP-2 and cAMP signaling influences the expression of SA lineage-determining genes, namely, the transcription factors ASH-1, Phox2a, and Phox2b. Our data show that the SApromoting effect of cAMP signaling involves the induction of Phox2a gene expression and that the SA-antagonizing effect involves the inhibition of ASH-1 and Phox2 gene expression. cAMP signaling promotes and antagonizes SA cell development. Earlier studies examined the effects of cAMP-elevating agents on the in vitro development of quail neural crest cells and reported seemingly conflicting roles for the cAMP signaling pathway. Specifically, 8-Br-cAMP and IBMX were reported to inhibit the ability of reconstituted basal membranelike matrix to stimulate SA cell development (46). By contrast, forskolin and ␤-adrenergic ligands were shown to regulate the expression of the quail TH gene and promote the development of SA cells (9). More recent investigations also demonstrated that BMP-2, -4, and -7/OP-1 dramatically stimulate SA cell development (55, 68, 70) and established the causal link of a BMP ligand on the generation of SA cells (69).

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Based on these investigations and the model proposing that multiple signaling cues direct crest cell fate (3), we sought first to resolve the role of cAMP signaling in SA cell development and second to investigate the combinatorial effects the BMP-2 and cAMP signaling pathways on SA cell development. Using primary cultures of quail neural crest cells, we examined SA cell development in cultures treated with combinations of BMP-2 and either IBMX, forskolin, or 8-Br-cAMP. We used Western blot assays of TH protein, a phenotypic marker of the SA lineage, to measure SA cell development (Fig. 1). We observed that the various cAMP-elevating agents had distinct abilities to stimulate the expression of TH protein. IBMX and forskolin increased the expression of TH protein, whereas 8-Br-cAMP had no detectable effect. Of even more interest, the various cAMP-elevating agents differentially influenced BMP-2-induced TH protein expression. IBMX acted in synergy with BMP-2 to increase the expression of TH protein, whereas forskolin and 8-Br-cAMP greatly reduced the ability of BMP-2 to stimulate TH protein expression. We reasoned that these differences might be due to the molecular mechanism by which each agent elevates intracellular cAMP. Specifically, forskolin directly stimulates de novo cAMP synthesis by activating the catalytic enzyme adenylate cyclase (37, 57). The 8-Br-cAMP analog mimics the effects of endogenous cAMP by specifically activating PKA but exhibits a significantly reduced rate of degradation by the cAMP-specific PDE (24, 48, 56). By contrast, IBMX increases cAMP levels by nonselectively inhibiting a variety of cyclic nucleotide PDEs, prolonging the intracellular half-life of endogenous cyclic nucleotides (6). Thus, agents efficiently increasing the intracellular concentration of cAMP, i.e., forskolin and 8-BrcAMP, are expected to produce a higher steady-state activation of the cAMP signaling pathway compared to an agent that passively sustains the levels of endogenous cAMP, i.e., IBMX. In support of this proposal, Table 3 shows that 8-Br-cAMP is the most potent activator of the cAMP signaling pathway. Although both BMP-2 and IBMX independently produced obvious increases in the development of TH-positive and CApositive SA cells, their combined effects were striking (Fig. 2). Data obtained from analyzing clonal cultures of crest cells (Table 2) further corroborate the dramatic, synergistic influence of BMP-2 and IBMX on the development of SA cells. Despite the common usage of IBMX in cAMP signaling studies, IBMX is a nonspecific inhibitor of PDE. Our studies confirmed that the effects of IBMX were specifically dependent on the cAMP signaling pathway by comparing the influence of several selective PDE inhibitors on TH protein expression (Fig. 3A). Importantly, we demonstrated that the cAMP-specific (type III) PDE inhibitor Ro 20-1724 (30, 54, 59) dramatically increased the expression of TH protein (Fig. 3A) and the development of SA cells (data not shown). In contrast, selective inhibition of either the Ca2⫹/calmodulin-dependent (type I) or the cGMP-specific (type V) PDE had no significant effect. We further tested the effects of cotreating neural crest cultures with Ro 20-1724 and either forskolin or 8-Br-cAMP (Fig. 3B). Independent of the presence of BMP-2, the combination of forskolin and PDE inhibitor decreased TH protein expression. Undoubtedly, the combination of Ro 20-1724 and forskolin results in higher intracellular cAMP levels than treatment with either agent alone. Accordingly, these data suggest that the specific function of the cAMP pathway to either promote or antagonize SA cell development is determined by the intracellular concentration of cAMP and, in turn, the activation level of the cAMP signaling pathway. cAMP signaling levels regulate SA cell development. To investigate this model, we tested the ability of the cAMP-

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FIG. 6. Synergy between the BMP-2 and cAMP signaling pathways regulates the SA lineage-determining gene Phox2a in neural crest cultures determined by RT-PCR of 1 ␮g of total RNA isolated from neural crest cells after 2 (A and B) days or 3 (C and D) days in culture. (A and C) Cycle-dependent amplification of TH and SA lineage-determining genes expressed by neural crest cultures grown in the presence of 10 ng of BMP-2 per ml. (B and D) Expression of TH and SA lineage-determining genes in neural crest cultures treated with combinations of 10 ng of BMP-2 per ml and the specified concentration (micromolar) of cAMP-elevating agent as indicated. Representative results of RT-PCR using 30 cycles of amplification to detect gene expression are shown. Amplification of the GAPDH gene was used as an internal control. Lane C is a negative control from which RNA was omitted in the RT-PCR.

elevating agents to activate the endogenous PKA enzyme (Table 3). IBMX or Ro 20-1724 produced small, detectable increases in PKA activity, whereas forskolin or 8-Br-cAMP markedly stimulated PKA activity. Importantly, the differential activation of PKA correlates with the ability of these cAMPelevating agents to either promote or antagonize SA cell development. Additionally, we related the effects of BMP-2, IBMX, and forskolin to changes in both intracellular cAMP levels and CREB phosphorylation. In experiments not shown, we found that the standard growth conditions required for neural crest cultures resulted in extremely high baselines, rendering both assays inadequate for the detection of changes in the respective components of the cAMP signaling pathway. Crest cells do not survive well in the absence of serum and/or chick embryo extract; culturing crest cells for 18 h in a 1:200 dilution of standard culture medium enabled us to maintain a reasonable degree of cell survival and to marginally reduce the assay baselines. Under these conditions, 100 ␮M forskolin produced a sustained elevation in intracellular cAMP and induced the phosphorylation of CREB (data not shown); however, such changes were not detectable in treatments with IBMX or 0.1 ␮M forskolin. Likewise, BMP-2 alone or in combination with a cAMP-elevating agent produced no detectable effects on cAMP levels or CREB phosphorylation. In summary, the high content of serum and embryo extract in the medium required for the maintenance of crest cells in vitro precluded the quantitative measurement of small changes in cAMP levels and CREB phosphorylation produced by treatment with IBMX or a low dose of forskolin. However, taken with results of our

PKA experiments (Table 3), these observations fully support the view that the various cAMP-elevating agents exhibit different capacities for activating cAMP signaling mechanisms. From the analyses described above, we cannot conclude whether BMP-2 regulates cAMP signaling in crest cells. Studies on chondrogenesis (41) and renal branching (20) have revealed that BMP-2 stimulates the activation of PKA and the phosphorylation of CREB by a mechanism not yet understood. Investigations using primary rat neural crest cultures observed that the combination of 10 ng of BMP-2 per ml and 5 ␮M forskolin weakly induced TH protein expression, but the combination of 0.1 ng of BMP-2 per ml and 5 ␮M forskolin stimulated a robust induction of TH protein (42). If BMP-2 does regulate the cAMP signaling pathway in crest cells, then indeed this decreased dosage of BMP-2 is akin, in part, to a reduced level of cAMP signaling promoting SA cell development. Additionally, we are puzzled by the biphasic nature of BMP-2 (and BMP-4) dosage on SA cell development (55, 68) and by our data indicating that 10 ␮M forskolin is converted by BMP-2 from an SA-promoting agent to an SA-antagonizing agent (Fig. 1). A finding that BMP-2 activates the cAMP signaling pathway in crest cells would provide a logical explanation for the attenuated effect of high dose of BMP-2 on SA cell development (55, 68) and the SA-antagonizing conversion of 10 ␮M forskolin by BMP-2 (Fig. 1). To confirm by an another method that the cAMP signaling pathway functions to either promote or antagonize SA cell development as a consequence of the level of its activation, we examined the dose-dependent effects of the various cAMPelevating agents on the BMP-2-induced expression of TH pro-

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tein (Fig. 4). These data conclusively demonstrate that TH protein expression is promoted by low levels of cAMP signaling, induced by 0.1 to 1.0 ␮M forskolin or 8-Br-cAMP, and that TH protein expression is antagonized by high levels of cAMP signaling, induced by 100 ␮M forskolin or 8-Br-cAMP. Accordingly, our investigations support a mechanism whereby cAMP signaling functions as a bimodal switch on SA cell development. The antagonistic effect of the cAMP signaling pathway is temporally defined. To verify that the observed antagonistic effect of cAMP-elevating agents on SA cell development is authentic, we performed two types of analyses. First, we demonstrated that this negative effect displays specific temporal requirements (Fig. 5A). Importantly, the inhibitory capacity of 100 ␮M forskolin is fully established within the first 48 h of culturing and is essentially lost if it is applied after the initial 24 h of culturing. This suggests that cAMP signaling mechanisms interfere with pathways required for crest cells to commit to the SA lineage. Second, we demonstrated that this negative effect is reproduced by the addition of a natural effector of the cAMP signaling pathway (Fig. 5B). Similar to 100 ␮M forskolin, 100 ␮M norepinephrine has an inhibitory capacity that is lost if it is applied after the initial 24 h of culturing. The major difference between the two agents is that the negative effect of norepinephrine on SA cell development requires the continuous treatment of the cultures for at least 72 h. Treatment with norepinephrine for only the initial 48 h of culturing increases TH protein expression. Based on earlier studies (60, 73, 74), we interpret this transient positive effect to involve the norepinephrine transporter and intracellular calcium which, in turn, may induce the expression of necessary molecular links, i.e., adrenergic autoreceptors, for norepinephrine to antagonize SA cell development. In conclusion, the brief but specific temporal requirements of forskolin as well as the ability of a natural cAMP signaling effector such as norepinephrine to inhibit TH protein expression further validate the significance of a mechanism whereby cAMP signaling opposes SA cell development. Integrating BMP-2 and cAMP signaling pathways in SA cell development. The stimulatory and inhibitory capacities of cAMP signaling (Fig. 4) suggested that the pathway regulates the expression of genes necessary for crest cell commitment to the SA lineage, i.e., genes acting upstream from the expression of TH and DBH. Gene knockout studies in the mouse have demonstrated that the ASH-1 gene (7, 19, 25, 36) and the Phox2 genes, Phox2a (49) and Phox2b (52, 53), are essential for development of SA progenitor cells. Accordingly, we investigated whether the cAMP signaling mechanisms regulate the expression of these SA lineage-determining genes. Importantly, we showed by RT-PCR that the combinatorial effect of BMP-2 and moderate levels of cAMP signaling, i.e., IBMX or 0.1 ␮M forskolin, is exerted not only on the increased expression of TH but also on the expression of the SA lineagedetermining gene Phox2a (Fig. 6D). This moderate level of cAMP signaling does not affect induction of the ASH-1 or Phox2b gene (Fig. 6B). Together with the recent reports that the Phox2 proteins are necessary (42) and sufficient (62) for development of the SA lineage, these data suggest that the BMP-2 and cAMP signaling pathways converge on Phox2a gene expression to promote SA cell differentiation. Additionally, we demonstrated that a high level of cAMP signaling, i.e., 100 ␮M forskolin, blocks the expression of both TH and the SA lineage-determining genes (Fig. 6B and D). These observations lend further support to the crucial role of the ASH-1 and Phox2 genes for development of the SA lineage (17) and

MOL. CELL. BIOL.

FIG. 7. Model for the BMP-2 and cAMP signaling pathways in SA cell development. The diagram illustrates the possible molecular mechanisms regulating the observed synergy and antagonism between the BMP-2 and cAMP signaling pathways. Arrows indicate direct and indirect relationships. Differentiation of the SA lineage requires activation of the ASH-1 and Phox2 genes, which, in turn, regulate the neurogenic program, neurotransmitter identity, and progenitor survival (17). BMP-2 in combination with moderate intracellular levels of cAMP promotes differentiation of SA progenitor cells by inducing expression of ASH-1 and Phox2 genes. Conversely, high intracellular levels of cAMP activate a yet to be delineated mechanism antagonistic to development of the SA lineage which opposes BMP-2 signaling and the consequent expression of SA lineage-determining genes.

suggest that the cAMP signaling pathway antagonizes SA cell differentiation by regulating other pathways mediating the expression of the SA lineage-determining genes. In conclusion, our results demonstrate the dynamic role of the cAMP signaling pathway in the development of SA cells, activating either a mechanism that promotes phenotypic specification or a mechanism that opposes crest cell commitment to the SA lineage. Although the nature of each mechanism remains to be elucidated, Fig. 7 depicts a model for how intracellular cAMP levels govern the bimodal switch on SA cell development. Thus, moderate levels of intracellular cAMP promote the expression of Phox2a, which, in turn, drives differentiation (62), and probably survival (10, 44, 49), of the SA progenitor cell. In contrast, high levels of intracellular cAMP activate a pathway that opposes BMP-2 signaling and inhibits the induction of genes necessary for SA lineage determination, i.e., the ASH-1 and Phox2 genes. ACKNOWLEDGMENTS We express gratitude to G. Maxwell and J. Varley, University of Connecticut Health Center, for sharing their techniques and culture conditions for primary neural crest cells; the Genetics Institute for providing BMP-2; J.-F. Brunet for communicating the partial sequences of chicken Phox2a and Phox2b; P. Robinson for sharing the

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epifluorescence microscope and digital imaging equipment; and the Poultry Research Center for maintaining the Japanese quail flock. This work is supported by grants from the Whitehall Foundation and NIH DK 44533 to O.M.A. REFERENCES 1. Ahn, H. S., W. Crim, M. Romano, E. Sybertz, and B. Pitts. 1989. Effects of selective inhibitors on cyclic nucleotide phosphodiesterases of rabbit aorta. Biochem. Pharmacol. 38:3331–3339. 2. Allan, I. J., and D. F. Newgreen. 1977. Catecholamine accumulation in neural crest cells and the primary sympathetic chain. Am. J. Anat. 149:413– 421. 3. Anderson, D. J. 1997. Cellular and molecular biology of neural crest cell lineage determination. Trends Genet. 13:276–280. 4. Anderson, D. J. 1993. Molecular control of cell fate in the neural crest: the sympathoadrenal lineage. Annu. Rev. Neurosci. 16:1291–1258. 5. Anderson, D. J., J. F. Carnahan, A. Michelsohn, and P. H. Patterson. 1991. 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