PACAP promotes sensory neuron differentiation: blockade by neurotrophic factors

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 25 (2004) 629 – 641

PACAP promotes sensory neuron differentiation: blockade by neurotrophic factors Katherine M. Nielsen, a,1 Martha Chaverra, a Sharon J. Hapner, a Branden R. Nelson, a Valerie Todd, a Richard E. Zigmond, b and Frances Lefcort a,* a b

Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717, USA Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106, USA

Received 7 February 2003; revised 1 December 2003; accepted 2 December 2003

Developing neurons encounter a panoply of extracellular signals as they differentiate. A major goal is to identify these extrinsic cues and define the mechanisms by which neurons simultaneously integrate stimulation by multiple factors yet initiate one specific biological response. Factors that are known to exert potent activities in the developing nervous system include the NGF family of neurotrophic factors, ciliary neurotrophic factor (CNTF), and pituitary adenylate cyclase-activating peptide (PACAP). Here we demonstrate that PACAP promotes the differentiation of nascent dorsal root ganglion (DRG) neurons in that it increases both the number of neural-marker-positive cells and axonogenesis without affecting the proliferation of neural progenitor cells. This response is mediated through the PAC1 receptor and requires MAP kinase activation. Moreover, we find that, in the absence of exogenously added PACAP, blockade of the PAC1 receptor inhibits neuronal differentiation. These data coupled with our finding that both PACAP and the PAC1 receptor are expressed during the peak period of neuronal differentiation in the DRG suggest that PACAP functions in vivo to promote the differentiation of nascent sensory neurons. Interestingly, we also demonstrate that the neurotrophic factors NT-3 and CNTF completely block the PACAP-induced neuronal differentiation. This points to the intricate integration of cellular signals by nascent neurons and, to our knowledge, is the first evidence for neurotrophic factor abrogation of a pathway regulated by G-protein-coupled receptors (GPCRs). D 2004 Elsevier Inc. All rights reserved.

Introduction The dorsal root ganglion (DRG) is composed of a heterogeneous mixture of functionally and chemically diverse sensory neurons and two types of glial cells (Schwann cells and satellite cells). These cells are all derived from a subset of neural crest cells that migrate ventrolaterally and aggregate adjacent to the neural tube (Lallier and Bronner-Fraser, 1988; LeDouarin, 1982; Weston,

* Corresponding author. Fax: +1-406-994-7077. E-mail address: [email protected] (F. Lefcort). 1 Current address: University of California-San Francisco, San Francisco, CA 94143, USA. Available online on ScienceDirect (www.sciencedirect.com.) 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.12.004

1970). Subsequent to the cessation of migration, DRG progenitor cells proliferate to give rise to both the neurons and glia of the ganglion. In the chick, two waves of neurogenesis occur, with the large diameter proprioceptors and mechanoreceptors being generated and differentiating first, followed by the smaller diameter pain and temperature receptors (Carr and Simpson, 1978; Frank and Sanes, 1991; Oakley et al., 1997; Pannese, 1974; Rifkin et al., 2000). Thus, the first wave of neurons are differentiating while the second wave of neurons are being born at a stage when these cell populations are spatially intermixed and often adjacent to one another (Farinas et al., 2002; Rifkin et al., 2000). Not only are neurogenesis and neuronal differentiation occurring contemporaneously, but so is the programmed cell death of the first wave of neurons generated within the DRG. In fact, apoptotic cells are often directly juxtaposed to mitotically active progenitor cells and nascent post-mitotic neurons (Carr and Simpson, 1978; Rifkin et al., 2000). Thus, neural precursors in the immature DRG are exposed simultaneously to signals that regulate mitogenesis, growth, survival, and cellular differentiation. The molecular mechanisms by which immature DRG cells integrate these disparate signals and yet ultimately initiate one particular response remain to be elucidated (Edlund and Jessell, 1999; Morrison, 2001; Sommer, 2001). As a first step towards addressing this question, the extrinsic signals active within the DRG must be identified and the repertoire of behavioral responses each elicits be determined. Some of the factors that influence particular stages of sensory neuron development have been well characterized. For example, members of the neurotrophin family of growth factors exert profound effects on post-mitotic sensory neurons during the process of target-mediated programmed cell death (Huang and Reichardt, 2001; Lindsay, 1996). Neurotrophins also modulate earlier events during DRG development, including cell proliferation (Geffen and Goldstein, 1996) and neuronal maturation (Memberg and Hall, 1995; Wright et al., 1992). Additional growth factors have been identified that can influence DRG development, including ciliary neurotrophic factor (CNTF), TGFh, GDNF, FGF, and LIF, and many of these have been shown to be expressed in the immature DRG during the peak period of neurogenesis and differentiation (Heuer et al., 1990; Holst et al., 1997; Kalcheim and Neufeld, 1990; Memberg and Hall, 1995; Murphy et al., 1993; Paratore et al., 2002; Pinco et al., 1993; Sieber-Blum, 2000; Wright

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Table 1 Percentage of TUNEL+ cells Control DRG +PACAP +PACR1 antagonist + PACAP + PACR1 antagonist

Results 5.7 8.8 8.3 9.3

F F F F

0.7% 0.3% 0.9% 0.5%

DRGs were dissociated and cultured for 6 h as described. TUNEL assay was performed (Experimental methods) and the percentage of TUNEL+ nuclei was determined from quadruplicate wells (500 cells counted/well). Results are the mean F SEM.

et al., 1992). Considering the panoply of cellular interactions that must occur during DRG development to generate the ca. 20 different functional and molecular subclasses of sensory neurons (Hall et al., 1997; Scott, 1992), it is likely that all of the environmental signals that regulate the proliferation, growth, survival, and differentiation of neurons and glia during DRG development have yet to be identified. One factor that regulates early events in the developing central and peripheral nervous systems is the neuropeptide, pituitary adenylate cyclase-activating peptide (PACAP; for review, see Waschek, 2002). PACAP belongs to the glucagon/secretin/VIP peptide family and exists in two active forms, PACAP1 – 38 and PACAP1 – 27, which are generated by alternative splicing of a single gene product. The sequences of these peptides have been remarkably well conserved during evolution, across vertebrates and extending even to tunicates, suggesting that they have an important function(s). PACAP is produced by neurons in the central and peripheral nervous systems and has been shown to modulate proliferation, survival, and differentiation of both neural and nonneural cells (Tischler et al., 1995; Waschek et al., 1998). Three PACAP receptors have been identified, all of which belong to the seven transmembrane domain, G-protein-coupled receptor (GPCR) superfamily. Once activated, these receptors stimulate adenylate cyclase, MAP kinase, and, in the case of one of the receptors, PAC1, phospholipase C (Vaudry et al., 1998, 2002; Waschek, 2002). Several splice variants of the PAC1 receptor have been identified, with different isoforms conferring activation of distinct downstream pathways (Spengler et al., 1993). PACAP is expressed in late embryonic rat DRG (E16) and promotes survival and neurite outgrowth of a subset of DRG neurons in vitro and regulates their expression of calcitonin gene-related peptide (CGRP; Lioudyno et al., 1998). In chick embryos, exogenously applied PACAP between the ages of E3.5 and E8.5 reduced programmed cell death for a subset of DRG neurons (Arimura et al., 1994). Both DRG studies were designed to determine the survival promoting activity of PACAP on sensory neurons during the period of target innervation and target-mediated programmed cell death. Much less is known about the factors that regulate earlier events integral to DRG development. PACAP and one of its receptors, PAC1, have been shown to be expressed by another major neural crest derivative, sympathetic precursor cells, where they function to regulate the genesis and differentiation of sympathetic neuroblasts (DiCiccoBloom and Deutsch, 1992; DiCicco-Bloom et al., 2000). The goal of our study was to determine whether PACAP regulated early events in the DRG such as neurogenesis and neuronal differentiation, and if so, to investigate how DRG neural precursors respond to simultaneous exposure to multiple signals that induce diametrically opposed responses and yet are present in the normal environment in situ.

PACAP promotes neuronal differentiation To determine whether PACAP influences early events in sensory neurogenesis, the response to PACAP was determined in primary dissociated cultures of immature chick DRG removed during the peak period of sensory neurogenesis (E4.5, Carr and Simpson, 1978; Pannese, 1974; Rifkin et al., 2000). At this stage, about 30 – 50% of the cells in the DRG in ovo are mitotically active as defined by BrdU labeling (Lefcort, unpublished; Geffen and Goldstein, 1996). To allow us to distinguish proliferative and differentiative effects of exogenous growth factors from those on survival, cells were only cultured for 6 h after plating, during which time cell death is minimal (Table 1). Proliferation was assessed by BrdU incorporation, a measure of DNA synthesis. We examined two aspects of neuronal differentiation: whether cells

Fig. 1. PACAP38 promotes neuronal differentiation. DRGs were dissociated and cultured as described (Experimental methods) for 6 h and the total percentage of neurons was determined as defined by the percentage of cells that were immunopositive with Tuj-1 antibodies regardless of whether they had extended a process or not. The total percentage of neurons with axons of at least one cell diameter was also determined. PACAP increased both the total percentage of neurons relative to control and the percentage of neurons that had undergone axonogenesis relative to control. It consequently reduced the percentage of neurons that had not yet extended an axon. The numbers represent the mean percent change relative to control for quadruplicate wells for two separate experiments. The means for each category were compared between control and PACAP-treated wells and statistical significance was determined by t tests: total neurons, P V 0.01; neurons with axons, P V 0.001; neuronal cell bodies without axons, P V 0.003.

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Fig. 2. PACAP38 promotes neuronal differentiation and has no affect on the proliferation of DRG progenitor cells. DRGs were dissociated and cultured for 6 h. PACAP and BrdU were added at the time of plating. Values represent the mean difference in the percent of positive cells (neurofilament, tuj-1, or BrdU) for each treatment relative to the control F SEM. For the neural differentiation assay, only neurofilament or tuj-1+ cells with a process of at least one cell diameter were counted.

expressed neuronal markers, that is, neurofilament or h3 tubulin (recognized by the Tuj-1 antibody), and whether cells had undergone axonogenesis (bearing a process of at least one cell diameter). When measured at the end of these short 6-h cultures, about 10% of the cells were post-mitotic neurons and immunopositive with neurofilament (NF) and Tuj-1 antibodies; ca. 30% of the cells incorporated BrdU and, hence, were mitotically active progenitor cells. The remaining cells are a mixture of post-mitotic neural and glial precursors that have not yet begun to express neural or glial markers (respectively) and nonneuronal cells (e.g., fibroblasts) because chunks of attached peripheral nerve could not always be removed from the dissected DRG (Rohrer et al., 1985). PACAP

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Fig. 4. PAC1 receptor antagonist blocks both PACAP38-induced neuronal differentiation and ongoing neuronal differentiation in the absence of added peptide. DRGs were dissociated and cultured for 6 h. PACAP (50 nM) or its antagonist PACAP6 – 38 (1 Am) was added at the time of plating. Values as above, n = 2 experiments, quadruplicate wells for each experiment. * indicates p V 0.0001, ANOVA and Student – Newman – Keuls. Only neurofilament+ cells with a process of at least one cell diameter were counted.

significantly increased both the total percentage of neurons (cells that expressed neuronal markers with or without an axon: from 16 F 0.8% in control wells to 20 F 1% in PACAP-treated wells; P V 0.012) and the percentage of neurons with processes greater than one cell diameter (from 9.5 F 0.6% in control wells to 14.8 F 0.6% in PACAP-treated wells; P V 0.001; Fig. 1). This increase in process outgrowth was maximal at 50 nM PACAP (ca. 85% increase, n = 19, p V 0.0001) with an EC50 of ca. 1 nM (Fig. 2). The same response was obtained when assessing expression of either of the two independent neuronal markers. As there were significantly more neurons with axons in the presence of PACAP than in control wells, these results demonstrate that PACAP drives a subset of nascent neurons to undergo axonogenesis (Figs. 1 – 3). Consequently, the percentage of neurons devoid of axons is actually significantly decreased in wells incubated with PACAP compared to control cultures (Fig. 1). Although the increase was

Fig. 3. PACAP does not increase neuronal numbers by reducing neuronal apoptosis; PACR1 antagonist blocks neuronal differentiation. DRGs were dissociated as described and cultured without any PACAP (A), in 50 nM PACAP38 (B), or in the presence of both PACAP and the PAC1R antagonist PACAP6 – 38 (C). The cells were fixed after 6 h. For A and B, cells were processed for TUNEL; apoptotic nuclei are identified with arrowheads. All wells were also immunolabeled with anti-neurofilament antibodies and the nuclear marker, DAPI (purple in A and C; blue in B); neurofilament expression is detectable in B (green) and C (red), and only faintly in A due to the optics used. Many neurons with long neurites are observed in the presence of PACAP (B), while in the presence of the PAC1R antagonist (C), neural precursor cells are present that have either very short neurites or have not yet undergone axonogenesis.

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Table 2 PACAP receptor antagonist does not decrease total cell number nor number of BrdU+ cells Treatment

Number of cells

Number of BrdU+ cells

Control PACR1 antagonist PACR1 antagonist + PACAP

579 F 23 538 F 43 597 F 34

105 F 4 105 F 4 104 F 6

DRGs were dissociated and cultured for 6 h as described. Results are the mean F SEM from quadruplicate wells.

smaller yet still significant, PACAP also increased the total percentage of neurons as defined by expression of neural markers. In contrast to sympathetic neuroblasts, we found that PACAP produced no change in DNA synthesis in dissociated immature DRG (Fig. 2). The fact that the increase in percent of cells expressing neuronal markers was not accompanied by a decrease in mitotic activity would suggest that PACAP is promoting the maturation of post-mitotic neural precursor cells that had not yet begun to express neuronal markers, rather than driving mitotically active neural progenitor cells out of the cell cycle and inducing their differentiation into neurons. Furthermore, the increase in the percentage of neurons cannot be due to stimulation of neurogenesis because the length of the assay time (6 h) is insufficient to complete the cell cycle of a neural progenitor (which is ca. 12 h; Lefcort and Todd, unpublished), besides the fact that PACAP did not increase the percentage of BrdU+ cells. Nor is PACAP

increasing the percentage of neurons by promoting their survival as we conducted a TUNEL assay on 6-h cultures and found that the percentage of TUNEL-positive cells was not decreased in the presence of PACAP (Table 1; Fig. 3). Blockade of PAC1 receptor inhibits neuronal differentiation To identify the receptor mediating the PACAP response, we tested whether inclusion of the PAC1 receptor antagonist PACAP6 – 38 (Harmar et al., 1998; Robberecht et al., 1992) would perturb the PACAP-induced neural differentiative response. For this and the remaining experiments, neural differentiation was quantified by measuring the number of NF+ or Tuj-1+ cells with a process of at least one cell diameter. Not only did the PAC1 receptor antagonist completely block the ability of PACAP to induce neuronal differentiation, but also decreased the levels of neural differentiation significantly below that in the control wells which received no added PACAP (Fig. 4). Moreover, in the absence of exogenous PACAP, addition of the antagonist alone significantly reduced the endogenous levels of neuronal differentiation by ca. 50% (Figs. 3 and 4). We find no evidence that the negative effects of the antagonist are due to toxicity: (1) we see no significant decrease in absolute cell number (Table 2), (2) nor any difference in the percentage of TUNEL-positive cells compared to PACAP-treated wells (Table 1), (3) nor any perturbation of the ongoing levels of mitogenesis in cultures incubated with the antagonist (Table 2), (4) nor find any morphological evidence for cellular toxicity (Fig. 3C).

Fig. 5. PACAP and PAC1R are both expressed in immature (E4.5) and mature (E8.5) DRGs. (A) E4.5 embryos were cryosectioned and labeled with antiPACAP antibody (see Experimental methods). Control sections received no primary antibody, but were otherwise processed similarly. PACAP is expressed in the spinal cord (sc) and DRG, but note its absence in the mesenchyme ventral to the spinal cord. (B) RT-PCR demonstrates PACAP and PAC1R expression in both the immature and mature DRG: (lane 2) E8.5 DRG cDNA amplified for PAC1R; (lane 3) E4.5 DRG cDNA amplified for PAC1R; (lane 4) E8.5 DRG cDNA amplified for PACAP; (lane 5) E4.5 DRG cDNA amplified for PACAP; (lanes 6 and 7) negative (lane 6) and positive (lane 7) controls of E8.5 cDNA amplified with h-actin primers; (lanes 8 and 9) negative (lane 8) and positive (lane 9) controls of E4.5 cDNA amplified with h-actin primers.

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PACAP38 and PAC1R are expressed in the DRG during the peak period of neuronal differentiation To examine PACAP expression in DRG, we immunolabeled sections from E4.5 embryos with an antibody specific for PACAP (Hannibal et al., 1995; Nielsen et al., 1998). PACAP-immunopositive cells were detected in the DRG and spinal cord (Fig. 5A). At this age, the two major cell types are trkC+ neurons, which are in the interior core of the ganglion, and a ring of mitotically active progenitor cells, which circumscribe the ganglion and extend into the dorsal root. PACAP-immunoreactive cells are in both regions (gliogenesis has not yet begun, hence there are no glial cells within the ganglion itself at this stage although there are glial progenitor cells within the dorsal root; Bhattacharyya et al., 1991; Carr and Simpson, 1978). In situ hybridizations will be required to determine the exact cell types within the DRG which synthesize PACAP. Using RT-PCR, we also identified transcripts for both PACAP and the PAC1 receptor throughout the period of neuronal differentiation in the DRG (E4.5 and E8.5; Fig. 5B). The PCR results indicate that only the prototype PAC1 receptor (null), and not the hop variants, is expressed in the DRG during the peak period of neuronal differentiation (Journot et al., 1995; Pisegna and Wank, 1996; Spengler et al., 1993). PACAP’s actions are mediated via a MAP kinase pathway that does not require trk receptor activation To determine the role of MAP kinases in the PACAP-induced response, we first determined whether MAP kinase was phosphorylated in response to PACAP treatment. Fig. 6 shows that MAPK is phosphorylated in response to PACAP treatment of immature DRG cells. We then tested whether pretreatment with the MAP kinase inhibitor U0126 would block the PACAPinduced neuronal differentiation. Blocking MAP kinase activation completely prevented the neuronal differentiation induced by PACAP (Fig. 7). Similar results were obtained using the MAP kinase antagonist PD98059 (data not shown). Cell number was

Fig. 6. PACAP induces MAP kinase phosphorylation. DRGs were dissociated and cultured in the presence of PACAP, NT-3, or both for 1 h. Lysates were made as described and MAP kinase immunoprecipitated, run on an SDS gel, and immunoblotted with an anti-phosphorylated MAP kinase antibody. Bands were quantified by densitometry.

Fig. 7. PACAP38-induced neuronal differentiation is dependent upon MAP kinase activation. DRGs were cultured as described in the presence or absence of PACAP (50 nM) and the MAP kinase inhibitor, U0126 (25 AM), for 6 h, stained with Tuj-1 antibodies, and the percentage of neurons bearing a process of at least one cell diameter was determined relative to control. Values represent the mean F SEM for three experiments; P V 0.001.

not decreased nor did cells appear unhealthy, rather, the only measurable change in the presence of the MAP kinase antagonist was that there were significantly fewer neurons with processes (Figs. 8A and B). In fact, the total percentage of neurons (as defined by expression of neural markers) did not change (15.6 F 0.8% in control wells to 15.5 F 0.9% in PACAP + U0126-treated wells), rather just the percentage of neurons with PACAP-induced axons decreased in the presence of U0126. Furthermore, many PACAP-treated neurons were strongly immunopositive when labeled with anti-phosphorylated MAP kinase antibodies, indicating that PACAP increased MAP kinase phosphorylation in a subset of neurons (Figs. 8C – F). PACAP and neurotrophins can both promote many of the same responses in neurons including neuronal survival and differentiation suggesting that they both activate conserved signaling pathways. A recent report demonstrated that PACAP-induced neuronal survival was in fact mediated by and dependant upon transactivation of trk receptors in the absence of neurotrophins (Lee et al., 2002). In that study, inactivation of trk receptors with K252a blocked the PACAP-induced neuronal survival (Lee et al., 2002). All immature nascent DRG neurons express trk receptors (Rifkin et al., 2000), hence we sought to determine whether PACAP’s ability to induce neuronal differentiation was dependent upon trk receptor transactivation. We cultured DRG cells in the presence of K252a and PACAP and found that blockade of trk receptor signaling did not interfere with the ability of PACAP to promote neuronal differentiation (Fig. 9). As a control for K252a activity, in the same experiment we cultured dissociated DRG in the presence of NT-3, a trk receptor ligand. We have previously shown (Hapner et al., in preparation) that high concentrations of NT-3 (>25 ng/ml) induce neuronal differentiation as defined by

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indicating the functional efficacy of the K252a used in our experiments (Fig. 9). NT-3 and CNTF block PACAP-induced neuronal differentiation The immature DRG at E4.5 is composed of a heterogeneous mixture of dividing progenitor cells and nascent post-mitotic neurons (Carr and Simpson, 1978; Pannese, 1974; Rifkin et al., 2000; Wakamatsu et al., 2000). Given the concurrent signaling of proliferative and differentiative cues during this time period, we sought to determine the response of cells in the immature DRG to simultaneous exposure to cues that induce cell proliferation and those that induce neural differentiation. As stated above, we have previously found that high concentrations (>25 ng/ml) of NT-3 induce neuronal differentiation while lower concentrations of either NT-3 or CNTF (1 – 20 ng/ml) induce the proliferation of DRG progenitor cells (Hapner et al., in preparation). Thus, we determined the response of DRG cells to simultaneous exposure to PACAP and mitogenic concentrations of NT-3 (10 ng/ml) or CNTF (10 ng/ml). Concurrent exposure to either of those growth factors together with PACAP completely blocked the PACAP-induced neuronal differentiative response (Fig. 10A). Thus, intriguingly, pathways activated by NT-3 and CNTF directly interfere with downstream signaling cascades activated by PACAP. In contrast, PACAP did not interfere with the NT-3- and CNTF-induced proliferative response (Fig. 10B). Furthermore, higher concentrations of NT-3 that are no longer mitogenic but instead promote neuronal differentiation (>25 ng/ml) did not block PACAP-induced neuronal differentiation (Table 3). An obvious site of convergence

Fig. 8. PACAP promotes neuronal differentiation via activation of MAP kinase. (A and B) DRGs were cultured and stained as above with Tuj-1 antibodies: U0126 treatment (B) reduced the PACAP-induced increase in both neurite length and in the number of neurons that had initiated axonogenesis. (C – F) DRGs were cultured for 6 h as described in the presence (E and F) or absence (C and D) of PACAP. Cells were then double immunolabeled with antibodies to phosphorylated MAP kinase and neurofilament (not shown). Neurons (arrows) were only faintly immunopositive for pMAPK in control wells, but many neurons were strongly immunopositive for pMAPK in the presence of PACAP; compare the intensity of labeled cells in C and D to those in E and F.

the same criteria used for PACAP-induced neuronal differentiation: an increase in the number of neural marker-positive cells with a process of at least one cell diameter. Here we show that while K252a had no effect on PACAP-induced neuronal differentiation, K252a did significantly reduce the ability of high concentrations of NT-3 (50 ng/ml) to induce neuronal differentiation

Fig. 9. PACAP-induced neuronal differentiation is not mediated by transactivation of trk receptors. DRGs were cultured as described and treated for 6 h with PACAP or NT-3 (50 ng/ml) in the presence or absence of K252a. K252a significantly reduced NT-3-induced neuronal differentiation but had no affect on PACAP-induced neuronal differentiation. Only tuj-1+ cells with a process of at least one cell diameter were counted. Values represent the mean F SEM for two experiments; P V 0.001.

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for these pathways might be the activation of MAP kinase because both NT-3 and PACAP binding to cells can lead to activation of MAPK (Vaudry et al., 2002) and we found that blockade of MAPK activation abrogated the PACAP-induced neural differentiation. Because discrete biological responses can be elicited by varying levels of MAPK activation, we were interested in determining in our cells whether MAPK activation was altered in the presence of simultaneous exposure to PACAP and low concentrations of NT-3. We found that in the presence of both factors, MAPK activation was increased relative to stimulation by PACAP or NT-3 alone (Fig. 6). To examine how common was this effect of non-GPCR ligands interfering with GPCR-mediated responses, we also tested the response to combinations of EGF and PACAP on neuronal

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Table 3 High concentrations of NT-3 do not block PACAP-induced neuronal differentiation Treatment PACAP PACAP PACAP PACAP

(10 (10 (10 (10

Percentage increase in neurons above control nM) nM) + NT-3 (30 ng/ml) nM) + NT-3 (50 ng/ml) nM) + EGF (10 ng/ml)

57 53 65 14

DRGs were dissociated and cultured for 6 h as described. The number of Tuj-1+ cells with an axon of at least one cell diameter was determined in quadruplicate wells for each treatment. The percent change in neuronal number relative to the control condition (see Experimental methods) was determined for each treatment P < 0.001.

differentiation (Table 3). Surprisingly, we found that low concentrations of EGF could also block PACAP-induced neuronal differentiation. Thus, cross talk between receptor tyrosine kinase (RTK) and GPCR-activated pathways may be quite prevalent.

Discussion

Fig. 10. NT-3 and CNTF block PACAP-induced neuronal differentiation. DRGs were dissociated as described and cultured for 6 h in a single or a combination of factors (PACAP (50 nM), NT-3 (10 ng/ml), CNTF (10 ng/ ml)), all added at the time of plating. Values represent mean percentage of (A) neurons (neurofilament+ cells with a process of at least one cell diameter) relative to control F SEM from quadruplicate cultures from two experiments; P V 0.0001. (B) PACAP does not interfere with NT-3 and CNTF-induced proliferation: DRGs were dissociated and cultured for 6 h in the presence of BrdU. The percentage of BrdU+ cells was determined in all conditions, quadruplicate wells from two separate experiments; P < 0.002.

Our data demonstrate a striking ability for PACAP to promote the differentiation of a subset of nascent sensory neurons. This response requires the PAC1 receptor and activation of the MAP kinase pathway. Not only does exogenously applied PACAP promote neuronal differentiation, but our results suggest that endogenously produced PACAP, or a closely related peptide, within the DRG, is necessary for neural differentiation in vivo. A second major finding of this study is that members of distinct polypeptide growth factor families, NT-3 and CNTF, can each block the PACAP-induced neuronal differentiation activity. While it is now well established that ligands for GPCRs can transactivate receptor tyrosine kinases (RTKs; Lee et al., 2002; Maudsley et al., 2000), to our knowledge this is the first documentation of neurotrophic factors blocking GPCR-stimulated activity. Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/VIP family of peptides, was isolated by Arimura and Shioda (1995) as a mammalian hypothalamic-releasing factor. As with other such factors, PACAP was later found to be more widely distributed in the nervous system and to have additional biological actions including promoting cell proliferation, survival, maturation, and neuronal differentiation (Waschek, 2002). The idea that PACAP affects neural differentiation first arose from the demonstration that the peptide causes neurite outgrowth in neuroblastoma (Hoshino et al., 1993), PC12 (Colbert et al., 1994), and adrenal chromaffin (Wolf and Krieglstein, 1995) cells and subsequently in cerebellar granule (Gonzalez et al., 1997) and sympathetic (DiCicco-Bloom et al., 2000) neuroblasts. The sequences of PACAP and its selective receptor (PAC1) have been determined for several species including the chick (McRory et al., 1997; Peeters et al., 1999) and indicate that these proteins have been highly conserved during evolution (Sherwood et al., 2000). In our studies, we used the commercially available PACAP, which is the human/bovine/rat sequence, because chicken PACAP differs by only one amino acid (an isoleucine to serine substitution in position 2; Erhardt et al., 2001). In addition, as PACAP38 but not PACAP27 was detected in chick brain (Erhardt et al., 2001) and as the effects of the two are similar on the PAC1 receptor (Harmar et al., 1998), we limited our studies to PACAP38.

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The chicken PAC1 receptor is >80% homologous to the mammalian receptor, and within the brain, two splice variants were detected: one corresponding to the short form of the receptor and the other to the hop1 isoform (Peeters et al., 1999; Spengler et al., 1993). Both the peptide and mRNA for PACAP and the PAC 1 receptors have been identified in neuroblast-enriched primary cell cultures derived from embryonic day 3.5 chick brain (Erhardt et al., 2001; Peeters et al., 2000). In the present study, we have extended these findings by demonstrating the expression of the ligand and receptor at a comparable stage in the peripheral nervous system (embryonic day 4.5). At this stage, the DRG is composed of both mitotically active progenitor cells and post-mitotic neurons (Carr and Simpson, 1978; Pannese, 1974; Rifkin et al., 2000). Thus, we show that both PACAP and the PAC1R are expressed during the maximal periods of neurogenesis and differentiation in the immature DRG, and hence PACAP may function in an autocrine or paracrine manner to regulate these early events. Our finding that neuronal differentiation is reduced in DRG cells cultured in the presence of the PAC1 receptor antagonist provides evidence that endogenously produced PACAP, or a related peptide, is released by cells in the immature DRG, binds to the PAC1 receptor, and functions to promote neuronal differentiation. In addition to being expressed during the peak period of neurogenesis in the DRG, we also found that both PACAP and the PAC1R are expressed in the more mature DRG (embryonic day 8.5) during the period of target innervation and programmed cell death. Within the peripheral nervous system, PACAP has been shown to induce the proliferation of sympathetic neuroblasts (DiCicco-Bloom et al., 2000). Even though both sympathetic ganglia and DRG are derived from the same precursor cell population, the neural crest, we show that PACAP has no mitogenic activity on cells in the DRG. One potential explanation for this discrepancy in biological responses is the difference in PAC1R isoforms expressed in these two distinct ganglia. Sympathetic ganglia express only the hop receptor isoform (Lu et al., 1998) while we found only the short, noninsert-containing receptor in the DRG. Thus, because the hop isoform may confer activation of additional or alternative signaling pathways than those stimulated by the short canonical receptor, two distinct biological responses might be elicited following binding of a common ligand (Waschek, 2002). Another activity attributed to PACAP for both DRG sensory neurons and sympathetic neurons is cell survival (Arimura et al., 1994; DiCicco-Bloom et al., 2000; Lioudyno et al., 1998; Przywara et al., 1998). Within the DRG there is an extensive period of programmed cell death coincident with target innervation that results from a competition for limited amounts of target-derived growth factors (Huang and Reichardt, 2001; Lindsay, 1996). Previous studies have demonstrated a trophic effect of PACAP on DRG neurons during the period of programmed cell death (Arimura et al., 1994; Lioudyno et al., 1998), and our finding of both PACAP and PAC1R expression in the DRG at this stage (E8.5) would support a trophic role for PACAP. However, our study was designed to determine whether PACAP regulated neurogenesis and differentiation within the DRG, which occurs before the period of programmed cell death, and thus we examined the response of the more immature E4.5 DRG (Carr and Simpson, 1978; Hamburger and Levi-Montalcini, 1949) to determine whether PACAP had nontrophic functions on immature sensory neurons (Lu et al., 1996). Furthermore, our data indicate that during early

stages of DRG development, PACAP does not reduce programmed cell death. A major finding of our study is that non-GPCR ligand family members, NT-3 and CNTF, can block the ability of PACAP to induce neuronal differentiation. This is particularly interesting given the fact that cells within the immature DRG are exposed simultaneously to multiple extrinsic signals that activate distinct intracellular signaling pathways, yet then undergo one of many specific responses, for example, proliferation, survival, or differentiation. The temporal and spatial expression pattern of both NT-3 and PACAP is consistent with their functioning to regulate developmental events in the immature DRG (Fig. 5; DiCicco-Bloom et al., 2000; Farinas et al., 1996; Lioudyno et al., 1998; Pinco et al., 1993). Although whether CNTF is expressed within or near the immature DRG is not known, its specific receptor CNTFRa is robustly expressed during the peak period of neurogenesis in the immature DRG and CNTF is known to be expressed in other embryonic peripheral ganglia (Finn and Nishi, 1996; Holst et al., 1997). Thus, it is likely that progenitor cells and nascent neurons are exposed simultaneously to all three factors. An outstanding question is the functional availability of these factors to nascent neurons given the complex intercellular microenvironment(s) within the immature DRG (Hall et al., 2002; Pannese, 1974). The ability for particular combinations of extrinsic signals to modulate cellular biological responses provides an additional mechanism for the exquisite regulation of biological outcomes. Because progenitor cell proliferation and neuronal differentiation are occurring simultaneously in the immature DRG, it is unclear how cells would integrate such conflicting signals as PACAP and low levels of NT3. Because higher levels of NT-3 also promote neuronal differentiation, a parsimonious explanation might be that initially, levels of NT-3 are low, inducing proliferation. PACAP then acts to promote differentiation of these nascent neural precursors and as levels of NT-3 increase, NT-3 then contributes towards the promotion of neural differentiation. Another possibility is that PACAP promotes neural differentiation indirectly by stimulating the release of a factor from one subset of cells within the immature DRG that then promotes the differentiation of a subset of neural precursors. Traditionally, GPCRs and RTKs have been considered functionally distinct, although several previous reports have shown the interactive effects of these and other receptor or ligand systems including a convergence of signaling cascades activated by PACAP and NGF in mediating PC12 neuronal differentiation (Vaudry et al., 2002), antagonist actions of PACAP on BMP7 stimulation of sympathetic dendrite formation (Drahushuk et al., 2002), PACAP prevention of a reduction in BDNF levels induced by NMDA (Frechilla et al., 2001), and modulation of PACAP and PAC1 receptor expression by NT-3 and NGF (Jamen et al., 2000; Jongsma Wallin et al., 2001) and of Trk receptors by PACAP (Lazarovici and Fink, 1999). Our data also indicate that the abrogative effect of NT-3 on PACAP-induced differentiation might be shared by other RTK ligands because EGF also blocked the PACAP response. Interestingly, EGF, like NT-3, has both proliferative and differentiative effects in the chick nervous system: it can promote both proliferation of progenitor cells in the chicken retinal margin (Fischer and Reh, 2000) and induce neurite outgrowth of chick CNS neurons (Rosenberg and Noble, 1989). Furthermore, the erbB family of EGF receptors is prominently expressed in the developing PNS, including the DRG (Francoeur et al., 1995; Li and Loeb, 2001; Topilko et al., 1996; Lefcort, F. and Loeb, J.A. unpublished observations).

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The phenomenon of heterologous transactivation between GPCRs and RTKs has recently received considerable attention (Dalle et al., 2002; Lee et al., 2002; Maudsley et al., 2000). PACAP can transactivate TrkA and TrkB in both PC12 cells and hippocampal neurons, respectively (although see also Lazarovici et al., 1998). In our system, PACAP is not exerting its actions via transactivation of trk receptors because K252a failed to block PACAP-induced neural differentiation. Insulin, another RTK ligand, induces heterologous desensitization of GPCRs in fibroblasts via a ubiquitination-mediated downregulation of the h-arrestin-1 adapter protein, which subsequently leads to desensitization of downstream-signaling pathways normally activated by GPCR ligands (Dalle et al., 2002; Luttrell and Lefkowitz, 2002). It will be interesting to ascertain whether the NT-3 and CNTF abrogation of the PACAP-induced neuronal differentiation is mediated by a similar mechanism. Variations in MAPK activation levels have been shown to be critical in determining the biological responses of neurons: EGF induces a transient MAPK activation and stimulates mitogenesis of PC12 cells while NGF induces a prolonged activation of MAPK and stimulates neuronal differentiation (Heasley and Johnson, 1992; Marshall, 1995; Nguyen et al., 1993; Traverse et al., 1992). In the Drosophila eye, quantitative differences in EGFR signaling and in MAP kinase activity elicit distinct biological responses including the decision to proliferate or differentiate and alter the pattern of photoreceptor differentiation (Halfar et al., 2001; Lesokhin et al., 1999; Yang and Baker, 2001). Our results also point to distinct functions for MAP kinase during neuronal differentiation: it is required for PACAP-induced neurite outgrowth because this response was blocked by MAP kinase inhibitor U0126, but blockade of MAP kinase activation did not interfere with PACAP-induced expression of neural markers. Given that cells can apparently ‘‘read’’ quantitative differences in MAPK activity which then trigger distinct responses, it will be interesting to determine whether the increase in MAP kinase activation obtained when cells are exposed to both NT-3 and PACAP contributes to the abrogative effect of NT-3 on PACAPinduced neuronal differentiation. In summary, our data point to the intricate integration of extrinsic signals by nascent neurons as they establish their mature identities and implicate PACAP in mediating a key role in the differentiation of sensory neurons.

Experimental methods DRG cultures Fertilized White Leghorn chicken embryos were obtained from Truslow Farms (Chestertown, MD) and incubated at 37jC in a rocking incubator (Kuhl, Flemington, NJ). Embryos were staged according to Hamburger and Hamilton (1951). Dorsal root ganglia (DRG) from E4.5 (Stage 25) chick embryos were dissected into Hanks’ balanced salt solution, calcium and magnesium free (Sigma, St. Louis, MO). To obtain a single-cell suspension, the DRG were incubated in 0.25% trypsin – 1 mM EDTA (Gibco, Grand Island, NY) for 5 min at 37jC and then triturated with pulled glass pipettes. The culture media consisted of Nutrient Mixture F-12 (Sigma) supplemented with Hybrimax Antibiotic/Antimycotic (1:100, Sigma), 0.4 mg/ml bovine albumin A-7638 (BSA, Sigma), T3 (10 ng/ml, Sigma), T4 (25 ng/ml, Sigma), transferrin (250 Ag/ ml, Gibco), and selenium (100 ng/ml, Gibco). Cells were plated on

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8-well Nunc glass chamber slides that were coated with poly-Dlysine (10 Ag/ml in F-12, Sigma) for 30 min at room temperature, rinsed twice with F-12, and coated with mouse laminin (20 Ag/ml in F-12, Gibco) overnight at 37jC. Approximately equal number of cells (roughly 10,000) were plated in each well. Immediately after plating, if proliferation was being assayed, bromodeoxyuridine (BrdU, Sigma) was added at 10 Ag/ml to each well. The growth factor in question was added at the stated concentration to each well with each experimental condition repeated in quadruplicate. The cells were then cultured for 6 h at 37jC, 7% CO2. The growth factors used were NT-3 (Genentech, South San Francisco, CA), ciliary neurotrophic factor (CNTF, R&D Systems; Minneapolis, MN; a kind gift of Dr. C. Paden, Montana State University), and recombinant human EGF (Invitrogen). The long form of PACAP, that is, PACAP1 – 38, was used throughout this study and was obtained from Peninsula Laboratories, Inc.; San Carlos, CA., as was PACAP6 – 38, an antagonist to PACAP. The MAP kinase antagonist, U0126, was purchased from Promega (Madison, WI). The TUNEL assay was performed according to the manufacturer’s instructions (Roche). Immunocytochemistry and immunoblotting For immunodetection of PACAP expression, embryos were cryosectioned and labeled with a monoclonal antibody to PACAP, a kind gift from Dr. Jan Fahrenkrug (Bispebjerg Hospital, Copenhagen; Hannibal et al., 1995; Nielsen et al., 1998) as described (Rifkin et al., 2000). To assay MAP kinase phosphorylation by immunoblot, E4.5 DRG were dissociated and cells were cultured on 35 mm plastic dishes coated with laminin 20 Ag/ml for 3 – 4 h at 37jC with equal numbers of cells plated per well. The wells were rinsed once with F-12 and then F-12 plus 0.4 Ag/ml BSA was added (quiescent medium) and the cells were incubated for 30 min. The quiesced cells were activated with either NT-3 (10 ng/ml), PACAP38 (10 nM), or both for 1 h and then washed 2 with F-12, placed on ice, and extracted at 4jC. The extraction buffer used was 0.05 M Tris pH 7.6, 0.1 M NaCl, 0.05 M NaFl, 0.01 M Na pyrophosphate, 1% TX-100, 0.1% SDS, 0.001 M EDTA plus protease and phosphatase inhibitor cocktails (Sigma) added to the buffer at 1:100 just before the extraction. Extracts were microfuged for 10 min. The supernatants (containing equal concentrations of protein) were incubated with nonphosphorylated MAP kinase Ab according to the manufacturer’s instructions (Cell Signaling Technology) followed by immunoprecipitation with Protein Asepharose (Pharmacia). The beads were washed 4 with extraction buffer plus inhibitors and then eluted by boiling in Laemmli sample buffer with h-mercaptoethanol. Proteins were separated on 12% SDS polyacrylamide gels followed by electrotransfer to polyvinylidene membranes (Sigma). After blocking with TBS plus 0.1% Tween 20, 6% BSA, and 1% casein, membranes were incubated overnight at 4jC with phosphorylated MAP kinase Ab (Cell Signaling Technology) diluted 1:1000 in TBS plus 0.1% Tween 20, 1% BSA, and 1% casein, followed by incubation for 1 h in goat anti-rabbit IgG-peroxidase (Cell Signaling Technology) diluted 1:2000 in the same buffer. The immune complexes were detected using a chemiluminescent detection substrate (Cell signaling Technology) and Hyperfilm ECL (Amersham Pharmacia Biotech). The blots were scanned and quantified as pixel number per band. For the detection of cells immunopositive for

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phosphorylated MAP kinase, cells were cultured and fixed as described and labeled with anti-phosphorylated MAP kinase antibody (Cell Signaling Technology), followed by incubation with Alexa-conjugated secondary antibody (Jackson Labs). Cells were photographed with equal exposure times. Proliferation assay Cells were rinsed twice with F-12 and then fixed with methanol/5% acetic acid at 20jC for 10 min. The slides were then washed four times with F-12 and two times with TBS (10 mM Tris, 150 mM NaCl, pH 7.4), 5 min per wash. They were then treated with 2 N HCl for 15 min at room temperature, followed by four 10min washes with TBS. The slides were then placed in blocking buffer (10% normal goat serum, 1% glycine, 0.4% Triton X-100 in 30 mM Tris, 150 mM NaCl) for 1 h at room temperature, followed by overnight incubation in primary antibody at 4jC. For BrdU staining, an anti-BrdU monoclonal antibody (Novacastra, Vector Laboratories, Burlingame, CA) was used at 1:300 in blocking buffer, overnight. Secondary antibodies included Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-Mouse (Jackson ImmunoResearch Laboratories, Inc.) used at 1:300 in blocking buffer. To visualize chromatin, 4V6 diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Eugene, OR) was added to this incubation at 1:1000. After the incubation, the slides were washed with TBS and then mounted in Prolong Antifade (Molecular Probes). Differentiation assay At the end of the 6-h culture period, FBS (Hyclone, Logan, UT) was added to each well at a 1:5 concentration. Cells were then immediately fixed for 30 min at room temperature by adding 8% paraformaldehyde in a buffer (0.1 M PO4, 0.15 N NaCl, pH 7.4) at a 1:1 concentration. Adding FBS, followed by the addition of paraformaldehyde solution directly to the wells without first removing the culture media, leads to the retention of the majority of neurons during the fixation procedure (Hapner, personal communication). The slides were washed for 2 min with TBS, then placed in blocking buffer (see Proliferation assay) for 20 min at room temperature, followed by overnight incubation in either an antibody to the 180-kDa neurofilament subunit (1:500 in blocking buffer, a kind gift of Dr. B. Granger, Montana State University) or Tuj-1, an antibody to a neural h-tubulin isoform (1:700 in blocking buffer, a kind gift of Dr. Robert Oakley, George Washington University). When both differentiation and proliferation were assayed in the same experiment, cells were fixed as in the differentiation assay and then processed for BrdU staining as described in the proliferation assay.

filters and noting the immunopositive cells. Although 10,000 cells were plated per well, a total of 500 cells per well were counted. Each experimental treatment was conducted in quadruplicate wells per experiment, and each experiment was repeated at least once and several were repeated multiple times (except for the MAP kinase phosphorylation assay). Each experiment included a control treatment which did not receive any growth factors. Data from the experiments were compiled as follows: first, the four control wells within an experiment were averaged; second, the percent change of each treatment well (four treatment wells per experiment) relative to the control average was determined; and, finally, t tests or ANOVAs and Student – Newman – Keuls tests, depending on the comparison in question, were run on these normalized percentage values (with n equal to the total number of experiments, see figure legends). The statistical significance of the combined data coincides with the statistical significance found within each experiment (with four wells per treatment). RT-PCR To determine whether PACAP and the PAC1R were expressed in immature (E4.5) and mature (E8.5) DRG, RT-PCR with primers designed to amplify these sequences was performed. E4.5 and E8.5 DRGs were collected and total RNA was isolated with standard techniques. Genomic DNA contamination was removed by digesting with RQ1-RNase-free DNase (Promega) for 30 min at 37jC, phenol – cholorofrm extracting, and isopropanol precipitating. Total RNA was resuspended in 25 Al nucleasefree H2O and 1 Al of which was used for single-step RT-PCR (ACCESS System, Promega). A gene-specific primer set for PAC1R was designed based on the available chicken PAC1R gene sequence (Peeters et al., 1999): forward 5V-GCTTCACTTTGATGATACAGGC, reverse 5V-CTTGCTCAGAATGGAGAGTTGTGTC. Additionally published primer sequences for PAC1R were also used for RT-PCR analysis, with the same results (Erhardt et al., 2001; Peeters et al., 1999). Published PACAP primers were used for RT-PCR analysis (Erhardt et al., 2001). Thirty-five cycles of PCR were performed and 5 Al was analyzed on 2% Ag EtBr gels. Single bands of the predicted size for the PACAP reaction and PAC1R reactions were observed, and in all PAC1R reactions the sizes of the products were consistent with the insert ( ) or short, isoform of PAC1R.

Acknowledgments F.L. was supported by NS035714 and HD40343. B.R.N. was supported by Kopriva Foundation (Montana State University) and NCRR P20-RR15583 (University of Montana). R.E.Z. was supported by NS12651.

Quantitation and statistical analyses All slides were examined on a Nikon FXA microscope. For each well, cells were first identified by their chromatin (DAPI) staining, which gives no indication of whether a cell is neuronal or nonneuronal. Every cell within the field of view was counted. The number of neurons (defined as neurofilament+ or Tuj-1+ and bearing at least one process one cell diameter in length) or the total number of BrdU+ cells was then determined by switching

References Arimura, A., Shioda, S., 1995. Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors: neuroendocrine and endocrine interaction. Front. Neuroendocrinol. 16 (1), 53 – 88. Arimura, A., Somogyvari-Vigh, A., Weill, C., Fiore, R.C., Tatsuno, I., Bay, V., Brenneman, D.E., 1994. PACAP functions as a neurotrophic factor. Ann. N.Y. Acad. Sci. USA 739, 228 – 243.

K.M. Nielsen et al. / Mol. Cell. Neurosci. 25 (2004) 629–641 Bhattacharyya, A., Frank, E., Ratner, N., Brackenbury, R., 1991. P0 is an early marker of the Schwann cell lineage in chickens. Neuron 7 (5), 831 – 844. Carr, V.M., Simpson, S.B., 1978. Proliferation and degeneration events in the early development of chick dorsal root ganglia. J. Comp. Neurol. 182, 727 – 740. Colbert, R.A., Balbi, D., Johnson, A., Bailey, J.A., Allen, J.M., 1994. Vasoactive intestinal peptide stimulates neuropeptide Y gene expression and causes neurite extension in PC12 cells through independent mechanisms. J. Neurosci. 14 (11 Pt. 2), 7141 – 7147. Dalle, S., Imamura, T., Rose, D.W., Worrall, D.S., Ugi, S., Hupfeld, C.J., Olefsky, J.M., 2002. Insulin induces heterologous desensitization of G protein-coupled receptor and insulin-like growth factor I signaling by downregulating h-arrestin-1. Mol. Cell, 6272 – 6285. DiCicco-Bloom, E., Deutsch, P., 1992. Pituitary adenylate cyclase activating peptide (PACAP) potently stimulates mitosis, neurogenesis and survival in cultured rat sympathetic neuroblasts. Regul. Pept. 37, 319. DiCicco-Bloom, E., Deutsch, P., Maltzman, J., Zhang, J., Pintar, J., Zheng, J., Friedman, W., Zhou, X., Zaremba, T., 2000. Autocrine expression and ontogenetic functions of the PACAP ligand/receptor system during sympathetic development. Dev. Biol. 219, 197 – 213. Drahushuk, K., Connell, T.D., Higgins, D., 2002. Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide inhibit dendritic growth in cultured sympathetic neurons. J. Neurosci. 22 (15), 6560 – 6569. Edlund, T., Jessell, T.M., 1999. Progression from extrinsic to intrinsic signaling in cell fate specification: a view from the nervous system. Cell 96, 211 – 224. Erhardt, N.M., Fradinger, E.A., Cervini, L.A., Rivier, J.E., Sherwood, N.M., 2001. Early expression of pituitary adenylate cyclase-activating polypeptide and activation of its receptor in chick neuroblasts. Endocrinology 142 (4), 1616 – 1625. Farinas, I., Yoshida, C.K., Backus, C., Reichardt, L.F., 1996. Lack of neurotrophin-3 results in death of spinal sensory neurons and premature differentiation of their precursors. Neuron 17 (6), 1065 – 1078. Farinas, I., Cano-Jaimez, M., Bellmunt, E., Soriano, M., 2002. Regulation of neurogenesis by neurotrophins in developing spinal sensory ganglia. Brain Res. Bull. 57, 809 – 816. Finn, T.P., Nishi, R., 1996. Expression of a chicken ciliary neurotrophic factor in targets of ciliary ganglion neurons during and after the celldeath phase. J. Comp. Neurol. 366 (4), 559 – 571. Fischer, A.J., Reh, T.A., 2000. Identification of a proliferating marginal zone of retinal progenitors in postnatal chickens. Dev. Biol. 220 (2), 197 – 210. Francoeur, J.R., Richardson, P.M., Dunn, R.J., Carbonetto, S., 1995. Distribution of erbB2 and erbB4 in the developing avian nervous system. J. Neurosci. Res. 41 (6), 836 – 845. Frank, E., Sanes, J.R., 1991. Lineage of neurons and glia in chick dorsal root ganglia: analysis in vivo with a recombinant retrovirus. Development 111 (4), 895 – 908. Frechilla, D., Garcia-Osta, A., Palacios, S., Cenarruzabeitia, E., Del Rio, J., 2001. BDNF mediates the neuroprotective effect of PACAP-38 on rat cortical neurons. NeuroReport 12 (5), 919 – 923. Geffen, R., Goldstein, R.A., 1996. Rescue of DRG that are programmed to degenerate in normal development: evidence that NGF can increase proliferation of neurectodermally-derived cells in vivo. Dev. Biol. 178, 51 – 62. Gonzalez, B.J., Basille, M., Vaudry, D., Fournier, A., Vaudry, H., 1997. Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts. Neuroscience 78 (2), 419 – 430. Halfar, K., Rommel, C., Stocker, H., Hafen, E., 2001. Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity. Development 128, 1687 – 1696. Hall, A.K., Ai, X., Hickman, G.E., MacPhedran, S.E., Nduaguba, C.O., Robertson, C.P., 1997. The generation of neuronal heterogeneity in a rat sensory ganglion. J. Neurosci. 17 (8), 2775 – 2784.

639

Hall, A.K., Burke, R.M., Anand, M., Dinsio, K.J., 2002. Activin and bone morphogenetic proteins are present in perinatal sensory neuron target tissues that induce neuropeptides. J. Neurobiol. 52 (1), 52 – 60. Hamburger, V., Hamilton, H.L., 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49 – 92. Hamburger, V., Levi-Montalcini, R., 1949. Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J. Exp. Zool. 111, 457 – 501. Hannibal, J., Mikkelsen, J.D., Clausen, H., Holst, J.J., Wulff, B.S., Fahrenkrug, J., 1995. Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus. Regul. Pept. 55 (2), 133 – 148. Harmar, A.J., Arimura, A., Gozes, I., Journot, L., Laburthe, M., Pisegna, J.R., Rawlings, S.R., Robberecht, P., Said, S.I., Sreedharan, S.P., Wank, S.A., Waschek, J.A., 1998. International Union of Pharmacology: XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol. Rev. 50 (2), 265 – 270. Heasley, L.E., Johnson, G.L., 1992. The beta-PDGF receptor induces neuronal differentiation of PC12 cells. Mol. Biol. Cell 3 (5), 545 – 553. Heuer, J.G, von Bartheld, C.S., Kinoshita, Y., Evers, P.C., Bothwell, M., 1990. Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system. Neuron 5 (3), 283 – 296. Holst, A., Heller, S., Junghans, D., Geissen, M., Ernsberger, U., Rohrer, H., 1997. Onset of CNTFRalpha expression and signal transduction during neurogenesis in chick sensory dorsal root ganglia. Dev. Biol. 191 (1), 1 – 13. Hoshino, M., Li, M., Zheng, L.Q., Suzuki, M., Mochizuki, T., Yanaihara, N., 1993. Pituitary activating peptide and vasoactive intestinal polypeptide: differentiation effects on human neuroblastoma NB-OK-1 cells. Neurosci. Lett. 159 (1 – 2), 35 – 38. Huang, E.J., Reichardt, L.F., 2001. Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24, 677 – 736. Jamen, F., Laden, J.C., Bouschet, T., Rodriguez-Henche, N., Bockaert, J., Brabet, P., 2000. Nerve growth factor upregulates the PAC1 promoter by activating the MAP kinase pathway in rat PC12 cells. Ann. N. Y. Acad. Sci. 921, 390 – 394. Jongsma Wallin, H., Danielsen, N., Johnson, J.M., Gratto, K.A., Karchewski, L.A., Verge, V.M., 2001. Exogenous NT-3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation. Eur. J. Neurosci. 14 (2), 267 – 282 (July). Journot, L., Waeber, C., Pantaloni, C., Holsboer, F., Seeburg, P.H., Bockaert, J.I., Spengler, D., 1995. Differential signal transduction by six splice variants of the pituitary adenylate cyclase-activating peptide (PACAP) receptor. Biochem. Soc. Trans. 23, 133 – 137. Kalcheim, C., Neufeld, G., 1990. Expression of basic fibroblast growth factor in the nervous system of early avian embryos. Development 109 (1), 203 – 215. Lallier, T.E., Bronner-Fraser, M., 1988. A spatial and temporal analysis of dorsal root and sympathetic ganglion formation in the avian embryo. Dev. Biol. 127, 99 – 112. Lazarovici, P., Fink Jr., D., 1999. Heterologous upregulation of nerve growth factor-TrkA receptors in PC12 cells by pituitary adenylate cyclase-activating polypeptide (PACAP). Mol. Cell Biol. Res. Commun. 2 (2), 97 – 102. Lazarovici, P., Jiang, H., Fink, D., 1998. The 38-amino-acid form of pituitary adenylate cyclase-activation polypeptide induces neurite outgrowth in pc12 cells that is dependent on protein kinase c and extracellular signal-regulated kinase but not on protein kinase a, nerve growth factor receptor tyrosine kinase, p21ras g protein, and pp60c-src cytoplasmic tyrosine kinase. Mol. Pharmacol. 54, 547 – 558. LeDouarin, N., 1982. The Neural Crest. Cambridge Univ. Press, London. Lee, F.S., Rajagopal, R., Kim, A.H., Chang, P.C., Chao, M.V., 2002. Activation of trk neurotrophin receptor signaling by pituitary adenylate cyclase-activation polypeptides. J. Biol. Chem. 277, 9096 – 9102.

640

K.M. Nielsen et al. / Mol. Cell. Neurosci. 25 (2004) 629–641

Lesokhin, A.M., Yu, S.Y., Katz, J., Baker, N.E., 1999. Several levels of EGF receptor signaling during photoreceptor specification in wild-type, Ellipse, and null mutant Drosophila. Dev. Biol. 205 (1), 129 – 144. Li, Q., Loeb, J.A., 2001. Neuregulin – heparin – sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle. J. Biol. Chem. 276 (41), 38068 – 38075. Lindsay, R., 1996. Role of neurotrophins and trk receptors in the development and maintenance of sensory neurons: an overview. Philos. Trans. R. Soc. London, Ser. B 351, 365 – 373. Lioudyno, M., Skoglosa, Y., Takei, N., Lindholm, D., 1998. Pituitary adenylate cyclase-activation polypeptide (PACAP) protects dorsal root ganglion neurons from death and induces calcitonin gene-related peptide (CGRP) immunoreactivity in vitro. J. Neurosci. Res. 51, 243 – 256. Lu, N., Black, I.B., DiCicco-Bloom, E., 1996. A paradigm for distinguishing the roles of mitogenesis and trophism in neuronal precursor proliferation. Dev. Brain Res. 94, 31 – 36. Lu, N., Zhou, R., DiCicco-Bloom, E., 1998. Opposing mitogenic regulation by PACAP in sympathetic and cerebral cortical precursors correlates with differential expression of PACAP receptor (PAC1-R) isoforms. J. Neurosci. Res. 53 (6), 651 – 662. Luttrell, L.M., Lefkowitz, R.J., 2002. The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J. Cell Sci. 115 (Pt 3), 455 – 465. Marshall, C.J., 1995. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179 – 185. Maudsley, S., Pierce, K., Musa Zamah, A., Miller, W., Ahn, S., Daaka, Y., Lefkowitz, R., Luttrell, L., 2000. The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J. Biol. Chem. 275, 9572 – 9580. McRory, J.E., Parker, R.L., Sherwood, N.M., 1997. Expression and alternative processing of a chicken gene encoding both growth hormonereleasing hormone and pituitary adenylate cyclase-activating polypeptide. DNA Cell Biol. 16 (1), 95 – 102. Memberg, S.P., Hall, A.K., 1995. Proliferation, differentiation, and survival of rat sensory neuron precursors in vitro require specific trophic factors. Mol. Cell. Neurosci. 6, 323 – 335. Morrison, S.J., 2001. Neuronal potential and lineage determination by neural stem cells. Curr. Opin. Cell Biol. 13, 666 – 672. Murphy, M., Reid, K., Brown, M.A., Bartlett, P.F., 1993. Involvement of leukemia inhibitory factor and nerve growth factor in the development of dorsal root ganglia neurons. Development 117, 1173 – 1182. Nguyen, T.T., Scimeca, J.C., Filloux, C., Peraldi, P., Carpentier, J.L., Van Obberghen, E., 1993. Co-regulation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1, and the 90-kDa ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotrophic factor, nerve growth factor, and the mitogenic factor, epidermal growth factor. J. Biol. Chem. 268 (13), 9803 – 9810. Nielsen, H.S., Hannibal, J., Fahrenkrug, J., 1998. Prenatal expression of pituitary adenylate cyclase activating polypeptide (PACAP) in autonomic and sensory ganglia and spinal cord of rat embryos. Ann. N. Y. Acad. Sci. 11 (865), 533 – 536. Oakley, R.A., Lefcort, F.B., Clary, D.O., Reichardt, L.F., Prevette, D., Oppenheim, R.W., Frank, E., 1997. Neurotrophin-3 promotes the differentiation of muscle spindle afferents in the absence of peripheral targets. J. Neurosci. 17 (11), 4262 – 4274. Pannese, E., 1974. The histogenesis of the spinal ganglia. Advances in Anatomy Embryology and Cell Biology. Springer-Verlag, New York. Paratore, C., Hagedorn, L., Floris, J., Hari, L., Kleber, M., Suter, U., Sommer, L., 2002. Cell-intrinsic and cell-extrinsic cues regulating lineage decisions in multipotent neural crest-derived progenitor cells. Int. J. Dev. Biol. 46 (1), 193 – 200. Peeters, K., Gerets, H., Princen, K., Vandesande, F., 1999. Molecular cloning and expression of a chicken pituitary adenylate cyclase-activation polypeptide receptor. Mol. Brain Res. 71, 244 – 255.

Peeters, K., Gerets, H., Arckens, L., Vandesande, F., 2000. Distribution of pituitary adenylate cyclase-activation polypeptide and pituitary adenylate cyclase-activating polypeptide type I receptor mRNA in the chicken brain. J. Comp. Neurol. 423, 66 – 82. Pinco, O., Carmeli, C., Rosenthal, A., Kalcheim, C., 1993. Neurotrophin-3 affects proliferation and differentiation of distinct neural crest cells and is present in the early neural tube of avian embryos. J. Neurobiol. 24 (12), 1626 – 1641. Pisegna, J.R., Wank, S.A., 1996. Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. J. Biol. Chem. 271, 17267 – 17274. Przywara, D.A., Kulkarni, J.S., Wakade, T.D., Leontiev, D.V., Wakade, A.R., 1998. Pituitary adenylyl cyclase-activating polypeptide and nerve growth factor use the proteasome to rescue nerve growth factor-deprived sympathetic neurons cultured from chick embryos. J. Neurochem. 71 (5), 1889 – 1897. Rifkin, J.T., Todd, V.J., Anderson, L.W., Lefcort, F., 2000. Dynamic expression of neurotrophin receptors during sensory neuron genesis and differentiation. Dev. Biol. 227, 465 – 480. Robberecht, P., Gourlet, P., De Neef, P., Woussen-Colle, M.C., Vandermeers-Piret, M.C., Vandermeers, A., Christophe, J., 1992. Structural requirements for the occupancy of pituitary adenylate-cyclase-activating-peptide (PACAP) receptors and adenylate cyclase activation in human neuroblastoma NB-OK-1 cell membranes. Discovery of PACAP(6 – 38) as a potent antagonist. Eur. J. Biochem. 207 (1), 239 – 246. Rohrer, H., Henke-Fahle, S., el-Sharkawy, T., Lux, H.D., Thoenen, H., 1985. Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence. EMBO J. 4 (7), 1709 – 1714. Rosenberg, A., Noble, E.P., 1989. EGF-induced neuritogenesis and correlated synthesis of plasma membrane gangliosides in cultured embryonic chick CNS neurons. J. Neurosci. Res. 24 (4), 531 – 536. Scott, S.A., 1992. Sensory Neurons: Diversity, Development and Plasticity, vol. 7(11). Oxford Univ. Press, New York, pp. 3739 – 3748. Sherwood, N.M., Krueckl, S.L., McRory, J.E., 2000. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon superfamily. Endocr. Rev. 21 (6), 619 – 670. Sieber-Blum, M., 2000. Factors controlling lineage specification in the neural crest. Int. Rev. Cytol. 197, 1 – 33. Sommer, L., 2001. Context-dependent regulation of fate decisions in multipotent progenitor cells of the peripheral nervous system. Cell Tissue Res. 305, 211 – 216. Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P.H., Journot, L., 1993. Differential signal transduction by five splice variants of the PACAP receptor. Nature 365, 170 – 175. Tischler, A.S., Riseberg, J.C., Gray, R., 1995. Mitogenic and antimitogenic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) in adult rat chromaffin cell cultures. Neurosci. Lett. 189, 135 – 138. Topilko, P., Murphy, P., Charnay, P., 1996. Embryonic development of Schwann cells: multiple roles for neuregulins along the pathway. Mol. Cell. Neurosci. 8, 71 – 75. Traverse, S., Gomez, N., Paterson, H., Marshall, C., Cohen, P., 1992. Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem. J. 288 (Pt. 2), 351 – 355. Vaudry, D., Gonzalez, B.J.J., Basille, M., Anouar, Y., Fournier, A., Vaudry, H., 1998. Pituitary adenylate cyclase-activating polypeptide stimulates both c-fos gene expression and cell survival in rat cerebellar granule neurons through activation of the protein kinase A pathway. Neuroscience 84 (3), 801 – 812. Vaudry, D., Stork, P.J.S., Lazarovici, P., Eiden, L.E., 2002. Signaling pathways for PC12 cell differentiation: making the right connections. Science 296, 1648 – 1649.

K.M. Nielsen et al. / Mol. Cell. Neurosci. 25 (2004) 629–641 Wakamatsu, Y., Maynard, T.M., Weston, J.A., 2000. Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis. Development 127 (13), 2811 – 2821. Waschek, J.A., 2002. Multiple actions of pituitary adenylate cyclase activation peptide in nervous system development and regeneration. Dev. Neurosci. 24, 14 – 23. Waschek, J.A., Casillas, R.A., Nguyen, T.B., DiCicco-Bloom, E.M., Carpenter, E.M., Rodriguez, W.I., 1998. Neural tube expression of pituitary adenylate cyclase-activation peptide (PACAP) and receptor: potential role in patterning and neurogenesis. Proc. Natl. Acad. Sci. U. S. A. 95, 9602 – 9607.

641

Weston, J.A., 1970. The migration and differentiation of neural crest cells. Adv. Morphog. 8, 41 – 114. Wolf, N., Krieglstein, K., 1995. Phenotypic development of neonatal rat chromaffin cells in response to adrenal growth factors and glucocorticoids: focus on pituitary adenylate cyclase activating polypeptide. Neurosci. Lett. 200 (3), 207 – 210. Wright, E.M., Vogel, K.S., Davies, A.M., 1992. Neurotrophic factors promote the maturation of developing sensory neurons before they become dependent on these factors for survival. Neuron 9, 139 – 150. Yang, L., Baker, N., 2001. Role of the EGFR/Ras/Raf pathway in specification of photoreceptor cells in the Drosophila retina. Development 128 (7), 1183 – 1191.