The AP-2 family proteins are basic helix-span-helix transcription factors that recognize the palindromic 5′-GCCNNNGGC-3′ motif or its related GC-rich sequences (reviewed in Refs. 10 and 11). Since the discovery of AP-2α expressed in the neural crest and neuroectoderm (12, 13), four additional members, AP-2β, AP-2γ, AP-2δ, and AP-2ε, have been added to the AP-2 family (14-19). AP-2 proteins share unique structures consisting of an N-terminal transactivation domain and a C-terminal DNA binding and dimerization domain. Although AP-2 proteins are coexpressed in some developing organs, they show different spatiotemporal expression during development, and gene inactivation studies indicated that they may have quite distinct roles in development (11). Based on initial studies of AP-2α and its expression in the neural crest (12, 13), it was generally assumed that AP-2α regulates differentiation of neural crest-derived cells such as SA cells. In line with this notion, mutant zebrafish deficient in AP-2α (tfAP-2a) showed defect in NA neuron development in both the LC and sympathetic neurons (20). However, because AP-2α inactivation in mice causes an early defect leading to neural tube closure, its precise role for NA neuron development was not clearly analyzed in mammals (21, 22).

Immunohistochemistry and in Situ Hybridization—E10.5-13.5 mouse embryos were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline at 4 °C. The embryos were treated with 30% sucrose overnight at 4 °C prior to embedding in OCT compound and stored at -70 °C. The embryos were sectioned with 16-μm thickness, and immunohistochemistry or in situ hybridization were performed. Transverse and parasagittal sections of embryos were used to analyze SG and LC, respectively. Coronal sections of the brain of newborn mice were used to analyze LC. The antibodies were detected using the Vectastain kit (Vector Labs), and the signal was visualized using 3,3′-diaminobenzidine. The following primary antibodies were used: rabbit anti-TH, 1:2000 for 3,3′-diaminobenzidine and 1:200 for immunofluorescence (Pel-Freeze, Rodgers, AR); mouse anti-TH, 1:200 for immunofluoroscence (Chemicon, Temecula, CA); rabbit anti-AP-2α, 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-AP-2β, 1:1000 (Santa Cruz Biotechnology); rabbit anti-AP-2γ, 1:1000 (Santa Cruz Biotechnology); rabbit anti-DBH, 1:5,000 (gift from Dr. Eipper); and rabbit anti-Phox2b, 1:10,000 (gift from Dr. Brunet). The specificity of anti-AP-2β antibody was confirmed (supplemental Fig. S1). For immunofluoroscence microscopy Alex Fluor 488 or 594-conjugated antibodies (Molecular Probes, Carlsbad, CA) were used after primary antibody treatment. Antisense RNA probes were made from pGEM-Easy T vector (Promega, Madison, WI) in which PCR products were cloned using primer sets described in Table 1. Hybrids with digoxigenin-labeled probes were visualized by treatment with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate.

Count of Phox2b-positive Cells and Measurement Expression of TH and DBH in SG—To measure the size of SG, regions between the forelimbs and hindlimbs of E13.5 embryos were sectioned with 20-μm thickness. Every tenth section was collected and stained with Phox2b-specific antibodies. Total Phox2b-positive cells in the SG from the collected sections were counted. The trunk regions of E13.5 embryos were sectioned with 16-μm thickness. Then expression of TH and DBH was detected by their specific antibodies and visualized using 3,3′-diaminobenzidine as described in the previous section. The intensity of signal was measured using the National Institutes of Health Image program (ImageJ). At least four different sections in each embryo were analyzed.

NA Measurement—E16.5 embryos or brains from newborn mice were frozen and stored at -70 °C. The samples were homogenized in the presence of 0.2 m perchloric acid and 0.1 mm EDTA. The samples were disrupted by ultrasonication and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatants were extracted with a syringe filter and used to measure the amount of NA by HPLC. The pellets were resuspended in phosphate buffer (10 mm potassium phosphate containing 0.2% Triton X-100), and the amount of protein was determined.

NCSC Cultures—Full-length AP-2α, AP-2β, and AP-2γ cDNA was cloned into pSlax13 (24). The cDNAs were cloned into the ClaI site of RCASBP, an avian replication-competent retroviral vector (24). The viruses were made in DF1 chicken fibroblast cells (ATCC), and titers were determined using the anti-gag protein monoclonal antibody AMV-3C2 (Developmental Studies Hybridoma Bank, Iowa, IA) (25). Primary culture of the trunk region of quail eggs (Conturnix japonica, CBT Farms, Chestertown, MD) was performed as described (25). NCSC from the trunk region were infected with each RCAS virus at a multiplicity of infection of 5. After 7 days in culture, the neural crest cells were analyzed by quantitative real time PCR or immunohistochemistry.

Quantitative Real Time PCR Analysis—cDNA was made from 5 μg of total RNA, which was isolated using TRIzol reagent (Sigma), using Superscript II (Invitrogen) with oligo(dT)_12-18 as primer. Real time PCR analyses were performed in duplicate using SYBR green I using DNA engine Opticon™ (MJ Research, Waltham, MA) to analyze mRNA expression levels. The primer sets used to detect mRNA are shown in Tables 1 and 2.

Development and Neurotransmitter Phenotype Specification of NA Neurons Are Compromised in AP-2β Null Mice—To further investigate the role of AP-2β in SA neuronal development as well as in NA phenotype specification in vivo, we tested whether NA neurons exhibit any developmental abnormalities in the AP-2β^-/- mouse. Although previous knock-out studies revealed the critical role of AP-2β in kidney development (27), its role for the nervous system development is not known. We isolated the trunk regions and hind-brains of embryos to investigate noradrenergic lineage-specific gene expression in the PNS and CNS, respectively. Quantitative real time PCR analysis demonstrated that the expression of DBH mRNA was reduced by ~78% in the PNS of AP-2β^-/- embryos compared with their wild type littermates (Fig. 2A). In addition, expression of other NA markers, e.g. GATA-3, Phox2a, and Phox2b, was significantly reduced in the trunk region of AP-2β^-/- embryos (Fig. 2A and data not shown). Analysis of NA maker genes, TH (Fig. 3, B and D) and DBH (Fig. 3F), and pan-neuronal markers of the PNS, peripherin (Fig. 3H) and SCG10 (Fig. 3J), indicated that size of the SG in trunk region is reduced in AP-2β^-/- embryos. Gangliogenesis of the SG occurs in a nonsynchronized fashion, depending on the location during development. Also the size of SG is varied depending on the sectional level. To precisely determine the size of SG, we collected every tenth section of trunk region and counted the total number of NA neurons in the SG by staining Phox2b, which is a NA neuron-specific transcription factor. At earlier embryonic stages (i.e. E10.5, E11.5, and E12.5) Phox2b-positive cell counting in the SG of AP-2β^-/- embryos showed 88, 80, and 71% of wild type littermates as controls, respectively (data not shown). Analysis of SG of E13.5 mouse embryo trunk region showed that Phox2b-positive neurons of AP-2β^-/- embryos were diminished to 40% of wild type littermates (Fig. 3K and supplemental Table S1). However, the mRNA level of TH was only marginally affected in the trunk of the embryos (Fig. 2A), although the size of SG was significantly reduced in AP-2β^-/- embryos (Fig. 3). This apparent discrepancy is likely because TH is highly expressed in mouse DRG, which seems to be intact in AP-2β^-/- embryos (supplemental Fig. S2) (28). We analyzed the expression level of TH and DBH genes in the NA neurons of SG by comparing the mean intensity (expression level per area), which was calculated using the National Institutes of Health ImageJ program. Expression of DBH in the SG of AP-2β^-/- embryos was diminished to 62% of wild type littermates (Fig. 3L), whereas expression of TH in the SG of AP-2β^-/- embryos was marginally diminished to 83% of wild type littermates (Fig. 3M).

Surprisingly, we found that DBH mRNA expression was even more dramatically diminished in the hindbrain of AP-2β^-/- embryos (Figs. (Figs.2B2B and and4).4). Indeed, DBH was almost nondetectable in the LC of AP-2β^-/- mice at both E13.5 (Fig. 4D) and P0 (Fig. 4J). In addition, expression of TH and another NA marker, norepinephrine transporter, was significantly diminished in the LC of AP-2β^-/- embryos (Fig. 4, F and L). In line with this observation, we also observed significant decrease of TH mRNA in the hindbrain of AP-2β^-/- embryos by PCR analysis (Fig. 2B). Most importantly, the level of NA was dramatically diminished (by >95%) in the CNS of mice at both E16.5 and P0 (Fig. 5). NA was also reduced by ~30% in the PNS (Fig. 5).

DOPS Treatment during Pregnancy Significantly Rescues the Neonatal Lethality of AP-2β^-/- Mice—AP-2β^-/- mice die during neonatal days 1 and 2, and this lethality has been attributed to kidney failure (27). In our hands, a small fraction (~10%) of newborn AP-2β^-/- mice survive beyond P2, but none of them survived past P20 (Fig. 6A and data not shown). We asked whether NA deficiency directly causes the neonatal lethality of AP-2β^-/- mice. To address this question, we treated pregnant AP-2β^-/- mice with DOPS, which can be converted to NA by l-aromatic amino acid decarboxylase and allows the animals to bypass the DBH deficiency. Newborn mice were genotyped after birth, and their survival was scored each day until P7. As previously reported, the body weight of AP-2β^-/- mice was about 60% of the wild type or heterozygous control littermates at P7 (10). DOPS treatment did not change the body weight of AP-2β^-/- mice (Fig. 6C and data not shown), suggesting that the body weight defect is not caused by NA deficiency. Strikingly, 100% of AP-2β^-/- pups survived with DOPS treatment at P0, whereas 71% survived without DOPS treatment (Fig. 6A). By P4, only 10.4% of untreated AP-2β^-/- pups survived, whereas 52% survived with DOPS treatment. Taken together, these results strongly suggest that AP-2β critically regulates the development and neurotransmitter phenotype of NA neurons in both the PNS and CNS of developing mice and that NA deficiency is one of the main causes for neonatal lethality observed in AP-2β^-/- mice.

Distinctive Roles of AP-2 Transcription Factors for Differentiation of Diverse Neural Crest-derived Cell Lineages—The AP-2 family transcription factors play essential roles for development and morphogenesis of diverse tissues and organs. In mammalian species, e.g. human and rodents, five members of AP-2 factors (α-ε) have been reported (11). Among three AP-2 members (AP-2α, β, and γ) expressed in the nervous system, we show that AP-2β specifically regulates development of NA neurons. These results are rather surprising because previous in vitro studies from this and other laboratories showed that AP-2α regulates SA-specific gene promoters such as TH, DBH, and phenylethanolamine N-methyltransferase (30-32). Nevertheless, using the gain-of-function analyses, we found that AP-2-α, β, and γ exhibit clearly distinctive roles in cell fate determination of NCSC. Whereas AP-2α appears to play an important role in melanocyte differentiation from NCSC, AP-2β favors differentiation of NCSC to the SA cell fate. In contrast, exogenous expression of AP-2γ did not affect generation of melanocyte or SA cells. Therefore, it seems that these AP-2 family transcription factors may have very distinctive mechanisms in vivo despite the fact that they can similarly transactivate the promoter activities in vitro. Indeed, all three factors (AP-2-α, β, and γ) exhibited similar transactivation and DNA binding activities in vitro to the TH and DBH promoters (data now shown). However, we cannot exclude the possible role of other AP-2 proteins, which may be expressed in a specific stage during development, in the differentiation of neural crest-derived SA lineages. Strikingly, our gain-of-function results showed that AP-2α dramatically favors melanocyte formation from NCSC at the expense of SA cells, which is consistent with the recent AP-2α conditional knock-out studies showing that AP-2α is critical for melanocyte differentiation (33). These findings are intriguing when considering the finding that human melanoma is associated with a loss of AP-2α (34) and suggest the possibility that AP-2α may have a crucial role in controlling both differentiation and maintenance of melanocytes. Notably, in the mutant zebrafish lacking AP-2α (tfAP-2a), NA neurons could not develop properly in both the LC and sympathetic neurons (20). Thus, tfAP-2a of zebrafish may represent a functional counterpart (orthologue) of AP-2β of vertebrates like mouse for NA neuron development.