©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Expression of the Mouse Necdin Gene
IDENTIFICATION OF A POSTMITOTIC NEURON-RESTRICTIVE CORE PROMOTER (*)

(Received for publication, October 2, 1995; and in revised form, October 26, 1995)

Taichi Uetsuki (1) (2) Keiich Takagi (1) Hiroko Sugiura (1) Kazuaki Yoshikawa (2)(§)

From the  (1)Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6, Fuchu, Tokyo 183, Japan and the (2)Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Necdin is a 325 amino acid residue protein encoded by a cDNA clone isolated from neurally differentiated embryonal carcinoma cells. In situ hybridization histochemistry revealed that necdin mRNA-containing cells in vivo coincided with postmitotic neurons in the mouse brain from early periods of neurogenesis until adulthood. To study the regulation of necdin gene expression, we have isolated and characterized the necdin gene from a mouse genomic DNA library. The necdin gene contains no intron, and its upstream region lacks canonical TATA and CAAT boxes. To assess promoter activity, the 5`-flanking sequence (844 base pairs) of the necdin gene was fused to the LacZ reporter gene and transiently transfected into retinoic acid-treated P19 embryonal carcinoma cells. Most of the transfectants expressing high levels of LacZ immunoreactivity were postmitotic neurons differentiated from P19 cells. Deletion analysis using luciferase reporter genes demonstrated that a neuron-restrictive core promoter is localized to positions -80 to -35, in which a G+C-rich domain and a putative binding site for transcription factors with PAS (per, arnt, and single-minded) dimerization domain are comprised. These results suggest that postmitotic neuron-restrictive expression of the necdin gene is mediated by the specific cis-acting elements and that this promoter is applicable to postmitotic neuron-targeted expression of various transgenic systems.


INTRODUCTION

Neurons in the central nervous system withdraw permanently from the cell cycle immediately after differentiation from their proliferative progenitors. To study the molecular mechanism whereby neurons become postmitotic, cultured cell lines that differentiate into postmitotic neurons are indispensable. The murine embryonal carcinoma P19 cells differentiate into postmitotic neurons in response to retinoic acid treatment(1, 2) . We have previously isolated a novel cDNA sequence encoding a 325-amino acid residue protein, designated necdin, from a subtraction cDNA library of retinoic acid-treated P19 cells(3) . Immunohistochemical studies have shown that necdin is a nuclear protein expressed in differentiated neurons in the mouse brain (3, 4) . Necdin mRNA is expressed in neurons differentiated from embryonal carcinoma cells but not in proliferative neuron-like cells originating from tumors such as neuroblastoma and pheochromocytoma(4) . These findings suggest that necdin is expressed in postmitotic neurons that are irreversibly differentiated from proliferative stem cells. More recently, we have demonstrated that ectopic expression of necdin in NIH3T3 cells suppresses the cell growth without affecting cell viability(5) . Therefore, it is likely that necdin is involved in intranuclear events by which neurons become permanently quiescent.

The mechanisms underlying transcriptional regulation of neuron-specific genes are just beginning to be elucidated. A common mechanism of transcriptional control of neuron-specific genes has been recently proposed. The silencer elements existing in the promoter regions of neuron-specific genes encoding type II sodium channel(6) , SCG10(7) , and synapsin I (8) define neuronal specificity. Furthermore, it has been shown that these elements interact with a tissue-specific negative regulator, which represses the expression of neuronal genes in non-neuronal cells(9, 10) . These findings led us to examine whether expression of the necdin gene in postmitotic neurons is mediated by specific cis-acting elements in the promoter region.

In this report, we demonstrate, by in situ hybridization histochemistry, that necdin mRNA is expressed endogenously in virtually all postmitotic neurons in the central nervous system from early stages of development until adulthood. To elucidate the mechanism underlying transcriptional regulation of necdin in postmitotic neurons, we have characterized the necdin gene isolated from a mouse chromosomal DNA library. To analyze the promoter function, we have employed a transient transfection system in which reporter genes are introduced into differentiating P19 cells, which later become postmitotic neurons. By using this system, specific cis-acting elements involved in postmitotic neuron-restrictive expression were defined.


EXPERIMENTAL PROCEDURES

In Situ Hybridization Histochemistry

Adult male ddY mice (90-day-old) and timed pregnant mice were sacrificed under deep diethylether anesthesia. Embryos were immersed in a 4% paraformaldehyde in phosphate-buffered saline, embedded in O.C.T. compound (Miles Laboratories), and quickly frozen on dry ice powder. Adult mouse brains were perfused and fixed with the above solution. Frozen tissues were sectioned 10-20-µm thickness in a cryostat and mounted onto a poly-D-lysine-coated slide glass. Antisense necdin RNA was prepared as a hybridization probe by transcribing full-length mouse necdin cDNA in SmaI-digested p4BFL (3) by T3 RNA polymerase in the presence of digoxigenin-labeled UTP (Boehringer Mannheim). Antisense RNA probe for neurofilament-L (or 68-kDa neurofilament) (NF-L) (^1)was synthesized by transcribing NF-L cDNA(11) , a gift from Dr. N. J. Cowan, which was inserted into the EcoRI site of Bluescript II, with T3 RNA polymerase after digestion with BamHI at the 3`-end. Synthesized RNA probes were digested into average 150-base fragments by treating RNA at 60 °C for 55 min with NaHCO(3). Prior to hybridization, sections were immersed at 4 °C in the following solutions; 0.2% Triton X-100 for 5 min, 0.2 N HCl for 15 min (followed by incubation with 1 µg/ml proteinase K at 37 °C for 5 min), 4% paraformaldehyde for 5 min, 2 mg/ml glycine for 30 min, and 2 times SSC (1 times SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7), 50% formamide for 1 h. The sections were then hybridized with the synthetic probes (0.4 µg/ml each) for 3 h at 70 °C in a premixed hybridization solution (Rapid hybridization buffer, Amersham) and washed at 56 °C with 2 times SSC, 50% formamide for 30 min and then with 1 times SSC, 50% formamide for 30 min. Hybridization signals were detected by the alkali phosphatase-mediated detection system (Boehringer Mannheim). Hybridization with necdin sense RNA revealed that endogenous alkali phosphatase was completely inactivated in the process described above.

Cloning of Mouse Necdin Chromosomal DNA

Mouse genomic DNA library (129SV mouse strain in the FIX II vector system) was purchased from Stratagene. About 10^6 plaques were screened with the mouse necdin cDNA probe(3) . Five positive clones were isolated, and the clone containing the largest 12.5-kb insert (nd5) was further analyzed. NotI-BamHI (0.9 kb) and BamHI-XbaI (4 kb) fragments of the nd5 insert were hybridized with necdin cDNA by Southern blot analysis and were subcloned into pBluescript II (Stratagene). Nested deletions for DNA sequencing were made using a kit supplied by Pharmacia Biotech Inc. DNA sequence was determined by either the dye terminator method or the dye primer method using an automatic DNA sequencer (model 373A) according to the protocol recommended by Applied BioSystems. The transcription start site was determined by the polymerase chain reaction (PCR)-based cDNA cloning method (12) using primers complimentary to positions +248 to +269 and positions +225 to +246, respectively, of necdin mRNA(3) . The PCR products were cloned into the pGEM-T vector (Promega), and their 5`-ends were sequenced.

Construction of Reporter Plasmids and Transfection into P19 Cells

To construct reporter plasmids, the LacZ (beta-galactosidase) cassette was excised from pCH110 (Pharmacia), an SV40 promoter-driven LacZ reporter plasmid, by digesting with HindIII and BamHI and inserted into pBluescript to make the promoter-less vector pBSZ. The 5`-flanking region (-844 to +63) of the necdin gene was then inserted, after constructing HindIII sites at both ends by PCR, into the HindIII site of pBSZ to make the reporter plasmid pNDZ. P19 embryonal carcinoma cells were cultured and induced to differentiate as described previously(1, 2) . For induction of neural differentiation, cultures of P19 cells were aggregated by plating at a density of 1 times 10^5 cells/ml into bacteria-grade culture dishes (Falcon 1029, Becton Dickinson) and incubated in the presence of 0.5 µM all-trans retinoic acid (Sigma) for 4 days. The aggregates were then trypsinized, passed through a 100-µm nylon mesh (Falcon 2360), and plated onto a 6-well plate (Nunc) at a density of 5 times 10^5 cells per well, in which polyethlenimine-coated coverslips were placed for immunocytochemistry. After 12 h, reporter genes (2 µg) were transfected into the cultured cells by polycationic liposome-mediated DNA transfection using Lipofectamine reagent (Life Technologies, Inc.) according to the protocol recommended by the manufacturer. The cells were fixed for immunostaining 3-4 days after transfection.

Fluorescent Immunocytochemistry

P19 cells transfected with reporter plasmids were examined by immunocytochemistry (4) . The cells were fixed in 4% formaldehyde for 4 °C and in methanol-acetone (1:1) for 15 min at -20 °C. The fixed cells were incubated with rabbit polyclonal anti-LacZ antibody (1:200) (Boehringer Mannheim) and mouse monoclonal antibody against microtubule-associated protein 2 (MAP2) (1:100) (Sigma) in phosphate-buffered saline for 30 min at 37 °C. After washing with phosphate-buffered saline, the cells were incubated with fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulins (1:50) (Tago) and Rhodamine B-conjugated anti-mouse immunoglobulins (1:50) (Tago). After washing with phosphate-buffered saline, immunoreactive cells were visualized and photographed with a fluorescence microscope (Nikon Microphot FXA-FL) using Kodak TMAX 400 film. Differentiated neurons and non-neuronal cells among the LacZ-positive cells were identified by their morphological characteristics under the fluorescence microscope.

Deletion Analysis of the Necdin Promoter

The necdin 5`-flanking region was inserted into the HindIII site of the luciferase reporter plasmid (PGV-B) (Toyo Ink, Tokyo). The promoter region was deleted by digestion with exonuclease III from the 5`-end of the promoter region using the kit and method recommended by Pharmacia. Deleted fragments were subcloned into pBluescript for sequencing. The reporter plasmids were transfected into retinoic acid-treated P19 cells according to the same method as used for the LacZ reporter transfection described above. The transfected P19 cells were cultured for further 3-4 days and lysed in a cell lysis solution. Luciferase activities were measured using a luminometer (Lumat LB9501, Berthold)(13) . The cell lysis solution and reagents for luciferase assay were purchased from Toyo Ink. The LacZ reporter gene pCH110 (2 µg) was cotransfected with the luciferase reporter genes, and luciferase activity in each experiment was normalized against LacZ activity measured with the reagents provided by Promega.


RESULTS

Expression of Necdin mRNA in the Mouse Brain in Vivo

The distribution of necdin mRNA in the mouse brain at the stage of postnatal day 90 was analyzed by in situ hybridization histochemistry (Fig. 1). In the cerebral hemisphere, necdin mRNA-positive cells were abundant in neuron-enriched areas. The cells containing high levels of necdin mRNA were present in the hypothalamus, piriform cortex, amygdaloid nuclei, and hippocampus (Fig. 1A). In the neocortex, the cells in inner cortical layers contained higher levels of necdin mRNA than those in the outer layers (Fig. 1, A and C). A gross regional distribution of necdin mRNA-positive cells in the sagittal section showed that necdin mRNA-positive cells were dense in the hypothalamus, midbrain, pons, and medulla oblongata, while hybridization signals of necdin mRNA were relatively low in the olfactory bulb, neocortex, cerebellum, and thalamus (Fig. 1B). In deep layers (layers V-VI) of the neocortex (Fig. 1C) and the spinal cord (Fig. 1D), larger neurons expressed higher levels of necdin mRNA. The hybridization signal was hardly detected in the areas where neuronal cell bodies are scantly distributed (for example, the corpus callosum, outermost layer of the neocortex, internal capsule, and white matter of the spinal cord (Fig. 1, A, B, and D). These results suggest that necdin mRNA is expressed predominantly in the neuronal population throughout the central nervous system of adult mice.


Figure 1: Expression of necdin mRNA in adult mouse brain. Frozen sections from the brain of 90-day-old mice were used for the detection of necdin mRNA by in situ hybridization histochemistry. A, coronal section of the cerebral hemisphere. B, sagittal section of the central nervous system. The regions such as the hippocampus, piriform cortex, amygdaloid nuclei, and hypothalamus contain high levels of necdin mRNA (A). Note the scanty distribution of necdin mRNA-positive cells in the white matter (A and B). C, deep layers (IV-VI) of the neocortex. D, transverse section of the spinal cord. Large neurons in layer V of the neocortex and anterior horn of the spinal cord contain high levels of necdin mRNA (arrowheads in C and D). NC, neocortex; CC, corpus callosum; HC, hippocampus; PI, piriform cortex; AM, amygdaloid nuclei; IC, internal capsule; HT, hypothalamus: TH, thalamus; CP, caudate nucleus/putamen; OB, olfactory bulb; C, colliculi of the midbrain; CB, cerebellum; P, pons; M, medulla oblongata; AH, anterior horn; PH, posterior horn. Scale bars = 500 µm (A, B, and D) 100 µm (C).



Fig. 2shows the distribution of necdin mRNA in the forebrain (telencephalon plus diencephalon) in comparison with that of NF-L mRNA, a typical marker for neuronal differentiation, during fetal and perinatal periods. At the stage of embryonic day (E) 14.5, necdin mRNA-positive cells were localized to the outermost layer (mantle layer) of the dorsal telencephalic region, where young migratory neurons accumulate (Fig. 2A). Necdin mRNA was undetected in the ventricular zone (germinal zone) of the forebrain vesicle, in which proliferative neuroepithelial stem cells are present. In addition, cells in the basal ganglia and the hypothalamic nuclei expressed high levels of necdin mRNA. Necdin mRNA was first detected in the forebrain area at E10.5 (data not shown). On the other hand, NF-L mRNA was absent from the telencephalic regions at E14.5 (Fig. 2D). At this stage, expression of NF-L mRNA was restricted to the midbrain, hindbrain, and spinal cord (data not shown). At E17.5, intense hybridization signals of necdin mRNA were detected in the hypothalamus, thalamus, amygdaloid nuclei, and pyramidal cell layer of the hippocampus (Fig. 2B), whereas moderate hybridization signals of NF-L mRNA were observed only in the thalamic region of the forebrain (Fig. 2E). At the stage of postnatal day 3, necdin mRNA was expressed in most of the forebrain regions. High levels of necdin mRNA were distributed in the hypothalamus, amygdaloid nuclei, pyramidal cell layer of the hippocampus, and cortical plate of the neocortex (Fig. 2C). At this stage, the distribution pattern of NF-L mRNA-positive cells was similar to that of necdin mRNA-positive cells, but the levels of NF-L mRNA in the hypothalamus and amygdaloid nuclei were lower than those of necdin mRNA (Fig. 2F). These results suggest that expression of the necdin gene precedes that of the NF-L gene and thus represents a good indication of neuronal differentiation at early stages of neurogenesis in the forebrain. Necdin mRNA was also detected in the peripheral nervous system (for example, the sympathetic ganglia, dorsal root ganglia, and enteric autonomic nerve plexus) but not in non-neuronal tissues except for skeletal myoblasts that transiently expressed necdin mRNA during the period of myotube formation. (^2)


Figure 2: Expression of necdin mRNA and NF-L mRNA in developing forebrain. Adjacent frozen sections of the mouse forebrain were used for detecting necdin mRNA at E14.5 (A), E17.5 (B), and P3 (C) and NF-L mRNA at E14.5 (D), E17.5 (E), and P3 (F). A and D, sagittal sections; B, C, E, and F, coronal sections. At E14.5 (A), necdin mRNA is abundantly expressed at the mantle layer of the telencephalon (arrowheads) but undetected at the germinal zone (arrows) where the neuroepithelial stem cells continue to proliferate. NF-L mRNA is detected only in the thalamic nuclei during prenatal periods (D and E), whereas intense hybridization signals of necdin mRNA are present in the hypothalamus, thalamus, amygdaloid nuclei, and hippocampus (A-C). NC, neocortex; TH, thalamus; GE, ganglionic eminence; HT, hypothalamus; AM, amygdala; HC, hippocampus. Scale bars = 0.5 mm for each pair of panels (panels A and D, panels B and E, and panels C and F).



Mouse Necdin Genomic DNA

Since the necdin gene is expressed in postmitotic neurons in vivo, we have investigated the regulatory mechanism of necdin gene expression in postmitotic neurons. We first isolated and characterized the necdin genomic DNA from a lambda phage library of mouse chromosomal DNA. Five clones showed positive hybridization signals, and restriction mapping of these clones revealed that the clone designated nd5 contained the largest 12.5-kb sequence including the entire necdin cDNA sequence (Fig. 3). The restriction map of necdin cDNA (3) completely matched that of the corresponding part of the genomic sequence, suggesting that this gene lacks an intron. The inserts of the other clones were all included in the nd5 DNA insert. We have found out, by Southern blot analysis of mouse genomic DNA, that the necdin gene is present as a single copy in the mouse haploid genome. (^3)Therefore, we inferred that the nd5 insert comprises the necdin gene.


Figure 3: Restriction maps of mouse necdin genomic DNA and sequencing strategy. A, the necdin genomic DNA encoded in the nd5 insert (12.5 kb). B, the NotI-XbaI region and sequencing strategy. Each arrow represents the direction and the length of nucleotides sequenced. The box represents the exon, in which the open reading frame is shadowed. Note that the necdin gene contains no intron. Scale bars = 1 kilobase pair (for A) and 200 bp (for B).



The intron-less nature of the necdin gene was confirmed by comparing the genomic DNA sequence with the necdin cDNA sequence (Fig. 4); the nucleotide sequence of the genomic clone completely matched the corresponding part of the cDNA sequence (3) except for one base mismatch in the coding region. This mismatch, presumably owing to individual variations or mouse strain difference, gives rise to one amino acid substitution (Val-17 Ala).


Figure 4: Nucleotide sequence of the mouse necdin gene. The nucleotide sequence of the NotI-XbaI region, which includes the entire exon and the flanking regions, is shown. The amino acid sequence in the coding region is presented by the single-letter amino acid notation. The transcription initiation sites, indicated as S1 and S2, were determined by the PCR-based 5`-end extension method(6) , and S1 is the major initiation site (see text for details). The arrow indicates the position of one base substitution (T to C). The polyadenylation signal (underlined) and the polyadenylation start site (poly A) are shown. Note the absence of canonical TATA and CAAT elements upstream from S1.



We then determined the transcription initiation site by the 5`-extension method using PCR(12) . Among 20 clones analyzed, 11 clones started at position 871 (S1 in Fig. 4), 5 clones at position 862 (S2 in Fig. 4), and the rest had various starting sites downstream from S1. Since the frequency of S1 is the highest, it is likely that S1 is the major transcription start site. Neither a canonical TATA box nor a CAAT element was found upstream from S1.

Neuron-specific Expression Driven by the Necdin Promoter

We then constructed a reporter plasmid (pNDZ) containing the 5`-flanking region (844 bp sequence upstream from S1) fused to the LacZ (beta-galactosidase) gene. To examine whether the necdin promoter is active in postmitotic neurons derived from P19 cells, pNDZ was transfected into retinoic acid-treated P19 cells at the proliferative stage, and its expression was determined by LacZ immunostaining after differentiation into postmitotic neurons. The promoter-less reporter vector pBSZ and the SV 40 promoter-driven LacZ reporter vector pCH110 were also transfected into P19 cells. Although LacZ immunoreactive cells in the pBSZ-transfected cells were undetected (data not shown), differentiated P19 cells transfected with pNDZ and pCH110 were positively stained with the anti-LacZ antibody (Fig. 5). Expression of MAP2 was utilized as a marker for neuronal differentiation. In addition, the postmitotic neurons in differentiated P19 cells were identified by their morphological characteristics including small round perikarya, scanty cytoplasm, and extension of neurites. Most of the reporter-expressing cells in pNDZ-transfected cells coincided with postmitotic neurons (Fig. 5, A and B), whereas about 50% of the LacZ-positive cells in pCH110 (SV 40-promoter)-transfected cells were non-neuronal cells with large nuclei and enriched cytoplasm (Fig. 5, C and D). This indicates that the necdin promoter shows a preference for postmitotic neurons that are differentiated from P19 cells.


Figure 5: Preferential expression of the necdin promoter-driven LacZ reporter gene in postmitotic neurons. LacZ reporter genes fused to the 844-bp 5`-flanking region of the necdin gene (A and B) and the SV 40 promoter (C and D) were transfected into retinoic acid-treated P19 cells, and cell type-specific expression of LacZ was morphologically examined. LacZ (A and C) and MAP2 immunoreactivities (B and D) were detected by double fluorescent immunocytochemistry. Identical visual fields (A and B, C and D) are shown. Arrows (A-D) and arrowheads (C and D) indicate postmitotic neurons and non-neuronal cells, respectively. Postmitotic neurons are identified by their typical morphological characteristics described in the text. Scale bar = 100 µm for A-D.



Localization of Neuron-specific Core Elements

To localize neuron-restrictive elements in the necdin promoter, we quantified the reporter activities in differentiated P19 cells transfected with luciferase reporter genes fused to various deleted sequences of the upstream region (Fig. 6). The promoter activity of the 154-bp upstream sequence (construct A) was about 60% of the undeleted control. The most pronounced reduction was noted when the sequence from -80 (construct D) to -63 (construct E) was deleted (the activity decreased to 12% of the undeleted control). Further deletion to -34 (construct G) decreased the activity to 4% of the undeleted control level. These results suggest that two elements (between -80 and -64 and between -47 and -35) mainly contribute to the promoter activity. In this region, xenobiotic responsive element (XRE) (CACGC(A/T)) (14, 15) and a GC-rich region were found at positions -70 to -64 and positions -47 to -33, respectively.


Figure 6: Deletion analysis of the necdin promoter. A, the necdin promoter sequence. Positions A-H indicate the 5`-ends of deletion mutants (constructs A-H). The positions are numbered, beginning with experimentally determined transcription initiation site S1 (designated mRNA +1) (see Fig. 2). GCR, G+C-rich region; M, translation start codon (ATG). B, relative luciferase activities of the deletion mutants. The efficiency of each transfection was normalized against LacZ activity derived from cotransfected pCH110 reporter gene. The promoter activity of each construct is presented as a relative value (percent) of that of undeleted 844-bp necdin promoter (UD). The promoter activity driven by the reverse-oriented 844-bp promoter (RV) is the lowest. Each value represents the mean of three independent experiments using different cultures.



When the 80-bp fragment (construct D) fused to the LacZ gene was transfected into retinoic acid-treated P19 cells to examine the cell type-specific promoter activity, more than 90% of LacZ positive cells coincided with postmitotic neurons (Table 1). The preference for neurons of the 80-bp promoter was similar to or slightly greater than that of the 844-bp promoter and much greater (about 10-fold) than that of the SV 40 promoter, which showed less cell type specificity. These data suggest that the 80-bp proximal region still retains the neuronal specificity.



We have transfected the luciferase reporter gene fused to the 844-bp promoter into non-neuronal cell lines (COS-1 cells and NIH3T3 fibroblasts) and undifferentiated P19 stem cells. The luciferase activities in these cells were less than 10% of those in differentiated P19 cells. Moreover, a specific negative regulatory element was undetected within the 844-bp promoter region when examined by transfecting the deleted constructs into non-neuronal cells (data not shown). These results suggest that the necdin promoter lacks a neuron-restrictive silencer element like those reported previously (6, 7, 8) and that specific cis-acting elements comprised in the proximal 80-bp region positively regulate the gene expression in postmitotic neurons.


DISCUSSION

This study has shown that necdin gene expression is specific to postmitotic neurons and thus serves as a suitable indication of neuronal differentiation. To date, expression of NF has been commonly utilized for neuronal differentiation. However, NF-L mRNA is expressed at very low levels in the forebrain during the prenatal period (see Fig. 2). The onset of NF expression is concomitant with axon elongation and correlates extremely well with neurofibrillar development(16) . Therefore, NF expression is not a representative indication of the neuronal differentiation per se but rather marks neurofibrillar differentiation. As shown here, the necdin gene is expressed in virtually all postmitotic neurons in the central nervous system from the very beginning of neuronal differentiation until adulthood but not in the undifferentiated stem cells. Therefore, necdin gene expression serves as a good indication of neuronal differentiation from the stem cells both in vivo and in vitro(3, 4) .

The necdin promoter can be applicable to postmitotic neuron-targeted expression of cloned genes of interest in a wide range of vertebrate species by using various transgenic systems such as transgenic animals and viral vector-mediated transgenes. We have recently demonstrated that reporter genes fused to the 844-bp necdin promoter are expressed exclusively in differentiated neurons of zebrafish embryos when injected into the fertilized eggs(17) . This indicates that the necdin promoter defines the neuronal specificity in vivo as well as in vitro and that the murine necdin promoter directs the neuron-specific gene expression even in the zebrafish, despite the evolutionary distance separating the two species. However, a -86 to +63 sequence, which comprises the 80-bp core promoter that confers the neuronal specificity (see Table 1), gave preferential skin expression in the zebrafish(17) . This discrepancy raises the possibility that additional cis-acting elements upstream of the core promoter region are required for neuron-restrictive expression in the zebrafish in vivo. Another possibility is that the neuronal specificity of the 80-bp core promoter is overestimated in the present system in which retinoic acid-treated P19 cells may differentiate into few skin cells. The additional cis-acting elements that increase the neuronal specificity in vivo remain to be defined in future studies.

It is unlikely that expression of the necdin gene in neurons is controlled by neuron-restrictive silencer elements(6, 7, 8) . We were unable to find such negative regulatory elements in the upstream region of the necdin gene. Silencer-dependent neuron-specific genes such as the type II sodium channel gene and the SCG10 gene are expressed in cell lines derived from neuroblastoma and pheochromocytoma, and these cells lack the silencer-binding factor(9, 10) . Unlike these genes, necdin mRNA is undetected in such cell lines originating from tumors(4) . Thus, the fashion of the transcriptional control of necdin may differ from those of neuron-specific genes whose expression is dependent on the neuron-restrictive silencer elements.

In the 80-bp proximal region of the necdin gene, a consensus motif of the XREs (CACGC(A/T)) (11, 12, 13, 14) and a G+C-rich region are present at positions -70 to -64 and positions -47 to -33, respectively (Fig. 6A). XRE was originally characterized as a binding element for the aryl hydrocarbon receptor, which is involved in the transcriptional induction of the cytochrome P4501A1 enzyme that metabolizes xenobiotics and procarcinogens(14) . It has been demonstrated that a G+C-rich element adjacent to XRE interacts with another binding factor that depends on the presence of a functional aryl hydrocarbon receptor(15) . Therefore, it is possible that XRE and the G+C-rich element in the necdin promoter cooperatively regulate the expression of the necdin gene in postmitotic neurons.

The aryl hydrocarbon receptor is a member of the transcription factor family with the PAS (per, arnt, and single-minded)-dimerization domain (18, 19) . The single-minded gene product (SIM) and the period gene product (PER) of Drosophila also belong to this family(20) . Moreover, it is suggested that these PAS transcription factors interact with target sequences resembling XRE(21, 22) . SIM is expressed specifically in the embryonic midline neuroepithelium of Drosophila, and mutations in this gene result in loss of the midline cell precursors in the central nervous system(23) , suggesting that SIM plays a key role in the neurogenesis and morphogenesis of the Drosophila central nervous system. Although neither vertebrate homologs of SIM nor neuron-specific genes regulated by PAS transcription factors in the vertebrate central nervous system have been reported yet, it is likely that the necdin gene is transcriptionally activated by mammalian homologs of SIM or related PAS transcription factors, which interact with XRE in the core promoter region. Detailed analyses of the interaction between the neuron-specific core element and endogenous binding factors are currently in progress.

Necdin is expressed in postmitotic neurons from the very beginning of neurogenesis until adulthood but not in neuroepithelial stem cells. Ectopic expression of necdin suppresses the growth of proliferative NIH3T3 cells(5) . Furthermore, we have found out that necdin binds to SV 40 large T antigen. (^4)Since tumor suppressor gene products such as p53 and Rb form complexes with the large T antigen, it is suggested that necdin suppresses cell proliferation in a fashion analogous to p53 and Rb. These findings raise the possibility that necdin is physiologically involved in the mechanism underlying induction and maintenance of the permanent arrest of cell division displayed by differentiated neurons.

Most of the neurons in the central nervous system enter the postmitotic state during fetal periods when the numbers of neurons in respective brain regions are primarily fixed. Therefore, the temporal and spatial patterns of growth arrest of neuronal precursors in different brain regions must be strictly controlled during development, preparing each particular brain area to acquire specific functions. It is tempting to speculate that a differential expression of the necdin gene along the neural tube induces its allometric growth that gives rise to morphological and functional changes of the central nervous system. Thus, further studies on transcriptional control of the necdin gene will lead to a better understanding of physiological roles of necdin in neurogenesis and morphogenesis of the central nervous system in vertebrates.


FOOTNOTES

*
This work was supported in part by Grants-in-Aid 05454670 and 04268102 from the Ministry of Education, Science, and Culture of Japan (to K. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D76440[GenBank].

§
To whom correspondence should be addressed. Tel.: 81-6879-8621; Fax: 81-6879-8623; yoshikaw@protein.osaka-u.ac.jp.

(^1)
The abbreviations used are: NF-L, neurofilament-L; kb, kilobase(s); PCR, polymerase chain reaction; SV 40, simian virus 40; MAP2, microtubule-associated protein 2; E, embryonic day; bp, base pair(s); XRE, xenobiotic responsive element.

(^2)
K. Takagi and K. Yoshikawa, unpublished observations.

(^3)
K. Maruyama and K. Yoshikawa, unpublished observations.

(^4)
H. Taniura and K. Yoshikawa, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. Haruo Okado and Yoshio Yaoita (Tokyo Metropolitan Institute for Neuroscience) for helpful advice and Tomoko Kohno for technical assistance. We are grateful to Drs. Michael McBurney (University of Ottawa) and Hiroshi Hamada (Osaka University) for the gift of P19 cells and to Dr. Nicholas Cowan for the gift of NF-L cDNA.


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