Department of Neurobiology, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, La Jolla, California 92037
The cell adhesion molecule L1 mediates neurite outgrowth and fasciculation during embryogenesis and mutations in its gene have been linked to a number of human congenital syndromes. To identify DNA sequences that restrict expression of L1 to the nervous system, we isolated a previously unidentified segment of the mouse L1 gene containing the promoter, the first exon, and the first intron and examined its activity in vitro and in vivo. We found that a neural restrictive silencer element (NRSE) within the second intron prevented expression of L1 gene constructs in nonneural cells. For optimal silencing of L1 gene expression by the NRSE-binding factor RE-1-silencing transcription factor (REST)/NRSF, both the NRSE and sequences in the first intron were required. In transgenic mice, an L1lacZ gene construct with the NRSE generated a neurally restricted expression pattern consistent with the known pattern of L1 expression in postmitotic neurons and peripheral glia. In contrast, a similar construct lacking the NRSE produced precocious expression in the peripheral nervous system and ectopic expression in mesenchymal derivatives of the neural crest and in mesodermal and ectodermal cells. These experiments show that the NRSE and REST/NRSF are important components in restricting L1 expression to the embryonic nervous system.
CELL adhesion molecules (CAMs)1 play fundamental roles in the development of the nervous system. During embryogenesis, CAMs participate in
neurite extension, fasciculation, axon guidance, and synapse formation (9, 10), and in the adult, they have roles in synaptic plasticity (34, 39). The neural cell adhesion molecule,
N-CAM, is expressed in the neural tube before differentiation of neuroepithelial precursors, appears on the surface
of neurons and glia, and is found on a number of nonneural tissues (9). In contrast, some CAMs appear at the time
of neural differentiation and are expressed solely or predominantly in the nervous system (2, 12, 14, 21, 26, 36, 57).
These observations raise an important question: What
restricts the expression of certain CAMs to the nervous
system? To answer this question, we have attempted to
identify the DNA elements and protein factors that control expression of two particular CAMs that are restricted
to the nervous system during development: the avian neuron-glia cell adhesion molecule (Ng-CAM) and mammalian L1 (2, 36). Ng-CAM and L1 are integral membrane
proteins of the N-CAM subfamily characterized by six immunoglobulin-like domains and five fibronectin type-III
repeats. Ng-CAM was first isolated from embryonic
chicken brain (13), and L1 was identified by monoclonal
antibodies prepared against mouse cerebellar membranes (44, 49). Both proteins have similar patterns of expression and appear on the surface of postmitotic neurons and peripheral glial cells (7, 24, 32, 38).
In previous work, we identified some of the regulatory
regions of the chicken Ng-CAM gene (19), including the
promoter and a region of the first intron that was found to
contain five contiguous neural restrictive silencer elements
(NRSEs). When linked to a reporter gene, the Ng-CAM
promoter drove expression in both N2A neuroblastoma
and NIH3T3 fibroblast cell lines. However, when the five
NRSEs were included in these constructs, reporter gene expression was found in neuroblastoma cells, but not in fibroblasts (19).
The NRSE has been found in a number of genes for proteins that are restricted to the nervous system, including
the type II sodium channel (33), synapsin I (46), and
BDNF (51). A zinc finger protein known as the neural restrictive silencer factor (NRSF) or the RE-1-silencing
transcription factor (REST) binds to the NRSE and silences expression of genes containing NRSEs in nonneural
cells (4, 47). REST/NRSF is expressed ubiquitously in
nonneural cells and in neuronal precursors but not in postmitotic neurons (4, 47). Thus, downregulation of REST/
NRSF is likely to be a key event in neural development in
that it releases silencing and allows expression of several
genes that are important for the establishment of the phenotype of neural cells.
Recently, a putative NRSE has been identified within an
intron of the human and mouse L1 gene sequences (19,
48). Given the observations that the Ng-CAM and L1
genes both contain NRSEs, and that the expression patterns of these genes in their respective organisms are similar, we examined the role of the NRSE in regulating L1
gene expression both in vitro and in vivo. In the present
study, we identify an additional segment of the mouse L1
gene containing the promoter and show that the NRSE
within the L1 gene is critical for a neurally restricted pattern of L1 gene expression in the embryonic nervous system.
Isolation of L1 Genomic and cDNA Clones
Corresponding to the 5 Genomic clones for mouse L1 were isolated from a library prepared from
genomic DNA isolated from mouse embryonic stem cells in bacteriophage P1 (Genome Systems, St. Louis, MO) by PCR using primers derived from the mouse L1 gene sequence (22). Rapid amplification of
cDNA ends (RACE) (11) on the L1 mRNA was performed using the
Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Palo
Alto, CA). RACE products and L1 genomic DNA fragments were subcloned into the pBluescriptII vector (Stratagene, La Jolla, CA). The sequence of both strands of L1 clones was determined (50). Sequencing data
were compiled using the GCG package (University of Wisconsin, Madison,
WI) and comparisons were made using FASTA (41). RNA start sites were
located using an RNAse protection assay (Ambion, Inc., Austin, TX).
Preparation of Luciferase and lacZ Reporter Constructs
L1-1 through L1-6 were constructed by insertion of L1 genomic fragments
into the XmaI and XhoI sites of pGL2basic (Promega Corp., Madison,
WI). To prepare L1-5N and L1-5Nr plasmids, double-stranded oligonucleotides containing two copies of the NRSE from the L1 gene were inserted
into the XmaI site upstream of the L1-5 promoter fragment. L1-7 through
L1-12, L1lacZ, and L1lacZ Large L1 gene fragments were assembled as follows: A fragment containing the 5 Preparation of REST/NRSF Expression Vectors and
Cellular Transfection Experiments with L1 Constructs
The cDNA for REST/NRSF (4, 47) was generated by PCR from total human RNA isolated from HeLa and Jurkat cells using High Fidelity Taq
polymerase (Boehringer-Mannheim Corp., Indianapolis, IN) and was inserted into the pCRII vector (Invitrogen, San Diego, CA). Expression of
REST/NRSF was confirmed using an in vitro transcription/translation system (Promega Corp.). The REST/NRSF cDNA or a 5 NIH3T3, COS-1, and N2A cells were cultured in DMEM supplemented
with either newborn or fetal calf serum and were transfected in equimolar
amounts of DNA using lipofectamine (Life Technologies, Gaithersburg,
MD). The lacZ reporter CMV The activities of L1 luciferase constructs were determined in three separate sets of experiments. In the first set of experiments (see Fig. 2), the
promoterless luciferase vector pGL2basic (Promega Corp.) served as the
negative control, and the vector pGL2control (Promega Corp.), containing an SV-40 early promoter upstream of the luciferase gene, provided a
positive control for promoter activity. The activities for pGL2control and
constructs L1-1 through L1-6 were calculated by subtracting background
activity in raw light units (RLU) produced by pGL2basic from the RLU
values for each construct. For each cell line, the RLU values for
pGL2basic and pGL2control were set at 0 and 100% activity, respectively. The relative luciferase activities for constructs L1-1 through L1-6 were
then expressed as a percentage of SV-40 early promoter activity. For the
second set of experiments (see Fig. 3 A), the background activity from
pGL2basic vector was subtracted from the values for L1-5, L1-5N, and L1-5Nr. Activity levels for pGL2basic and the unsilenced L1 promoter construct L1-5 were set at 0 and 100%, respectively, in each of the four cellular transfection conditions (NIH3T3, N2A, NIH3T3 + D-REST, and N2A + REST). The relative luciferase activities for L1-5N and L1-5Nr were then expressed as a percentage of L1-5 promoter activity. For the third set of
experiments (see Fig. 3 B), the background activity produced by the promoterless vector lucpA was subtracted from the raw RLU values for L1-7
through L1-12. The activity levels for lucpA and L1-7 were set at 0 and
100%, respectively, in each of the four transfection conditions. The relative luciferase activities were then expressed as a percentage of L1-7 promoter activity. The values for all of the relative luciferase activities in each
experimental set are the average of at least six independent experiments.
Generation and Analyses of Transgenic Mice
Containing lacZ Constructs
To prepare transgenic mice, L1lacZ and L1lacZ Animals positive for the transgenes were mated to establish individual
lines. Males from transgenic lines were mated with C57BL6 females, and
pregnant females were sacrificed at different days of gestation to obtain
embryos at different stages of development. At least one litter from each
of the transgenic lines produced in these studies was analyzed for Characterization of the 5 We previously characterized the structures of the genes
for two avian neural CAMs that are closely related to
mammalian L1: Ng-CAM (19) and Nr-CAM (unpublished
data). Comparison of the 5
To obtain cDNA sequences corresponding to the full-length 5 To characterize the 5 Using RNAse protection assays, multiple transcription
initiation sites were mapped to the 5 The sequences of 97 bp of the proximal promoter, the
first exon, and a portion of the first intron are shown in
Fig. 1 B. The RNA start sites initiated within a region containing several trinucleotide repeats of GCC and CAG
(Fig. 1 B). Searches of the promoter and first exon for elements known to regulate gene expression revealed a binding site for the transcription factor SP1 (8) that overlaps
the 5 Comparison of the Mouse and Human L1
Promoter Regions
A portion of the human L1 gene sequence is available
from GenBank/EMBL/DDBJ under accession number
U52112 and is part of a larger sequence from the X chromosome. We compared the sequence of the human X
chromosome upstream of the existing human L1 gene with
our sequence of the mouse L1 promoter and first exon and found that there was a high degree of similarity between
the 5 The NRSE and the First Intron Are Required for
Silencing of the L1 Gene Expression by REST/NRSF in
Cellular Transfection Experiments
To examine whether the DNA sequences upstream of
exon 1 in the mouse L1 gene had promoter activity, we
prepared six luciferase reporter constructs containing
exon 1 together with varying lengths of 5 The fact that reporter constructs
containing the L1 promoter and the first exon were
equally active in neural (N2A) and nonneural (NIH3T3)
cells indicated that cell type-specific expression required other regions of the gene. To assess whether the NRSE
within the second intron of the L1 gene played a role in
the silencing of L1 promoter in nonneural cells, we prepared a number of L1 gene constructs containing or lacking the NRSE and examined their activity in cellular transfection experiments.
Two constructs, L1-5N and L1-5Nr, were prepared by
inserting two copies of the NRSE from the L1 gene in either forward or reverse orientation upstream of the most
active L1 promoter, L1-5. These constructs were transfected into NIH3T3 or N2A cells and examined for activity. The presence of NRSEs in either orientation led to a
significant reduction in L1 promoter activity in NIH3T3 cells, but not in N2A cells (Fig. 3 A). These data indicate
that the NRSEs are sufficient for silencing L1 promoter
activity in a cell type-specific manner.
To assess the role of the NRSE in the context of the native L1 gene, six constructs containing up to 18 kb of the
mouse L1 gene (L1-7 through L1-12; Fig. 3 B) were prepared and examined for their activity in NIH3T3 and N2A
cells. To compare the relative level of silencing by downstream segments of the gene, the activity of L1-7 was set at
100%. In NIH3T3 cells, addition to the promoter of an L1
gene segment containing exons 2-4 including introns and the NRSE (L1-8) reduced the activity to 24% of control
(L1-7). Deletion of the NRSE in L1-8 (L1-9) resulted in
50% of the control activity, indicating that the NRSE was
responsible for only a portion of the silencing found in this
DNA fragment. Addition of the first intron and second
exon of the L1 gene (L1-10) showed a reduction in activity
(21% of L1-7) that was approximately equal to inserting
the L1 genomic segment containing the NRSE (L1-8). This result suggests that the first intron contains a silencer that is as effective as the NRSE. Addition of the entire L1
genomic region between intron 1 through exon 4 (including the NRSE) (L1-11) resulted in the most significant reduction in activity (3% of L1-7). Deletion of the NRSE in
L1-11 yielded L1-12. L1-12 had reduced activity (19% of
L1-7) that was approximately equal to L1-10, further indicating that a silencing activity within the first intron was
still effective even in the absence of the NRSE.
In N2A cells, the activities of L1-8 through L1-12 were
indistinguishable from that of L1-7 (Fig. 3 B). Overall, the
results from the transfection experiments indicate that the
NRSE and an additional silencer present in the first intron, both of which function in NIH3T3 cells, are not active in N2A cells.
To demonstrate that the NRSE in the L1 gene was a target
for silencing via REST/NRSF, we performed two types of
cellular cotransfection experiments. In the first experiment, a plasmid directing the expression of a truncated
REST/NRSF protein (called D-REST) containing the
DNA-binding zinc fingers but not the silencer domain (4, 47) was cotransfected into NIH3T3 cells together with various L1 gene constructs to examine whether a dominant
negative form of the REST/NRSF protein could prevent
silencing of L1 promoter activity. In the second experiment, N2A cells were cotransfected with L1 constructs and
a plasmid expressing the fully active REST/NRSF protein
to determine whether ectopic expression of REST/NRSF
in cells that normally contain low levels of REST/NRSF
activity could reduce the activity from the L1 constructs.
D-REST released silencing of all L1 gene constructs
that showed silencer activity in NIH3T3 cells (L1-5N, L1-5Nr, L1-8, L1-9, L1-10, L1-11, and L1-12) (Fig. 3, A and
B). Expression of the full-length REST protein in N2A
cells led to partial silencing of the activities of L1-5N, L1-5Nr, L1-8, L1-10, L1-11, and L1-12 but did not affect the
activities of L1-7 and L1-9 (Fig. 3, A and B). Thus, REST
reduced the activity of all L1 gene constructs containing
the NRSE, the first intron alone, or the two elements in
combination.
We conclude that (a) the NRSE in the L1 gene responds
to REST/NRSF; (b) the first intron contains a silencer that
can function without the NRSE, but nonetheless acts in response to REST/NRSF; and (c) optimal silencing of the L1
gene by REST/NRSF is achieved when both the first intron and the NRSE are combined. These in vitro findings
prompted an analysis in vivo of the modulation of L1 gene
expression by the NRSE.
Production of Transgenic Mice Containing L1
Gene Constructs
To determine whether the 5
33 transgenic lines were established for the L1lacZ
transgene. 15 showed no expression of For the L1lacZ To examine in detail the differences between neurally
restricted and unsilenced patterns of L1 gene expression,
one L1lacZ line showing the neurally restricted pattern I
and one L1lacZ
Expression of the L1lacZ Transgene Is Coincident
with Neural Differentiation and Is Restricted to the
Nervous System
Expression of the L1lacZ transgene was not detected before neural differentiation at E8.5 (Fig. 5 A) and was first
observed at E9.5 in the central nervous system (CNS)
within the midbrain and in the peripheral nervous system
(PNS) within the trigeminal ganglion (Fig. 5 B; mb). At
E10.5, the punctate Expression of the L1lacZ Expression of L1lacZ The most dramatic differences in the intensity of Between E10.5 and E12.5 the L1lacZ Expression of the L1lacZ As an example of ectopic extraneural expression, the pattern of appearance of the L1lacZ
Deletion of the NRSE Releases Silencing of L1 Gene
Expression in Nonneural Derivatives of Neural Crest
and in Mesodermal and Ectodermal Cells
To provide further histological analysis of the cell populations within differentiating tissues in which the L1lacZ and
L1lacZ
Table I.
Cellular Expression of L1lacZ and L1lacZ
Materials and Methods
End of the mRNA
N were prepared using the vector CMV
(CLONTECH Laboratories, Inc.), after replacing the human cytomegalovirus promoter with a polylinker containing the XmaI and XhoI sites,
which allowed the insertion of the L1 genomic fragments. lacZ constructs
were converted into luciferase reporters by removing the lacZ gene by digestion with NotI and replacing it with a modified luciferase gene cassette
from pGEMluc (Promega Corp.) containing an SV-40 polyadenylation
signal. A promoterless version of this vector, called lucpA, was also prepared to provide a negative control for the luciferase constructs L1-7 through L1-12.
untranslated sequences of exon 2 was inserted into the XhoI
site downstream of exon 1. Fragments containing intron 1 were added at
the BlpI site at the 3
terminus of exon 1. A partial XbaI digest generated
a construct that included the L1 promoter, exon 1, intron 1, and exon 2. The region containing the translated portion of exon 2, intron 2, exon 3, intron 3, and exon 4 was used in the generation of L1-8, L1-9, L1-11, and L1-12. The resulting fragment had Sse8387I restriction sites at either end
and contained a SnaBI site at the 3
end of exon 4. A similar fragment
lacking a segment of 20 bp that deleted the NRSE within intron 2 was generated by PCR. These fragments were inserted into L1 genomic constructs
at the Sse8387I site downstream of the SV-40 polyA signal. The nuclear
localization signal from the SV-40 large T antigen was inserted upstream
of the lacZ gene by replacing a XhoI-ClaI fragment with a SalI-ClaI fragment from the pnlacF vector (from J. Peschon, University of Washington, Seattle, WA).
segment encoding the NH2-terminal 423 amino acids containing the zinc finger DNA-binding domain (D-REST) was cloned in frame with the hemagglutinin (HA) tag in the mammalian expression vector SR
3. Expression of REST/ NRSF and D-REST proteins was confirmed after transfection of COS-1 cells followed by immunoblot analysis of cell extracts with a monoclonal
antibody to the HA tag. D-REST was ~50 kD, which corresponds to the
predicted size of this protein fragment plus the HA tag. Expression of
REST/NRSF produced two bands, one migrating at 200 kD and another
at 120 kD, a size that corresponds to the reported size of REST/NRSF.
The 200-kD band might represent a posttranslationally modified REST/
NRSF protein, as suggested in previous studies (4).
gal (CLONTECH Laboratories, Inc.) was
cotransfected to normalize for transfection efficiency. For cotransfections,
between 50- and 100-fold molar excess of either the REST/NRSF or D-REST
expression vector was added to the transfection mixture. Cells were harvested after 48 h, and extracts were prepared, normalized for
-galactosidase activity, and assayed for luciferase activity, as described (17).
Fig. 2.
Activity of L1 promoter constructs in cellular transfection experiments of NIH3T3 and N2A cells. The relative luciferase activities of L1 constructs are expressed as a percentage
of SV-40 promoter activity produced by the PGL2control vector.
The values for pGL2control and pGL2basic were set at 100 and
0% activity, respectively. The corrected values for activities were
derived by subtracting the background activity in RLU produced
by the promoterless vector pGL2basic from the RLU values for
each construct. The numbers in parentheses are the mean RLU
values produced by each construct and the standard errors for
both percentage activity and RLU are shown (n = 6).
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Fig. 3.
Relative luciferase
activities of L1 gene constructs in transfection experiments of NIH3T3 and N2A
cells and in cotransfection experiments with constructs
expressing either a dominant
negative form of REST/NRSF
(designated D-REST) or the
full-length REST/NRSF. The
relative activities represent a
percentage within the range that is established between
the background activity of a
promoterless control vector
(which is set at 0%) and an
unsilenced L1 promoter construct (which is set at 100%).
The numbers in parentheses
are the mean values for RLU
produced by each construct
in each of the four experimental conditions (NIH3T3,
N2A, NIH3T3 + D-REST, and N2A + REST). To
derive the relative activities
shown in A, the RLUs for
the promoterless pGL2basic
vector were subtracted from the values of L1-5, L1-5N, and L1-5Nr in each of the four experimental conditions. The activity levels
produced by pGL2basic were set at 0%, and the levels for the unsilenced L1 promoter construct L1-5 were set at 100%. (B) The
levels of background activity produced by the control vector lucpA were subtracted from the RLU values for each construct, L1-7
through L1-12. The RLU values for the promoterless vector lucpA were set at 0% activity, and values for the unsilenced L1 promoter
construct L1-7 were set at 100% activity. Standard errors for both percentage activity and RLUs are shown, n = 6 for A, and n = 10 for B.
[View Larger Version of this Image (35K GIF file)]
N transgenes were introduced into the RC6 mouse genome by standard oocyte microinjection techniques (16). Transgenes were excised from plasmids by digestion with
restriction enzymes XmaI and SnaBI. Genomic DNA isolated from tails
of F0 progeny was screened for the presence of the transgene by PCR using the TissueAmp kit (Qiagen, Inc., Chatsworth, CA). Two different
primer sets were designed for the PCR to discriminate between mice carrying the wild-type NRSE and those in which the NRSE was deleted.
-galactosidase expression. To stain whole mounts, embryos were fixed in 0.2%
glutaraldehyde/1% formaldehyde in PBS, washed in PBS, and immersed
in staining solution containing 0.02% deoxycholate, 30 mM K3Fe(CN)6,
30 mM K4Fe(CN)6, and 0.5 mg/ml Bluogal (Life Technologies) in PBS
overnight at 37°C. To detect
-galactosidase activity in tissue sections, embryos were fixed, transferred through an ascending gradient of sucrose to
24% sucrose/PBS, frozen in Tissue Tek (Miles, Inc., Elkhart, IN), and sectioned (40 µm) on a cryomicrotome. Sections were attached to poly-
L-lysine-coated microscope slides and stained for
-galactosidase as described above, mounted with 50% glycerol, and photographed with bright
field optics.
Results
End of the Mouse L1 Gene
sequences of the Ng-CAM
and Nr-CAM genes with those reported for the mouse L1
gene sequence (22) revealed a close correspondence in intron/exon structure (see Fig. 1 A). However, in both the Ng-CAM and Nr-CAM genes, the ATG codon is situated
in the second exon, whereas in the L1 gene the ATG
codon was located in a region proposed to be the 5
-most
exon, called exon A (22). These contrasting comparisons
suggested to us that there may be an additional exon and a
promoter region upstream of exon A in the L1 gene.
Primer extension analyses of mouse L1 mRNA transcripts
in the original study (22) were in accord with this supposition.
Fig. 1.
(A) Schematic representations of the mouse and human L1 genes. (Top) Diagram of the 5-most sequences established in previous studies (22). The nomenclature for the exons
are those given by Kohl et al. in their study (22). The extended
portion of the L1 gene that we describe in the present study is
shown below. The lengths of the exon sequences in base pairs are
indicated in parentheses. Coding and 5
untranslated sequences
are indicated by black boxes and open boxes, respectively. The
arrow indicates the 5
-most site of transcription initiation. The
positions of XbaI (X) restriction sites are also indicated. The line
segments shown above the promoter for the mouse L1 gene labeled B and C indicate regions that have a high degree of similarity with the human L1 gene. (B and C) Comparison of the sequences at the 5
end of the mouse (mL1) and human (hL1) L1
genes. Putative binding sites for NF-1/CBP and SP1 are indicated
by boxes. First exon sequences in the mouse and human L1 genes
are indicated by upper case letters. Arrowheads define transcription initiation sites for the mouse L1 gene. The trinucleotide repeats are underlined. The 3
splice junction is boxed and indicated by an arrow.
[View Larger Version of this Image (37K GIF file)]
end of the L1 messenger RNA, we performed a
RACE experiment (11) on RNA isolated from N2A cells.
DNA sequencing of RACE clones revealed an additional
segment of 5
untranslated sequences not detected in previous studies (22). No RACE products terminated at the
5
end of exon A, suggesting that, contrary to previous conclusions (22), the 5
end of exon A is not a major site
for initiation of L1 gene transcription in N2A cells.
end of the L1 gene, we cloned and
sequenced a 13-kb region of genomic DNA immediately
upstream of the 5
-most sequences previously reported
(22). These sequence data are available from GenBank/
EMBL/DDBJ under accession number U91929. They include a 2,943-bp segment of 5
flanking sequence, the 119-bp first exon, and 9,429 bp of the first intron. The location of this gene segment relative to the previously characterized
portion of the L1 gene (22) is shown in Fig. 1 A. The additional 5
untranslated sequences found in RACE experiments mapped the first exon ~10 kb upstream of exon A
(Fig. 1 A). Given these new findings, we have redesignated
exon A as exon 2.
end of the first exon.
No transcripts were detected initiating at exon 2. Exon 1 was defined to be the 119 bp between the 5
-most RNA
start site and the beginning of the 3
splice junction. When
combined with the results from our RACE experiments,
these data provide additional support for the conclusion
that exon 2 (previously exon A) is not a site of transcription initiation.
-most transcription initiation site. In previous studies
of the L1 gene (22), a binding site for homeodomain proteins was also identified 170 bp upstream of exon A (3, 22). Our analysis, however, now places this DNA element
within the 3
end of the first intron. Most significant for the
present experiments, the mouse L1 gene sequence contains
a single NRSE immediately downstream of exon 2 (Fig. 1 A).
end of the mouse and human L1 genes (Fig. 1 A). As
is found in the mouse gene, the first exon of the human L1
gene is ~10 kb upstream of exon 2. As shown in Fig. 1, B
and C, the sequences of the first exon, the 3
splice junctions, and the proximal promoters containing the SP1 sites
are highly conserved between the mouse and human L1
genes. A region of the L1 promoter further upstream, containing a putative binding site for transcription factors of the NF-1 and CBP families, is also highly conserved between the two species (Fig. 1 C). The single NRSE in the
second intron of the mouse L1 gene is also found in a comparable position in the human L1 gene. No other NRSEs
were found in searches of the mouse and human L1 gene
sequences.
flanking sequence from the gene and examined their activity in either
NIH3T3 fibroblasts or N2A neuroblastoma cells. All six L1 gene fragments (containing as few as 70 and as many as
2,943 bp upstream from the transcription initiation sites)
showed promoter activity (Fig. 2). L1-5 and L1-1 were the
most active promoter constructs in NIH3T3 and N2A
cells, respectively. In general, the luciferase activities of
L1-1 through L1-6 were comparable or greater than the
activity of the SV-40 early promoter and on average were
between 13- and 8-fold greater than those produced by the
promoterless luciferase vector in NIH3T3 cells and N2A cells, respectively. These findings indicate that there is a
promoter upstream of exon 1 that is constitutively active
in both NIH3T3 and N2A cells.
end of the L1 gene was sufficient to direct expression of the gene to the nervous system in vivo and to examine the effect of NRSE removal on
L1 expression, transgenic mice were generated containing
two different L1-lacZ reporter constructs (Fig. 4). The two
transgenes, designated L1lacZ and L1lacZ
N, were identical to L1-11 and L1-12 (Fig. 3 B), except that the luciferase gene was replaced by a lacZ gene cassette containing a nuclear localization signal. The only difference between these L1 transgenes was that L1lacZ contained
the NRSE, whereas L1lacZ
N did not.
Fig. 4.
(Top) Diagram of the L1lacZ
and L1lacZN constructs that were
used to generate transgenic mice. A
detailed description of the construction
of these transgenes is provided in Materials and Methods. (Bottom)
-galactosidase expression patterns of individual lines carrying either the L1lacZ or
L1lacZ
N transgenes in whole mounts of E10.5 embryos. All transgenic lines (lines I, Ia, and II, as examples) showed identical expression patterns in structures within the central
and peripheral nervous system. Most
L1lacZ lines showed a completely
neurally restricted pattern of
-galactosidase expression (line I). Some
L1lacZ lines also showed some nonneural expression at a reduced level in
a subset of tissues in which the
L1lacZ
N transgene was strongly expressed (line Ia). All L1lacZ
N lines
(II, as an example) had extensive extraneural staining patterns of
-galactosidase that were identical. Abbreviations for anatomical structures: bw, body wall; cg, cranial ganglia; cm, cephalic mesenchyme; de, dorsal ectoderm; drg, dorsal root
ganglia; h, heart; pn, peripheral nerve; sc, sympathetic chain; t, telencephalon.
[View Larger Version of this Image (50K GIF file)]
-galactosidase,
suggesting that in these cases, the transgene most likely integrated into genomic regions that completely silenced its
expression. Of the 18 expressing lines, 12 showed neurally
restricted
-galactosidase expression patterns that were
identical (Fig. 4, pattern I). The remaining six lines showed
the same neural pattern but in addition displayed some
staining outside of the nervous system. For example, the line shown in Fig. 4 called Ia has the same neural pattern
as I but has additional staining in the cephalic mesenchyme. The other lines were similar to Ia but differed
slightly in the locations in which the nonneural staining
was seen. In all cases, the nonneural staining appeared in
locations that were a subset of those carrying the L1lacZ
N
transgene (see below). Such patterns might arise from a
partial release of silencing of L1 gene expression upon integration of the transgene in a particularly active region of the genome.
N transgene that lacked the NRSE,
seven transgenic lines were obtained, and four of these
showed
-galactosidase expression. All four lines had an
identical expression pattern that included the neural pattern characteristic of the L1lacZ lines, but in addition had
intense and expansive
-galactosidase staining outside of
the nervous system (Fig. 4, pattern II).
N line showing the extensive nonneural
staining pattern II were selected. The expression patterns
of both transgenes in whole mounts were first compared at
each day of embryonic development between E8.5 and
E12.5 (Fig. 5).
Fig. 5.
Analyses of the -galactosidase staining patterns in whole mount from mouse embryos carrying either L1lacZ (A-E) or
L1lacZ
N (F-J) transgenes. The stages of embryonic development are as follows: E8.0-8.5 (A and F), E9.5 (B and G), E10.5 (C and H),
E11.5 (D and I), and E12.5 (E and J). Abbreviations for anatomical structures: b1, branchial arch 1; b2, branchial arch 2; bw, body wall;
cg, cranial ganglia; cm, cephalic mesenchyme; ct, circumpharyngeal tract; de, dorsal ectoderm; drg, dorsal root ganglia; h, heart; mb, midbrain; p, prosencephalon; pm, posterior mesoderm; pn, peripheral nerve; sc, sympathetic chain; t, telencephalon; tg, trigeminal ganglion.
Bars, 1 mm (apply to each stage of development).
[View Larger Version of this Image (77K GIF file)]
-galactosidase expression in the midbrain became more intense, showing a distinct posterior
boundary at the mesometencephalic fold (Fig. 5 C; mf). At
E11.5, expression of the L1lacZ transgene extended to
more rostral locations of the CNS and was observed in the
telencephalon (Fig. 5 D; t). In the PNS starting at E10.5,
expression of
-galactosidase was observed in cranial and
dorsal root ganglia and along the nerves emanating from
these ganglia (Fig. 5 C; cg and drg). Between E11.5 and
12.5, the cranial and dorsal root ganglia and the sympathetic chain showed strong L1lacZ transgene expression
(Fig. 5, D and E; cg, drg, and sc).
N Transgene Occurs
before Neural Differentiation and Appears at Several
Nonneural Sites
N transgene was observed first at
E8.5, a full day earlier than in embryos carrying the
L1lacZ transgene (compare Fig. 5, F and G, with A and
B). This initial expression of the L1lacZ
N transgene was
found within the first branchial arch and in the prosencephalon (Fig. 5 F; b1 and p). These areas contain migratory neural crest cells from the regions of the developing hindbrain and mesencephalon that differentiate into both
PNS and craniofacial mesenchymal tissues.
-galactosidase expression patterns between the L1lacZ and
L1lacZ
N transgenes were apparent at E9.5. While L1lacZ
embryos showed faint expression of
-galactosidase in the
midbrain and trigeminal ganglion (Fig. 5 B), L1lacZ
N
embryos showed intense
-galactosidase expression in the
head and trunk (Fig. 5 G). In the head, the L1lacZ
N
transgene was expressed in the prosencephalic region surrounding the eye and in the frontonasal mass. Expression
was widespread in the cephalic mesenchyme, branchial
arches 1 and 2, and in the circumpharyngeal tract (Fig. 5
G; cm, b1, b2, and ct). In the trunk, expression of
L1lacZ
N was also evident in the posterior mesoderm
(Fig 5 G; pm). In the PNS, expression of the L1lacZ
N
was observed earlier than that of the L1lacZ transgene appearing at E9.5-10.5, when neural crest cells were condensing into the primordia of the cranial and dorsal root
ganglia (Fig. 5, G and H). In the CNS, expression of the
L1lacZ
N transgene appeared in the midbrain, telencephalon, and spinal cord in a pattern that was very similar to
that of the L1lacZ transgene (compare Fig. 5, H-J, to C-E).
N transgene continued to be expressed during the differentiation of neural
crest cells into both neural and mesenchymal tissues. Expression of the transgene persisted in the periocular region, the snout, and in dorsal cranial regions, particularly
within an area overlapping the telencephalon and midbrain, and over the hindbrain (Fig. 5, H-J). In the PNS between E11.5 and E12.5, the L1lacZ
N transgene was expressed in the fully differentiated cranial and dorsal root
ganglia, the sympathetic chain, and in peripheral nerves
(Fig. 5, I and J; drg, sc, and pn). Between E10.5 and E12.5,
expression of L1lacZ
N was prominent in the dorsal ectoderm, particularly over the hindbrain and cervical spinal
cord, and extended over the entire ventral body wall and
into the limbs (Fig. 5, H-J; de and bw).
N Transgene during
Limb Development
N transgene was followed in more detail in limbs that were isolated from embryos between E10.5 and E13.5 (Fig. 6, A-D). In the
anterior limb bud at E10.5, expression was seen in the apical ectodermal ridge (Fig. 6 A; aer). At E11.5, expression
became more prominent in the anterior portion of the limb and also along the nerves (Fig. 6 B; am, pn). At
E12.5, expression of
-galactosidase was observed in both
anterior and posterior portions of the limb (Fig. 6 C; am,
pnz). At this time, extensive staining was observed in the
nuclei of cells along peripheral nerves that penetrate and
ramify within the limb. Since the lacZ reporter gene contained the nuclear localization signal, much of the staining
along the nerves is likely to be in the nuclei of the precursors of peripheral glial cells ensheathing the nerves. By
E13.5, expression of the L1lacZ
N transgene was prominent in the interdigital mesenchyme and persisted in cells
ensheathing the nerves (Fig. 6 D; im). Such apical ectodermal ridge and interdigital
-galactosidase staining was not
observed in embryos carrying the L1lacZ transgene (data
not shown).
Fig. 6.
Analyses of the -galactosidase staining patterns produced by the L1lacZ
N transgene during the development of the forelimb. The dorsal side of the limb is shown at each stage of development: E10.5 (A), E11.5 (B), E12.5 (C), and E13.5 (D). Abbreviations for structures: aer, apical ectodermal ridge; am, anterior mesoderm; im, interdigital mesenchyme; pn, peripheral nerve; pnz, posterior necrotic zone. Bar, 1 mm.
[View Larger Version of this Image (40K GIF file)]
N transgenes showed differences in their expression, transverse sections were taken from embryos at various positions along the anteroposterior axis and examined
for cellular expression of
-galactosidase. All tissue sites
showing differences in the cellular expression patterns between these two transgenes are illustrated in Fig. 7. The
common and ectopic sites of expression for the two transgenes are also summarized in Table I.
Fig. 7.
Comparison of the patterns of -galactosidase expression in sections of embryos carrying either the L1lacZ transgene (A-C, E, G, I, K, M, O, and Q) or the L1lacZ
N transgene (D, F, H, J, L, N, P, and R) transgenes. All transverse sections were taken from
E12.5 embryos, except for those taken at the level of the mandibular and maxillary processes (I and J), which were taken from E13.5
embryos. Sections were taken at the level of the telencephalon (A), the olfactory nerve (B), the midbrain (C and D), diencephalon and
Rathke's pouch (E and F), eye and trigeminal ganglion (G and H), heart (K and L), thoracic spinal cord (M and N), hindlimbs (O and
P), and the kidneys (Q and R). Abbreviations for anatomical structures: bw, body wall; c, cornea; de, dorsal ectoderm; drg, dorsal root
ganglia; ec, endocardial cushion; gt, genital tubercle; hl, hindlimb; k, kidney; lv, left ventricle of the heart; man, mandibular process; max,
maxillary process; mg, midgut; mn, motoneurons; mnr, motoneuron roots; mt, metanephric tubule; mz, marginal zone; ob, olfactory
bulb; oe, olfactory epithelia; on, olfactory nerve; po, periocular skeleton; rmb, roof of midbrain; rp, Rathke's pouch; tg, trigeminal ganglion; tp, tooth primordium.
[View Larger Version of this Image (130K GIF file)]
N
Transgenes
Embryos carrying the L1lacZ transgene showed a neurally restricted pattern of -galactosidase expression that
is consistent with the known pattern of L1 expression in
postmitotic neurons and peripheral glia. The L1lacZ transgene was expressed in the marginal zones of the telencephalon and midbrain (Fig. 7, A and C; mz), in the olfactory nerve (Fig. 7 B; on), and in the intermediate zone of
the olfactory bulb (Fig. 7, B and G; ob). In the spinal cord,
L1lacZ was expressed in the mantle layer in motoneurons and in cells surrounding the motor and sensory roots (Fig.
7 M; mn and mnr). The pattern of L1lacZ
N transgene expression was identical to the L1lacZ pattern in all of these
neural cells (for examples, see Fig. 7, D, H, and N; mz, ob,
mn, and mnr). In addition to these common regions of expression, the L1lacZ
N transgene showed intense expression in the neuroepithelia within the roof of the midbrain
and in Rathke's pouch (Fig. 7, compare C and D, E and F;
rm and rp).
In the PNS, both transgenes were expressed in the
trigeminal (Fig. 7, G and H; tg), facio-acoustic, and glossopharyngeal ganglia. Expression of both transgenes was
observed in the sympathetic chain and the vagus nerve, although the expression by the L1lacZN was significantly
more intense than that of the L1lacZ transgene (data not
shown). Both transgenes were also expressed in dorsal root ganglia and in the nuclei of cells ensheathing fiber
bundles that project toward the periphery (Fig. 7, M and
N; drg).
In contrast to L1lacZ embryos, mice carrying the
L1lacZN transgene showed extensive
-galactosidase
staining in nonneural tissues. The L1lacZ
N transgene
showed intense expression in craniofacial mesenchymal
tissues, particularly in the periocular skeleton and the cornea, and in the mandibular and maxillary processes.
L1lacZ embryos showed no
-galactosidase expression in
these areas (Fig. 7, compare G and H, and I and J; po, c,
man, and max). The heart revealed little if any expression
of the L1lacZ transgene (Fig. 7 K) but showed intense expression of the L1lacZ
N transgene in the endocardial
cushion tissue and in the wall of the left ventricle (Fig. 7 L;
ec and lv). In addition to these sites, the L1lacZ
N transgene was expressed in the dorsal ectoderm overlying the
spinal cord (Fig. 7 N; de).
In the abdominal region, both transgenes were expressed in the presumptive enteric ganglia surrounding the
esophagus, stomach, midgut, and duodenum. However,
the L1lacZN transgene showed more intense expression
in the cells surrounding these tissues (Fig. 7, compare O
and P; mg). The L1lacZ
N transgene was also expressed in the mesoderm of the abdominal body wall, the genital
tubercle, and the hindlimb (Fig. 7 P; bw, gt, and hl). Expression of both transgenes was observed in the abdominal paraganglia and later (at E13.5) in the adrenal medulla
(data not shown). At E12.5, the L1lacZ
N transgene was
expressed in the kidney within the metanephric tubules
and the ureter (Fig. 7 R; k and mt). Such expression was
not observed in embryos carrying the L1lacZ transgene (Fig. 7 Q).
The present studies provide evidence that the tissue-specific expression of the L1 adhesion molecule is modulated
by an NRSE that silences the promoter of the L1 gene. In
initial studies, we identified and sequenced a previously
uncharacterized segment of 13 kb at the 5 end of the
mouse L1 gene. Our results show that the first exon of the
L1 gene is actually located 10 kb further upstream of exon
A, the previously identified first exon (22). Two types of
analysis indicate that this is in fact the first exon of the L1
gene. First, all of the sequences obtained from cDNA
clones generated by RACE were included in this exon.
Second, RNAse protection analyses showed multiple start
sites in the L1 genomic sequence that were immediately
upstream of the sequence found in our cDNA clones produced by RACE.
By searching the human L1 gene sequence, we identified an exon comparable to the one we defined for mouse;
it too is about 10 kb upstream of the 5-most exon that was
assigned previously. The mouse and human L1 genes are
very similar in the region of the proximal promoter and
first exon. Both genes contain binding sites for the transcription factor SP1 and share a segment of ~60 bp that include binding sites for nuclear factor 1 (NF-1) and the
CCAAT-binding protein (CBP). Further analyses of these DNA elements and use of transcription initiation sites will
be required to assess their role in the regulation of L1 gene
expression.
The human L1 gene is located on the X chromosome near the region known to cause the fragile X syndrome, and mutations in the human L1 gene have recently been linked to three syndromes: X-linked spastic paraplegia (SPG1); mental retardation, aphasia, shuffling gait, and adducted thumbs (MASA); and X-linked hydrocephalus (HSAS) (18, 45, 53). So far, the L1 mutations that give rise to these disorders have been mapped to the coding exons of the L1 gene. It may be of interest to determine whether mutations in the promoter might also contribute to some of these syndromes. For example, transcriptional start sites for the L1 gene include two sets of trinucleotide repeats, and expansion of such repeats has been associated with a number of congenital disorders of the nervous system (25).
The NRSE and REST/NRSF Silence Expression of the Mouse L1 Gene In Vitro
In cellular transfection experiments, the L1 promoter by itself was expressed in both neural (N2A) and nonneural (NIH3T3) cells. However, when combined with downstream segments of the gene that included the first intron and the NRSE, the promoter was active only in neural (N2A) cells. Two copies of the NRSE were sufficient to silence the activity of L1 gene constructs in NIH3T3 cells. This silencing was eliminated upon expression of a truncated version of REST/NRSF (called D-REST), which is known to release silencing by the NRSE (4). Similarly, expression of the full-length REST/NRSF protein in N2A cells resulted in a reduction in the activity of NRSE-containing constructs. These data are in accord with the conclusion that the NRSE mediates silencing of L1 gene expression in nonneuronal cells that normally express REST/NRSF.
In addition to the NRSE, sequences in the first intron partially silenced L1 gene expression in NIH3T3 cells, and optimal silencing was observed when both the NRSE and the first intron were included in L1 gene constructs. The DNA sequence of the entire first intron does not contain any additional NRSEs, but expression of REST/NRSF silenced the activity of a construct containing this intron alone. These results suggest that there may be an unidentified sequence in the first intron, not related to the NRSE, that binds to REST/NRSF. Alternatively, a silencer protein other than REST/NRSF may bind to sequences in the first intron and silence L1 gene expression by interacting with REST/NRSF.
The Transgene L1lacZ Has a Neurally Restricted Pattern of Expression and Deletion of the NRSE Leads to Premature and Ectopic Expression in Nonneural Cells
L1 is found predominantly on postmitotic neurons in the
CNS and PNS and is also expressed by premyelinating
Schwann cells in the peripheral nervous system (35, 38, 44,
49). A segment of the L1 gene that included the promoter,
the first four exons, and the first three introns (L1lacZ)
was sufficient in 12 independent lines of transgenic mice to
give an identical neurally restricted pattern consistent with
the normal pattern of L1 expression. In addition to the
neurally restricted pattern obtained with L1lacZ, six lines
showed some nonneural expression of -galactosidase in a
subset of the tissues (primarily the cephalic mesenchyme) in which the construct lacking the NRSE, L1lacZ
N, was
also expressed. These results suggest that silencing of L1lacZ
expression may be partially released when the transgene is
integrated in some genomic locations.
Absence of the NRSE in the L1lacZN construct led to
extensive extraneural expression of
-galactosidase in the
embryo. All four of the L1lacZ
N lines had identical patterns of expression in nonneural cells; none of these lines
showed the neurally restricted pattern that was seen with
the L1lacZ transgene. These cells included nonneural derivatives of the neural crest and some cells of mesodermal and ectodermal origin (see Table I). However, in the CNS,
expression of the L1lacZ
N transgene was indistinguishable from expression of the unmutated L1lacZ transgene,
and both transgenes were expressed in neural derivatives
of the neural crest, including neurons and glia of the dorsal
root, sympathetic, parasympathetic, and enteric ganglia
and chromaffin cells of the adrenal medulla.
The L1lacZN transgene was expressed earlier than the
L1lacZ transgene in the neural crest cells of the head and
trunk, before their differentiation into the peripheral nervous system and mesenchymal tissues. This precocious expression was accompanied by the ectopic expression of the
L1 gene in mesenchymal derivatives of the neural crest,
such as the connective tissue cells of the head and face and
the cells that make up part of the aorticopulmonary septum of the heart. In addition, our preliminary studies of
newborn mice indicate that the L1lacZ
N, but not the
L1lacZ transgene, is expressed in the precursors of melanocytes (unpublished data). These results indicate that
the NRSE and REST/NRSF are likely to play a key role in
the further differentiation of the neural lineage from the
ectomesenchymal lineage of neural crest.
The precocious expression of the L1lacZN transgene
in neural crest cells also resulted in a more intense level of
-galactosidase expression than the L1lacZ transgene
along several tracts of peripheral nerves, such as those innervating the limb and gut. Although more thorough analyses using specific molecular markers are required, the
cells showing the elevated
-galactosidase expression appear to be Schwann cell precursors. Recent studies have
shown that L1 is expressed early during the differentiation
of immature, premyelinating Schwann cells but is downregulated in mature Schwann cells (15). The more intense expression of L1 that we observed in the PNS of the limb upon
deletion of the NRSE may represent a release from silencing of the L1 gene in myelinating Schwann cells. The NRSE
in the L1 gene may therefore play a role in the downregulation of L1 expression that occurs in mature Schwann cells.
Multiple Silencers May Be Required to Prevent Expression of L1 in the Full Spectrum of Nonneural Tissues
REST/NRSF mRNA and protein are present in a majority
of nonneuronal cell types (4, 23, 37, 47) and in undifferentiated neural precursors (4, 47), suggesting that the presence of REST/NRSF prevents precocious or full expression of the neuronal phenotype during early neurogenesis.
Deletion of the NRSE sequence from neuronal genes
might therefore be expected to lead to expression of neuronal genes in both neuronal precursors and nonneuronal cells. Previous studies of the NRSE in the SCG10 gene
(54) and the rat gene for the Na,K-ATPase 3 subunit (40)
showed that deletion of the element led to expression of
these genes in a number of nonneural tissues, such as
heart, liver, kidney, lung, and ovaries. However, analyses
of the expression of these genes at the cellular level were
not performed.
As indicated above, our results demonstrate that deletion of the NRSE in the L1 gene leads to precocious expression of L1 in those neural crest cells that ultimately form the peripheral nervous system and to ectopic expression in all nonneural derivatives of the neural crest as well as in cells of mesodermal and ectodermal origin. The data suggest that the REST/NRSF protein may play a role in the differentiation of neural crest cells, restricting the expression of certain genes to neural and glial derivatives. We did not see ectopic expression in a number of other tissues, such as liver, spleen, or ovaries, or in neural precursors of the central nervous system, all of which express REST/NRSF. Therefore, elimination of the NRSE does not lead to L1 expression in the full spectrum of tissues in which REST/NRSF is expressed. Recent evidence suggests that multiple restriction points are likely to exist during the differentiation of the CNS (5, 28, 52). It is therefore likely that REST/NRSF is only one of the many factors that restrict the expression of certain genes to neurons. These observations and our in vitro studies suggest that there are additional silencers in the L1 gene that act either independently or in concert with the NRSE and REST/ NRSF. At least one such element is localized to the first intron. It will be of interest to identify this silencer and the regulatory proteins to which it binds. Such studies may reveal specific combinations of factors required for silencing of L1 gene expression in different tissues.
A question arises about the factors that activate the L1
promoter in the nonneural tissues of the mice containing
the transgene in which the NRSE is deleted. There is a
striking correlation between the tissue patterns and cells
that express the L1lacZN transgene and the expression
patterns of the Msx homeodomain proteins (29). A
tenable hypothesis is that in the absence of the NRSE, expression of the L1 gene may be induced directly by Msx
homeodomain proteins or indirectly by factors that induce
the expression of Msx genes, such as members of the bone
morphogenetic protein (BMP) family (1, 6, 27, 56). In support of this possibility, BMP-2, -4, and -7 have been shown
to induce expression of L1 in neuroblastoma-glioma NG-108-15 cell line (42, 43). In addition, deletion of the NRSE
may allow the activation of L1 gene expression by BMPs
outside of the nervous system. For instance, BMP-4 and its
receptors are expressed in a pattern that closely resembles the expression of L1lacZ
N transgene in apical ectodermal ridge, the anterior mesenchyme, and interdigital zone
of the limb (20, 55, 56).
The present studies provide a transgenic mouse model for understanding the mechanisms underlying activation of the L1 gene by such factors and for further analysis of how this activation might be silenced by the NRSE and REST/NRSF. They also provide a basis for future studies of the components that positively regulate the neuronal expression of the L1 gene as well as those that may misregulate its expression, resulting in abnormal neural development and function.
Received for publication 3 April 1997 and in revised form 19 June 1997.
Address all correspondence to Frederick S. Jones, Department of Neurobiology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: (619) 784-2600. Fax: (619) 784-2646. e-mail: fjones{at}scripps.eduWe are grateful to Madhu Katragadda for excellent technical assistance. We thank Drs. Bruce Cunningham, Kathryn Crossin, Joseph Gally, Leslie Krushel, and George Miklos for critical reading of the manuscript.
This work was supported by U.S. Public Health Service Grants HD33576 (to G.M. Edelman) and NS34493 (to F.S. Jones) and a grant from the G. Harold and Leila Mathers Charitable Trust. G.M. Edelman is a consultant to Becton Dickinson and Company.
BMP, bone morphogenetic protein; CAM, cell adhesion molecule; CBP, CCAAT-binding protein; CNS and PNS, central and peripheral nervous system; E, embryonic day; HA, hemagglutinin; N-CAM, neural cell adhesion molecule; NF-1, nuclear factor-1; Ng-CAM, neuron-glia cell adhesion molecule; NRSE, neural restrictive silencer element; NRSF, neural restrictive silencer factor; RACE, rapid amplification of cDNA ends; REST, RE-1-silencing transcription factor; RLU, raw light units.
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