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From the
SNAP-25 is a presynaptic nerve terminal protein which is also
essential for the process of neurite outgrowth in vivo and in vitro. However the processes regulating its expression have
not been characterized previously. We show that the gene encoding this
protein, SNAP, is strongly activated by the Brn-3a POU
(Pit-Oct-Unc) family transcription factor. Expression of both Brn-3a
and SNAP-25 increases when ND7 neuronal cells are induced to extend
neurite processes by serum removal. Inhibition of Brn-3a expression in
these cells inhibits SNAP-25 expression and abolishes the neurite
outgrowth that normally occurs in response to serum removal. These
results identify Brn-3a as the first transcription factor having a role
in process outgrowth in neuronal cells acting, at least in part, via
the activation of SNAP-25 gene expression.
The POU (Pit-Oct-Unc) family of transcription factors was
originally defined on the basis of a common DNA binding domain present
in the mammalian transcription factors Pit-1, Oct-1, and Oct-2 and in
the nematode Unc-86 protein. These factors play a critical role in the
development of specific cell types, particularly neuronal cells (for
reviews, see Verrijzer and Van der Vliet(1993) and Wegner et
al.(1993)). In particular, the Pit-1 factor is known to be
essential for pituitary gland development, and its inactivation results
in dwarfism in both mice (Li et al., 1990) and humans
(Radovick et al., 1992). Similarly, the unc-86
mutation in the nematode leads to a failure to develop specific cell
types particularly sensory neurons (Desai et al., 1988; Finney et al., 1988).
Following the isolation of the original POU
proteins, He et al.(1989) used a degenerate PCR
These three members of the Brn-3 family are the most similar
mammalian factors to unc-86 and, together with the Drosophila POU factors I-POU and tI-POU (Treacey et
al., 1991, 1992), constitute the POU IV subfamily (Wegner et
al., 1993). To obtain evidence on the potential role of the Brn-3
factors in mammalian sensory neurons, we initially studied their
expression in the ND7 neuronal cell line from which we originally
cloned Brn-3b and which expresses all three factors (Lillycrop et
al., 1992).
This cell line was originally prepared by the
immortalization of rat dorsal root ganglion neurons by fusion with the
C1300 mouse neuroblastoma cell line (Wood et al., 1990).
Although these cells proliferate indefinitely in culture, they can be
induced to cease dividing and extend neuritic processes of up to 500
µM in length by transfer to serum free medium (Suburo et al., 1992). This differentiation event is accompanied by
the movement of secretory granule components and subsequently synaptic
vesicle components to the tips of the processes (Wheatley et
al., 1992). We showed that the levels of Brn-3a are greatly
elevated in the nondividing, process bearing cells compared with the
level in undifferentiated cells (Lillycrop et al., 1992). In
contrast, the levels of other POU family transcription factors such as
Brn-3c or Oct-2 remain unchanged, whereas the level of Brn-3b decreases
in the differentiated cells (Lillycrop et al., 1992).
This
increased expression of Brn-3a during the in vitro differentiation of ND7 cells to a neuronal-like phenotype
suggested it might play a role in this process paralleling the role of
the unc-86 gene product in the nematode. Interestingly, in
co-transfection experiments, Brn-3a can activate a test promoter
bearing a suitable binding site for the Brn-3 factors, whereas Brn-3b
cannot do so (Budhram-Mahadeo et al., 1994; Morris et
al., 1994). Moreover treatments which result in the
differentiation of ND7 cells with an associated rise in Brn-3a also
result in the activation of this test promoter (Budhram-Mahadeo et
al., 1994). Hence changes in the expression of endogenous Brn-3a
can modulate the expression of a target promoter exactly as in
co-transfection experiments with exogenous Brn-3a.
These findings
suggested, therefore, that the rise in Brn-3a expression which occurs
upon differentiation of ND7 cells may play a role in producing the
cellular changes which are observed during this process, acting via the
activation of specific target genes. We have therefore used an
antisense approach to inhibit this increase in Brn-3a expression. We
show that this results in a lack of process formation by the ND7 cells
which is associated with a reduced expression of SNAP-25 (Oyler et
al., 1989; De Camilli, 1993), a protein which is essential for
neurite outgrowth (Osen-Sand et al., 1993). Moreover, we show
that the SNAP gene encoding this protein is directly activated
by Brn-3a.
Expression vectors containing either antisense
or sense POU domains, together with a vector conferring resistance to
neomycin, were introduced into ND7 cells by the calcium phosphate
transfection method of Gorman(1985). Stable transfectants were selected
by supplementing media with G418 to a final concentration of 800
µg/ml 48 h after transfection. Independent clones were isolated
after approximately 1 week of selection when individual foci of cells
were evident and grown in full growth medium supplemented with G418.
Putative clones capable of expressing either antisense or sense POU
domains were treated with dexamethasone at a final concentration of 1
µM for 24 h to induce expression of the MMTV promoter (Lee et al., 1981).
In initial screening experiments to confirm that the
exogenous constructs were producing sense or antisense POU domain RNAs
in the cell lines, PCR was performed using a primer internal to the
vector sequence and a primer internal to the POU domain so as not to
amplify endogenous Brn.3 mRNA. In subsequent experiments shown in Fig. 1, the level of the sense Brn-3 mRNA in each line was
determined by using a primer at the 5`-end of the POU domain
(5`-GAC/CCTC/GGAG/AGCGTTCGCCGAGC-3`) in conjunction with a primer
internal to the POU homeodomain (5`-GATGGCC/GGCGATCTTCTC-3`). Control
reactions using primers which detect ribosomal protein L6 mRNA
(5`-ATCGCTCCTCAAACTTGACC-3` and 5`-AACTACAACCACCTCATGCC-3`) were
performed in parallel. Following amplification with each pair of
primers the PCR products were run on a 2% agarose gel, which was
blotted onto a Hybond N nitrocellulose filter (Amersham) and hybridized
with homologues probes to detect either total Brn.3 or L6 PCR products.
Cultures were incubated in differentiation media for 48 h before
cells were photographed to allow scoring of neurite outgrowth. Cultures
were scored blind by a second observer unaware of the treatment to
which they had been subjected.
For
Western blot analysis, cells differentiated in either the absence or
presence of dexamethasone were harvested and 5
To determine whether the change in Brn-3a levels played any
direct role in the differentiation of the ND cells, we prepared a
construct in which the antisense strand of the Brn-3a POU
domain-encoding region was expressed under the control of the
glucocorticoid-inducible MMTV promoter. A similar construct expressing
the sense strand of the POU domain, but including a stop codon to
prevent any protein production, was used as a control.
Two
independent cell lines isolated by stable transfection of ND7 cells
with the antisense construct showed a clear decrease in the level of
endogenous Brn-3 mRNA when treated with dexamethasone to induce the
MMTV promoter (Fig. 1). In contrast no decrease was observed when
the control cell line containing the construct in the sense orientation
was treated in this way indicating that this reduction was due to the
induction of the antisense RNA in response to dexamethasone treatment.
Similar results were obtained using three independent RNA preparations
derived from each of the cell lines with all assays being carried out
in the linear range of the PCR assay (see ``Materials and
Methods'').
Interestingly the basal level of the Brn-3 mRNA in
the antisense lines was somewhat lower than in the parental ND7 cells
or the sense cell line (Fig. 1), indicating that the basal
expression directed by the MMTV promoter in the absence of induction
was having some effect on Brn-3 levels. As expected the levels of the
mRNAs encoding either the L6 ribosomal protein (Fig. 1) or
another POU family transcription factor, Oct-2, were unaltered in any
of the cell lines (data not shown). Although the antisense Brn-3a probe
may also bind to the closely related Brn-3b and Brn-3c mRNAs, these
mRNAs are present at relatively low levels in differentiated ND7 cells
compared with the Brn-3a mRNA (Lillycrop et al., 1992),
indicating that the predominant effect is likely to be on this mRNA. In
agreement with this a clear decline in the Brn-3a mRNA level in the
antisense lines was also observed with PCR primers specific for this
mRNA (data not shown). Hence the level of the Brn-3a mRNA has been
specifically reduced in the treated cells presumably by RNase
H-mediated degradation of RNA-RNA heteroduplexes (Walder and Walder,
1988; Agrawal et al., 1990).
Having established that the
NDA.9 and NDA.10 antisense cell lines had lower levels of the Brn-3a
mRNA, we investigated the effect of transferring these cells and the
sense strand-expressing cell line to serum-free medium. In these
experiments (Fig. 2) all the cell lines showed a similar
inhibition of DNA synthesis upon transfer which was comparable with
that observed in the parental ND7 cells. Moreover, no further decrease
was observed in DNA synthesis upon treatment of the cell lines with
dexamethasone to induce expression of the antisense construct. In
contrast, the NDA.9 and NDA.10 showed a very significant decrease in
the percentage of cells which produced processes upon transfer to serum
free medium and this effect was observed even in the absence of
dexamethasone induction paralleling the reduction in Brn-3a levels
observed under this condition (Fig. 3a).
Both we (Lillycrop et al., 1992) and others (He et al., 1989; Gerrero et al., 1993) have shown that
the Brn-3a POU family transcription factor is highly expressed both in
neuronal cells in vivo as well as in neuronal cell lines in vitro with expression being regulated during
differentiation both in vivo and in vitro. In
addition this factor can trans-activate artificial test promoters
containing appropriate target sites such as the octamer motif bound by
many POU family transcription factors (Budhram-Mahadeo et al.,
1994; Morris et al., 1994) or a high affinity DNA binding site
found in the corticotrophin-releasing hormone gene promoter (Gerrero et al., 1993). Moreover, such trans-activation can also be
observed on natural gene promoters such as those for the genes encoding
pro-opiomelanocortin (Gerreo et al., 1993) or
When taken together with
the relationship between Brn-3a and the nematode Unc-86 protein (He et al., 1989), these findings suggest that Brn-3a is likely to
play a key role in some aspect of neuronal development and
differentiation. To date, however, no such role has been identified.
However, the significant rise in Brn-3a levels when ND7 cells are
treated with serum-free medium (Lillycrop et al., 1992) or
cyclic AMP (Budhram-Mahadeo et al., 1994) suggests a possible
association with the events which occur during this process.
Thus
these treatments result in the cessation of cell division and the
extension of neuritic processes (Suburo et al., 1992; Wheatley et al., 1992). In addition a proportion of the cells in
serum-free medium undergo programmed cell death by apoptosis, although
this effect is not observed in the cells treated with cyclic AMP
(Howard et al., 1993).
By using an antisense approach we
show that although the rise in Brn-3a expression plays no apparent role
in growth arrest or apoptosis, it appears to be involved in the
extension of neurite processes. Moreover, it appears to achieve this
effect, at least in part, by activating expression of the SNAP-25 gene
which increases during the normal differentiation process of ND7 cells.
Thus Brn-3a can activate the SNAP-25 gene promoter, and the
up-regulation of this factor in ND7 cell differentiation may therefore
be responsible for the corresponding increase in SNAP-25 expression
during this process. Similarly the inhibition of Brn-3a expression
using an antisense approach would result in the observed decrease in
SNAP-25 expression and hence the failure of neurite outgrowth. These
results therefore implicate Brn-3a as the first example of a
transcription factor which regulates neurite outgrowth acting via the
modulation of SNAP-25 gene expression.
Interestingly in our
antisense cell lines we also observed a decrease in the expression of
two synaptic vesicle proteins rab3A and synaptophysin (Bennett and
Scheller, 1993) in response to dexamethasone treatment, an effect which
was not observed in the sense cell line (data not shown). As the
inhibition of SNAP-25 expression using a direct antisense approach to
target the SNAP-25 mRNA also results in decreased synaptophysin
expression (Osen-Sand et al., 1993), it is possible that these
effects are an indirect consequence of the direct effect of Brn-3a on
SNAP-25 expression. Alternatively, the ability of Brn-3a to activate
the synapsin I gene promoter (data not shown) suggests that it may be
able to directly activate the genes encoding several synaptic proteins
involved in neurite outgrowth.
Our transfection experiments raise
the possibility that Brn-3a directly activates the SNAP-25 promoter by
interacting with the region of the promoter between -288 to
-126. Interestingly, both the octamer consensus sequence (for
review, see Falkner et al.(1986)) and a distantly related
sequence in the corticotrophin-releasing hormone promoter have been
shown to mediate activation by Brn-3a (Budhram-Mahadeo et al.,
1994; Gerreo et al., 1993). Inspection of the region of the
SNAP-25 promoter from -288 to -126 which is critical for
activation by Brn-3a does not, however, reveal any motifs closely
related to these sequences. It is also possible, therefore, that Brn-3a
may act indirectly on the SNAP-25 gene, perhaps by activating the gene
encoding another trans-activating factor which in turn activates the
SNAP-25 promoter.
Whatever the case, it is clear that Brn-3a can
directly or indirectly activate the SNAP-25 gene promoter via sequences
located between -288 and -126 of the promoter. In turn such
induction would allow the production of the SNAP-25 gene product
enabling it to fulfill its essential role in the exocytosis required
for neuronal process outgrowth and potentially in synaptic vesicle
exocytosis and nerve terminal plasticity.
Volume 270,
Number 26,
Issue of June 30, pp. 15858-15863, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
()approach to isolate cDNA clones encoding further POU
proteins. One of these, originally known as Brn-3 and now renamed
Brn-3a (Lillycrop et al., 1992; Theil et al., 1994)
or Brn-3.0 (Gerrero et al., 1993), was highly expressed in
sensory neurons and showed strong homology to unc-86. It was
therefore suggested to be the mammalian homologue of this nematode
factor and to play a similar critical role in the development of
mammalian sensory neurons (He et al., 1989). Subsequently
using a similar approach, two other closely related mammalian factors
were also isolated from neuronal cells and were named Brn-3b (Lillycrop et al., 1992; Xiang et al., 1993), also known as
Brn-3.2 (Turner et al., 1994), and Brn-3c (Ninkina et
al., 1993) also known as Brn-3.1 (Gerrero et al., 1993).
Construction of Antisense Cell Lines
The POU
domain of Brn-3A was excised from bluescript with HincII and XbaI and inserted in the antisense orientation under the
control of the glucocorticoid-inducible MMTV promoter via the SmaI and XbaI sites in the mammalian expression
vector pJ5 (Morgenstern and Land, 1990). The POU domain sense
construct, which contains a single nucleotide insertion 9 codons into
the POU domain that induces a shift in the reading frame of this
protein, resulting in the introduction of a stop codon 38 codons into
the POU domain), was similarly inserted into compatible sites in the
linker region of pJ5.
RNA Isolation and PCR Analysis
RNA was isolated
from cells by the guanidinium thiocyanate method (Chirgwin et
al., 1979) and used as template for production of cDNA using
random hexanucleotide primers (Pharmacia). To ensure no contaminating
DNA was present, RNA samples were treated with DNase prior to cDNA
synthesis. The resulting cDNA was amplified by PCR essentially
according to the method of Kawasaki(1990). Typically 0.125 µg of
cDNA was included in each PCR and 15, 20, or 25 cycles of PCR performed
to ensure conditions in which the PCR product signal was quantitatively
related to input RNA. Controls using RNA samples without reverse
transcription were used to demonstrate that contaminating DNA was
absent.
Figure 1:
a, PCR amplification of
the endogenous mRNAs encoding Brn-3 or the L6 ribosomal protein in
parental ND7 cells (ND7), the cell lines (NDA.9 and NDA.10) containing
the Brn-3a POU domain in the antisense orientation, or the control cell
line containing the POU domain in the sense orientation with an added
stop codon (NDF.5). Prior to differentiation in serum-free medium,
cells were either treated with 1 µM dexamethasone (+)
or left untreated (-). b, levels of the Brn-3 mRNA in
the different cell lines as determined by densitometric scanning of
data of the type illustrated in a and equalized on the basis
of the level of the L6 ribosomal protein mRNA as determined in parallel
amplifications.
Cell Line Differentiation
Induction of the MMTV
promoter was achieved by supplementing full growth media with
dexamethasone to a final concentration of 1 µM (Lee et
al., 1981). Approximately 12-15 h after addition of
dexamethasone to cultures, cells were induced to undergo
differentiation by incubation in serum-free media consisting of a 1:1
mix of Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) and nutrient mix Ham's F-12 (Life
Technologies, Inc.) supplemented with human transferrin (5 µg/ml),
bovine insulin (250 ng/ml), sodium selenite (20 nM), and
dexamethasone (1 µM) (Suburo et al., 1992;
Wheatley et al., 1992)). In all experiments control uninduced
cultures were analyzed in parallel with experimental cultures. These
cultures were identical to experimental cultures, with the exception
that MMTV promoter activity was not induced by addition of
dexamethasone to either full growth media or differentiation media.
Assay of Cellular DNA Synthesis
To measure
cellular DNA synthesis, [
H]thymidine was added to
proliferating cells in 10% fetal calf serum or to cells incubated in
differentiation medium for 72 h in the presence or absence of
dexamethasone. Cells were harvested 4 h after
[H]thymidine addition to cultures and the
incorporation of [H]thymidine determined by
precipitating cellular DNA with 10% trichloroacetic acid. Values shown
represent the average [H]thymidine incorporation
of triplicate cultures.
Assay of SNAP-25 Levels
PCR amplifications of the
SNAP-25 mRNA were carried out as described above using the primers:
5`-TGACCAGCTGGCTGATGAGTC-3` and 5`-CCCATGTCTAGGGCCATATGA-3`.
10
cells of each culture submitted to analysis by SDS-polyacrylamide
gel electrophoresis. Gels were transferred to nitrocellulose by Western
blotting as described previously (Dhillon et al., 1993) and
SNAP-25 detected by probing filters using a rabbit polyclonal antisera
raised against SNAP-25. To ensure an equal amount of each sample was
loaded onto gels, filters were stripped and re-probed using a control
antibody, in this case a mouse monoclonal antisera raised against
actin.
Transient Transfection and Chloramphenicol
Acetyltransferase Assay
Transfection of DNA was carried out
according to the method of Gorman(1985). Routinely, 1 10
ND7 cells (Wood et al., 1990) were transfected with 10
µg of the reporter plasmid and 10 µg of the Brn-3 expression
vectors (Theil et al., 1994). In all cases cells were
harvested 72 h later. The amount of DNA taken up by the cells in each
case was measured by slot blotting the extract and hybridization with a
probe derived from the ampicillin resistance gene in the plasmid vector
(Abken and Reifenrath, 1992). This value was then used to normalize the
values obtained in the chloramphenicol acetyltransferase assay as a
control for differences in uptake of plasmid DNA in each sample. Assays
of chloramphenicol acetyltransferase activity were carried out
according to the method of Gorman(1985) using samples which had been
equalized for protein content as determined by the method of
Bradford(1976).
Figure 2:
[H]Thymidine
incorporation in parental ND7 cells, the antisense Brn-3 cell lines
(NDA.9 and NDA.10), or the sense cell line (NDF.5) when proliferating
in serum-containing medium (P) or following transfer to
differentiation-inducing serum free medium (D) in the presence
(+) or absence (-) of 1 µM dexamethasone.
Figure 3:
a, percentage of parental ND7 cells or the
NDF.5, NDA.9, or NDA.10 clones which formed neuritic processes in
serum-free medium when maintained in the presence (+) or absence
(-) of 1 µM dexamethasone. b,
representative fields of the control NDF.5 or the antisense NDA.9 and
NDA.10 cells in serum-free medium in the presence of absence or 1
µM dexamethasone.
In response
to dexamethasone treatment, a further reduction in process formation
was observed in both antisense cell lines paralleling the further
reduction in Brn-3a levels (Fig. 3a), and the processes
which did form were much shorter (Fig. 3b). In contrast
a small enhancement in process formation was observed when the sense
cell line was treated with dexamethasone (Fig. 3a), and
no effect was observed in the parental ND7 cells (data not shown). No
significant differences in cell survival as assayed by the ability of
cells to exclude trypan blue were noted in any of the cultures
following transfer to serum-free medium either before or after
dexamethasone induction. The antisense cells therefore continue to
survive without extending processes (Fig. 4). Hence the
inhibition of Brn-3a synthesis in the antisense lines is associated
with reduced neurite outgrowth.
Figure 4:
Cell viability in the different cell lines
following transfer to serum free medium with (+) or without
(-) 1 µM dexamethasone.
In view of the ability of Brn-3a to
activate the expression of specific target genes, it is likely that the
inhibition of process outgrowth in the antisense lines is dependent
upon a failure to activate specific target genes whose protein products
are required for neurite outgrowth. One obvious candidate gene is that
encoding SNAP-25 (synaptosomal-associated protein of M
25,000; Oyler et al., 1989) which plays a critical role
in the process of vesicle exocytosis which is required for axon
outgrowth (De Camilli et al., 1993. Osen-Sand et al.,
1993; Huttner, 1993). Thus the direct inhibition of SNAP-25 expression
using an antisense approach results in a failure of axon outgrowth both in vitro and in vivo and specifically affects neurite
elongation as opposed to the formation of small spikes (Osen-Sand et al., 1993). Interestingly, the levels of SNAP-25 increase
in undifferentiated ND7 cells following serum removal (Fig. 5),
whereas synaptic vesicle components such as synaptophysin redistribute
to the tips of the processes but do not increase in abundance (Suburo et al., 1992; Wheatley et al., 1992). We therefore
measured the levels of SNAP-25 in the antisense cell lines. As shown in Fig. 5, both the antisense lines showed reduced levels of the
SNAP-25 mRNA and protein when treated with dexamethasone compared with
the sense cell line or parental ND7 cells. These effects were
consistently observed with several independent RNA and protein samples
isolated from each of the cell lines.
Figure 5:
a, representative PCR amplification of the
SNAP-25 mRNA in proliferating (P) or differentiated (D) ND7 cells or in the NDF.5, NDA.9, or NDA.10 cell lines
following differentiation by transfer to serum-free medium in the
presence (+) or absence (-) of 1 µM
dexamethasone. b, Western blot of protein samples from the
various cell lines (as in a) with a polyclonal antibody to
SNAP-25. Arrows indicate molecular weight markers of the sizes
indicated. c, levels of the SNAP-25 protein in the different
cell lines following differentiation in the presence (+) or
absence (-) of dexamethasone as obtained by densitometric
scanning of data of the type illustrated in b and equalization
on the basis of the level of the actin protein in each sample as
determined in parallel blots.
Hence the reduction in neurite
outgrowth in the antisense cell lines is paralleled by a reduction in
SNAP-25 expression. In order to determine whether the Brn-3a
transcription factor was able to activate the SNAP-25 gene promoter, we
co-transfected ND7 cells with a Brn-3a expression vector (Theil et
al., 1994; Morris et al., 1994) together with a construct
containing the promoter of the SNAP-25 gene (Hess et al.,
1992)
()from -2200 base pairs upstream of
the transcriptional start site to +106 bases downstream. As shown
in Fig. 6, the full SNAP-25 promoter was strongly stimulated by
co-transfection with the Brn-3a expression vector compared with the
level of promoter activity observed upon co-transfection with the empty
expression vector alone. Similar stimulation was also observed with a
construct containing the sequence from -288 to +106 of the
SNAP-25 promoter. In contrast a truncated promoter containing only the
sequence from -126 to +106 was not transactivated (Fig. 6), indicating that this effect is specific and requires
sequences between -288 and -126 in the SNAP-25 promoter.
Figure 6:
Chloramphenicol acetyltransferase (CAT) assay of constructs containing either 2200 (construct
A), 288 (construct C), or 126 (construct D) bases of DNA 5` to the
transcriptional start site of the SNAP-25 gene linked to the CAT gene.
The target plasmids were co-transfected into ND7 cells with either
empty expression vector (V) or the expression vector encoding
Brn-3a (3). a shows a representative assay of constructs A and
D, whereas b shows the mean of three experiments with
constructs A, C, and D whose standard deviation is shown by the bars.
-interexin
(Budhram-Mahadeo et al., 1995).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 V. Budhram-Mahadeo, M. Parker, and D. S. Latchman POU Transcription Factors Brn-3a and Brn-3b Interact with the Estrogen Receptor and Differentially Regulate Transcriptional Activity via an Estrogen Response Element Mol. Cell. Biol., February 1, 1998; 18(2): 1029 - 1041. [Abstract] [Full Text]
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M. D. Smith, P. J. Morris, S. J. Dawson, M. L. Schwartz, W. W. Schlaepfer, and D. S. Latchman Coordinate Induction of the Three Neurofilament Genes by the Brn-3a Transcription Factor J. Biol. Chem., August 22, 1997; 272(34): 21325 - 21333. [Abstract] [Full Text] [PDF]
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 Y. Qi, J. K. T. Wang, M. McMillian, and D. M. Chikaraishi Characterization of a CNS Cell Line, CAD, in which Morphological Differentiation Is Initiated by Serum Deprivation J. Neurosci., February 15, 1997; 17(4): 1217 - 1225. [Abstract] [Full Text] [PDF]
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M. D. Smith, S. J. Dawson, and D. S. Latchman Inhibition of Neuronal Process Outgrowth and Neuronal Specific Gene Activation by the Brn-3b Transcription Factor J. Biol. Chem., January 10, 1997; 272(2): 1382 - 1388. [Abstract] [Full Text] [PDF]
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J. Sze, Y Liu, and G Ruvkun VP16-activation of the C. elegans neural specification transcription factor UNC-86 suppresses mutations in downstream genes and causes defects in neural migration and axon outgrowth Development, January 3, 1997; 124(6): 1159 - 1168. [Abstract] [PDF]
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E. Ensor, M. D. Smith, and D. S. Latchman The BRN-3A Transcription Factor Protects Sensory but Not Sympathetic Neurons from Programmed Cell Death/Apoptosis J. Biol. Chem., February 9, 2001; 276(7): 5204 - 5212. [Abstract] [Full Text] [PDF]
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ABSTRACT
