From the
We report the molecular characterization of a mouse basic
helix-loop-helix factor, designated MATH-1, structurally related to the
product of the Drosophila proneural gene atonal.
MATH-1 mRNA is first detected in the cranial ganglions and the dorsal
part of the central nervous system on embryonic day 9.5 (E9.5). From
E10.5 onward, prominent expression of MATH-1 continues in the dorsal
part of the central nervous system but becomes restricted to the
external granular layer of the cerebellum by E18 and is undetectable in
the adult nervous system. MATH-1 activates E box-dependent
transcription in collaboration with E47, but the activity is completely
antagonized by the negative regulator of neurogenesis HES-1. These
results suggest that MATH-1 may be a target of HES-1 and play a role in
the differentiation of subsets of neural cells by activating E
box-dependent transcription.
In mammals, the embryonic ectoderm gives rise to the neural tube
and the neural crest, both of which are the origins of the nervous
system (Hamilton et al., 1962). In the neural tube, which
generates the central nervous system (CNS),
One useful
approach to dissect the genetic program of mammalian neurogenesis is
the isolation of genes structurally related to those involved in
Drosophila neurogenesis, which provides a powerful model
system. Many genes involved in Drosophila neurogenesis have
been genetically and molecularly characterized. Among them,
helix-loop-helix (HLH) factors play an essential role in the early
stages of neural development (Jan and Jan, 1993; Campos-Ortega and Jan,
1991). The HLH factors encoded by the Drosophila proneural
genes, achaete-scute complex and daughterless, are
positive regulators for sensory organ formation, whereas those encoded
by hairy, Enhancer of split, and extramacrochaetae are negative regulators (Moscoso del Prado and Garcia-Bellido,
1984; Rushlow et al., 1989; Klämbt et al., 1989;
Ellis et al., 1990; Garrell and Modolell, 1990; Skeath and
Carroll, 1991; Campuzano and Modolell, 1992). Mammalian factors
homologous to these Drosophila regulators have recently been
analyzed and have given us a useful clue for understanding the
molecular nature of mammalian neurogenesis. For example, Mash1 (Johnson
et al., 1990), a mammalian homologue of Drosophila achaete-scute complex, has been shown to be essential for
the generation of olfactory and autonomic neurons (Guillemot et
al., 1993). Mash1 forms a heterodimer with E12/E47 (Murre et
al., 1989), mammalian homologues of the Drosophila
daughterless protein, and activates transcription by binding to
the E box (CANNTG) (Johnson et al., 1992). In addition, HES-1,
a mammalian homologue of Drosophila hairy and Enhancer of
split, is expressed at high levels in the developing nervous
system and represses Mash1-induced transcription (Sasai et
al., 1992; Takebayashi et al., 1994). These results show
that HLH factors structurally related to the Drosophila factors are involved in developmental processes of mammalian
neurogenesis.
HES-1 is expressed at high levels in neural precursor
cells, but its expression level decreases as neural differentiation
proceeds (Sasai et al., 1992). Persistent expression of HES-1
prevents neural differentiation in the CNS (Ishibashi et al.,
1994). The mechanism of how HES-1 prevents neurogenesis is not known,
but because HES-1 also inhibits myogenesis by antagonizing the HLH-type
myogenic determination factor MyoD (Sasai et al., 1992), it is
reasonable to hypothesize that HES-1 may prevent neurogenesis by
antagonizing HLH-type neural determination factors. Here, we have
attempted to identify the HLH factors that are involved in mammalian
neurogenesis. Whereas Mash1 acts as a neural determination factor in
the peripheral nervous system (Guillemot et al., 1993) and is
expressed at high levels in the developing CNS (Lo et al.,
1991; Guillemot and Joyner, 1993), Mash1-deficient mice exhibited no
apparent abnormalities in the CNS (Guillemot et al., 1993),
suggesting that other HLH factors may compensate or be necessary for
CNS development. Recently, a novel Drosophila proneural gene,
atonal, was molecularly characterized and shown to encode an
HLH factor essential for the differentiation of the chordotonal organs
(Jarman et al., 1993). These results prompted us to search for
novel mammalian HLH factors structurally related to the product of
atonal in order to investigate the molecular mechanisms of
mammalian neurogenesis. We have found that a mammalian HLH factor
structurally related to the product of Drosophila atonal is
expressed in restricted regions of the developing nervous system,
activates E box-dependent transcription, and is antagonized by the
negative regulator HES-1.
For
Northern blot analysis, 5 µg of poly(A)
The E box probes were prepared as follows. For the E1
probe, two oligonucleotides, 5`-GCCACCCTTGAACCAGGTGGACTTTTTGG-3` and
5`-GCCAAAAAGTCCACCTGGTTCAAGGGTGG-3`, were annealed and labeled at both
ends by filling with Klenow enzyme in the presence of
[
Reactions of the gel
mobility shift assay were carried out as described previously (Sasai
et al., 1992).
The luciferase reporter (0.5 µg) and the
eukaryotic expression plasmids (0.5 µg each) were cotransfected
into C3H10T1/2 cells using the calcium phosphate coprecipitation
method. The total amounts of DNA were adjusted to 2 µg with
pSV-CMV. Two days after transfection, luciferase activities were
determined as described previously (Ow et al., 1986).
Using the PCR fragment as a probe, we screened a mouse
genomic library. The
As shown in Fig. 9, MATH-1
significantly activated transcription from the promoter containing the
E box element (Fig. 9, lane 3) but did not activate
transcription without the E box (Fig. 9, lane 9). This E
box-dependent trans-activation may result from either the
formation of a homodimer of MATH-1 or a heterodimer of MATH-1 and the
endogenous E12/E47, which is ubiquitously expressed. The latter
hypothesis seems more likely, because MATH-1 alone did not interact
with the E box sequence (Fig. 8). In addition, when E47
expression vector was cotransfected with MATH-1, MATH-1-induced
trans-activation was enhanced (Fig. 9, lane 4), further suggesting that the heterodimer of MATH-1 and
E12/E47 may be involved in this trans-activation.
MATH-1 expression in the
nervous system is transient and not detected in the adult nervous
system, suggesting that MATH-1 may be involved in the differentiation
process but not in the maintenance of subsets of the neural cells. In
our preliminary experiments, we infected the neural precursor cells
prepared from E10 mouse embryos with MATH-1-transducing retrovirus.
However, we did not observe induction of neural
differentiation.
It
has recently been shown that diffusible factors from the notochord,
floor plate, and roof plate play a major role in the determination of
dorsal-ventral polarity within the neural tube (Basler et al.,
1993; Yamada et al., 1993). These diffusible factors may
induce transcription factors that activate ventral- or dorsal-specific
genes. For example, Sonic hedgehog secreted from the notochord induces
the expression of the transcription factor HNF-3
In the adult, MATH-1 is expressed in the epithelial cells of the
gastrointestinal tract but not in the neural cells. Northern blot
analysis demonstrated exactly the same size of MATH-1 transcript in the
adult and embryonal tissues. Furthermore, we did not detect any
additional MATH-1 products in the reverse transcriptase-mediated PCR
experiments when the adult and embryonal RNAs were used (data not
shown). Thus, a single MATH-1 protein may be expressed in both embryos
and adults, although the possibility that different molecules could be
produced by alternative splicing has not yet been excluded.
Equally important is that neurogenesis
may be negatively regulated by multiple HLH factors. HES-1 acts as a
transcriptional repressor and is down-regulated as neural
differentiation proceeds (Sasai et al., 1992). Forced
expression of HES-1 prevents mammalian neurogenesis in the CNS
(Ishibashi et al., 1994). Interestingly, the two activators
MATH-1 and Mash1 are negatively regulated by HES-1 (this study and
Sasai et al., 1992). We thus speculate that HES-1 prevents
neurogenesis by negatively regulating positive factors such as MATH-1
and Mash1. Another example concerns Id-1 and Id-2, negative regulators
of myogenesis. They are also expressed at high levels in the developing
nervous system (Duncan et al., 1992; Neuman et al.,
1993), suggesting that these factors also participate in the negative
regulation of neurogenesis. Thus, the balance between multiple positive
and negative regulators may play an important role in mammalian
neurogenesis, as in the Drosophila system.
Transcription
factors that do not belong to the HLH family are also required for
proper development of the nervous system. For example, the paired box
Pax factors play an essential role in neural development (Gruss and
Walther, 1992). In particular, the relationship between MATH-1 and
Pax-3 may be interesting, because Pax-3 is produced in the dorsal half
of the neural tube (Goulding et al., 1991) and therefore
co-expressed with MATH-1. Mutation of Pax-3 ( splotch) affects
development of the neural tube, resulting in spina bifida and
exencephaly (Epstein et al., 1991). Pax-3 expression occurs
just prior to closure of the neural tube, before MATH-1 is expressed.
Thus, Pax-3 may regulate neurogenesis by activating MATH-1 expression,
or Pax-3 and MATH-1 may cooperatively activate neural-specific genes.
Further studies of the functions and relationship of these factors will
lead to an understanding of the complex mechanisms of mammalian
neurogenesis.
The nucleotide sequence reported in this paper has been
submitted to the GSDB, DDBJ, EMBL, and NCBI Data Banks with accession
number D43694.
We thank Dr. L. J. Richards for critical reading of
the manuscript and useful discussions, Dr. N. Mizuno for useful
discussions, and Dr. K. Tomita for technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
mitotic germinal cells present in the ventricular zone next
to the lumen determine their fate, migrate toward the outer zones, thus
forming the mantle layer, and undergo terminal differentiation
(Jacobson, 1991). During this process, these differentiating cells may
receive signals from the floor plate and/or the roof plate, which are
the ventral and dorsal end point of the neural tube, respectively, and
these cells gain ventral or dorsal phenotypes (Basler et al.,
1993; Yamada et al., 1993). Thus, the ventral (basal plate)
and dorsal parts (alar plate) of the mantle layer, the boundary of
which is delineated by the sulcus limitans near the midpoint of the
lateral wall of the neural tube, contain distinct populations of neural
precursor cells. The underlying molecular mechanisms of these complex
developmental processes are not yet well understood.
Cloning of the MATH-1 Gene
After reverse
transcription with oligo(dT) priming of poly(A)RNA
from the CNS of embryonic day 16 (E16) mouse embryos, the cDNA was
subjected to 30-40 cycles of polymerase chain reaction (PCR)
under standard conditions (Lee et al., 1988; Saiki et
al., 1988), except that the annealing temperature was lowered to
45 °C during the first five cycles. The fully degenerate primers
corresponding to the amino acid sequences NARER and TLQMA were
synthesized for 5`- and 3`-primers, respectively. The third codon
positions of 4-fold degeneracy were substituted by inosine.
BamHI and EcoRI sites were introduced at the 5` end
of the 5`- and 3`-primers, respectively. Amplified fragments were
subcloned into pBluescript and sequenced. Out of 24 clones sequenced, 3
encoded the MATH-1 sequence. The SacI- KpnI fragment
containing the MATH-1 cDNA isolated from the subclones was used as a
probe for screening the mouse genomic library (Stratagene). One
positive clone was obtained, and the
2-kilobase pair
HindIII- XhoI fragment of the positive clone was
sequenced.
In Situ Hybridization and Northern Blot
Analyses
In situ hybridization experiments were
performed, essentially as described previously (Akazawa et
al., 1992, 1994). S- or digoxigenin-labeled antisense
RNA (
1.5 kilobases) corresponding to the
SphI- SacI fragment, which contained most of the
MATH-1 coding region, was synthesized in vitro. The probes
were hybridized to whole embryos or 10-µm cryostat sections.
RNA and 20
µg of total RNA were electrophoresed on a formamide/1.2% agarose
gel and transferred to a nylon membrane. The filter was hybridized with
the
P-labeled SphI- SacI fragment at 42
°C in 50% formamide, 5
SSC, 5
Denhardt's
solution, 50 m
M sodium phosphate buffer, pH 6.8, 0.1% SDS, and
100 µg/ml heat-denatured salmon sperm DNA and washed at 65 °C
in 0.1
SSC and 0.1% SDS.
DNA Binding Analysis
The proteins were expressed
in Escherichia coli and prepared as described previously
(Kageyama et al., 1991). The cDNA fragments of MATH-1 (amino
acid residues 153-218) and E47 (residue 473 to the end) were
subcloned into pGEMEX-1 and pMNT T7 expression plasmids, respectively.
JM109 (DE3) cells transformed by the expression plasmids were grown,
and the protein expression was induced by treatment of 1 m
M isopropyl-1-thio--
D-galactopyranoside. The cells
were suspended in a solution of 30 m
M Tris-HCl, pH 7.5, 1
m
M EDTA, and 20% sucrose and were applied to an
SDS-polyacrylamide gel. The expressed proteins were eluted from the
gel, treated in 6
M guanidine HCl for 20 min, and dialyzed
against 0.1
M KCl/HM (20 m
M HEPES, pH 7.9, 1 m
M MgCl
, 2 m
M dithiothreitol, and 17% (v/v)
glycerol).
-
P]dCTP. The E2 and E3 probes contained
the same sequence as the E1 probe except for the 6-nucleotide E box
element (underlined); the E2 and E3 probes contained CAAATG and CAGCTG,
respectively, instead of the CAGGTG sequence.
Transcriptional Analysis
For eukaryotic expression
plasmids, the HindIII- XhoI fragment of the MATH-1
gene and full-length E47 and HES-1 cDNAs were cloned into the
eukaryotic expression vector (pSV-CMV) containing the cytomegalovirus
promoter and enhancer, as described previously (Sasai et al.,
1992). The luciferase reporter plasmid pE7-A-luc was made by
inserting seven repeats of the E box (CAGGTG) into the XhoI
site (-273 relative to the transcription initiation site) of the
p
actin-luc.
Isolation and Structural Analysis of a Novel HLH
Factor, MATH-1
To isolate a novel HLH factor structurally
related to the Drosophila proneural gene atonal, we
amplified cDNAs from the CNS of mouse embryos by PCR using degenerate
oligonucleotide primers (see ``Experimental Procedures''). We
obtained two closely related but distinct clones and further
characterized one of them (designated MATH-1), the deduced amino acid
sequence of which showed the higher homology to the atonal product.
2-kilobase pair
HindIII- XhoI fragment of the mouse MATH-1 genomic
clone was sequenced (Fig. 1 A), and we found one open reading
frame that contained the MATH-1 sequence. This open reading frame
started from the methionine codon at nucleotide residue 195 (nucleotide
residue 1 is the 5`-end of the HindIII site) and ended at the
stop codon at 1248 (Fig. 1 B). Preceding the methionine
codon at 195, there was an in-frame stop codon at 39
(Fig. 1 B). Reverse transcription-mediated PCR analysis
using the primers upstream of the stop codon at 39 and downstream of
the stop codon at 1248 showed that this region was transcribed without
any introns (data not shown), suggesting that MATH-1 consists of 351
amino acid residues with the calculated molecular size of 37.9 kDa
(Fig. 1 B). The feature of an intronless coding region is
the same as that of the Mash1 genome, a gene related to the MATH-1 gene
(Guillemot and Joyner, 1993).
Figure 1:
Schematic structure and nucleotide
sequence of the mouse MATH-1 gene. A, schematic structure of
the mouse MATH-1 gene. The open box represents an intronless
coding region, and the closed box shows the bHLH domain.
Abbreviations are as follows: B, BglII; H,
HindIII; S, SphI; X, XhoI.
B, nucleotide and its deduced amino acid sequence of the mouse
MATH-1 gene. The nucleotide sequence of the coding strand together with
the deduced amino acid sequence of MATH-1 are indicated. The putative
bHLH domain is shown by a bar above the amino acid sequence.
The in-frame stop codon in the 5`-flanking region is
underlined.
MATH-1 showed 70% identity to the
atonal protein in the basic HLH (bHLH) domain (Fig. 2) but had no
significant homology outside of this domain. MATH-1 also exhibited
similarity to the bHLH domains of other factors such as Mash1 (49%
identity) (Johnson et al., 1990), Mash2 (51% identity)
(Johnson et al., 1990), and Xash3 (48% identity) (Zimmerman
et al., 1993) (Fig. 2). Whereas the bHLH domain of
atonal is located at its extreme carboxyl terminus (Jarman et
al., 1993), that of MATH-1 is present in the middle of the
protein, like Mash proteins. Another feature is that MATH-1 is rich in
proline (36 out of 351 amino acid residues). These proline residues may
be involved in protein-protein interaction (Mermod et al.,
1989). MATH-1 is also rich in serines, which could be phosphorylated,
in the carboxyl terminus (33% in the region between 325 and 351). For
example, Ser-328 and Ser-331 are potential protein kinase C
phosphorylation sites. Outside of this region, Ser-193 could be
phosphorylated by protein kinase A.
Figure 2:
Sequence comparison of MATH-1 and other
bHLH factors. The positions of the basic region, the putative
amphipathic helices 1 and 2, and the loop are shown above. The
conserved amino acid residues among the bHLH factors are
boxed. The conserved residues between MATH-1 and the
atonal product are designated by an asterisk (*). Sources for
sequences are as follows: Mash1 and Mash2 (Johnson et al.,
1990); Xash3 (Zimmerman et al., 1993); HES-1 (Sasai et
al., 1992); MyoD (Davis et al., 1987); and N-myc (Kohl
et al., 1986).
Spatial and Temporal Distribution of MATH-1
mRNA
To determine the temporal and spatial distribution of
MATH-1 mRNA, we performed in situ hybridization analysis. We
did not detect any MATH-1 transcript in mouse embryos at E8.5 before
the process of turning (data not shown). MATH-1 mRNA was first
expressed at E9.5 in the cranial ganglions, such as the trigeminal
ganglion, as well as in the dorsal wall of the neural tube (Fig. 3,
a and b). The expression in the cranial ganglions was
transient, becoming much weaker by E10.5 (Fig. 3 c). In
contrast, the transcript was much more prominent in the CNS of E10.5
(Fig. 3, c and d) and E11.5 embryos
(Fig. 3 e), where high levels of expression were observed
throughout the dorsal part of the neural tube. On E12.5, MATH-1
expression was prominent in the alar plate of the hindbrain, which is
located dorsolateral to the basal plate at this level (Fig. 4, a and b). High expression of the MATH-1 transcript was also
present in the ventrally and dorsally migrating cells of the alar plate
of the hindbrain (Fig. 4 b, arrows). The
ventrally migrating cells shown are the bulbopontine extension (Fig.
4 b, thin arrows), which later forms the pontine
nuclei rostrally and the olivary nuclear complex caudally. The dorsally
migrating cells shown form the superficial cortex of the cerebellum
(the external granular layer) (Fig. 4 b, thick
arrows), which gives rise to the granule cells. In the developing
spinal cord, MATH-1 expression remained at high levels on E12.5 and was
restricted to the dorsal ventricular zone and alar plates with highest
levels in the most dorsal region (Fig. 4, a and
c). In contrast, MATH-1 expression became weaker in the dorsal
part of the midbrain and was almost undetectable in the forebrain on
E12.5 (Fig. 4 a).
Figure 3:
Whole mount in situ hybridization
analysis of E9.5, E10.5, and E11.5 embryos. Digoxigenin-labeled
antisense MATH-1 RNA probe was hybridized to embryos. Abbreviations are
as follows: Mes, mesencephalon; Met, metencephalon;
Myel, myelencephalon; TG, trigeminal ganglion.
a, lateral view of E9.5 embryo showing MATH-1 expression in
the trigeminal ganglion. b, dorsal view of E9.5 embryo. MATH-1
is expressed in the dorsal region of the CNS. c, lateral view
of E10.5 embryo. Expression becomes weaker in the trigeminal ganglion
but more prominent in the CNS. d, dorsal view of E10.5 embryo
showing high levels of expression in the CNS. e, lateral and
dorsal views of E11.5 embryos. Expression persists throughout the
dorsal region of the CNS.
Figure 4:In situ hybridization analysis of
E12.5 embryos. S-Labeled antisense MATH-1 RNA probe was
hybridized to the frozen sections of E12.5 embryos. a,
sagittal section. Prominent MATH-1 expression is observed in the dorsal
part of the hindbrain and spinal cord. b, coronal section.
Thick and thin arrows indicate dorsally and ventrally
migrating cells of the alar plate, respectively. Large and
small triangular arrowheads show the roof and alar plates of
the spinal cord and the alar plate of the metencephalon, respectively.
c, higher magnification of the spinal cord. Dorsal is toward
the bottom. CC, central canal. Planes of sections a and b are schematically exhibited on the upper right.
On E13, MATH-1 expression was
observed in the dorsal part of the hindbrain and spinal cord with the
highest levels in the metencephalon (Fig. 5 a). However, by
E18, MATH-1 expression had disappeared in the spinal cord and became
restricted to the cerebellum (Fig. 5 b). On postnatal day
3 (P3), MATH-1 transcripts were observed in the external granular layer
of the cerebellum (Fig. 6 A). From the external granular layer,
granule cells migrate through the molecular layer and Purkinje cell
layer to form the internal granular layer of the cerebellum. MATH-1
expression was only found in the external granular layer and not in the
migrating granule cells or the mature granule cells of the internal
granular layer (Fig. 6 B). As the external granular layer
gradually disappeared during the postnatal periods, MATH-1 expression
was also decreased (Fig. 6 A). On P28, MATH-1 mRNA was no
longer detected in the nervous system (Fig. 6 A, d).
Furthermore, the adult brain, including the cerebellum, produced no
detectable levels of the MATH-1 transcript (Fig. 7 A). The
finding that MATH-1 is expressed in subsets of differentiating neural
cells but subsequently disappears suggests that MATH-1 may be involved
in the developmental process of neurogenesis but may not be required
for the maintenance of the terminally differentiated neurons.
Figure 5:In situ hybridization analysis of
E13 and E18 embryos. a, sagittal section of E13 embryo.
Expression persists in the dorsal part of the metencephalon,
myelencephalon, and spinal cord but disappears in the forebrain and
midbrain. Met, metencephalon; SC, spinal cord.
a`, Nissl staining of the section shown in a.
b, sagittal section of E18 embryo showing restricted
expression in the cerebellum. MATH-1 expression is now observed in the
gastrointestinal tract. Cb, cerebellum; GI,
gastrointestinal tract. b`, Nissl staining of the section
shown in b.
Figure 6:In situ hybridization analysis of
postnatal brains. A, in situ hybridization analysis
of P3 ( a), P11 ( b), P14 ( c), and P28
( d) brains. MATH-1 expression in the CNS is restricted to the
external granular layer of the cerebellum. Expression gradually
decreases postnatally and disappears by P28. a`-d`, Nissl
staining of postnatal brains. Cb, cerebellum; Hc,
hippocampus; OB, olfactory bulb. B, higher
magnification of P3 ( a) and P14 ( b) cerebelli. MATH-1
is expressed only in the external granular layer and not in the
migrating granule cells or the mature granule cells in the internal
granular layer. a` and b`, Nissl staining of P3 and
P14 cerebelli.
MATH-1
expression seemed specific to the developing nervous system in early
embryos, but MATH-1 transcript was detected in the gastrointestinal
tract on E18 (Fig. 5 b). In the adults, MATH-1 expression
was evident in the gastrointestinal tract (Fig. 7 A). In
situ hybridization analysis suggested that this MATH-1 expression
was confined to the epithelial cells but not to the neural cells of the
gastrointestinal tract (Fig. 7 B). Thus, MATH-1 appears to be
expressed only in non-neural tissues in the adults, suggesting that
MATH-1 may have different functions in early embryos and adults.
DNA Binding Analysis of MATH-1
To characterize the
function of MATH-1, we next examined its DNA binding activity. The
MATH-1 protein was expressed in E. coli and subjected to gel
mobility shift analysis. It was previously shown that the
Drosophila atonal product binds to several E box sequences as
a heterodimer with the daughterless protein (Jarman et
al., 1993). We thus tested several E box sequences for MATH-1
binding with or without E47, a mammalian homologue of the
Drosophila daughterless product. As shown in Fig. 8, whereas
E47 bound to these E box elements with different affinities
(Fig. 8, lanes 3, 7, and 11), MATH-1
did not bind to any of the E box sequences tested (Fig. 8,
lanes 2, 6, and 10 and data not
shown). However, MATH-1 significantly activated the DNA binding of E47
(Fig. 8, lanes 4, 8, and 12). It is
possible that this enhanced binding was the result of the formation of
a heterodimer of MATH-1 and E47, in a manner similar to that of the
atonal and daughterless proteins. Among the E box
sequences tested, the mixture of MATH-1 and E47 bound to the CAGGTG
sequence with the highest affinity (Fig. 8, lane 4), like the atonal and daughterless heterodimer (Jarman et al., 1993). Thus, these results
suggest that MATH-1 interacts with the E box elements as a heterodimer
with E47.
Figure 8:
DNA binding analysis of MATH-1. The DNA
binding activity of MATH-1 was examined by gel mobility shift assay
with or without E47, as indicated above each lane. The
double-stranded oligonucleotide probes contained an E box sequence:
lanes 1-4, E1 (CAGGTG); lanes 5-8, E2
(CAAATG); and lanes 9-12, E3 (CAGCTG). Whereas E47 bound
to these E box sequences with various affinities, MATH-1 alone did not.
The mixture of MATH-1 and E47 efficiently interacted with these E box
elements (relative affinity, E1 > E3 >
E2).
Transcriptional Analysis of MATH-1
To characterize
the transcriptional activity of MATH-1, we performed DNA-mediated gene
transfer experiments using C3H10T1/2 cells. Previously, these cells
have been used to show that the neuronal determination factor Mash1 and
the muscle determination factor MyoD activate transcription from the E
box-containing promoters (Benezra et al., 1990; Johnson et
al., 1992; Sasai et al., 1992). MATH-1 expression was
directed under the control of the cytomegalovirus enhancer and
promoter, and reporter plasmids were comprised of the luciferase gene
under the control of the -actin promoter with or without seven
repeats of the E box element (CAGGTG).
Figure 9:
Transcriptional analysis of MATH-1. 0.5
µg of the plasmid containing the luciferase ( Luc) reporter
gene under the control of the -actin promoter with ( lanes
1-6) or without ( lanes 7-12) seven repeats of
the E box element (CAGGTG) was transfected into C3H10T1/2 cells. 0.5
µg each of the MATH-1, E47, or HES-1 expression vector was also
cotransfected, as indicated on the left. Total amounts of DNA
were adjusted to 2 µg with the control vector pSV-CMV. Cells were
harvested 48 h later, and luciferase activities were measured. Each
value of relative luciferase activities is the average of at least
three independent experiments that were done in
duplicate.
Recently,
we have shown that persistent expression of HES-1 prevents neural
differentiation in the CNS (Ishibashi et al., 1994). To
determine whether the activity of MATH-1 is regulated by HES-1, we
co-expressed HES-1 with MATH-1 and E47. Cotransfection of the HES-1
vector resulted in complete inhibition of the MATH-1/E47-induced
transcriptional activation (Fig. 9, lane 5),
suggesting that the E box-dependent transcriptional activation of
MATH-1 and E47 is negatively regulated by HES-1.
MATH-1 Is a Transcriptional Activator That May Be
Involved in Mammalian Neurogenesis
In this study, we have
described the molecular characterization of MATH-1, a novel mammalian
factor that has a significant sequence homology in the bHLH domain to
the product of the Drosophila proneural gene atonal.
MATH-1 does not bind to the DNA template by itself, but, in
collaboration with E47, it can interact with some of the E box
elements. The sequence recognized by the mixture of MATH-1 and E47 with
the highest affinities is the same as the one recognized by
Atonal-Daughterless complex. Thus, MATH-1 is also similar in function
to atonal. Furthermore, MATH-1 is specifically expressed in
the developing nervous system, like atonal. These results
raise the possibility that MATH-1 may have a similar function in neural
differentiation to that of atonal.
(
)
Thus, MATH-1 may not be
involved in the steps where the neural precursor cells determine their
fate and undergo their initial differentiation. Rather, because MATH-1
expression seems specific to subsets of differentiating neurons, it is
possible that MATH-1 may be required for the process where the neural
cells undergo the neuronal type-specific maturation. Further studies,
such as the identification of target genes for MATH-1, will be
necessary to understand its exact functions during neurogenesis.
in the floor
plate, which may then activate ventral-specific genes and repress the
dorsal-specific genes (Echelard et al., 1993; Krauss et
al., 1993; Riddle et al., 1993; Sasaki and Hogan, 1994).
It is possible that diffusible factors from the roof plate may induce
the expression of MATH-1, which may then activate dorsal-specific gene
expression and therefore regulate the development of dorsal neurons.
Multiple Positive and Negative Factors May Be Involved in
Mammalian Neurogenesis
Accumulating evidence suggests that, like
myogenesis, mammalian neurogenesis is controlled by multiple HLH
factors (Johnson et al., 1990; Sasai et al., 1992;
Akazawa et al., 1992; Guillemot et al., 1993;
Ishibashi et al., 1993; Feder et al., 1993; Ishibashi
et al., 1994; Sakagami et al., 1994; Takebayashi
et al., 1995). Mash1 is expressed in the developing nervous
system and is essential for peripheral nervous system development
(Guillemot et al., 1993). Although Mash1 is present at high
levels in the developing CNS, null mutation of this factor does not
cause any apparent abnormalities in the CNS (Guillemot et al.,
1993). Thus, it is possible that other factors may compensate, as in
the case of the muscle determination factors MyoD and Myf-5; whereas
null mutation of MyoD or Myf-5 gives rise to apparently normal skeletal
muscles, lack of both factors shows a complete absence of skeletal
muscles (Rudnicki et al., 1992; Braun et al., 1992;
Rudnicki et al., 1993). Because MATH-1 and Mash1 seem
co-expressed, at least in some regions of the developing nervous
system, and have similar transcriptional activity (both activate
transcription from E box-containing promoters), MATH-1 may substitute
for Mash1 in the development of some regions of the nervous system.
Further studies, such as an analysis of the null mutation of MATH-1,
will clarify this problem.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.