©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mammalian Helix-Loop-Helix Factor Structurally Related to the Product of Drosophila Proneural Gene atonal Is a Positive Transcriptional Regulator Expressed in the Developing Nervous System(*)

Chihiro Akazawa (§) , Makoto Ishibashi (1), Chikara Shimizu , Shigetada Nakanishi , Ryoichiro Kageyama (¶)

From the (1) Institute for Immunology Department of Anatomy, Kyoto University Faculty of Medicine, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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),() 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.

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.


EXPERIMENTAL PROCEDURES

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.

For Northern blot analysis, 5 µg of poly(A)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).

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 [-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.

Reactions of the gel mobility shift assay were carried out as described previously (Sasai et al., 1992).

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 pactin-luc.

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).


RESULTS

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.

Using the PCR fragment as a probe, we screened a mouse genomic library. The 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).

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.


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.


DISCUSSION

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.

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.() 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.

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 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.

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.

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.

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.


FOOTNOTES

*
This work was supported by research grants from the Ministry of Education, Science, and Culture of Japan, the Sankyo Foundation, the Yamanouchi Foundation, and the Inamori Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence reported in this paper has been submitted to the GSDB, DDBJ, EMBL, and NCBI Data Banks with accession number D43694.

§
Present address: The Salk Institute for Biological Studies, La Jolla, CA 92037.

To whom correspondence should be addressed: Inst. for Immunology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel.: 81-75-753-4438; Fax: 81-75-753-4404.

The abbreviations used are: CNS, central nervous system; HLH, helix-loop-helix; bHLH, basic HLH; E, embryonic day; P, postnatal day; PCR, polymerase chain reaction.

C. Akazawa, M. Ishibashi, C. Shimizu, S. Nakanishi, and R. Kageyama, unpublished data.


ACKNOWLEDGEMENTS

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.


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