2323
Development 127, 2323-2332 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 DEV2535
Hes genes regulate sequential stages of neurogenesis in the olfactory epithelium Elise Cau1,*, Gérard Gradwohl1,*, Simona Casarosa1, Ryoichiro Kageyama2 and François Guillemot1,‡ 1IGBMC, CNRS/INSERM/Université Louis Pasteur, BP 163, 67404 Illkirch Cédex, CU de Strasbourg, France 2Laboratory of Growth Regulation, Institute for Virus Research, Kyoto University, Shogoin-Kawaracho,Sakyo-ku,
Kyoto 606, Japan
*These authors contributed equally to the work ‡Author for correspondence (e-mail:
[email protected])
Accepted 8 March; published on WWW 10 May 2000
SUMMARY We have characterised the functions of the bHLH transcriptional repressors HES1 and HES5 in neurogenesis, using the development of the olfactory placodes in mouse embryos as a model. Hes1 and Hes5 are expressed with distinct patterns in the olfactory placodes and are subject to different regulatory mechanisms. Hes1 is expressed in a broad placodal domain, which is maintained in absence of the neural determination gene Mash1. In contrast, expression of Hes5 is restricted to clusters of neural progenitor cells and requires Mash1 function. Mutations in Hes1 and Hes5 also have distinct consequences on olfactory placode neurogenesis. Loss of Hes1 function leads both to expression of Mash1 outside of the normal domain of neurogenesis and to increased density of MASH1-positive progenitors within this domain, and results in an excess of neurons after a delay. A mutation in Hes5 does not produce any apparent defect. However, olfactory placodes that are double mutant for Hes1 and
Hes5 upregulate Ngn1, a neural bHLH gene activated downstream of Mash1, and show a strong and rapid increase in neuronal density. Together, our results suggest that Hes1 regulates Mash1 transcription in the olfactory placode in two different contexts, initially as a prepattern gene defining the placodal domain undergoing neurogenesis and, subsequently, as a neurogenic gene controlling the density of neural progenitors in this domain. Hes5 synergises with Hes1 and regulates neurogenesis at the level of Ngn1 expression. Therefore, the olfactory sensory neuron lineage is regulated at several steps by negative signals acting through different Hes genes and targeting the expression of different proneural gene homologs.
INTRODUCTION
damage (Graziadei and Graziadei, 1979; Crews and Hunter, 1994). These properties have facilitated the identification and characterisation of OSN progenitors, which are present in the embryonic as well as in the adult OE. Recent evidence suggests the existence of at least two types of progenitors, representing sequential steps in the OSN lineage. A rare population of slowdividing cells with the self-renewing capacity of stem cells has been shown to generate a much more abundant population of rapidly dividing precursors committed to neuronal differentiation (Calof and Chikaraishi, 1989; Calof et al., 1996; Mumm et al., 1996). Several genes of the basic helix-loop-helix (bHLH) class have been implicated in neurogenesis in the olfactory placode (Cau et al., 1997). Mash1 is the first of these genes to be expressed in OSN progenitors and analysis of the OE phenotype of mice mutant for Mash1 shows that it plays a central role during early stages of the OSN lineage (Guillemot et al., 1993; Cau et al., 1997). Loss of Mash1 function results in a lack of expression of the downstream bHLH genes Neurogenin1 (Ngn1) and NeuroD, and in a failure to generate OSN precursors in the OE (Cau et al., 1997; our unpublished
The generation of the numerous types of neurons that constitute the adult nervous system from an initially homogenous population of neurectodermal progenitor cells is an extremely complex process, which has been thoroughly investigated, first in Drosophila (Campos-Ortega, 1993; Jan and Jan, 1994) and, more recently, in vertebrates (Tanabe and Jessell, 1996; Chan and Jan, 1999). In vertebrates, the early steps of specification and differentiation of neuronal precursors have been studied in several models, including the primary neurons of lower vertebrates (Chitnis and Kintner, 1995), the spinal cord (Tanabe and Jessell, 1996), the neural crest (Anderson et al., 1997) and the retina (Cepko, 1999). The olfactory sensory epithelium (OE) has also been a useful model to identify transcription factors and extracellular signals regulating neurogenesis in the mouse (Calof et al., 1996). The value of the OE model stems from the simple nature of this tissue, where primary olfactory sensory neurons (OSNs) are the main neuronal type, and from its capacity to renew its neuronal population during adulthood and to regenerate it after
Key words: Mouse, Olfactory placode, Mash1, Prepattern, Notch signalling, Lateral inhibition
2324 E. Cau and others data). Mash1 has therefore a neural determination function in the OE similar to that of its Drosophila counterparts of the achaete-scute complex (AS-C) (Ghysen and DamblyChaudiere, 1988; Jan and Jan, 1994). A preliminary analysis of the OE phenotype of Ngn1 mutant mice indicates that Ngn1 is required at a later step, in the differentiation of OSNs (E. C., Q. Ma, D. Anderson and F. G., unpublished data). To get further insights into the regulation of OSN development, we have examined the function of genes that are potential regulators of Mash1 and Ngn1 in this tissue. In Drosophila imaginal discs, expression of the AS-C proneural genes is controlled by upstream regulators, or prepattern genes (Ghysen and Dambly-Chaudiere, 1989; Skeath and Carroll, 1994; Simpson, 1996), whose products bind to site-specific enhancers found around the coding sequence of AS-C genes (Gomez-Skarmeta et al., 1995; Modolell, 1997). One of these upstream regulators, hairy, is a direct repressor of achaete expression in the wing and leg imaginal discs (Ohsako et al., 1994; Van Doren et al., 1994). In absence of hairy, the achaete expression domain expands and ectopic neural precursors are generated (Skeath and Carroll, 1991; Orenic et al., 1993). Proneural genes, which are initially expressed in groups of cells, called proneural clusters, become later restricted to single cells which thereby acquire a neural fate. This change in proneural gene expression involves a process of lateral inhibition mediated by the Notch receptor (Simpson, 1997). Interaction of Notch with its ligand Delta activates a signalling pathway resulting in transcriptional activation of the genes of the Enhancer of split (Espl) complex (Jennings et al., 1994; Artavanis-Tsakonas et al., 1995; Bray, 1997; Simpson, 1997). Activity of the Espl genes, which encode bHLH transcriptional repressors structurally related to hairy, inhibits neural development by antagonizing proneural gene function (Ohsako et al., 1994; Van Doren et al., 1994; Nakao and Campos-Ortega, 1996; Jennings et al., 1999). The components of the Notch signalling pathway are conserved in vertebrate species (Lewis, 1996; Gridley, 1997). Vertebrate Notch, Delta and Serrate/Jagged (which, like Delta, encode Notch ligands), have been implicated in processes of lateral inhibition controlling cell fate specification in numerous tissues, including the neural plate, inner ear, retina, skin and pancreas (Chitnis and Kintner, 1995; Chitnis et al., 1995; Henrique et al., 1997; Crowe et al., 1998; Lanford et al., 1999). Four genes homologous to the Drosophila Hairy and Espl genes, named Hes1-Hes3 and Hes5, have been identified in mouse (Akazawa et al., 1992; Sasai et al., 1992). Genetic analysis have shown that Hes1 and Hes5 in particular have important roles in neurogenesis (Ishibashi et al., 1994, 1995; Tomita et al., 1996; Ohtsuka et al., 1999; reviewed in Kageyama and Nakanishi, 1997). However it has remained unclear whether these genes were regulating neural development through a process of lateral inhibition, or in a different context. To better define the functions of Hes1 and Hes5 in neurogenesis, we have examined in details the regulation of expression and function of these two genes, using the development of the olfactory placode as a model. We have analysed the OE phenotype of Hes1 null mutant mice, and we have generated mice null mutant for the gene Hes5 to examine its function in neurogenesis and its possible synergy with Hes1. Our results show that Hes1 activity in OE neurogenesis can be divided in two distinct phases. Hes1 functions initially to
delimit a domain of proneural gene expression at the onset of neurogenesis in the placode. Hes1 subsequently functions in a process of lateral inhibition which regulates the recruitment of neural precursors in the OE. Therefore, Hes1 appears to cumulate the functions of a prepattern gene and of a neurogenic gene. Mash1 is a target of Hes1 for these two activities. In contrast, Hes5 appears to function exclusively as a neurogenic gene and it regulates the expression of the differentiation gene Ngn1. Thus, development of the OSN lineage is negatively regulated by Hes genes at several sequential stages. MATERIALS AND METHODS Generation of Hes 5 mutant animals To generate a Hes5 targeting vector, a PGK-neor cassette was flanked by 5′ and 3′ arms consisting of a 3 kb XhoI-SmaI fragment and a 3 kb NotI-SmaI fragment from the mouse (129/Sv) Hes5 locus (unpublished data), respectively. The mutation introduced by this vector is a deletion of 724 bp, including the coding region from Met1 to Ala76 and comprising the bHLH domain. The vector was electroporated in the ES cell lines H1 (from IGBMC, Strasbourg, unpublished) and R1 (Nagy et al., 1993), and selection was performed as described (Fode et al., 1998). Homologous recombination was detected by Southern blotting of ES cell genomic DNA, using a 0.9 kb SmaI-BamHI fragment and a 0.2 kb DraIII-XhoI fragment as 3′ and 5′ external probes, respectively. Ten independent targeted ES clones were obtained out of 252 clones analysed. Three of them were used to generate chimeras by blastocyst injection. Germline transmission was obtained by crossing the chimeras with C57bl6 females and the colony was expended on a CD1 genetic background. Generation of compound mutant animals for Hes1, Hes5 and Mash1 For staging of embryos, mid-day of the vaginal plug was considered as E0.5. The Mash1 and Hes1mutant mouse lines have been described in Guillemot et al. (1993) and Ishibashi et al. (1995), respectively. Compound Hes1;Hes5 embryos were obtained by intercrossing Hes1+/−;Hes5+/− or Hes1+/−;Hes5−/− mice. At E10.5, the 8 expected genotypes resulting from these crosses were obtained with Mendelian ratios (see Results section). Whole-mount in situ hybridization The Mash1, Ngn1 and SCG10 probes have been described in Cau et al. (1997). The Hes1 probe was transcribed from a 0.9 kb cDNA clone described in Ishibashi et al. (1995). The Hes5 probe was transcribed from a 1.3 kb cDNA clone described in Akazawa et al. (1992). The protocol for whole-mount RNA in situ hybridization has been described in Cau et al. (1997). The protocol for two-colour wholemount RNA in situ hybridization is available upon request. Immunohistochemistry with anti-MASH1 antibody Detection of the MASH1 protein was performed on 14 µm cryosections of E10.5 embryos frozen in OCT, using a monoclonal anti-MASH1 antibody (Lo et al., 1991). After postfixation in 4% paraformaldehyde at room temperature for 10 minutes, and 3 washes of 5 minutes in PBS with 0.05% Tween-20, immunohistochemistry was performed as described in Porteus et al. (1994).
RESULTS Expression of Hes genes in the embryonic olfactory epithelium In an effort to understand how neurogenesis is regulated in the embryonic OE, we examined the expression in this tissue of
Hes genes and olfactory epithelium neurogenesis 2325 genes belonging to the Hes (Hairy and Enhancer of split) in the Mash1 mutant olfactory placode (Fig. 1E), except in a family of transcriptional repressors. We performed this study small domain of the placode where neurogenesis is not affected at E12.5, a stage when the OE has begun to acquire an by loss of Mash1 (Cau et al., 1997). organisation in cell layers characteristic of the mature tissue. At this stage, the OE contains two populations of dividing progenitors, located respectively on the apical and basal sides of the epithelium. Cells dividing apically are already present in the olfactory placode, whereas basal progenitors appear in the OE only at around E12.5. As development of the OE proceeds, the proportion of basal progenitors then increases progressively, and that of apical progenitors decreases (Smart, 1971; Cuschieri and Bannister, 1975). Two genes of the Hes family, Hes1 (Sasai et al., 1992) and Hes5 (Akazawa et al., 1992), are expressed in the embryonic OE with distinct patterns. At E12.5, Hes1 transcripts are present in most cells on the apical side of the OE and are absent from the basal side (Fig. 1G). Hes5 in contrast is expressed in scattered cells, which are present only on the basal side of the OE (Fig. 1I). The two genes are already expressed with different patterns when neurogenesis begins in the olfactory placode. At E10.5, Hes1 expression is widespread throughout the placode, but is highest at the periphery and lower in a large central area (Fig. 1A). The area of high Hes1 expression abuts the more centrally located placodal territory, which contains neuronal precursors marked by expression of the neural determination gene Mash1 (Fig. 1C; Cau et al., 1997). In contrast to the broad expression of Hes1, Hes5 expression is restricted to cell clusters (Fig. 1D), which are either intermingled with Mash1-expressing cells or have a more central position (Fig. 1F). At this early stage, there is no distinct cell layering in the olfactory placode, and Hes1-positive cells and Hes5-positive cells are found in both apical and basal positions (data not shown). At E15.5, Hes5 OE expression is not detectable and that of Hes1 is low and restricted to a few apical cells (data not shown). Hes1 and Hes5 have been shown to be important effectors of the Notch signalling pathway in the embryonic central nervous system (Kageyama and Nakanishi, 1997; Ohtsuka et al., 1999). If Hes genes are also regulated by Notch signalling during OE neurogenesis, their expression patterns should be Fig. 1. Hes1 and Hes5 expression in the olfactory epithelium requires Mash1 affected by a mutation in Mash1, which results in an function, whereas Hes1 expression at the placodal stage is independent of Mash1. early block in OE neurogenesis (Cau et al., 1997). (A,B,D,E) Flat mounts of olfactory placodes from wild type (A,D) and Mash1 Indeed, expression of Hes1 (Fig. 1H) and Hes5 (Fig. mutant (B,E) E10.5 embryos hybridized with cRNA probes for Hes1 (A-C) and 1J) is absent in most of the OE of Mash1 mutant Hes5 (D-F). Medial is to the left, lateral to the right; dorsal is up and ventral is embryos at E12.5. Therefore, activation of these down. In the placode, Hes1 is expressed broadly (A) and this pattern is not genes is part of a program of neurogenesis in the affected in a Mash1 mutant placode (B), while Hes5 expression is restricted to cell clusters (D) and requires Mash1 function (E). (C,F) Flat mounts of wild-type OE. Examination of Hes1 expression in Mash1 mutant E10.5 placodes double labelled for (C) Mash1 (brown) and Hes1 (blue) and (F) embryos at E10.5 revealed that, in contrast with its Mash1 (blue) and Hes5 (brown). The periphery of the placode is to the left, the center to the right. The domain of high Hes1 expression is peripheral to the later expression (Fig. 1H), the early expression of Mash1-positive cell clusters (C) and Hes5-positive cell clusters are either Hes1 in the olfactory placode is largely unaffected by intermingled with, or more centrally located than, Mash1-positive cells (F). the loss of Mash1 (Fig. 1B). This result suggests that (G-J) Sections of wild type (G,I) and Mash1 mutant (H,J) OE at E12.5 hybridized different mechanisms regulate Hes1 expression at the with Hes1 (G,H) and Hes5 (I,J) cRNA probes. At this stage, both expression of onset of neurogenesis and at later stages in the OE. Hes1 in apical cells (G) and of Hes5 in basal cells (I) require Mash1 function As expected, expression of Hes5 is severely reduced (H,J). Hes1 is also expressed in the mesenchyme surrounding the OE (G,H).
2326 E. Cau and others Table 1. Clustering of MASH1-positive cells in Hes1 mutant olfactory placodes __________________________________________________ MASH1-positive cells Cells isolated or in pairs ‘small clusters’ (3-4 cells) ‘large clusters’ (5-8 cells)
WT (n=3)
Hes1−/− (n=3)
92.5±6.6 7.5±6.6 0.0±0.0
68.2±6.6 27.1±3.7 4.79±3.8
To quantify the clustering of MASH1-positive cells in Hes1 mutant and wild-type placodes, the percentage of MASH1-positive cells falling in the following three categories: cells isolated or in pairs, ‘small clusters’ (3-4 cells) and ‘large clusters’ (5-10 cells), are shown. In wild-type olfactory placodes, most MASH1-positive cells are either isolated or in pairs, and a small fraction is grouped in small clusters of less than 5 cells. In Hes1 mutant placodes, there is a larger percentage of cells in clusters, and the size of clusters reaches up to up to 8 cells. The percentages of cells are average±s.d, and the numbers of animals counted are indicated in parenthesis. For each animal, three sections were counted using a minimum of 50 cells.
Hes1 mutant phenotype in the olfactory epithelium The distinct expression patterns of Hes1 and Hes5 in the developping OE suggested that these genes may regulate distinct steps in OE neurogenesis. To address this possibility, we examined the loss-of-function phenotypes of Hes1 and Hes5 in the OE, starting with the study of embryos carrying a null mutation in Hes1 (Ishibashi et al., 1995). Previous studies have shown that loss of Hes1 results in premature neuronal differentiation and upregulation of Mash1 in the forebrain and in morphological defects affecting with a low penetrance the cranial neural tube and the eye (Ishibashi et al., 1995; Tomita et al., 1996). The first indication that loss of Hes1 affects neurogenesis in the OE was a large increase in the number of Mash1-positive cells in the olfactory placodes of Hes1 mutant embryos. Expression of Mash1 starts in wildtype olfactory placodes at the onset of neurogenesis (E10.0 or 25 somites) and, by E10.5, Mash1 is expressed in clusters of cells located in the medial side and to a lesser extent in the lateral side of the placode (Fig. 2A; Cau et al., 1997). In Hes1 mutant placodes, ectopic Mash1 expression was observed in ventral and dorsal areas, which normally do not express the gene at this stage (Fig. 2B.). There was also a marked increase in number of Mash1-positive cells in the lateral side of the placode, and scattered Mash1-positive cells in the center of the placode. This enlargement of the Mash1-positive area in Hes1 mutant placodes shows that Hes1 is involved in defining the area of the placode that activate Mash1 expression. In addition to this enlargement of the Mash1-expressing area, Hes1 mutant placodes also present a change in level of Mash1 expression in the medial domain of the placode, where Mash1 is normally expressed. This defect is characterised by an increase in Mash1 expression, in particular in cells that normally express the gene at low level, so that Mash1 reaches a rather uniformly high expression (compare insets in Fig. 2A and B). To examine this phenotype with a better resolution, we studied Mash1 expression at the protein level, on sections of olfactory placodes. In wild-type placodes, cells were mostly isolated or in groups of two, whereas larger clusters of up to eight MASH1-positive neighbouring cells were observed in Hes1 mutant placodes (Fig. 2C,D; Table 1). The increase in level of Mash1 expression and the presence of clusters of MASH1-positive cells in the neurogenic domain of Hes1
mutant placode suggest that, in addition to an early function in defining the limits of the neurogenic domain of the placode, Hes1 is also involved in a process of lateral inhibition that regulates the number and density of Mash1-positive OSN precursors generated in this domain. We then examined whether the generation of extra Mash1positive precursors leads to an increase in the number of neurons differentiating in Hes1 mutant placodes. Using SCG10 as a pan-neuronal marker, scattered neurons were found in the center of wild-type placodes at E10.5 (Fig. 2E; Cau et al., 1997). There was no apparent change in number of neurons in Hes1 mutant placodes at the same stage (Fig. 2F), whereas a significant increase was observed at E12.5 (Fig. 2I,J). The delay between the appearance of additional Mash1-positive precursors and of additional SCG10-positive neurons suggests that ectopic Mash1 expression may not be sufficient to promote neurogenesis in Hes1 mutant placodal cells and that negative regulators other than Hes1 may inhibit neurogenesis at a step downstream of Mash1 expression. Hes5 is expressed in the olfactory placode and OE (Fig. 1D,F) and is upregulated in Hes1 mutant placodes (Fig. 2G,H), suggesting that a functional compensation could take place between Hes1 and Hes5. Lack of neural defects in Hes5 mutant embryos We generated a null mutation in the Hes5 gene to study its function and possible interaction with Hes1 in the OE and other parts of the nervous system (Fig. 3). The protein-coding sequence of the Hes5 gene is divided in three exons (Takebayashi et al., 1995). We built a targeting construct in which the first two exons of Hes5, which encode the bHLH domain, and part of the third exon are deleted and replaced by a PGK-neor cassette. This mutation, which should result in a null allele, was introduced in ES cells and clones carrying the targeted mutation were used to generate three independant mutant mouse strains (see Materials and Methods). Animals heterozygous for the Hes5 null mutation were viable and fertile. When interbred, they generated progenies with a normal ratio of homozygous mutant mice (32 out of 149, or 21.5%). These animals do not present defects, are fertile and have a normal life span. Histological examination of their brain did not reveal any abnormality (data not shown). To determine if neurogenesis in the OE is affected in absence of Hes5, we examined the expression of Mash1 and SCG10 in the placode and OE of Hes5 mutant embryos. No difference in number and distribution of Mash1-positive OSN progenitors and SCG10-positive neurons could be detected between wildtype and mutant placode and OE (Fig. 4G,H and data not shown), suggesting that lack of Hes5 alone is not sufficient to significantly perturb neurogenesis in the OE.
Hes1 and Hes5 synergize to regulate neurogenesis in the OE Hes1 and Hes5 have similar activities of transcriptional repressors and are both expressed in the embryonic nervous system, including the OE (Fig. 1; Akazawa et al., 1992; Sasai et al., 1992). Therefore these genes could functionally compensate for one another in animals mutant for one of the two genes. To examine this possibility, embryos carrying both Hes1 and Hes5 mutations were generated. When the progeny of Hes1+/−;Hes5+/− and Hes1+/−;Hes5−/− intercrosses were harvested at E10.5, the eight possible genotypes were obtained
Hes genes and olfactory epithelium neurogenesis 2327 Table 2. Synergy between Hes1 and Hes5 in regulation of brain morphogenesis WT
Hes1+/−
Hes1−/−
Hes5−/− Hes1+/−;Hes5−/− Hes1−/−;Hes5−/−
0% (n=54)
0% (n=82)
27.3% (n=55)
0% (n=48)
7.75% (n=129)
71.2% (n=59)
The numbers are percentages of embryos with neural tube defects (open anterior neural tube and reduced size of telencephalic vesicles) in the progeny of intercrossed Hes1;Hes5 double mutant animals at E10.5. Hes1−/− mutant embryos exhibit with a low penetrance defects in closure of the neural tube and in brain morphology, as previously reported (Ishibashi et al., 1995). The penetrance of these defects is strongly increased in double Hes1;Hes5 homozygous mutant embryos. In addition, Hes1+/−;Hes5−/− embryos present at low frequency neural tube defects which are never observed in Hes1+/− embryos. Numbers of embryos of each genotype are shown in parenthesis.
at Mendelian ratios. In contrast, no live double homozygous embryos were recovered at E11,5 (Hes1+/+;Hes5−/−, n=8; Hes1+/−;Hes5−/−, n=15, Hes1−/−;Hes5−/−, n=0, recovered from Hes1+/−;Hes5−/− intercrosses), suggesting that loss of Hes5 function increases the severity of the Hes1 mutant phenotype, which alone leads to embryonic lethality only between E12.5 and birth (Ishibashi et al., 1995). As previously reported, a small fraction of Hes1−/− embryos displayed neural tube defects, including lack of neural tube closure at cranial level (Table 2; Ishibashi et al., 1995). The frequency of these defects was strongly increased in E10.5 embryos homozygous for both Hes1 and Hes5 mutations (Table 2). Hes1+/−;Hes5−/− embryos also showed morphological anomalies which were not observed in Hes1+/− single mutants (Table 2). Thus Hes1 and Hes5 synergize to regulate neural tube morphogenesis. We then examined whether Hes1 and Hes5 also synergize in the regulation of neurogenesis in olfactory placodes. We first examined the distribution of Mash1 transcripts and protein in the placodes of Hes1 and Hes5 single and double mutant embryos. As described above, Mash1 expression was not affected in Hes5 single mutant placodes (data not shown), and there was a clear increase in number of Mash1-positive cells, detectable at both RNA and protein levels, in Hes1 mutant placodes (Fig. 2A-D). In double mutant Hes1−/−;Hes5−/− placodes, there was an increase in Mash1 expression, but it was not significantly different from that observed in Hes1−/− single mutant placodes (data not shown). This result suggests that Hes5 does not synergize with Hes1 to regulate Mash1 expression. We have previously shown that activation of Mash1 is the earliest step in olfactory placode neurogenesis and that its expression in OE progenitors is followed by the expression of another neural bHLH gene, Ngn1 (Cau et al., 1997). The sequential activation of these two genes is reflected in their requirement at distinct steps of OE neurogenesis. In absence of Mash1, basal OE progenitors are not produced (Cau et al., 1997; our unpublished data), whereas in Ngn1 mutant OE, basal progenitors are generated but they fail to differentiate in OSNs (E. C., Q. Ma, D. J. Anderson and F. G., unpublished data). We examined Ngn1 expression in Hes1 and Hes5 mutant placodes to determine whether OE neurogenesis was affected at a step downstream of Mash1 expression. At E10.0, scattered Ngn1-positive cells were found throughout wild-type placodes (Fig. 4A) and the number and distribution of Ngn1-positive cells appeared similar in Hes5 mutant placodes (data not shown). In contrast, the number and density of Ngn1-positive
Table 3. Increase in number and clustering of Ngn1positive cells in Hes1 and Hes1;Hes5 mutant olfactory placodes. Ngn1-positive cells Isolated cells ‘small clusters’ (2 to 5 cells) ‘large clusters’ (6 to 20 cells)
WT (n=4)
Hes1−/− (n=3)
Hes1−/−;Hes5−/− (n=3)
49.5±5.4 50.6±5.4 0.0±0.0
33.5±3.68 56.2±4.3 10.2±2.67
17.8±3.1 49.2±4.1 33.0±4.1
To quantify the clustering of Ngn1-positive cells in Hes mutant placodes, the percentages of Ngn1-positive cells falling in the following three categories: ‘isolated cells’ (1 cell), ‘small clusters’ (2 to 5 cells) and ‘large clusters’ (6 to 20 cells), are shown. In wild-type placodes, a similar number of Ngn1-positive cells is found isolated or in small clusters, and no cluster contains more than 5 cells. In Hes1 single mutants, the proportion of cells in small clusters is the same but there are less isolated cells and larger clusters are found. In Hes1;Hes5 double mutant placodes, the number of cells in small clusters is again the same, but there are even less isolated cells and more cells in large clusters. The percentages of cells are average±s.d., and the numbers of embryos analysed are in parenthesis. For each animal, three sections at different levels of the placode (rostral, medial and caudal) were analysed, and at least 50 cells were counted per section.
cells appeared higher in Hes1 mutant placodes (Fig. 4B) and further increased in Hes1;Hes5 double mutant placodes (Fig. 4C). The clearest defect was in the size and number of clusters of Ngn1-positive cells. The size of these clusters was increased (maximum size of 5 cells in WT and Hes5 mutant placodes, of 8 cells in Hes1 mutant placodes and of 20 cells in Hes1;Hes5 mutant placodes), and a larger fraction of Ngn1-positive cells was found in these clusters rather than isolated, in Hes single and double mutant placodes (Table 3). These results show that there is a gradual increase in Ngn1 expression from wild type to Hes1 single to Hes1;Hes5 double mutant placodes, and suggest that Hes5 and Hes1 synergize to repress Ngn1 expression in olfactory placodes. We next examined whether the upregulation of Ngn1 observed in Hes1;Hes5 double mutant placodes results in an increase in number of neurons in these placodes. As shown earlier, the number of SCG10-positive neurons is not significantly different in wild-type, Hes1 single and Hes5 single mutant placodes at E10.5 (Figs 2E,F, 4D-H and not shown). In contrast, there is a large increase in number of SCG10-positive cells in the placodes of Hes1;Hes5 double mutant embryos at this stage (Fig. 4F,I). Therefore, Hes1 and Hes5 synergize to regulate the number and density of neurons in olfactory placodes. DISCUSSION We have examined the role of two related transcriptional repressors, HES1 and HES5, in neurogenesis in the olfactory epithelium, by examining the phenotype in this tissue of Hes1 and Hes5 single and double mutant embryos. Together, our results suggest the following interpretation of the Hes olfactory placode phenotypes. Loss of Hes1 results in an increase in number of cells expressing the determination gene Mash1 in the placode, and a higher level of Mash1 expression in placodal cells that normally express it. The additional Mash1-positive cells activate expression of the downstream differentiation gene Ngn1, with limited efficiency in absence of Hes1 only, and in more cells in absence of both Hes1 and Hes5. The
2328 E. Cau and others Fig. 2. Neurogenesis defects in Hes1 mutant olfactory placodes. (A,B) Wild-type (A) and Hes1 mutant (B) flat-mounted placodes at E10.5 hybridized with a Mash1 cRNA probe. In Hes1 mutant placodes (B), Mash1 is ectopically expressed in cells around the placode and its expression is more uniformly high in the lateral side of the placode, where high levels of Mash1 expression are normally restricted to cell clusters (see high magnifications in insets). (C,D) Wild type (C) and Hes1 mutant (D) sections of placodes at E10.5 immunostained with an anti-MASH1 antibody. MASH1positive nuclei (arrowheads in C,D) are only found isolated or in pairs in wild-type placodes, whereas larger clusters of labelled nuclei are found in Hes1 mutant placodes (see also Table 2). (E-H) Flat mounts of wild-type (E,G) and Hes1 mutant (F,H) olfactory placodes at E10,5 hybridized with cRNA probes for the pan-neuronal marker SCG10 (E,F) and for Hes5 (G,H). No obvious difference in number or distribution of SCG10-positive neurons is observed between the wild-type and Hes1 mutant placodes (E,F). Hes5 is ectopically expressed in Hes1 mutant placodes (G,H). (I,J) Expression of SCG10 in wild-type (I) and Hes1 mutant (J) OE at E12.5. The density of neurons is significantly increased in the mutant OE.
increase in Ngn1 expression then results in additional neurons in the mutant placodes. These data suggest that both Hes1 and Hes5 negatively regulate neurogenesis, but at different stages in the OSN lineage. The main level of Hes1 control is on Mash1 expression, which marks an early stage in olfactory epithelium neurogenesis. In contrast, Hes5 does not appear to regulate Mash1 expression, but it synergizes with Hes1 at a downstream step, the regulation of Ngn1 expression, to control neuronal production. The observation that Mash1 and Hes1 are both expressed in apical cells whereas Ngn1 and Hes5 are expressed in basal cells in the OE (Fig. 1; Cau et al., 1997)
supports the interpretation that Hes1 and Hes5 act at distinct steps in OE neurogenesis and have distinct molecular targets. Fig. 5 presents our current interpretation of the interactions taking place between the negative regulators Hes1 and Hes5 and the positive regulators Mash1 and Ngn1 during OE neurogenesis. The genes of the Hes family encode bHLH transcription factors that are most closely related to two groups of negative regulators of neurogenesis in Drosophila, hairy and the products of the Enhancer of Split complex (Espl). Both hairy and Espl products have been shown to directly repress transcription of the proneural gene achaete (Ohsako et al., 1994; Van Doren et al., 1994), but their activity is required in different contexts. hairy is a prepattern gene (Skeath and Carroll, 1991, 1994; Fisher and Caudy, 1998). It is required in large areas of the wing and leg imaginal discs to prevent ectopic expression of the proneural gene achaete and the formation of ectopic bristles (Skeath and Carroll, 1991; Orenic et al., 1993; Fischer and Caudy, 1998). The genes of the Espl complex are neurogenic genes that are activated by Notch signalling in a process of lateral inhibition during embryonic and adult neurogenesis (Jennings et al., 1994). Activation of the Espl genes blocks the accumulation of high amounts of proneural protein in most cells of the proneural clusters, thereby preventing them from adopting a neural fate (Bray, 1997). In this report, we show that Hes1 regulation and mutant phenotype in the developping OE suggest that Hes1 has a dual role, acting as a prepattern (hairy-like) gene at the onset of neurogenesis in the olfactory placode and subsequently as a neurogenic (Espl-like) gene regulating Mash1 expression in OE progenitors. In addition, the regulation of Hes5 expression in the olfactory placode and the placodal phenotype of Hes1;Hes5 double mutants support a role for Hes5 as a neurogenic gene acting at a later step in the OSN lineage.
Hes1 has characteristics of a prepattern gene in the olfactory placode Hes1 is expressed in a broad domain of the olfactory placode before the onset of Mash1 expression and of neurogenesis. Once Mash1 expression has been initiated (at the 24- to 26somite stage), Hes1 expression is maintained at high level
Hes genes and olfactory epithelium neurogenesis 2329 throughout the periphery of the placode and is reduced in more central areas, where expression of Mash1 and other neural bHLH genes has been induced (Fig. 1; Cau et al., 1997). This early expression pattern is independent of the emergence of neural precursors, and thus of lateral inhibition, as it is unaffected in Mash1 mutant embryos in which most OSN progenitors are missing. Therefore, earlier signals involved in olfactory placode patterning must be responsible for the induction of Hes1 expression. The phenotype of the Hes1 mutant olfactory placodes, which present a vast enlargement of Mash1expressing areas, provide further evidence that the early function of Hes1 is unrelated to Notch-mediated lateral inhibition. Together, these results support the notion that Hes1 is involved in delimiting the domains of Mash1 expression in the placode, and has therefore a function similar to that of the prepattern gene hairy (Skeath and Carroll, 1991; Orenic et al., 1993). Hairy has been shown to directly repress achaete transcription through binding to a unique CACGCG binding site in its promoter (Ohsako et al., 1994; Van Doren et al., 1994). How HES1 represses Mash1 transcription in the mouse is not known, but human HES1 has been shown to bind to a CACGCA sequence in the promoter of the Hash1 gene, and thereby to repress its expression in carcinoma cells (Chen et al., 1997), suggesting that a direct interaction between these two genes could be a conserved mechanism regulating neurogenesis in vertebrates.
Hes1 has also characteristics of a neurogenic gene Several observations indicate that, after this early function as a prepattern gene, Hes1 might be transcribed in response to Notch signalling and be involved in repression of neurogenesis in a process of lateral inhibition. Activation of Notch1 has been shown to induce Hes1 expression in a variety of cell lines and in retinal explants (Jarriault et al., 1995, 1998; Ohtsuka et al., 1999). This induction of Hes1 expression requires the binding of the mouse homolog of suppressor of Hairless, RBP-J to a consensus site in the Hes1 promoter (Jarriault et al., 1995, Fig. 4. Hes1 and Hes5 synergise for the regulation of neuronal differentiation in the olfactory placode. (A-F) E10.5 placodes from wild-type (A,D,G), Hes1−/− (B,E), Hes5−/− (H) and Hes1−/−;Hes5−/− (C,F,I) embryos hybridized with cRNA probes for Ngn1 (A-C) or SCG10 (D-I) and either sectioned (A-F) or flat mounted (G-I). Compared with wildtype placodes, Hes1 mutant placodes show an increase in both the percentage of Ngn1-positive cells found in clusters, and in the size of the clusters. This effect is more pronounced in Hes1−/−;Hes5−/− placodes (see insets in A-C; Table 3). SCG10positive neurons are present in similar numbers in the placodes of wild-type (D,G), Hes1 mutant (E; Fig. 2J) and Hes5 mutant embryos (H). In contrast, the number and density of neurons is greatly increased in the placodes of Hes1;Hes5 double mutant embryos (F,I).
Fig. 3. Generation of Hes5 mutant mice. (A) Schematic representation of the endogenous Hes5 locus (top), the targeting vector (middle) and the recombined Hes5 null allele (bottom). The genomic fragments used as 5′ and 3′ external probes are indicated at the top of the figure. (B) Analysis of the genotype of five ES cell lines, one wild-type (WT) and four with a targeted Hes5 allele (lanes 1-4). Southern blot analysis was performed on genomic ES cell DNA digested with BamHI or EcoRV and hybridized with 3′ and 5′ external probes, respectively. The 3′ probe detects a 7 kb wild-type and a 4 kb mutant band. The 5′ probe recognizes a wild-type band of more than 12 kb and a 7 kb mutant band. Restriction enzymes: RV, EcoRV; Xh, XhoI; B, BamHI; S, SmaI; N, NotI.
Hes1-/-
2330 E. Cau and others 1998) and thus appears to involve the same mechanism as for transcription of Espl genes in Drosophila and Xenopus (Artavanis-Tsakonas et al., 1995; Wettstein et al., 1997). In the OE at E12.5, we have shown that Hes1 is expressed in apical cells and that this expression is lost in Mash1 mutants. These results suggest that Hes1 participates in a regulatory loop between Mash1 and a Notch-mediated lateral signalling pathway in apical cells of the OE, similar to the loop which is involved in the selection of bristle precursors in the Drosophila PNS (Heitzler et al., 1996). The analysis of the Hes1 mutant phenotype also supports the notion that Hes1 participates in a process of lateral signalling in the OE. Loss of Hes1 results in an increase in Mash1 transcripts levels in areas of the placode that normally express the gene, in addition to an expansion of these areas mentioned earlier. Moreover, we have observed with an antibody that MASH1-positive precursors are found grouped in clusters of adjacent cells in Hes1 mutant placodes, whereas they remain scattered in wild-type placodes, suggesting that Hes1 is required to prevent adjacent placodal cells from expressing MASH1. Finally, the density of neurons in the placode is greatly increased in embryos double mutant for Hes1 and Hes5. These alterations in proneural gene expression and in distribution of neurons appear similar to the phenotypes observed in the nervous system of Drosophila or vertebrate embryos in which Notch signalling has been disrupted (Chitnis et al., 1995; Henrique et al., 1997; Simpson, 1997). Our results therefore support the idea that Hes1 regulates neurogenesis in the OE in two clearly distinct contexts. Apparently conflicting results have previously been reported on whether Hes1 expression and function depend on Notch signalling. Hes1 has been shown to be transcribed in response to Notch activation and Hes1 activity to be important for Notch signalling in embryonic neural tissues (Ohtsuka et al., 1999). In contrast, Hes1 transcript levels are not detectably affected in the neural tube and other tissues of embryos mutant for RBPJ or Notch1, suggesting on the contrary that the transcription of Hes1 is largely independent of Notch signalling (de la Pompa et al., 1997). Therefore Hes1 may also have both Notch-dependent and Notch-independent activities in embryonic tissues other than the OE and particularly in the neural tube.
Hes5 is a neurogenic gene In contrast to the complexity of Hes1 expression patterns and functions, our results and published data provide strong evidence that Hes5 is transcribed in the embryonic nervous system solely in response to Notch signalling and that Hes5 functions only in lateral inhibition. Firstly, as for Hes1, the Hes5 gene contains consensus binding sites for RBP-J, and Notch signalling activates the Hes5 promoter in cell lines, and induces endogenous Hes5 expression in retinal explants (Kageyama and Nakanishi, 1997; Ohtsuka et al., 1999). Secondly, Hes5 transcripts normally present in the brain and spinal cord are almost completely eliminated in RBP-J mutant embryos and severely reduced in Notch1 mutant embryos, supporting the view that Hes5 transcription requires Notch signalling (de la Pompa et al., 1997). Similarly, Hes5 expression in olfactory placodes is greatly reduced in Mash1 mutant embryos and is thus likely controlled by Notch activity in this tissue as well.
Although Hes5 mutant animals do not show any gross abnormalities, the sharp increase in the frequency of neurulation defects in double Hes1;Hes5 mutant embryos, and the neurogenic phenotype of their olfactory placodes (increased size and number of Ngn1-positive cell clusters and increased neuronal density), argue that Hes5 is a negative regulator of neurogenesis in the mouse embryo, and that Hes1 and Hes5 can to some extent functionally compensate for the loss of one another. This conclusion is strongly supported by results of experiments in which forced expression of an activated form of Notch1 was able to accelerate the differentiation of neuroepithelial cells from wild-type embryos as well as Hes1 and Hes5 single mutants, but not from Hes1;Hes5 double mutant embryos (Ohtsuka et al., 1999). Therefore, either Hes1 or Hes5 appear to be required in the neural tube of mouse embryos to mediate Notch signalling. However, the neurogenic phenotype that we observe in the OE of double Hes mutants appears to be only partial, since the majority of MASH1-positive cells remain isolated and only a subset of them are grouped in clusters, suggesting that other genes mediating lateral inhibition must exist in this tissue. Regulation of the OSN lineage by Notch signalling at several steps Distinct stages of OSN progenitor maturation have been defined by the sequential expression of the three neural bHLH genes Mash1, Ngn1 and NeuroD (Cau et al., 1997). Mash1 is involved in the generation of basal OSN progenitors (Cau et al., 1997 and our unpublished data), whereas Ngn1 is required for their differentiation (E. C., Q. Ma, D. J. Anderson and F. G., unpublished data), and the function of NeuroD in this lineage has not yet been characterized. Analysis of the Hes1 mutant phenotype demonstrates that Hes1 regulates OSN development at the level of Mash1 expression. Despite the enlargement of the Mash1-positive cell population in Hes1 mutant olfactory placodes, there is only a relatively small increase in expression of Ngn1, suggesting that the step of Ngn1 expression is also subject to negative regulation. Indeed, the phenotype of Hes1;Hes5 double mutant placodes shows a further increase in number of Ngn1-positive cells and of SCG10-positive neurons without change in expression of Mash1, indicating that Hes5 is likely to regulate OSN development at the level of Ngn1 expression. Altogether, these data suggest that Notch signalling, acting through different Hes genes, regulates the production of OSNs by targeting the expression of two bHLH genes which control the development of the lineage at two distinct steps at least (Fig. 5). This complex negative regulation of the OSN lineage at two levels, the determination and the differentiation of basal progenitors, allows for a fine tuning of the rate of neuronal production in the OE. The observation that Notch signalling regulates neurogenesis at several levels, proneural gene transcription and a downstream step, is not unprecedented. It has been proposed that Drosophila E(Spl) proteins antagonize proneural genes not only by binding to their promoter and repressing their transcription, but also by dimerizing with proneural proteins, and also possibly by competing with them for binding to the promoters of common target genes (Nakao and CamposOrtega, 1996; Jennings et al., 1999). Similarly, HES1 represses Mash1 transcription but also interferes with MASH1 activity
Hes genes and olfactory epithelium neurogenesis 2331 REFERENCES
?
Hes1 Delta/Serrate Notch ?
Mash1
Ngn1
neuronal differentiation
Delta/Serrate Notch ?
Hes5 APICAL CELLS
BASAL CELLS
Fig. 5. A model of the interactions between the negative regulators Hes1 and Hes5 and the positive regulators Mash1 and Ngn1 during OSN lineage development. The arrows represent regulations at the transcriptional level. Mash1 controls the expression of both Hes1 and Hes5 by a mechanism which, by analogy with other systems where achaete-scute and enhancer of split genes interact, is likely to involve the Notch pathway. The arrow with a question mark refers to the earlier, Mash1-independent, regulation of Hes1. There is an interesting correlation between the stage in the OSN lineage at which positive and negative regulators act and their expression pattern in the apical-basal axis of the OE. The early activity of Mash1 in OSN precursor determination (Cau et al., 1997) and of Hes1 in Mash1 regulation (this paper) correlate with the apical expression of these two genes, while the later function of Ngn1 in OSN differentiation (our unpublished data) and of Hes5 in Ngn1 regulation (this paper) correlate with the basal expression of these two genes. This suggests a model whereby Mash1-positive apical cells would represent an early progenitor stage in the OSN lineage, regulated by Hes1. These cells would give rise to more mature progenitors which migrate basally, express Ngn1, and are regulated by Hes5. To test this model will require studying the lineal relationships between apical and basal precursors, and between apical precursors and OSNs. Also, our analysis of the Hes1 mutant and Hes5 mutant phenotypes has not addressed whether these genes were acting in apical cells and basal cells, respectively, because it focused on placodes at E10.5, when the two populations of progenitors are not yet segregated. The synergy between Hes1 and Hes5 in Ngn1 regulation (Fig. 4; Table 3) could be due to Hes5 directly repressing Ngn1, and Hes1 repressing the positive regulator Mash1. Alternatively, Hes1 could also act directly on Ngn1 regulation (shown in the diagram by a dashed line). The comparison between the modest increase in Ngn1 expression and the strong increase in number of SCG10-positive neurons in Hes1;Hes5 double mutant placodes also suggests that Hes5 could regulate a step in OSN differentiation downstream of Ngn1 expression (dashed line).
in hippocampal neuron cultures, possibly by titration of a dimerization partner of MASH1 (Castella et al., 1999). The exact mechanisms by which Hes1 and Hes5 regulate the transcription of Mash1 and Ngn1 and the activity of their products in the OSN lineage remain to be examined. We thank David Anderson the generous gift of the MASH1 antibody, and Valérie Meyer for help with histology. This work was supported by grants from the European Biotech Program, the Ministère de l’Enseignement et de la Recherche and the Association pour la Recherche sur le Cancer, and funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique and the Centre Hospitalier Universitaire Régional. C. S. was supported by a long-term fellowship from the Human Frontier Science Program.
Akazawa, C., Sasai, Y., Nakanishi, S. and Kageyama, R. (1992). Molecular characterization of a rat negative regulator with a basic helix-loop-helix structure predominantly expressed in the developing nervous system. J. Biol. Chem. 267, 21879-21885. Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M. and Sommer, L. (1997). Cell lineage determination and the control of neuronal identity in the neural crest. Cold Spring Harb. Symp. Quant. Biol. 62, 493504. Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M. E. (1995). Notch signaling. Science 268, 225-232. Bray, S. J. (1997). Expression and function of Enhancer of split bHLH proteins during Drosophila neurogenesis. Perspect. Dev. Neurobiol. 4, 313-323. Calof, A. L. and Chikaraishi, D. M. (1989). Analysis of neurogenesis in a mammalian neuroepithelium: proliferation and differentiation of an olfactory neuron precursor in vitro. Neuron 3, 115-127. Calof, A. L., Hagiwara, N., Holcomb, J. D., Mumm, J. S. and Shou, J. (1996). Neurogenesis and cell death in olfactory epithelium. J. Neurobiol. 30, 67-81. Campos-Ortega, J. A. (1993). Early neurogenesis in Drosophila melanogaster. in Development of Drosophila melanogaster. pp. 1091-1130. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Castella, P., Wagner, J. A. and Caudy, M. (1999). Regulation of hippocampal neuronal differentiation by the basic helix- loop-helix transcription factors HES-1 and MASH-1. J. Neurosci. Res. 56, 229-240. Cau, E., Gradwohl, G., Fode, C. and Guillemot, F. (1997). Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors. Development 124, 1611-1621. Cepko, C. L. (1999). The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates. Curr. Opin. Neurobiol. 9, 37-46. Chan, Y. M. and Jan, Y. N. (1999). Conservation of neurogenic genes and mechanisms. Curr. Opin. Neurobiol. 9, 582-588. Chen, H., Thiagalingam, A., Chopra, H., Borges, M. W., Feder, J. N., Nelkin, B. D., Baylin, S. B. and Ball, D. W. (1997). Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete- scute homolog-1 expression. Proc. Natl. Acad. Sci. USA 94, 5355-5360. Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C. (1995). Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375, 761-766. Chitnis, A. and Kintner, C. (1995). Neural induction and neurogenesis in amphibian embryos. Perspect. Dev. Neurobiol. 3, 3-15. Crews, L. and Hunter, D. (1994). Neurogenesis in the olfactory epithelium. Perspect. Dev. Neurobiol. 2, 151-161. Crowe, R., Henrique, D., Ish-Horowicz, D. and Niswander, L. (1998). A new role of Notch and Delta in cell fate decisions: patterning the feather array. Development 125, 767-775. Cuschieri, A. and Bannister, L. H. (1975). The development of the olfactory mucosa in the mouse: electron microscopy. J. Anat. 119, 471-498. de la Pompa, J. L., Wakeham, A., Correia, K. M., Samper, E., Brown, S., Aguilera, R. J., Nakano, T., Honjo, T., Mak, T. W., Rossant, J. and Conlon, R. A. (1997). Conservation of the Notch signalling pathway in mammalian neurogenesis. Development 124, 1139-1148. Fisher, A. and Caudy, M. (1998). The function of hairy-related bHLH repressor proteins in cell fate decisions. BioEssays 20, 298-306. Fode, C., Gradwohl, G., Morin, X., Dierich, A., LeMeur, M., Goridis, C. and Guillemot, F. (1998). The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons. Neuron 20, 483-494. Ghysen, A. and Dambly-Chaudiere, C. (1988). From DNA to form: the achaete-scute complex. Genes Dev. 2, 495-501. Ghysen, A. and Dambly-Chaudiere, C. (1989). Genesis of the Drosophila peripheral nervous system. Trends Genet. 5, 251-255. Gomez-Skarmeta, J. L., Rodriguez, I., Martinez, C., Culi, J., FerresMarco, D., Beamonte, D. and Modolell, J. (1995). Cis-regulation of achaete and scute: shared enhancer-like elements drive their coexpression in proneural clusters of the imaginal discs. Genes Dev. 9, 1869-1882. Graziadei, G. A. and Graziadei, P. P. (1979). Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reconstitution of the olfactory sensory neurons after axotomy. J. Neurocytol. 8, 197-213. Gridley, T. (1997). Notch signaling in vertebrate development and disease. Mol. Cell. Neurosci. 9, 103-108.
2332 E. Cau and others Guillemot, F., Lo, L. C., Johnson, J. E., Auerbach, A., Anderson, D. J. and Joyner, A. L. (1993). Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 75, 463-476. Heitzler, P., Bourouis, M., Ruel, L., Carteret, C. and Simpson, P. (1996). Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signalling in Drosophila. Development 122, 161-171. Henrique, D., Hirsinger, E., Adam, J., Le Roux, I., Pourquie, O., IshHorowicz, D. and Lewis, J. (1997). Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina. Curr. Biol. 7, 661-670. Ishibashi, M., Moriyashi, K., Sasai, Y., Shiota, K., Nakanishi, S. and Kageyama, R. (1994). Persistent expression of helix-loop-helix factor Hes1 prevents mammalian neural differentiation in the central nervous system. EMBO J. 13, 1799-1805. Ishibashi, M., Ang, S. L., Shiota, K., Nakanishi, S., Kageyama, R. and Guillemot, F. (1995). Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helixloop-helix factors, premature neurogenesis, and severe neural tube defects. Genes Dev. 9, 3136-3148. Jan, Y. N. and Jan, L. Y. (1994). Genetic control of cell fate specification in Drosophila peripheral nervous system. Annu. Rev. Genet. 28, 373-393. Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R. and Israel, A. (1995). Signalling downstream of activated mammalian Notch. Nature 377, 355-358. Jarriault, S., Le Bail, O., Hirsinger, E., Pourquie, O., Logeat, F., Strong, C. F., Brou, C., Seidah, N. G. and Isra l, A. (1998). Delta-1 activation of notch-1 signaling results in HES-1 transactivation. Mol. Cell. Biol. 18, 7423-7431. Jennings, B., Preiss, A., Delidakis, C. and Bray, S. (1994). The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo. Development 120, 3537-3548. Jennings, B. H., Tyler, D. M. and Bray, S. J. (1999). Target specificities of Drosophila enhancer of split basic helix-loop- helix proteins. Mol. Cell. Biol. 19, 4600-4610. Jimenez, G. and Ish-Horowicz, D. (1997). A chimeric Enhancer-of-split transcriptional activator drives neural development and achaete-scute expression. Mol. Cell. Biol. 17, 4355-4362. Kageyama, R. and Nakanishi, S. (1997). Helix-loop-helix factors in growth and differentiation of the vertebrate nervous system. Curr. Opin. Genet. Dev. 7, 659-665. Lanford, P. J., Lan, Y., Jiang, R., Lindsell, C., Weinmaster, G., Gridley, T. and Kelley, M. W. (1999). Notch signalling pathway mediates hair cell development in mammalian cochlea. Nat.Genet. 21, 289-292. Lewis, J. (1996). Neurogenic genes and vertebrate neurogenesis. Curr. Opin. Neurobiol. 6, 3-10. Lo, L. C., Johnson, J. E., Wuenschell, C. W., Saito, T. and Anderson, D. J. (1991). Mammalian achaete-scute homolog 1 is transiently expressed by spatially restricted subsets of early neuroepithelial and neural crest cells. Genes Dev. 5, 1524-1537. Modolell, J. (1997). Patterning of the adult peripheral nervous system of Drosophila. Perspect. Dev. Neurobiol. 4, 285-296. Mumm, J. S., Shou, J. and Calof, A. L. (1996). Colony-forming progenitors
from mouse olfactory epithelium: evidence for feedback regulation of neuron production. Proc. Natl. Acad. Sci. USA 93, 11167-11172. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J. C. (1993). Derivation of completely cell culture-derived mice from earlypassage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424-848. Nakao, K. and Campos-Ortega, J. A. (1996). Persistent expression of genes of the enhancer of split complex suppresses neural development in Drosophila. Neuron 16, 275-286. Ohsako, S., Hyer, J., Panganiban, G., Oliver, I. and Caudy, M. (1994). Hairy function as a DNA-binding helix-loop-helix repressor of Drosophila sensory organ formation. Genes Dev. 8, 2743-2755. Ohtsuka, T., Ishibashi, M., Gradwohl, G., Nakanishi, S., Guillemot, F. and Kageyama, R. (1999). Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation. EMBO J. 18, 2196-2207. Orenic, T. V., Held, L. I., Jr., Paddock, S. W. and Carroll, S. B. (1993). The spatial organization of epidermal structures: hairy establishes the geometrical pattern of Drosophila leg bristles by delimiting the domains of achaete expression. Development 118, 9-20. Porteus, M. H., Bulfone, A., Liu, J. K., Puelles, L., Lo, L. C. and Rubenstein, J. L. (1994). DLX-2, MASH-1, and MAP-2 expression and bromodeoxyuridine incorporation define molecularly distinct cell populations in the embryonic mouse forebrain. J. Neurosci. 14, 6370-6383. Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R. and Nakanishi, S. (1992). Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev. 6, 2620-2634. Simpson, P. (1996). A prepattern for sensory organs. Drosophila development. Curr. Biol. 6, 948-950. Simpson, P. (1997). Notch signalling in development: on equivalence groups and asymmetric developmental potential. Curr. Opin. Genet.Dev. 7, 537542. Skeath, J. B. and Carroll, S. B. (1991). Regulation of achaete-scute gene expression and sensory organ pattern formation in the Drosophila wing. Genes Dev. 5, 984-995. Skeath, J. B. and Carroll, S. B. (1994). The achaete-scute complex: generation of cellular pattern and fate within the Drosophila nervous system. Faseb J. 8, 714-721. Smart, I. H. (1971). Location and orientation of mitotic figures in the developing mouse olfactory epithelium. J. Anat. 109, 243-251. Takebayashi, K., Akazawa, C., Nakanishi, S. and Kageyama, R. (1995). Structure and promoter analysis of the gene encoding the mouse helix- loophelix factor HES-5. Identification of the neural precursor cell- specific promoter element. J. Biol. Chem. 270, 1342-1349. Tanabe, Y. and Jessell, T.M. (1996). Diversity and pattern in the developping spinal cord. Science 15, 1115-1123. Tomita, K., Ishibashi, M., Nakahara, K., Ang, S. L., Nakanishi, S., Guillemot, F. and Kageyama, R. (1996). Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron 16, 723-734. Van Doren, M., Bailey, A. M., Esnayra, J., Ede, K. and Posakony, J. W. (1994). Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete. Genes Dev. 8, 2729-2742. Wettstein, D. A., Turner, D. L. and Kintner, C. (1997). The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124, 693-702.
