Vertebrate bHLH Genes and the Determination of Neuronal Fates

Experimental Cell Research 253, 357–364 (1999) Article ID excr.1999.4717, available online at http://www.idealibrary.com on

REVIEW Vertebrate bHLH Genes and the Determination of Neuronal Fates Franc¸ois Guillemot 1 IGBMC, CNRS/INSERM, Universite´ Louis Pasteur, BP163, 67404 Illkirch Ce´dex, CU de Strasbourg, France

INTRODUCTION

The assembly of neuronal circuits requires that neurons with the appropriate phenotype are generated at defined positions and in correct number. Rapid progress is being made in deciphering the cellular and molecular mechanisms regulating neurogenesis in the vertebrate nervous system [1, 2]. In vivo cell lineage tracing studies and in vitro cultures have demonstrated the existence of various types of neural progenitor cells, including multipotent stem cells that can generate neurons, astrocytes, and oligodendrocytes, and precursors with more restricted proliferation and differentiation potentials [3, 4]. A number of secreted molecules regulate the proliferation, lineage commitment, and/or differentiation of these different types of progenitors in culture [5], and Notch signaling has been shown to influence whether progenitors enter a differentiation pathway or remain undifferentiated [6]. A large number of transcription factors are sequentially and transiently expressed in neural precursor cells as neurogenesis proceeds. Gain-of-function studies performed in Xenopus or chick embryos and loss-of-function studies carried out in mice have implicated different transcription factor families in distinct operations. Proteins of the basic helix-loop-helix (bHLH) class have a central role in the determination of neuronal lineages in the peripheral and central nervous system and in the acquisition of pan-neuronal traits by differentiating neurons. Many phenotypic characteristics of neurons are regulated by homeodomain proteins, but bHLH factors are also involved in the specification of certain aspects of the phenotype of neurons, in particular their neurotransmitter identity, thereby coupling generic and subtype-specific subprograms of differentiation. bHLH GENES AND THE DETERMINATION OF NEURAL LINEAGES

Vertebrate Homologs of Drosophila Proneural Genes The basic helix-loop-helix class of transcription factors was first defined in 1989 when genes as diverse as 1

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the oncogene Myc, the myogenic gene MyoD, and the proneural genes of the achaete-scute complex (as-c) were found to encode proteins sharing a structural motif with DNA binding and dimerization properties [7, 8]. The presence of MyoD and as-c genes among the founding members of the bHLH class was an indication that genes of this class play an important role in celltype specification. The subsequent findings that bHLH genes are involved in the development of a number of tissues, including hematopoietic cells, cardiac muscle, and squeletton [9 –11], confirmed this hypothesis. The role of the achaete-scute genes in neural development has been the focus of numerous studies. Neural development in Drosophila is a multistep process involving a number of sequential fate decisions (reviewed in [1, 12]). The achaete-scute genes and atonal, another bHLH proneural gene, initiate this process by providing neural competence to ectodermal cells. Proneural genes are also required to activate Notch signaling in neural competent cells, which results in the selection of the future neural precursors. A number of regulatory genes involved in the differentiation of neural precursors are then activated downstream of proneural genes. These genes have been grouped in two categories on the basis of their expression patterns and functions [12]: pan-neural precursor genes, which are expressed by all neural precursors and are thought to regulate generic aspects of the neuronal differentiation process, and neuronal-type selector genes, which are expressed by discrete subsets of neural precursors and are involved in the specification of neuronal identity. A number of bHLH genes related to Drosophila proneural genes have been isolated in vertebrates (reviewed in [13]). These genes have maintained a neuralspecific expression during evolution, and genetic experiments performed mostly in Xenopus and mouse have shown that their cellular functions and the regulatory pathways that they activate have been conserved as well. Vertebrate proneural gene homologs can be grouped in subfamilies encoding proteins with more highly related bHLH domains (Fig. 1). Their large number may reflect both the complexity of the neurogenesis process, as these genes are expressed at multiple stages in neural lineages, and the diversity of

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FIG. 1. Radial phylogenetic tree of vertebrate neural bHLH genes and their Drosophila relatives. Twelve bHLH genes related to Drosophila proneural genes are expressed in the nervous system of vertebrates (only mouse orthologs are shown, except for Xash3 and Cash4). These genes constitute four subfamilies with highly related bHLH domains. One of them groups orthologs of the ac-sc genes and includes ash1 (present in all vertebrate species studied [13]), Xash3 (found only in Xenopus [18, 19]), and Cash4 (found only in chick [20]). Another subfamily groups orthologs of atonal, including ath1 and ath5 [22, 33]. A third one groups the Neurogenin genes (Ngns) [16, 21, 72–74], which are more closely related to the recently isolated Drosophila tap/biparous gene [75, 76] than to the achaete-scute or atonal genes. The fourth subfamily includes NeuroD and related genes [15, 45– 48, 77], which have no known orthologs in Drosophila, but are distantly related to atonal. The neural bHLH genes Nscl1 and Nscl2 (not shown) are not related to proneural genes but to the SCL gene expressed in hemopoietic precursors [13]. The names of the Drosophila genes are shown in black, the vertebrate genes with a predominant expression in dividing progenitors (“determination genes”) are in red, and those with a predominant expression in postmitotic neurons (“differentiation genes”) are in orange.

neuronal lineages, as all are expressed only in subsets of neural lineages [14]. Some genes (namely Mash1, Neurogenins, and Math1) are expressed in dividing cells, suggesting that they are involved in the specification of neural precursor populations, while others (in particular, NeuroD and related genes) are expressed mostly in postmitotic precursors or differentiated neurons, suggesting roles in the differentiation of neuronal precursors or the maintenance of the differentiated state [13]. Studies of the function of these genes have, in general, supported the division of bHLH genes into early acting determination genes and late acting differentiation genes. Gain-of-Function Studies: bHLH Genes Have Proneural Activity Several bHLH genes are expressed in the neural plate of Xenopus embryos when primary neurons are being generated. The neurogenin gene X-ngnr-1 is ex-

pressed before overt neuronal differentiation in the three longitudinal domains of primary neurogenesis, while XneuroD is expressed in the same domains but only as primary neurons differentiate. Misexpression of either gene by RNA injection in Xenopus leads to the differentiation of ectopic neurons, both in the neural plate and in the regions of the ectoderm fated to become epidermis [15, 16]. Both genes thus have a proneural activity in this context, although based on their expression, only X-ngnr-1 is likely to normally have such an activity during primary neurogenesis [17]. The results of the misexpression of ac-sc-related genes (Xash1 and Xash3 in Xenopus) have been more difficult to interpret. One of the phenotypes obtained is an enlargement of the neural plate at the expense of surrounding tissues, neural crest, and epidermis [18, 19], which is unlikely to reflect the real function of these genes. Vertebrate proneural homologs are first expressed after formation of the neural plate and thus

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do not normally mediate a choice between neural and epidermal fates, but are rather involved in the generation of neural precursor populations within the neural plate. One possible exception is Cash4, a chicken gene with a broad expression during neural plate formation and before neurogenesis in a region of the ectoderm fated to generate the posterior CNS and some epidermis [20]. One of the most interesting outcomes of the misexpression experiments in Xenopus has been the identification of regulatory pathways that are activated by bHLH genes during neurogenesis. For example, injection of X-ngnr-1 mRNA causes ectopic expression of endogenous XneuroD, while the converse is not true, suggesting that the two genes are normally involved in a unidirectional cascade during neurogenesis [16]. Cascades of bHLH genes, which have been shown to take place during both primary and secondary neurogenesis and involve a number of bHLH genes [21–23], are likely to be essential in the establishment of a neural fate and the progressive differentiation of neural lineages. Loss-of-Function Studies: Mash1, Ngn1, and Ngn2 Are Determination Genes Many bHLH genes have now been knocked out in mice, and these experiments have been important for establishing where and when these genes are required and what their cellular functions are. The mutation of Mash1 has revealed a function of this gene in most of the regions of the nervous system in which it is expressed [21, 24 –29]. The olfactory epithelium and ventral forebrain present the most severe defects, characterized by a loss of progenitor populations [21, 28, 29]. These tissues also lack expression of several Notch ligands, suggesting that Mash1 has conserved the dual function of Drosophila proneural genes in the determination of neuronal lineages. The Mash1-mutant phenotype in sympathetic ganglia is less severe, as sympathetic neuron precursors are generated and begin to differentiate but are later arrested in their terminal differentiation [25]. This requirement for Mash1 at a late differentiation step in sympathetic neurogenesis could be due to the expression in neural crest precursors of another gene compensating for the loss of the determination function of Mash1. Ngn1- and Ngn2-mutant mice also show defects in neural tissues that suggest that these genes have neural determination functions. The mice lack sensory neurons in dorsal root and cranial ganglia [30 –32], and in the case of epibranchial placode-derived ganglia, this is due to a lack of delamination of neuronal precursors from the placodes and a failure to activate downstream genes, including NeuroD and the Notch ligand Delta1. Ngn genes and Mash1 have therefore similar determination functions in distinct neural lin-

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eages. The gene Math1 is similarly required for the production of granule cell precursors in the cerebellum [33], but no potential downstream target gene has yet been reported for Math1. Interactions between bHLH Genes and Notch Signaling Notch signaling in the nervous system of vertebrates is required to maintain neural progenitors in an undifferentiated state [6, 34]. Activation of Notch signaling is an essential function of proneural genes, and as their Drosophila counterparts, vertebrate determination genes induce the expression of Delta genes [16, 28, 30, 31]. Interaction between Delta molecules at the surface of differentiating precursors and Notch receptors in neighboring precursors activates Notch signaling in these later cells, resulting in inhibition of determination gene expression and inhibition of differentiation [28, 35, 36]. Notch signaling in neural progenitors is mediated by the genes Hes1 and Hes5, which belong to a family of bHLH repressors related to the Drosophila hairy and enhancer-of-split genes [37– 41]. Genetic analysis of Hes1 function in mouse has shown that, as expected for an effector of Notch signaling, it inhibits neurogenesis and acts at least in part through repression of the positive regulator Mash1 [41– 44]. There is evidence that Notch signaling also regulates neural lineages at steps downstream of the determination genes [36] but this important point needs to be further examined. bHLH GENES AND NEURONAL DIFFERENTIATION

The Ngn genes activate a differentiation program which includes the sequential expression of the bHLH genes NeuroD, Math3, Nscl1, and Nscl2 in regions of the nervous system as diverse as the cranial sensory ganglia, spinal cord, retina, and cerebral cortex [30, 31, 45; our unpublished data], while expression of the NeuroD-related genes Math2 and NeuroD2 is largely restricted to mature CNS neurons, particularly in the cerebral cortex [46 – 48]. There is, in contrast, no known bHLH gene activated downstream of Mash1 in the CNS (although NeuroD and the divergent HLH genes eHand/Hxt and dHand are expressed downstream of Mash1 in the olfactory epithelium and sympathetic ganglia, respectively [21, 49]). Therefore, unknown bHLH genes with differentiation functions may be expressed in Mash1-expressing regions of the CNS, such as the ventral telencephalon. The analysis of mice that are mutant for NeuroD supports the notion that this gene is required for neuronal differentiation. These mice die a few days after birth from diabetes, due to NeuroD function in the endocrine pancreas [50], but this phenotype can be rescued with a transgene encoding NeuroD under the

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insulin promoter. NeuroD-mutant mice then survive to adulthood but present a severe neurological phenotype, which is due to a failure of cerebellar and hippocampal granule cells to differentiate, resulting in their death [51]. Neuronal precursors must exit the cell cycle in order to terminally differentiate (except for sympathetic neurons) and NeuroD is involved in coupling the end of mitosis with differentiation in certain lineages. NeuroD, in association with the coactivator p300, has been shown to arrest cell division both in cell culture and in enteroendocrine cells in vivo [52]. One of the mechanisms involved could be the regulation by NeuroD of p21, a cyclin-dependent kinase inhibitor of the Cip/Kip family, shown to be important for cell cycle exit in various cell types [52, 53]. The specific role of each of the other genes sequentially activated in the bHLH differentiation cascade is not yet known. bHLH GENES AND THE SPECIFICATION OF NEURONAL PHENOTYPES

A differentiating neuron expresses some features common to all neurons and others that are restricted to defined neuronal populations. It has been proposed that pan-neuronal and neuronal subtype-specific components of the neuronal phenotype are controlled by distinct regulatory subprograms [1]. Because most bHLH genes are expressed in a number of phenotypically unrelated neuronal lineages and their loss-offunction mutations result in elimination rather than misspecification of neuronal lineages, they have been usually considered to promote generic neuronal differentiation. However, recent data have shown that bHLH genes also control some aspects of neuronal identity. The first evidence was obtained in Drosophila, in which ectopic expression experiments showed that as-c genes and ato promote the development of different types of sense organs [54 –56], but more recent data have ascribed similar functions to vertebrate bHLH genes. bHLH Genes and Cell-Type Specification in the Retina A number of bHLH genes are expressed with distinct temporal patterns in the retina [57], suggesting that they may be involved in the specification and/or differentiation of particular retinal cell types, and there is now strong experimental support for this hypothesis (reviewed in [58]). Forced expression of Xath5 and XneuroD, two genes expressed by progenitors of the Xenopus retina when they begin to differentiate [22], affect the cellular composition of the retina in different ways. Xath5 increases the number of retinal cell progenitors differentiating in ganglion cells, while XneuroD favors the differentiation of the later born interneurons [22]. An involvement of NeuroD in retinal

interneuron differentiation is also supported by the analysis of retina obtained from NeuroD-mutant mice or infected by NeuroD-expressing retroviruses, which together demonstrate that NeuroD expression promotes the amacrine cell fate at the expense of bipolar cells [59]. Analysis of Mash1-mutant retina suggests that Mash1 has an opposite function in the retina, promoting bipolar cell differentiation at the expense of amacrine cells [60]. The pathways that are regulated by bHLH genes in retinal progenitors and control the fate of these cells have not yet been identified. The Specificity of Mash1 Function Is Controlled by Regional Cues The regulation of the noradrenergic phenotype by Mash1 is the best documented example of a subtypespecific neuronal property specified by a bHLH gene. The expression of Mash1 in the PNS is restricted to precursors of sympathetic, parasympathetic, and enteric neurons, which share a noradrenergic phenotype of neurotransmission. Mash1 is also expressed in areas of the hindbrain that generate noradrenergic neurons, and in all these lineages, Mash1 expression is followed by that of the homeobox gene, Phox2a, an essential determinant of the noradrenergic phenotype [61, 62]. Phox2a is a likely direct transactivator of the genes encoding the noradrenaline-synthesizing enzymes dopamine b-hydroxylase (DBH) and tyrosine hydroxylase (reviewed in [62]) and it is required, in particular, for the generation of the locus ceruleus, the main noradrenergic center of the brain [63]. Interestingly, Mash1mutant mice lack expression of DBH in peripheral autonomic neurons and miss all noradrenergic neurons in the brain, including those in the locus ceruleus [27]. These defects in noradrenergic neuron development are due in part to Phox2a regulation by Mash1, as Phox2a expression is severely reduced or missing throughout the CNS and PNS of Mash1 mutants [27], and forced expression of Mash1 in neural crest cells leads to Phox2a expression, as well as expression of pan-neuronal differentiation markers [64]. Therefore, Mash1 appears to coordinate in autonomic and hindbrain precursors a generic program of neuronal differentiation and a subprogram specific for noradrenergic neurons [14, 62, 64] (Fig. 2). However, Mash1 function in the brain goes beyond the regulation of noradrenergic neuron development. Mash1 is also required for neurogenesis in several regions of the forebrain, including the ventral telencephalon, in which GABA is the main neurotransmitter [28, 29, 65]. We have recently obtained evidence that Mash1 is involved in the specification of the GABAergic phenotype, as its forced expression in the dorsal telencephalon results in ectopic expression of GAD67, which encodes a biosynthetic enzyme for GABA (C. Fode and F.G., unpublished data). Thus,

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FIG. 2. bHLH and homeobox genes interact to specify a neurotransmitter phenotype. In sympathetic precursors, the bHLH gene Mash1 activates in parallel a program of generic neuronal differentiation, resulting in expression of pan-neuronal genes (NF160 and peripherin) and a subprogram specific for autonomic neurons, which leads, in particular, to the expression of the noradrenergic-synthesizing enzyme dopamine b-hydroxylase (DBH). The homeobox genes Phox2a and Phox2b are the main determinants of the noradrenergic phenotype and are likely direct regulators of DBH [62]. Mash1 is an essential regulator of Phox2a in most central and peripheral noradrenergic precursors. However, the interactions between Phox2 genes, Mash1, and the noradrenergic phenotype in sympathetic neurons are complex, since Phox2b expression is independent of Mash1, Mash1 and Phox2b are both required for Phox2a expression, and Mash1 appears to be required for DBH expression not only through regulation of Phox2a but also through a Phox2-independent pathway [62, 64]. The differentiation genes activated by Mash1 in sympathetic precursors (marked ?) are not known.

Mash1 appears to control distinct subtype-specific differentiation programs in different divisions of the brain (noradrenergic differentiation in the hindbrain, GABAergic differentiation in the forebrain). The specificity of Drosophila proneural genes has been mapped to a few residues in the basic portion of the bHLH domain that may be involved in cofactor binding [55]. Similarly, the context-dependent activity of Mash1 may be due to its interaction with regionally expressed cofactors.

ganglia [66]. Similarly, expression of Phox2a and other autonomic specific genes in sympathetic precursors in vivo requires not only Mash1 activity but also diffusible signals generated by the notochord or the floor plate [67]. Studies of autonomic neuron differentiation in neural crest cultures have suggested an interesting model of how bHLH factors might cooperate with extrinsic signaling molecules to drive cell-type specification [68]. Exposure of neural crest cells to proteins of the bone morphogenetic protein (BMP) family induces Mash1 expression and eventually neurogenesis [69]. Forced expression of Mash1 in neural crest cells is, however, not sufficient to promote neurogenesis, but Mash1 maintains the competence of these cells to respond to BMPs, which they would rapidly lose otherwise [68]. A positive feedback loop between BMP2 and Mash1 is thus required to drive neuronal commitment in neural crest cells. These results suggest that, by controlling the ability of neural progenitors to respond to extrinsic factors, bHLH proteins may play an important role in integrating signals from the environment into transcriptional programs of differentiation. CONCLUSIONS

Ngns and Mash1 specify distinct fates in neural crest-derived precursors and thus couple the specification of neuronal identity with generic neuronal differentiation. These activities of bHLH genes are not strictly cell autonomous but depend on extrinsic signals that are likely to contribute to the spatial and temporal control of neurogenesis and to the acquisition of specific neuronal phenotypes. Recent data indicate that bHLH genes have similar functions in the determination and differentiation of neuronal lineages and the specification of neuronal phenotypes in the CNS. A number of signaling pathways control the generation of distinct cell types along the dorso-ventral and the antero-posterior axis of the neural tube [70, 71]. How these extrinsic signals and transcriptional programs of neuronal differentiation are integrated to generate neuronal diversity in the CNS, and to what extent bHLH genes are involved, remains to be examined.

bHLH Gene Function Depends on Extrinsic Signals The expression of Ngn genes in the PNS is restricted to sensory neuron precursors, and as for Mash1 and the autonomic phenotype, Ngns appear to specify certain features of the sensory phenotype. Ectopic expression of Ngns in neural crest and mesoderm-derived tissues results in induction of various combinations of pan-neuronal and sensory-specific markers [66]. Interestingly, Ngns activate some sensory-specific genes cell-autonomously, while the expression of other sensory markers appears to depend on Ngns as well as on environmental signals present near the dorsal root

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