InteractiveFly: GeneBrief

Hey: Biological Overview | References

BIOLOGICAL OVERVIEW

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. The single Drosophila Hey orthologue is primarily expressed in a subset of newly born neurons that receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed the study of Notch signalling during the genesis of secondary neurons in the larval central nervous system (Monastirioti, 2010).

Among the superfamily of basic-helix-loop-helix (bHLH) transcription factors, several structurally distinct classes are discerned. One of these, the bHLH-Orange (bHLH-O) class (Fischer, 2007; Iso, 2003), is characterized by the 'Orange' domain, a protein interaction domain perhaps serving as an extended dimerization surface (Taelman, 2004). bHLH-O proteins are important developmental and physiological regulators in processes ranging from neurogenesis to circadian rhythm control (Monastirioti, 2010).

In a number of invertebrate and vertebrate species, bHLH-O repressors are known to inhibit neural differentiation. In Drosophila, the seven E(spl) bHLH-O proteins are expressed in the neuroectoderm, where they inhibit cells from differentiating as neuroblasts (NBs). In vertebrates, a number of Hes bHLH-O proteins, notably Hes1, Hes3 and Hes5 in the mouse, are also expressed in the neuroectoderm; in this case it is the neural stem cells that express the Hes genes, which are subsequently downregulated in the differentiating neuronal progeny (Kageyama, 2008). Triple Hes1, Hes3, Hes5 knock-out causes premature neural differentiation, disruption of the neuroepithelium and a hypoplastic nervous system owing to stem cell depletion (Hatakeyama, 2004). In Drosophila, loss of the entire E(spl) locus results in supernumerary NB specification from the neuroectoderm and a hyperplastic nervous system. Despite these differences, owing to the different mode of neural precursor specification between vertebrates and insects, the generalization can be made that E(spl)/Hes proteins antagonize neuronal differentiation. At most developmental settings across metazoan phylogeny, neural expression of E(spl)/Hes genes is a direct response to Notch signalling (Monastirioti, 2010).

Expression of another subfamily of bHLH-O genes has been detected in the progenitor cell zones of the developing vertebrate central nervous system (CNS). These genes encode the three Hey proteins, so named after a characteristic tyrosine residue in their C-terminal domain (Hairy/enhancer-of-split like with a Y); they are also known as Hrt, Herp, Hesr, Chf or Gridlock. Although neural defects are minor in Hey knock-out mice, overexpression studies have suggested that Hey and Hes proteins might synergize with each other in suppressing neural differentiation and maintaining the neural stem cell fate (Sakamoto, 2003). Hey1 has even been linked to the pathogenesis and aggressiveness of gliomas (Hulleman, 2009). Hey knock-out mice have highlighted their roles in developmental processes outside the nervous system, in particular, heart and vasculature development (Fischer, 2004; Kokubo, 2005). In these contexts, all three mammalian Hey genes appear to respond to Notch signalling, similar to E(spl)/Hes genes in neurogenesis. Biochemical data support Hes-Hey heterodimer formation (Iso, 2001; Taelman, 2004), raising the possibility that these two subclasses of bHLH-O proteins might synergize in some developmental contexts as Notch effectors (Monastirioti, 2010).

The Drosophila genome contains a single Hey orthologue (Kokubo, 1999), which had not been studied to date. This study characterized it in the hope of better understanding the process of neural precursor specification, based on the assumption that, by analogy to vertebrates, Hey might display protein-protein interactions with E(spl). Surprisingly, Hey was not co-expressed with the E(spl) proteins in the neuroectoderm, rather was restricted to differentiating neurons, suggesting a radically different role in neurogenesis than had been assumed. Once NBs are specified in Drosophila, they undergo cycles of asymmetric cell divisions that give rise to a secondary precursor, called a ganglion mother cell (GMC), in addition to self-renewing. GMCs divide once to give rise to two neurons or, less often, glia. The majority of GMC divisions are asymmetric, with the fates of the two daughters dictated by unequal levels of Notch signalling. The 'A' sibling neuron requires high Notch signalling, whereas the 'B' sibling neuron downregulates Notch reception, which is usually achieved by asymmetric segregation of a Notch inhibitor, Numb, into the nascent 'B' neuron. This study describes a complex pattern of Hey expression in relation to these divisions during both neurogenic phases of the animal, early embryogenesis and larval life, where thousands of new neurons are added to generate the adult CNS. In all sibling pairs that were identified, Hey was expressed in the 'A' neuron. Genetic analysis confirmed that Hey is a Notch target gene in most instances. These results extend the Hey-Notch relationship to Drosophila in support of an ancient connection between bHLH-O genes and Notch activity and, for the first time, implicate a bHLH-O protein in the process of GMC asymmetric division (Monastirioti, 2010).

A full-length Hey cDNA was amplified from a Drosophila cDNA library, which was used as a probe for in situ hybridization, and for cloning in prokaryotic expression vectors. Bacterially expressed full-length Hey protein was used to raise anti-Hey antibodies. There were no obvious differences between the RNA and protein patterns. Hey protein showed nuclear accumulation, as expected for a transcription factor, and was primarily detected in a segmentally repeated pattern within the CNS starting at stage 10. Later, more Hey-positive cells gradually appear in the CNS. The neuroectodermal epithelium, where the related E(spl) bHLH-O proteins are expressed already starting at stage 8 (Jennings, 1994), is devoid of Hey expression, which instead is detected at deeper levels overlapping with the GMC/immature neuron marker Pros. From double-staining with the neuronal antigen Elav it was clear that the vast majority of Hey-positive cells represent neurons rather than GMCs, confirmed as lack of colocalization with the NB/GMC marker Asense. Besides neurons, Hey expression was detected in a subset of Repo-positive glia of the CNS and peripheral nervous system (PNS). Of note, Eve staining, which was used to visualize particular neurons, also marks the dorsally located pericardial cells. No Hey immunoreactivity was detected within or near these heart precursors, contrary to the strong expression of mammalian Hey genes during cardiogenesis. Finally, a few Hey-positive cells per segment were detected in the embryonic PNS. Most of these were also neurons, by virtue of being Elav-positive, but were not characterized further (Monastirioti, 2010).

Lineage-specific markers were used to characterize Hey expression in more detail. One was Even skipped, which marks a subset of neurons: the aCC/pCC sibling pair, the RP2 motoneuron, the cluster of U motoneurons and the cluster of EL interneurons. Another was the AJ96-lacZ enhancer trap, which marks the MP2 precursor and its progeny, the dMP2/vMP2 neurons. With AJ96-lacZ, strong Hey accumulation was detected in vMP2 but not in dMP2. Weak Hey expression was detected shortly before mitosis of the MP2 progenitor during late stage 10. Among the Eve-positive neurons, pCC and the U neurons expressed Hey. aCC, RP2 and the EL neurons were Hey-negative. At stage 11, the sibling of RP2, RP2sib, a smaller cell, which only transiently expresses Eve, was Hey-positive. Hey expression in all these neurons appeared transient. For example, whereas immunoreactivity in vMP2 was strong at stage 12, it was downregulated and barely detectable by stage 14. Similarly, by stage 14 no Hey could be detected in pCC cells, although it was still expressed strongly in some of the later-born U motorneurons. Transient Hey expression was also observed in the two identical progeny of MP1, a midline precursor, which are marked by Odd (Monastirioti, 2010).

Most of the neurons described above belong to well-characterized lineages, in which sibling fates arise through differential Notch signalling. In each of the RP2/RP2sib, aCC/pCC and dMP2/vMP2 pairs, the second cell requires Notch signalling in order to acquire the 'A' fate, distinct from that of its sibling cell ('B' fate). Also in the U lineages, which arise from sequential GMCs from neuroblast NB7-1, the U neurons require Notch, whereas their Eve-negative Usib neurons do not. All Notch-requiring cells, namely RP2sib, pCC, vMP2 and the U cells, robustly express Hey, whereas none of their 'B'-fate siblings do so. This raises the possibility that Hey is expressed in response to Notch (Monastirioti, 2010).

Thus Hey was detected almost exclusively in the CNS in young postmitotic neurons and glia, specifically those that receive a Notch signal at birth. It has long been appreciated that Notch signalling plays an important role in the acquisition of neuronal/glial cell fate after GMC division, with most GMCs producing two different progeny, an 'A' cell with high Notch activity and a 'B' cell with no Notch activity. Still, no Notch target genes had been identified in this process. This study shows that Hey is such a target gene in many, and perhaps all, GMC asymmetric divisions. These conclusions are based on the expression pattern of Hey, its response to Notch pathway perturbation and on the ability of ectopic Hey to block development of RP2 and dMP2, two 'B'-type neurons (Monastirioti, 2010).

Although these is good evidence that Hey expression can recapitulate the effect of Notch signalling, Hey loss-of-function has only a mild phenotype. The trivial possibility that the transposon insertion allele used has residual activity is unlikely as (1) no Hey protein is detectable in homozygous mutants and (2) the Hey^f06656 allele results in recessive lethality. Nevertheless, the issue will be permanently decided with the generation and analysis of more Hey alleles. The alternative hypothesis, which seems more probable, is that one or more additional factors besides Hey can also act as nuclear effectors downstream of Notch in the 'A' GMC progeny. No Hey paralogues exist in the D. melanogaster genome, but structurally divergent proteins, even outside the bHLH-O family, could share similar functional characteristics. At the moment, there are no good candidate for such a factor; however, a number of bHLH-O factors have been excluded that do not seem to be co-expressed with Hey in neurons, namely E(spl)mγ and m8, Hairy and Dpn (Monastirioti, 2010).

Besides GMCs, a number of other neural progenitors, namely NBs, sensory organ precursors (SOPs) and SOP progeny cells, all undergo asymmetric cell divisions with Notch involvement. No Hey expression was detected in either the NB/GMC pair or in the SOP progeny cells of external sensory organs, suggesting that Hey expression is turned on exclusively in GMC asymmetric divisions. Hey-positive glia could also be the progeny of asymmetrically dividing GMCs. It is yet unclear which cells might be the immediate progenitors of the few Hey-positive PNS neurons (Monastirioti, 2010).

Until the present work and the recent paper by Krejci (2009), the only Drosophila bHLH-O genes known to be targets of Notch were the seven of the E(spl) complex. Hey and two other bHLH-O genes, dpn and Her, had been predicted as candidate Notch targets based on nearby clustering of putative Su(H) binding sites, the DNA elements via which activated Notch is tethered to its target genes. Although HES-related (Her) does not seem to be a true Notch target (Rebeiz, 2002; Krejci, 2009) have shown that dpn is a Notch target in the muscle-progenitor-like Drosophila DmD8 cell line; an in vivo context for such a response has yet to be determined. Together with Hey, this makes a total of 9 out of 13 bHLH-O genes in the Drosophila genome which are regulated by Notch. It should be stressed that Notch has a number of additional (non-bHLH-O) targets, depending on the species and cellular context, but few, if any, show such widespread association as the bHLH-O genes. The latter are activated by Notch in a multitude of unrelated contexts, such as neuroectoderm, mesoderm, wing epithelium, leg segmentation (Bray, 2006; Lai, 2004) and now GMC asymmetric cell divisions in Drosophila, and in neural progenitors, presomitic mesoderm, cardiogenesis and vasculogenesis in vertebrates (Aulehla, 2008; High, 2008; Louvi, 2006; Monastirioti, 2010).

In addition to its widespread Notch-dependent expression, this study detected a clear instance of Notch-independent expression of Hey within the GMCs and neurons of the MB precursors. Other examples where Hey expression does not correlate with known events of Notch signalling are the MP2 NB and the two MP1 midline neurons. It is also clear that in embryos with severe Notch signalling defects, a small number of Hey-positive cells is still seen in the CNS, suggesting that there are additional neural lineages, where Hey is likely to be expressed independently of Notch. Analysis of the cis regulatory regions of Hey should shed light on Notch-dependent and Notch-independent enhancer elements (Monastirioti, 2010).

The bHLH-O family has undergone considerable diversification during evolution (Simionato, 2007). Although sequence analysis can unambiguously assign genes to this family, it cannot identify orthologues in distantly related species. A classic example is the Drosophila to mammals comparison, where no clear orthologue relationships exist between Hairy, Dpn and the seven E(spl) in Drosophila and Hes1, 2, 3, 5, 6 and 7 in mammals, suggesting that the diversification of these proteins occurred separately after divergence of protostomes and deuterostomes. Hey proteins are the singular exception, being particularly well conserved. The bHLH domain of Drosophila Hey shows 97-98% similarity to that of its mammalian counterparts. This might lead one to expect substantial conservation of Hey function, which, strangely enough, was not observed (Monastirioti, 2010).

First, mammalian Hey genes have a very broad expression pattern, including presomitic mesoderm, embryonic heart, vascular precursors, developing brain and spinal cord, neural crest etc (Kokubo, 1999; Leimeister, 1999). Fly Hey, by contrast, seems confined within the CNS and PNS. Although there is complexity in its expression, as documented in this study with its contextual Notch dependence/independence, the great majority of its expression pattern seems to be in the newly born Notch-dependent 'A'-type neurons. The absence of Hey expression from the developing Drosophila heart is most striking, given the foremost importance of Hey genes in vertebrate cardiogenesis. A second indicator of functional non-conservation comes from comparing the role of Hey within the nervous systems of mammals versus Drosophila. In the former, Hey has been proposed to act in the maintenance of progenitor fate and to antagonize neuronal differentiation, similar to Hes proteins (Sakamoto, 2003). In fact, it has been proposed that Hey-Hes heterodimers mediate these effects. In the fly, Hey expression was not detected within progenitor cells, with the few rare GMC exceptions, noted above. Hey-E(spl) or Hey-Dpn co-expression could not be detected, although all seven E(spl) genes were not tested for lack of specific reporter lines. To overcome any doubt, functional tests were made by ectopically expressing Hey. Instead of suppressing sensory organ formation, it mildly increased the number of bristles, showing an opposite phenotype from that of E(spl) or hairy ectopic expression. It can therefore be confidently said that Hey does not antagonize neural differentiation in the fly (Monastirioti, 2010).

This leaves a puzzle of why Hey is so strongly conserved. Perhaps some yet uncharacterized molecular aspect of its role in chromatin recognition/transcriptional regulation is conserved, despite considerable diversification in cellular and developmental contexts. These contexts have diverged greatly between insects and vertebrates, the only unifying theme being their regulation by Notch signalling. A homologous function might be that of promoting gliogenesis, as Hey2 was shown to promote Müller glia formation in the murine retina (Satow, 2001). Further comparative studies encompassing more species will no doubt shed light on the function of this highly conserved bHLH-O protein (Monastirioti, 2010).

Search PubMed for articles about Drosophila Hey

Aulehla A. and Pourquie O. (2008). Oscillating signaling pathways during embryonic development. Curr. Opin. Cell Biol. 20: 632-637. PubMed ID: 18845254

Bray S. J. (2006). Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7: 678-689. PubMed ID: 16921404

Fischer A., et al. (2004). The Notch target genes Hey1 and Hey2 are required for embryonic vascular development. Genes Dev. 18: 901-911. PubMed ID: 15107403

Hatakeyama, J., et al. (2004). Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation. Development 131(22): 5539-50. PubMed ID: 15496443

High F. A. and Epstein J. A. (2008). The multifaceted role of Notch in cardiac development and disease. Nat. Rev. Genet. 9: 49-61. PubMed ID: 18071321

Hulleman E., et al. (2009). A role for the transcription factor HEY1 in glioblastoma. J. Cell Mol. Med. 13: 136-146. PubMed ID: 18363832

Iso T., Kedes L. and Hamamori Y. (2003). HES and HERP families: multiple effectors of the Notch signaling pathway. J. Cell Physiol. 194: 237-255. PubMed ID: 12548545

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(12): 3537-48. PubMed ID: 7821220

Kageyama R., Ohtsuka T. and Kobayashi T. (2008). Roles of Hes genes in neural development. Dev. Growth Differ. 50 Suppl. 1: S97-S103. PubMed ID: 18430159

Kokubo H., Lun Y. and Johnson R. L. (1999). Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split. Biochem. Biophys. Res. Commun. 260: 459-465. PubMed ID: 10403790

Kokubo H., Miyagawa-Tomita S., Nakazawa M., Saga Y. and Johnson R. L. (2005). Mouse hesr1 and hesr2 genes are redundantly required to mediate Notch signaling in the developing cardiovascular system. Dev. Biol. 278: 301-309. PubMed ID: 15680351

Krejci A., Bernard F., Housden B. E., Collins S. and Bray S. J. (2009). Direct response to Notch activation: signaling crosstalk and incoherent logic. Sci. Signal 2: ra1. PubMed ID: 19176515

Lai E. C. and Orgogozo V. (2004). A hidden program in Drosophila peripheral neurogenesis revealed: fundamental principles underlying sensory organ diversity. Dev. Biol. 269: 1-17. PubMed ID: 15081353

Leimeister C., Externbrink A., Klamt B. and Gessler M. (1999). Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis. Mech. Dev. 85: 173-177. PubMed ID: 10415358

Louvi A., and Artavanis-Tsakonas S. (2006). Notch signalling in vertebrate neural development. Nat. Rev. Neurosci. 7: 93-102. PubMed ID: 16429119

Monastirioti, M., (2010). Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis. Development 137(2): 191-201. PubMed ID: 20040486

Rebeiz M., Reeves N. L. and Posakony J. W. (2002). SCORE: a computational approach to the identification of cis-regulatory modules and target genes in whole-genome sequence data. Site clustering over random expectation. Proc. Natl. Acad. Sci. 99: 9888-9893. PubMed ID: 12107285

Satow T., et al. (2001). The basic helix-loop-helix gene hesr2 promotes gliogenesis in mouse retina. J. Neurosci. 21: 1265-1273. PubMed ID: 11160397

Sakamoto M., et al. (2003). The basic helix-loop-helix genes Hesr1/Hey1 and Hesr2/Hey2 regulate maintenance of neural precursor cells in the brain. J. Biol. Chem. 278: 44808-44815. PubMed ID: 12947105

Simionato E., et al. (2007). Origin and diversification of the basic helix-loop-helix gene family in metazoans: insights from comparative genomics. BMC Evol. Biol. 7: 33. PubMed ID: 17335570

Taelman V., et al. (2004). Sequences downstream of the bHLH domain of the Xenopus hairy-related transcription factor-1 act as an extended dimerization domain that contributes to the selection of the partners. Dev. Biol. 276: 47-63. PubMed ID: 15531363

date revised: 4 April 2022

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