Proneural Proteins Research Articles

Two modes of recruitment of E(spl) repressors onto target genes.

The decision of ectodermal cells to adopt the sensory organ precursor fate in Drosophila is controlled by two classes of basic-helix-loop-helix transcription factors: the proneural Ac and Sc activators promote neural fate, whereas the E(spl) repressors suppress it. We show here that E(spl) proteins m7 and mgamma are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mgamma correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. Irrespective of whether E(spl) are recruited via direct DNA binding or interaction with proneural proteins, the co-repressor Groucho is always needed for target gene repression.

On the organisation of the regulatory region of the zebrafish deltaD gene.

DeltaD is one of the four zebrafish Delta homologues presently known. Experimental evidence indicates that deltaD participates in a number of important processes during embryogenesis, including early neurogenesis and somitogenesis, whereby the protein it encodes acts as a ligand for members of the Notch receptor family. In accordance with its functional role, deltaD is transcribed in several domains of mesodermal and ectodermal origin during embryogenesis. We have analysed the organisation of the regulatory region of the deltaD gene using fusions to the reporter gene gfp and germline transgenesis. Cis-regulatory sequences are dispersed over a stretch of 12.5 kb of genomic DNA, and are organised in a similar manner to those in the regulatory region of the Delta-like 1 gene of mouse. Germline transformation using a minigene comprising 10.5 kb of this genomic DNA attached to the 3' end of a full-length cDNA clone rescues the phenotype of embryos homozygous for the amorphic deltaD mutation after eight(AR33). Several genomic regions that drive transcription in mesodermal and neuroectodermal domains have been identified. Transcription in all the neural expression domains, with one exception, is controlled by two relatively small genomic regions, which are regulated by the proneural proteins neurogenin 1 and zash1a/b acting as transcriptional activators that bind to so-called E-boxes. Transcriptional control of deltaD by proneural proteins therefore represents a molecular target for the regulatory feedback loop mediated by the Notch pathway in lateral inhibition.

Mammalian Scratch participates in neuronal differentiation in P19 embryonal carcinoma cells

Mammalian Scratch (Scrt) is a Snail family zinc finger transcription factor that is specifically expressed in newly differentiating, post-mitotic central nervous system neurons. While Scrt-related genes appear essential for invertebrate neurogenesis, the role of Scrt in mammalian neural development is unknown. In this study, we found that neural differentiation of multipotent mouse P19 embryonal carcinoma cells by retinoic acid led to the appearance of Scrt together with neuron-specific class III β-tubulin (Tuj1), following the earlier elaboration of Mash1. Transient co-transfection in P19 cells with either Mash1 or NeuroD2 plus E12 also induced Scrt gene expression. Moreover, overexpression of Scrt alone was sufficient to confer Tuj1 immunoreactivity and neuronal morphology in a subset of P19 cells. Scrt thus appears to function downstream of proneural bHLH proteins in promoting mammalian neural differentiation in this model system.

Identification of NKL, a novel Gli-Kruppel zinc-finger protein that promotes neuronal differentiation.

The proneural basic helix-loop-helix proteins play a crucial role in promoting the differentiation of postmitotic neurons from neural precursors. However, recent evidence from flies and frogs indicates that additional factors act together with the proneural bHLH proteins to promote neurogenesis. We have identified a novel zinc finger protein, neuronal Kruppel-like protein (NKL), that positively regulates neurogenesis in vertebrates. NKL is expressed in Xenopus primary neurons and in differentiating neuronal precursors in the intermediate zone of the mouse and chick neural tube. In frog embryos, NKL is induced by overexpression of Neurogenin (Ngn), arguing that NKL is downstream of the proneural determination genes. Our results show that NKL and a NKL/VP16 fusion protein promote differentiation of neuronal precursors in the embryonic chick spinal cord. Following in ovo misexpression of NKL, neuroepithelial cells exit the cell cycle and differentiate into neurons. Similarly, NKL/VP16 induces extra primary neurons in frogs and upregulates expression of the neural differentiation factors, Xath3 and MyT1, as well as the neuronal markers, N-tubulin and elrC. Our findings establish NKL as a novel positive regulator of neuronal differentiation and provide further evidence that non-bHLH transcription factors function in the neuronal differentiation pathway activated by the vertebrate neuronal determination genes.

The EGF receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning.

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. Here we show that the proneural proteins trigger, in addition, positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (EGFR) and the Ras/Raf pathway. These interactions, which we denominate 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the EGFR/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess EGFR signalling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the EGFR and Notch pathways act antagonistically in notum macrochaetae determination.

Negative Autoregulation of Mash1 Expression in CNS Development

Mash1, a neural-specific bHLH transcription factor, is essential for the formation of multiple CNS and PNS neural lineages. Transcription from the Mash1 locus is elevated in mice null for Mash1, suggesting that MASH1 normally acts to repress its own transcription. This activity is contrary to the positive autoregulation of other proneural bHLH proteins. To investigate the mechanisms involved in this process, sequences flanking the Mash1 gene were tested for the ability to mediate negative autoregulation. A Mash1/lacZ transgene containing 36 kb of cis-regulatory sequence exhibits an increase in lacZ expression in the Mash1 mutant background, which phenocopies the observation of transcriptional autoregulation at the endogenous Mash1 locus. Using Mash1/lacZ lines with progressively less cis-acting sequence, autoregulatory responsive elements were demonstrated to colocalize with a previously characterized 1.2-kb CNS enhancer. Mutations of E-box sites within this enhancer did not result in an apparent loss of autoregulation, suggesting that MASH1 does not directly repress its own transcription. Interestingly, these mutations did not indicate any underlying positive auto- or cross-regulation of Mash1. Furthermore, the loss of autoregulation in the Mash1 mutant background is reminiscent of a loss of lateral inhibitory signaling. However, mutations in HES consensus sites, the likely purveyors of Notch-mediated lateral inhibition, do not support a role for these sites in negative autoregulation. We hypothesize that MASH1 normally inhibits its own expression indirectly, possibly through a HES-mediated repression of positive regulators or through novel HES binding sites.

Spatially Restricted Factors Cooperate with Notch in the Regulation of Enhancer of split Genes

Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologues in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is acheived and to find out whether there is shared or cross regulation between E(spl) genes, we have analysed the enhancer activity of sequences from the adjacent E(spl)mβ, E(spl)mγ and E(spl)mδ genes and made comparisons to E(spl)m8. We find that although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400–500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mγ and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. We conclude that additional factors besides the proneural proteins limit the response of E(spl)mγ in vivo. In contrast to the other genes, E(spl)mβ exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mβ enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators and, since the activation of target genes is critical in determining the developmental consequences of Notch activity, provide a framework for understanding Notch function in different developmental contexts.

Cato Encodes a Basic Helix-Loop-Helix Transcription Factor Implicated in the Correct Differentiation of Drosophila Sense Organs

In Drosophila neurogenesis, proneural genes encode bHLH proteins that are required for neural precursor selection. But many vertebrate homologues are expressed later and are postulated to have multiple roles during neurogenesis. We have isolated a new Drosophila gene, cato, which encodes a protein with a bHLH domain that is closely related to that of the proneural protein Atonal. cato expression is restricted to the developing PNS, where it is expressed in between the stages of precursor selection and terminal differentiation (and therefore later than the proneural genes). We present evidence from loss-of-function and misexpression experiments that cato is involved in sensory neurone morphology. Moreover, in prospero mutants, in which axon and dendrite outgrowth is defective, cato is strongly derepressed in the developing CNS.

Discrete Enhancer Elements Mediate Selective Responsiveness of Enhancer of split Complex Genes to Common Transcriptional Activators

In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell–cell signaling pathway, which is utilized in imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH) transcriptional repressors, while 4 others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Here we present strong evidence from promoter–reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes were known previously to be direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins achaete and scute. Our extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. We next show that the very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mγ are a property of small (200–300 bp), evolutionarily conserved transcriptional enhancer elements, which can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. We also demonstrate that the characteristic inactivity of the E(spl)mγ enhancer in the notum and margin territories of the wing disc can be overcome by elevated activity of the N receptor. We conclude that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in which these modes of regulation are operative.

Target specificities of Drosophila enhancer of split basic helix-loop-helix proteins.

Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5'-TGGCACGTG(C/T)(C/T)A-3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.

The function of hairy-related bHLH repressor proteins in cell fate decisions.

Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.

Proneural gene self-stimulation in neural precursors: an essential mechanism for sense organ development that is regulated by Notch signaling.

To learn about the acquisition of neural fate by ectodermal cells, we have analyzed a very early sign of neural commitment in Drosophila, namely the specific accumulation of achaete-scute complex (AS-C) proneural proteins in the cell that becomes a sensory organ mother cell (SMC). We have characterized an AS-C enhancer that directs expression specifically in SMCs. This enhancer promotes Scute protein accumulation in these cells, an event essential for sensory organ development in the absence of other AS-C genes. Interspecific sequence comparisons and site-directed mutagenesis show the presence of several conserved motifs necessary for enhancer action, some of them binding sites for proneural proteins. These and other data indicate that the enhancer mediates scute self-stimulation, although only in the presence of additional activating factors, which most likely interact with conserved motifs reminiscent of NF-kappaB-binding sites. Cells neighboring the SMC do not acquire the neural fate because the Notch signaling pathway effectors, the Enhancer of split bHLH proteins, block this proneural gene self-stimulatory loop, possibly by antagonizing the action on the enhancer of the NF-kappaB-like factors or the proneural proteins. These data suggest a mechanism for SMC committment.

A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system.

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

A chimeric enhancer-of-split transcriptional activator drives neural development and achaete-scute expression.

Drosophila melanogaster neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Here, we test if E(spl) proteins function as promoter-bound repressors by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. Our results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway.

Functional dissection of the Drosophila enhancer of split protein, a suppressor of neurogenesis.

The Enhancer of split [E(spl)] gene complex of Drosophila comprises seven related genes encoding a special type of basic helix-loop-helix proteins, the function of which is to suppress the neural developmental fate. One of these proteins is E(spl) itself. To gain insight into the structural requirements for E(spl) function, we have expressed a large number of deletion variants in transgenic flies. Three protein domains were identified as essential for suppression of bristle development: the carboxyl-terminal tetrapeptide WRPW, the region comprising the putative helix III and helix IV, and the region between helix IV and the WRPW motif. Lack of the basic helix-loop-helix domain, helix III or IV, only partially inhibits the suppressor activity of the protein. Truncated variants that lack all the regions carboxyl-terminal to helix IV elicit the development of additional neural progenitors, and thus act as dominant-negative variants. All these results suggest that E(spl) suppresses neural development by direct interaction with other proteins, such as groucho and the proneural proteins.

Lethal of Scute requires overexpression of Daughterless to elicit ectopic neuronal development during embryogenesis in Drosophila

Classical genetics indicates that the achaete-scute gene complex (AS-C) of Drosophila promotes development of neural progenitor cells. To further analyze the function of proneural genes, we have studied the effects of Gal 4-mediated expression of lethal of scute, a member of the AS-C, during embryogenesis. Expression of lethal of scute forces progenitor cells of larval internal sensory organs, which are normally committed to this fate independently of the activity of the AS-C, to take on features of external sensory organs. Supernumerary neural cells can be induced ectopically only if daughterless is overexpressed, either alone or together with lethal of scute: cells of the amnioserosa and the hindgut then express neuronal markers. Furthermore, cells of the proctodeal anlage, which normally lack neural competence, acquire the ability to develop as neuroblasts following transplantation into the neuroectoderm. We show here that activated Notch prevents the cells of the neuroectoderm from forming extra neural tissue when they express an excess of proneural proteins. Under the present conditions, lateral inhibition is thus dominant over the activity of proneural genes.

Control of neural precursor specification by proneural proteins in the CNS of Drosophila.

Formation of neural precursors in Drosophila is determined by proneural genes. The distinctive pattern of expression of some genes of the achaete-scute complex in the embryonic neuroectoderm has prompted the speculation that they could also function in the specification of neural precursor identity in the CNS. To test this hypothesis, we have analysed the capacity of different proneural proteins to promote the development of a particular CNS precursor, the MP2 precursor. Our results indicate that: (i) all known proneural proteins are similarly able to support the formation of a neural precursor at the position of MP2; (ii) different proneural proteins promote the expression of different characteristics of MP2; and (iii) a totally normal specification of the MP2 fate can only be attained by the proneural genes achaete or scute.

Evolution of proneural atonal expression during distinct regulatory phases in the developing Drosophila eye

Background Receptors of the Notch family affect the determination of many cell types. In the Drosophila eye, Notch antagonises the basic helix–loop–helix (bHLH) protein atonal, which is required for R8 photoreceptor determination. Similar antagonism between Notch and proneural bHLH proteins regulates most neural cell determination, however, it is uncertain whether the mechanisms are similar in all cases. Here, we have analyzed the sensitivity of atonal expression to Notch signalling using a temperature-sensitive Notch allele, by the expression of activated Notch or of the ligand Serrate, and by monitoring expression of the atonal-dependant gene scabrous and of the Notch-dependent Enhancer of split genes.Results The atonal expression pattern evolves from general ‘prepattern’ expression, through transient ‘intermediate groups’ to R8 precursor-specific expression. Successive phases of atonal expression differ in sensitivity to Notch. Prepattern expression of atonal is not inhibited. Inhibition begins at the intermediate group stage, corresponding to the period when atonal gene function is required for its own expression. At the transition to R8 cell-specific expression, Notch is activated in all intermediate group cells except the R8 cell precursor. R8 cells remain sensitive to inhibition in columns 0 and 1, but become less sensitive thereafter; non-R8 cells do not require Notch activity to keep atonal expression inactive. Thus, Notch signaling is coupled to atonal repression for only part of the atonal expression pattern. Accordingly, the Enhancer-of-split mδ protein is expressed reciprocally to atonal at the intermediate group and early R8 stages, but is expressed in other patterns before and after.Conclusions In eye development, inhibition by Notch activity is restricted to specific phases of proneural gene expression, beginning when prepattern decays and is replaced by autoregulation. We suggest that Notch signalling inhibits atonal autoregulation, but not expression by other mechanisms, and that a transition from prepattern to autoregulation is necessary for patterning neural cell determination. Distinct neural tissues might differ in their proneural prepatterns, but use Notch in a similar mechanism.

HairlessPromotes Stable Commitment to the Sensory Organ Precursor Cell Fate by Negatively Regulating the Activity of theNotchSignaling Pathway

InDrosophilaimaginal discs, the function of theHairless(H) gene is required at multiple steps during the development of adult sensory organs. Here we report the results of a series of experiments designed to investigate thein vivorole ofHin sensory organ precursor (SOP) cell specification. We show that the proneural cluster pattern of proneural gene expression and of transcriptional activation by proneural proteins is established normally in the absence ofHactivity. By contrast, single cells with the high levels ofachaete, scabrous,andneuralizedexpression characteristic of SOPs almost always fail to appear inHmutant proneural clusters. These results indicate thatHis required for a relatively late step in the development of the proneural cluster, namely, the stable commitment of a single cell to the SOP cell fate. We also show that expression of an activated form of the Notch receptor leads to bristle loss with the same cellular basis—failure of SOP determination—as loss ofHfunction and that simultaneous overexpression ofHsuppresses this effect. Finally, we demonstrate by epistasis experiments that the failure of stable commitment to the SOP fate inHnull mutants requires the activity of the genes of theEnhancer of splitcomplex, includinggroucho.Our results indicate thatHpromotes SOP determination by antagonizing the activity of theNotchpathway in this cell, thereby protecting it from inhibitory signaling by its neighbors in the proneural cluster. We propose a simple threshold model in which the principal role ofHin SOP specification is to translate a quantitative difference in the activity of theNotchpathway (in the SOP versus the non-SOP cells) into a stable binary cell fate decision.

Dual organisation of the Drosophila neuropeptide receptor NKD gene promoter

Neuropeptide function in the peripheral and central nervous systems has been described in mammals as well as in insects. We previously reported the cloning of the neuropeptide receptor NKD, a Drosophila melanogaster homologue of the mammalian tachykinin receptors. This receptor is expressed during Drosophila embryonic development and in the adult fly. Use of the NKD promoter region to drive β-galactosidase expression in transgenic flies reveals a bipartite promoter organisation: the distal region controls NKD expression in neurosecretory cells of the central nervous system during late embryogenesis, whereas the proximal region is responsible for transient expression in peripheral nervous system precursor cells early in development. This early NKD expression, first restricted to the sensory organ precursor cell, an atonal positive cell, is abolished in the ato 1 mutant. In addition, we show that the proneural protein atonal, in association with daughterless, transactivates the NKD promoter in Schneider S 2 cells via the proximal E box NKDE2. Furthermore, heterodimers of atonal and daughterless interact with this E box in gel shift assay.