The enhancer of split complex of Drosophila includes four Notch-regulated members of the bearded gene family.

During Drosophila development, transcriptional activation of genes of the Enhancer of split Complex (E(spl)-C) is a major response to cell-cell signaling via the Notch (N) receptor. Although the structure and function of the E(spl)-C have been studied intensively during the past decade, these efforts have focused heavily on seven transcription units that encode basic helix-loop-helix (bHLH) repressors; the non-bHLH members of the complex have received comparatively little attention. In this report, we analyze the structure, regulation and activity of the m1, m2 and m6 genes of the E(spl)-C. We find that E(spl)m2 and E(spl)m6 encode divergent members of the Bearded (Brd) family of proteins, bringing to four (m(alpha), m2, m4 and m6) the number of Brd family genes in the E(spl)-C. We demonstrate that the expression of both m2 and m6 is responsive to N receptor activity and that both genes are apparently direct targets of regulation by the N-activated transcription factor Suppressor of Hairless. Consistent with this, both are expressed specifically in multiple settings where N signaling takes place. Particularly noteworthy is our finding that m6 transcripts accumulate both in adult muscle founder cells in the embryo and in a subset of adepithelial (muscle precursor) cells associated with the wing imaginal disc. We show that overexpression of either m2 or m6 interferes with N-dependent cell fate decisions in adult PNS development. Surprisingly, while misexpression of m6 impairs lateral inhibition, overexpression of m2 potentiates it, suggesting functional diversification within the Brd protein family. Finally, we present our initial studies of the structure, expression and regulation of the newest member of the Brd gene family, Ocho, which is located in the recently identified Bearded Complex.

A notch-independent activity of suppressor of hairless is required for normal mechanoreceptor physiology.

Suppressor of Hairless [Su(H)]/Lag-1/RBP-Jkappa/CBF1 is the only known transducing transcription factor for Notch receptor signaling. Here, we show that Su(H) has three distinct functions in the development of external mechanosensory organs in Drosophila: Notch-dependent transcriptional activation and a novel auto-repression function, both of which direct cell fate decisions, and a novel auto-activation function required for normal socket cell differentiation. This third phase of activity, the first known Notch-independent activation function for Su(H) in development, depends on a cell type-specific autoregulatory enhancer that is active throughout adult life and is required for proper mechanoreception. These results establish a direct link between a broadly deployed cell signaling pathway and an essential physiological function of the nervous system.

Antagonism of notch signaling activity by members of a novel protein family encoded by the bearded and enhancer of split gene complexes.

Cell-cell signaling through the Notch receptor is a principal mechanism underlying cell fate specification in a variety of developmental processes in metazoans, such as neurogenesis. In this report we describe our investigation of seven members of a novel gene family in Drosophila with important connections to Notch signaling. These genes all encode small proteins containing predicted basic amphipathic (&agr;)-helical domains in their amino-terminal regions, as described originally for Bearded; accordingly, we refer to them as Bearded family genes. Five members of the Bearded family are located in a newly discovered gene complex, the Bearded Complex; two others reside in the previously identified Enhancer of split Complex. All members of this family contain, in their proximal upstream regions, at least one high-affinity binding site for the Notch-activated transcription factor Suppressor of Hairless, suggesting that all are directly regulated by the Notch pathway. Consistent with this, we show that Bearded family genes are expressed in a variety of territories in imaginal tissue that correspond to sites of active Notch signaling. We demonstrate that overexpression of any family member antagonizes the activity of the Notch pathway in multiple cell fate decisions during adult sensory organ development. These results suggest that Bearded family genes encode a novel class of effectors or modulators of Notch signaling.

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)mgamma 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)mgamma 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.

An essential role for the Drosophila Pax2 homolog in the differentiation of adult sensory organs.

The adult peripheral nervous system of Drosophila includes a complex array of mechanosensory organs (bristles) that cover much of the body surface of the fly. The four cells (shaft, socket, sheath, and neuron) which compose each of these organs adopt distinct fates as a result of cell-cell signaling via the Notch (N) pathway. However, the specific mechanisms by which these cells execute their conferred fates are not well understood. Here we show that D-Pax2, the Drosophila homolog of the vertebrate Pax2 gene, has an essential role in the differentiation of the shaft cell. In flies bearing strong loss-of-function mutations in the shaven function of D-Pax2, shaft structures specifically fail to develop. Consistent with this, we find that D-Pax2 protein is expressed in all cells of the bristle lineage during the mitotic (cell fate specification) phase of bristle development, but becomes sharply restricted to the shaft and sheath cells in the post-mitotic (differentiative) phase. Two lines of evidence described here indicate that D-Pax2 expression and function is at least in part downstream of cell fate specification mechanisms such as N signaling. First, we find that the lack of late D-Pax2 expression in the socket cell (the sister of the shaft cell) is controlled by N pathway activity; second, we find that loss of D-Pax2 function is epistatic to the socket-to-shaft cell fate transformation caused by reduced N signaling. Finally, we show that misexpression of D-Pax2 is sufficient to induce the production of ectopic shaft structures. From these results, we propose that D-Pax2 is a high-level transcriptional regulator of the shaft cell differentiation program, and acts downstream of the N signaling pathway as a specific link between cell fate determination and cell differentiation in the bristle lineage.

The K box, a conserved 3' UTR sequence motif, negatively regulates accumulation of enhancer of split complex transcripts.

Cell-cell interactions mediated by the Notch receptor play an essential role in the development of the Drosophila adult peripheral nervous system (PNS). Transcriptional activation of multiple genes of the Enhancer of split Complex [E(spl)-C] is a key intracellular response to Notch receptor activity. Here we report that most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3' untranslated regions (3' UTRs). We present three lines of evidence that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes. First, K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species. Second, the wild-type m8 3' UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences. Finally, m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, we find that K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, we present sequence analyses that implicate genes of the iroquois Complex (Iro-C) and engrailed as additional targets of K box-mediated regulation.

The Drosophila gene Bearded encodes a novel small protein and shares 3' UTR sequence motifs with multiple Enhancer of split complex genes.

Gain-of-function alleles of the Drosophila gene Bearded (Brd) cause sensory organ multiplication and loss phenotypes indistinguishable at the cellular level from those caused by loss-of-function mutations in the genes of the Notch pathway (Leviten, M. W. and Posakony, J. W. (1996). Dev. Biol. 176, 264-283). We have carried out a molecular analysis of the structure and expression of both wild-type and mutant Brd transcription units. We find that the Brd transcript is truncated and accumulates to substantially higher levels in the gain-of-function mutants, due to the insertion of a transposable element of the blood family in the Brd 3' untranslated region (UTR). The wild-type Brd 3' UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that we refer to as the 'Brd box'. Moreover, the 3' UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif we refer to as the 'GY box' (GTCTTCC). We find that both the Brd box and the GY box are also present in the 3' UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and two of the Brd boxes present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them. Finally, we find that Brd encodes a novel small protein of only 81 amino acids that is predicted to include a basic amphipathic alpha-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic alpha-helix, suggesting that the two proteins have related biochemical functions.

The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression.

During the development of the Drosophila adult peripheral nervous system (PNS), inhibitory cell-cell interactions mediated by the Notch receptor are essential for proper specification of sensory organ cell fates. We have reported previously (M. W. Leviten, E. C. Lai and J. W. Posakony (1997) Development 124, 4039-4051) that the 3' untranslated regions (UTRs) of many genes involved in Notch signalling, including Bearded (Brd) and the genes of the Enhancer of split Complex (E(spl)-C), contain (often in multiple copies) two novel heptanucleotide sequence motifs, the Brd box (AGCTTTA) and the GY box (GTCTTCC). Moreover, the molecular lesion associated with a strong gain-of-function mutant of Brd suggested that the loss of these sequence elements from its 3' UTR might be responsible for the hyperactivity of the mutant gene. We show here that the wild-type Brd 3' UTR confers negative regulatory activity on heterologous transcripts in vivo and that this activity requires its three Brd box elements and, to a lesser extent, its GY box. We find that Brd box-mediated regulation decreases both transcript and protein levels, and our results suggest that deadenylation or inhibition of polyadenylation is a component of this regulation. Though Brd and the E(spl)-C genes are expressed in spatially restricted patterns in both embryos and imaginal discs, we find that the regulatory activity that functions through the Brd box is both temporally and spatially general. A Brd genomic DNA transgene with specific mutations in its Brd and GY boxes exhibits hypermorphic activity that results in characteristic defects in PNS development, demonstrating that Brd is normally regulated by these motifs. Finally, we show that Brd boxes and GY boxes in the E(spl)m4 gene are specifically conserved between two distantly related Drosophila species, strongly suggesting that E(spl)-C genes are regulated by these elements as well.

Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signalling.

During imaginal development of Drosophila, Suppressor of Hairless [Su(H)], an evolutionarily conserved transcription factor that mediates intracellular signalling by the Notch (N) receptor, controls successive alternative cell fate decisions leading to the differentiation of multicellular sensory organs. We describe here the distribution of the Su(H) protein in the wing disc epithelium throughout development of adult sense organs. Su(H) was found to be evenly distributed in the nuclei of all imaginal disc cells during sensory organ precursor cells selection. Thus differential expression and/or subcellular localization of Su(H) is not essential for its function. Soon after division of the pIIa secondary precursor cell, Su(H) specifically accumulates in the nucleus of the future socket cell. At the onset of differentiation of the socket cell, Su(H) is also detected in the cytoplasm. In this differentiating cell, N and deltex participate in the cytoplasmic retention of Su(H). Still, Su(H) does not colocalize with N at the apical-lateral membranes. These observations suggest that N regulates in an indirect manner the cytoplasmic localization of Su(H) in the socket cell. Finally, the pIIb, shaft and socket cells are found to adopt invariant positions along the anteroposterior axis of the notum. This raises the possibility that tissue-polarity biases these N-mediated cell fate choices.

Gain-of-function alleles of Bearded interfere with alternative cell fate decisions in Drosophila adult sensory organ development.

We have isolated a novel class of gain-of-function mutations at the Bearded (Brd) locus which specifically affect the development of adult sensory organs in Drosophila. These Brd alleles cause bristle multiplication and bristle loss phenotypes resembling those described for the neurogenic genes Notch (N) and Delta (Dl). We have found that supernumerary sensory organ precursor (SOP) cells develop in the proneural clusters of Brd mutant imaginal discs; like normal SOPs, these are dependent on the function of the proneural genes achaete and scute, and express elevated levels of ac protein. At cuticular positions exhibiting the Brd bristle loss phenotype, we have found that the progeny of the multiplied SOPs develop aberrantly, in that neurons and thecogen (sheath) cells appear but not trichogen (shaft) and tormogen (socket) cells. This appears to represent a transformation of the pIIa secondary precursor cell within the SOP lineage to a pIIb secondary precursor cell fate. These results suggest that Brd gain-of-function alleles interfere with Notch pathway-dependent cell-cell interactions at two distinct stages of adult sensory organ development. We have also identified enhancers and suppressors of the Brd dominant phenotypes; these include both previously characterized mutations and alleles of apparently novel loci. Finally, we have found that Brd null mutants are viable and exhibit no mutant phenotypes, suggesting that Brd may be a component of an overlapping function.

Hairless promotes stable commitment to the sensory organ precursor cell fate by negatively regulating the activity of the Notch signaling pathway.

In Drosophila imaginal discs, the function of the Hairless (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 the in vivo role of H in 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 of H activity. By contrast, single cells with the high levels of achaete, scabrous, and neuralized expression characteristic of SOPs almost always fail to appear in H mutant proneural clusters. These results indicate that H is 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 of H function and that simultaneous overexpression of H suppresses this effect. Finally, we demonstrate by epistasis experiments that the failure of stable commitment to the SOP fate in H null mutants requires the activity of the genes of the Enhancer of split complex, including groucho. Our results indicate that H promotes SOP determination by antagonizing the activity of the Notch pathway 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 of H in SOP specification is to translate a quantitative difference in the activity of the Notch pathway (in the SOP versus the non-SOP cells) into a stable binary cell fate decision.

Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity.

We have investigated the functional relationships among three loci that are required for multiple alternative cell fate decisions during adult peripheral neurogenesis in Drosophila: Notch (N), which encodes a transmembrane receptor protein, Suppressor of Hairless [Su(H)], which encodes a DNA-binding transcription factor, and the Enhancer of split gene complex [E(spl)-C], which includes seven transcription units that encode basic helix-loop-helix (bHLH) repressor proteins. We describe several lines of evidence establishing that Su(H) directly activates transcription of E(spl)-C genes in response to N receptor activity. Expression of an activated form of the N receptor leads to elevated and ectopic E(spl)-C transcript accumulation and promoter activity in imaginal discs. We show that the proximal upstream regions of three E(spl)-C genes contain multiple specific binding sites for Su(H). The integrity of these sites, as well as Su(H) gene activity, are required not only for normal levels of expression of E(spl)-C genes in imaginal disc proneural clusters, but also for their transcriptional response to hyperactivity of the N receptor. Our results establish Su(H) as a direct regulatory link between N receptor activity and the expression of E(spl)-C genes, extending the known linear structure of the N cell-cell signaling pathway.

Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element.

The gap gene hunchback (hb) is required for the formation and segmentation of two regions of the Drosophila embryo, a broad anterior domain and a narrow posterior domain. Accumulation of hb transcript in the posterior of the embryo occurs in two phases, an initial cap covering the terminal 15% of the embryo followed by a stripe at the anterior edge of this region. By in situ hybridization with transcript-specific probes, we show that the cap is composed only of mRNA from the distal transcription initiation site (P1), while the later posterior stripe is composed of mRNA from both the distal and proximal (P2) transcription initiation sites. Using a series of genomic rescue constructs and promoter-lacZ fusion genes, we define a 1.4 kb fragment of the hb upstream region that is both necessary and sufficient for posterior expression. Sequences within this fragment mediate regulation by the terminal gap genes tailless (tll) and a huckebein, which direct the formation of the posterior hb stripe. We show that the tll protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by tll. We propose a model in which the anterior border of the posterior hb stripe is determined by tll concentration in a manner analogous to the activation of anterior hb expression by bicoid.

The sequence similarity of the Drosophila suppressor of hairless protein to the integrase domain has no functional significance in vivo.

Suppressor of Hairless [Su(H)] plays an essential role in neurogenesis in Drosophila by controlling successive alternative cell fate decisions in the developing adult epidermis. Analysis of the predicted amino acid sequence of the Su(H) protein revealed a weak similarity to the catalytic domain of a family of phage integrases and yeast recombinases. We present here the results of a site-directed mutagenesis of the integrase-related region of Su(H), which indicate that this sequence similarity has no functional significance in vivo. We suggest that the JK-RBP protein, encoded by the mouse homologue of Su(H), does not act as a recombinase, as originally proposed.

Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete.

hairy (h) acts as a negative regulator in both embryonic segmentation and adult peripheral nervous system (PNS) development in Drosophila. Here, we demonstrate that h, a basic-helix-loop-helix (bHLH) protein, is a sequence-specific DNA-binding protein and transcriptional repressor. We identify the proneural gene achaete (ac) as a direct downstream target of h regulation in vivo. Mutation of a single, evolutionarily conserved, high-affinity h binding site in the upstream region of ac results in the appearance of ectopic sensory organs in adult flies, in a pattern that strongly resembles the phenotype of h mutants. This indicates that direct repression of ac by h plays an essential role in pattern formation in the PNS. Our results demonstrate that HLH proteins negatively regulate ac transcription by at least two distinct mechanisms.

Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling.

In Drosophila imaginal discs, the spatially restricted activities of the achaete (ac) and scute (sc) proteins, which are transcriptional activators of the basic-helix-loop-helix class, define proneural clusters (PNCs) of potential sensory organ precursor (SOP) cells. Here, we report the identification of several genes that are direct downstream targets of ac-sc activation, as judged by the following criteria. The genes are expressed in the PNCs of the wing imaginal disc in an ac-sc-dependent manner; the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY; where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including Bearded, Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate.

Antagonistic activities of Suppressor of Hairless and Hairless control alternative cell fates in the Drosophila adult epidermis.

Successive alternative cell fate choices in the imaginal disc epithelium lead to the differentiation of a relatively invariant pattern of multicellular adult sensory organs in Drosophila. We show here that the activity of Suppressor of Hairless is required for both the sensory organ precursor (SOP) versus epidermal cell fate decision, and for the trichogen (shaft) versus tormogen (socket) cell fate choice. Complete loss of Suppressor of Hairless function causes most proneural cluster cells to accumulate high levels of the achaete and Delta proteins and to adopt the SOP fate. Late or partial reduction in Suppressor of Hairless activity leads to the apparent transformation of the tormogen (socket) cell into a second trichogen (shaft) cell, producing a 'double shaft' phenotype. We find that overexpression of Suppressor of Hairless has the opposite phenotypic effects. SOP determination is prevented by an early excess of Suppressor of Hairless activity, while at a later stage, the trichogen (shaft) cell is transformed into a second tormogen (socket) cell, resulting in 'double socket' bristles. We conclude that, for two different cell fate decisions in adult sensory organ development, decreasing or increasing the level of Suppressor of Hairless function confers mutant phenotypes that closely resemble those associated with gain and loss of Hairless activity, respectively. These results, along with the intermediate SOP phenotype observed in Suppressor of Hairless; Hairless double mutant imaginal discs, suggest that the two genes act antagonistically to commit imaginal disc cells stably to alternative fates.