Promoter Structure

The Drosophila snail gene is required for proper mesodermal development. Genetic studies suggest that it functions by repressing adjacent ectodermal gene expression including that of single-minded. snail encodes a protein with a zinc-finger motif. sim possesses early and late promoters: PE and PL. PE drives the mesectodermal transcription in the cellular blastoderm through germ band extention. PL is activated during germ band extension and later conrols midline glial expression. PE is proximal and PL is distal. Six Snail binding sites have been detected in the proximal region, 2.8 kb upstream of the structural gene (Kasai, 1992).

Two potential targets of sim regulation are the sim gene itself, and the 47F gene, which is expressed specifically in sim-expressing midline cells. The sim gene uses two distinct promoters with overlapping temporal specificities; expression from the late promoter is autoregulated by sim. Sim protein associates with the sim late promoter on polytene chromosomes. Sim protein also binds to a small number of additional chromosomal sites, among which are the sites of the 47F gene, and two other genes, Tl and cdi, whose expression also depend on sim function (Muralidhar, 1993).

Notch signal transduction appears to involve the ligand-induced intracellular processing of Notch, and the formation of a processed Notch-Suppressor of Hairless complex that binds DNA and activates the transcription of Notch target genes. This suggests that loss of either Notch or Su(H) activities should lead to similar cell fate changes. However, previous data indicate that, in the Drosophila blastoderm embryo, mesectoderm specification requires Notch but not Su(H) activity. The determination of the mesectodermal fate is specified by Single-minded (Sim), a transcription factor expressed in a single row of cells abutting the mesoderm. The molecular mechanisms by which the dorsoventral gradient of nuclear Dorsal establishes the single-cell wide territory of sim expression are not fully understood. Notch activity is required for sim expression in cellularizing embryos. In contrast, at this stage, Su(H) has a dual function. Su(H) activity is required to up-regulate sim expression in the mesectoderm, and to prevent the ectopic expression of sim dorsally in the neuroectoderm. Repression of sim transcription by Su(H) is direct and independent of Notch activity. Conversely, activation of sim transcription by Notch requires the Su(H)-binding sites. Thus, Notch signaling appears to relieve the repression exerted by Su(H) and to up-regulate sim transcription in the mesectoderm. A model is proposed in which repression by Su(H) and derepression by Notch are essential to allow for the definition of a single row of mesectodermal cells in the blastoderm embryo (Morel, 2000).

To gain insight into the molecular mechanisms by which Su(H) and Notch regulate sim expression, an examination was carried out to see whether Su(H) regulates sim expression in a direct manner. The regulatory elements necessary for mesectodermal expression of sim are contained within a 2.8-kb genomic DNA region. Sequence analysis has identified 10 putative Su(H)-binding sites, with 6 of these exactly matching the GTGRGAA consensus binding sites (Su4, Su5, Su7, Su8, Su9, and Su10). In gel shift experiments, Su(H) binds strongly to oligonucleotides corresponding to each of these sites. Two additional sites, Su2 and Su6, match the consensus RTGRGAR that accomodates nearly all sites that have been shown to bind Su(H) in vitro. These two sites bind weakly to Su(H), both in direct binding assays and in competition experiments. The ability of two noncanonical sites, Su1 and Su3, to bind Su(H) in vitro was also examined. Both Su1 and Su3 bind weakly to Su(H). Other sequences that differ from the RTGRGAR at a single position are not known to bind Su(H) in vitro. Thus, the sim regulatory sequences contain at least 10 binding sites for Su(H). Eight of these sites are clustered in a 500-bp region that contains functional binding sites for Dorsal, Twist, and Snail. Moreover, the organization of this regulatory region has been conserved throughout evolution between D. melanogaster and D. virilis. Together, these data strongly suggest that Su(H) regulates sim transcription directly (Morel, 2000).

Su(H) not only mediates the Notch-dependent activation of sim transcription, but also acts as a transcriptional repressor. This latter conclusion is supported by the following two findings: (1) a complete loss of Su(H) activity leads to weak ectopic expression of sim in the neuroectoderm; (2) the deletion of all of the Su(H)-binding sites from the sim regulatory region also results in ectopic activation of the sim promoter in the ventral neuroectoderm. In Notch mutant embryos, repression by Su(H) is observed not only in the neuroectoderm, but also in the mesectoderm. Because Su(H) is expressed maternally, it is speculated that uniformly localized Su(H) might repress the activation of sim transcription in all of the cells in which Notch is not activated (Morel, 2000).

This study provides the first evidence that Su(H) can act as a transcriptional repressor in Drosophila, and that its repression activity is inhibited by the activation of the Notch receptor. In mammals it has been suggested that the binding of processed Notch to CBF1 competes with the binding of corepressors to CBF1 to promote the formation of an activation complex. The results presented here suggest that Su(H) might mediate such a transcriptional switch at the sim promoter in mesectodermal cells (Morel, 2000).

This regulatory mechanism, in which transcriptional repression is inhibited by a signaling input, may be a general feature of Notch-mediated gene regulation. Consistent with this view, repression by Su(H) might contribute to the difference seen between Notch and Su(H) mutant cuticular phenotypes. Similarly, the cuticular phenotype associated with a deletion removing all of the bHLH-Enhancer of split genes, but not groucho, also appears to be more severe than the one associated with a complete loss of Su(H) function. Because the bHLH-Enhancer of split genes are direct transcriptional targets of Su(H) during neurogenesis, it has been suggested that Su(H) might also act as a transcriptional repressor of the Enhancer of split genes. The finding that Su(H) can repress a Notch target gene indicates that phenotypic differences between Notch and Su(H) mutations do not necessarily imply that Notch signals in a Su(H)-independent manner (Morel, 2000).

How is a single-cell wide territory of sim expression established on the basis of the nuclear gradient of Dorsal? The data presented here, together with previous studies, suggest the following model. In the mesoderm, transcriptional activation of sim by Dorsal and Twist is inhibited by Snail. Whether Su(H) and/or Notch play any role in these cells is not known. In more dorsal cells that do not accumulate Snail, it is proposed that positive regulation of sim by low levels of Dorsal and Twist is antagonized by Su(H). However, in cells bordering the mesoderm, negative regulation by Su(H) would be relieved locally by Notch signaling. This would lead to the specific expression of sim in these cells, which will then form the mesectoderm (Morel, 2000).

An important feature of this model is that Notch signaling overcomes repression by Su(H) only in the single row of cells abutting the mesoderm. One possible explanation for this is that Notch participates in the contact-dependent reception of a mesodermal signal. Results from nuclear transplantation experiments support the existence of a mesodermal signal. When transplanted into snail/twist double mutant embryos that do not express sim, wild-type nuclei can induce the expression of sim in neighboring mutant cells. This result suggests that, in wild-type embryos, mesodermal cells may produce an inductive signal that activates sim transcription in the mesectoderm. Although the molecular nature of this signal is not known, it is speculated that this mesodermal signal might participate in the activation of Notch (Morel, 2000).

Consistent with the view that Notch is specifically activated in ventral cells, changes in the subcellular distribution of both Notch and Delta have been observed ventrally in stage 5 embryos. (1) Lower levels of Notch are found in ventral cells as the ventral furrow forms. (2) In cellularized embryos, Delta is found at the cell membrane, except in ventral cells, in which it predominantly accumulates in vesicles. Both down-regulation of Notch and vesicular accumulation of Delta are consistent with Delta activating Notch in ventral cells in stage 5 embryos (because Snail represses sim transcription, activation of Notch in the mesoderm may have no effect on sim transcription). It will thus be of interest to determine whether these changes in the subcellular distribution of Notch and Delta can be observed in both mesodermal and mesectodermal cells, but not in the more dorsal neuroectodermal cells (Morel, 2000).

In conclusion, repression by Su(H) can be viewed as a refining mechanism ensuring that Notch target genes are expressed only in cells reaching a high threshold of Notch activation. In the early embryo, repression of sim expression allows for the definition of a single row of mesectodermal cells. In these cells, a high level of Notch activity might be induced by a juxtacrine (contact-dependent) inductive signal produced by the mesoderm. In view of this hypothesis, the sharp mesodermal boundary defined by snail expression would be shifted dorsally by one cell, thereby defining a single row of mesectodermal cells (Morel, 2000).

The activity of Notch is required for the transcriptional activation of the sim gene in the mesectoderm, and Su(H) directly regulates sim expression. However, both the sim gene and the simmut-lacZ construct that does not respond to activated Su(H) are expressed in mesectodermal cells in the complete absence of Su(H) activity. These results might suggest that Notch signals, at least in part, in a Su(H)-independent manner to activate sim expression in the mesectoderm. Alternatively, the observation that Su(H) acts to repress sim expression raises the possibility that Notch might be required to antagonize repression by Su(H). To distinguish between these two possibilities, the expression of simmut-lacZ was examined in Notch mutant embryos. simmut-lacZ is expressed at a low level both in the mesectoderm and ectopically in the dorsal neuroectoderm. This pattern is very similar to that observed for simmut-lacZ in wild-type embryos, and dramatically differs from the complete loss of sim-lacZ expression seen in Notch mutant embryos. This shows that the Su(H)-binding sites are required to repress sim transcription in the mesectoderm as well as in the neuroectoderm in the absence of Notch signaling. Furthermore, this demonstrates that repression of sim expression by Su(H), both in ventral neuroectodermal and mesectodermal cells, does not require Notch activity. It is concluded that Su(H) acts as a Notch-independent repressor. Thus, no evidence has been found for a Su(H)-independent function of Notch in the regulation of sim expression (Morel, 2000).

Evolution of the ventral midline in insect embryos; analysis of sim regulation

The ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D.mel.) and the mosquito Anopheles gambiae (A.gam.), the midline is narrow and spans just 1–2 cells. However, in the honeybee, Apis mellifera (A.mel.), the ventral midline is broad and encompasses 5–6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D.mel., but it is activated solely by Twi in A.mel. It is suggested that the Twi-only mode of regulation—and the broad ventral midline—represents the ancestral form of CNS patterning in Holometabolous insects (Zinzen, 2006).

Dorsoventral (DV) patterning of the D.mel. embryo is initiated by a nuclear gradient of the Dorsal (Dl) transcription factor, which differentially regulates at least 50 target genes in a concentration-dependent manner. Most of these genes encode sequence-specific transcription factors and components of cell signaling pathways that control gastrulation. Genetic analyses, microarray screens, and DNA-binding assays with defined DV enhancers have elucidated a gene network of functional interconnections among 40 Dl target genes. The goal of this study is to use this information to understand the evolution of DV patterning among divergent insects (Zinzen, 2006).

In D.mel., the Dl gradient leads to localized activation of Notch signaling in single rows of cells straddling the presumptive mesoderm (Bardin, 2006; De Renzis, 2006). This localized Notch signal works together with the bHLH factor Twi to activate sim expression. After invagination of the ventral furrow, the sim-expressing cells converge at the ventral midline, and the bHLH-PAS Sim transcription factor activates target genes required for midline differentiation (Zinzen, 2006).

The ventral midline is a source of localized signals that help pattern the nerve cord. For example, a transmembrane protease encoded by rhomboid (rho) produces a secreted source of the EGF ligand Spitz. Sim also leads to the expression of slit, which encodes a secreted repellant that binds the Roundabout receptor and inhibits the growth of axonal projections across the midline (Zinzen, 2006).

Sim target genes are highly conserved in A.mel., and in situ hybridization assays reveal that they are similarly expressed in the ventral midline of the developing honeybee nerve cord. However, their expression is significantly broader in A.mel. than in D.mel., 5–6 cells versus 1–2 cells, respectively. Evidence is presented that this broader midline is due to divergent regulation of sim expression. In A.mel., sim is regulated solely by Twi and does not depend on Notch signaling, whereas Notch is responsible for restricting sim to single rows of cells in the early D.mel. embryo (e.g., Bardin, 2006; De Renzis, 2006). It is proposed that the acquisition of Notch dependence at the sim locus is sufficient to account for restricted expression of sim and the narrow midline in D.mel (Zinzen, 2006).

The ventral midline in D.mel. embryos encompasses just 1–2 cells that express signaling molecules such as rho and slit. In contrast, orthologous genes are expressed in 5–6 cells in the honeybee embryo. Notably, the initial expression pattern of A.mel. sim is expanded, and the sim-staining pattern remains broad after convergence of the midline following the spreading of neurogenic ectoderm over the mesoderm. In addition to expression in the ventral midline, sim staining is also detected in more lateral clusters of cells exhibiting segmental periodicity in A.mel. embryos; these might be neurons or glial cells migrating away from the midline (Zinzen, 2006).

Previous studies suggest that sim functions as a 'master control gene' to direct differentiation of the ventral midline in D.mel. To determine whether the expanded sim pattern in honeybees can account for the broadening of the midline, whether ectopic sim expression is sufficient to induce transcription of target genes such as slit and rho. The D.mel. sim-coding sequence was placed under the control of the eve stripe 2 enhancer (eve.2) and expressed in transgenic embryos. There is transient sim expression in the stripe 2 domain of early (stages 5–7) embryos in addition to the endogenous pattern (mesectoderm) in the presumptive ventral midline (Zinzen, 2006).

The initial sim expression pattern is established by a distal 5′ enhancer that contains linked Dl-, Twi-, and Suppressor of Hairless [Su(H)]-binding sites. Expression is maintained by a separate autoregulatory enhancer containing Sim/Tango-binding sites; Tango is a ubiquitous bHLH-PAS transcription factor that forms heterodimers with Sim. Though the eve stripe 2 enhancer mediates transient activation, autoregulation maintains expression of the endogenous sim gene in the ventral neurogenic ectoderm of advanced-stage embryos, but not in the mesoderm or dorsal ectoderm (Zinzen, 2006).

Ectopic sim expression leads to the induction of various target genes, including rho, slit, sog, and the transcription factor otd. These results provide evidence that ectopic sim expression is sufficient to expand the ventral midline in D.mel. In principle, the altered midline seen in the honeybee embryo could be explained by a change in sim regulation. The distal 5′ enhancer that establishes sim expression is the most likely site of change, since the autoregulatory enhancer merely maintains expression within the limits of the established pattern (Zinzen, 2006).

To determine the basis for the distinct sim expression patterns in flies and honeybees, it was necessary to isolate the early sim enhancer from A.mel. However, the identification of homologous enhancers is complicated by the rapid turnover of noncoding DNA sequences in insect genomes. For example, the 5′ flanking regions of the sim loci in D.mel. and A.gam. lack simple sequence homology, even though they belong to the same order (Diptera). Nonetheless, it was possible to identify the early sim enhancer in A.gam. based on the clustering of Dl-, Twi-, and Su(H)-binding sites. The D.mel. and A.gam. enhancers are located in similar positions relative to the sim transcription unit (Zinzen, 2006).

A.mel. is a member of the order Hymenoptera and is highly divergent from D.mel. Computational methods used for the in silico identification of the A.gam. sim enhancer were further developed to ensure the accurate identification of the sim enhancer in A.mel. The current method (ClusterDraw2) employs position-weighted matrices (PWMs) to identify binding motif clusters (Zinzen, 2006).

The efficacy of the method was tested by surveying ~50 kb genomic intervals encompassing the sim loci of D.mel. and A.gam.. PWMs of Dl, Twi, Snail, and Su(H) were used in various combinations and individually. The best binding site clusters coincide exactly with the known sim enhancers (Zinzen, 2006).

ClusterDraw2 was used to survey a ~50 kb genomic DNA interval encompassing the sim locus of A.mel.. The best prediction occurs in the 5′ flanking region of the gene, similar to the locations of the fly and mosquito enhancers. However, while the D.mel. and A.gam. sim enhancers contain several optimal Su(H)-binding sites, the A.mel. cluster lacks such sites, but contains several high-scoring Twi sites. This is consistent with the possibility that A.mel. sim is regulated by Twi alone, rather than by the combination of Twi+Notch (Zinzen, 2006).

A 2.2 kb genomic DNA fragment encompassing the predicted A.mel. sim enhancer directs lateral stripes of lacZ expression in transgenic D.mel. embryos. A similar pattern was obtained with a 471 bp fragment containing the predicted Twi-binding sites. This pattern encompasses 3–4 cells on either side of the presumptive mesoderm, similar to the expression of the endogenous A.mel. sim gene, but distinct from the single-row sim patterns in D.mel. and A.gam. (Zinzen, 2006).

The fly, honeybee, and mosquito sim enhancers were crossed into various genetic backgrounds to determine the basis for their distinct expression patterns. The D.mel. m5/8 enhancer was also examined. It is located within the Enhancer of split (E(spl)) complex, where it controls the expression of the m5 and m8 genes within the mesectoderm. The m5/8 enhancer directs lacZ expression in a pattern that is virtually identical to that produced by the D.mel. sim enhance (Zinzen, 2006).

Transgenic D.mel. embryos carrying an eve.2::NICD fusion gene exhibit ectopic Notch signaling in the eve stripe 2 domain. The m5/8-lacZ transgene is strongly induced in the neurogenic ectoderm and dorsal ectoderm, but not in the mesoderm, where the Sna repressor is present. The D.mel. sim-lacZ transgene displays only modest ectopic induction by the eve.2::NICD transgene; this induction appears as a 'pyramid' limited to ventral regions of the neurogenic ectoderm. This pyramid coincides with the intersection of ectopic Notch signaling and the endogenous Twi gradient. The different patterns ('pyramid' versus 'column') seen for the sim and m5/8 enhancers appear to reflect activation by Notch+Twi or regulation by Notch alone, respectively. The m5/8 enhancer contains an SPS (Su(H) Paired Site) motif, and it has been suggested that the endogenous m8 gene is activated solely by Notch signaling (Zinzen, 2006).

The A.mel. sim enhancer is not activated by the eve.2::NICD transgene, consistent with the absence of Su(H) sites in this enhancer. To determine whether it is activated by Twi, the lacZ fusion gene was crossed into embryos carrying an hsp83::twi-bcd-3′UTR transgene that produces high levels of Twi transcripts at the anterior pole. The resulting ectopic anteroposterior Twi protein gradient induces intense expression of the lacZ reporter gene directed by the A.mel. sim enhancer. In contrast, neither the D.mel. sim enhancer nor the m5/8 enhancer is induced by this ectopic gradient. Finally, the D.mel. sim enhancer is inactive in mutant embryos derived from germline clones lacking Su(H) activity, whereas the honeybee sim enhancer is fully active. Thus, unlike the D.mel. sim enhancer, the A.mel. enhancer does not rely on Notch signaling (Zinzen, 2006).

The preceding analysis suggests that the D.mel. sim enhancer is activated by Twi and Notch signaling, whereas the A.mel. sim enhancer is activated solely by Twi. These distinct modes of regulation are reflected by the composition of binding sites in the different enhancers. The A.mel. enhancer contains several optimal Twi sites, but it lacks unambiguous Su(H) sites. In contrast, the D.mel. enhancer contains several optimal Su(H) sites, but just one optimal Twi site. Both enhancers contain binding sites for the Sna repressor, which inhibits expression in the mesoderm (Zinzen, 2006).

sim regulation was examined in the mosquito, A.gam., to determine whether the midline of ancestral dipterans might have been regulated solely by Notch signaling, as seen for the fly m5/8 enhancer. The A.gam. genome contains a clear ortholog of the sim gene, expressed in a single row of cells in the mesectoderm, similar to the pattern seen in D.mel. The A.gam. sim enhancer directs sporadic expression within the mesectoderm of transgenic D.mel. embryos, but it is strongly induced by the eve.2::NICD transgene. This response is similar to that obtained with the D.mel. m5/8 enhancer, but it is distinct from the 'pyramid' pattern seen for the D.mel. sim enhancer (Zinzen, 2006).

To determine whether the sim loci of other drosophilids are regulated by Twi+Notch, as seen in D.mel., or Notch alone, sim enhancers from D. pseudoobscura (D.pse.) and D. virilis (D.vir.) were tested in transgenic eve.2::NICD D.mel. embryos. Surprisingly, these enhancers behave like the A.gam. sim enhancer: they are expressed throughout the neurogenic ectoderm and dorsal ectoderm (“column”) in response to Notch signaling, rather than the “pyramid” pattern indicative of Notch+Twi regulation. These observations suggest that the evolution of sim regulation is highly dynamic, although there is no obvious difference in the number or quality of Su(H) and Twi sites in the different drosophilid enhancers. Perhaps a subtle shift in the organization of binding sites distinguishes regulation by Notch alone versus Notch+Twi (Zinzen, 2006).

Transcriptional Regulation

Differential activation of the Toll receptor leads to the formation of a broad Dorsal nuclear gradient that specifies at least three patterning thresholds of gene activity along the dorsoventral axis of precellular embryos. The activities of the Pelle kinase and Twist basic helix-loop-helix (bHLH) transcription factor in transducing Toll signaling have been investigated. Pelle functions downstream of Toll to release Dorsal from the Cactus inhibitor. Twist is an immediate-early gene that is activated upon entry of Dorsal into nuclei. Transgenes misexpressing Pelle and Twist were introduced into different mutant backgrounds and the patterning activities were visualized using various target genes that respond to different thresholds of Toll-Dorsal signaling. These studies suggest that an anteroposterior gradient of Pelle kinase activity is sufficient to generate all known Toll-Dorsal patterning thresholds and that Twist can function as a gradient morphogen to establish at least two distinct dorsoventral patterning thresholds. How the Dorsal gradient system can be modified during metazoan evolution is discussed and it is concluded that Dorsal-Twist interactions are distinct from the interplay between Bicoid and Hunchback, which pattern the anteroposterior axis (Stathopoulos, 2002).

The snail, sim, vnd and sog expression patterns represent four different Toll-Dorsal signaling thresholds. snail is activated only by peak levels of the Dorsal gradient; sim and vnd are activated by intermediate levels, and sog is activated by the lowest levels of the gradient. These expression patterns were visualized in mutant and transgenic embryos via in situ hybridization using digoxigenin-labeled antisense RNA probes (Stathopoulos, 2002).

Dorsal target genes are essentially silent in mutant embryos that lack an endogenous dorsoventral Dorsal nuclear gradient. Mutant embryos were collected from females that are homozygous for a null mutation in the gastrulation defective (gd) gene, which blocks the processing of the Spätzle ligand and the activation of the Toll receptor. These mutants permit the analysis of ectopic, anteroposterior Dorsal and Twist gradients in 'apolar' embryos that lack dorsoventral polarity. snail, vnd, and sog are sequentially expressed along the anteroposterior axis of mutant embryos that contain a constitutively activated form of the Toll receptor (Toll10b) misexpressed at the anterior pole using the bicoid (bcd) promoter and 3' UTR. These expression patterns depend on an ectopic anteroposterior Dorsal nuclear gradient. The repression of the vnd and sog patterns at the anterior pole is probably mediated by Snail, which normally excludes expression of these genes in the ventral mesoderm of wild-type embryos (Stathopoulos, 2002).

The activated Pelle-Tor4021 kinase also directs sequential anteroposterior patterns of snail, vnd, and sog expression in gd/gd mutant embryos. As in the case of Toll10b, the activated Pelle kinase was misexpressed at the pole using the bcd 3' UTR. The snail, vnd and sog expression patterns are similar to those obtained with the Toll10b transgene. The vnd and sog expression patterns are probably repressed at the anterior pole by Snail. These results suggest that the levels of Pelle kinase activity are sufficient to determine different Dorsal transcription thresholds (Stathopoulos, 2002).

sog is normally activated throughout the neurogenic ectoderm by the lowest levels of the Dorsal gradient. The low levels of Dorsal present in Tollrm9/Tollrm10 mutant embryos are sufficient to activate sog everywhere except the extreme termini. The twist-bcd transgene leads to the loss of sog expression in anterior regions, probably because of repression by Snail. Snail also appears to repress vnd and sog expression in anterior regions of transgenic embryos that contain the Toll10b or Pelle-Tor4021 transgenes (Stathopoulos, 2002).

The low levels of Dorsal present in Tollrm9/Tollrm10 mutant embryos are insufficient to activate sim, although there is occasional staining in the posterior pole. The twist-bcd transgene leads to the efficient activation of sim in anterior regions. Staining appears to be restricted to those regions where snail expression is lost. These results suggest that a Twist gradient is sufficient to generate multiple dorsoventral patterning thresholds (sim and snail) in the presence of low, uniform levels of Dorsal (Stathopoulos, 2002).

The twist-bcd transgene was introduced into mutant embryos that completely lack Dorsal. Without the transgene these mutants do not express twist, snail, sim, vnd or sog. Introduction of the twist-bcd transgene causes intense expression of twist in the anterior 40% of the embryo. This broad Twist gradient fails to activate snail, but succeeds in inducing weak expression of sim and somewhat stronger staining of vnd at the anterior pole. The activation of vnd in mutant embryos is comparable with the expression seen in wild-type and Tollrm9/Tollrm10 embryos. However, in both wild-type and mutant embryos the vnd pattern is transient, and lost after the completion of cellularization. These results indicate that Twist can activate dorsoventral patterning genes in the absence of Dorsal (Stathopoulos, 2002).

An anteroposterior Twist gradient generates at least two thresholds of gene activity in mutant embryos that contain decreased levels of Dorsal. High levels of Twist activate sim at the anterior pole, whereas lower levels are sufficient to induce the expression of snail in more posterior regions of embryos containing low, uniform levels of the Dorsal protein. These results demonstrate that twist gene activity is not dedicated to mesoderm formation. Instead, Twist supports expression of two regulatory genes, sim and vnd, which pattern ventral regions of the neurogenic ectoderm. The twist-bcd transgene was shown to induce weak expression of both genes even in mutant embryos that completely lack Dorsal (Stathopoulos, 2002).

Notch is necessary for midline activation of sim (Menne, 1994 and Martin-Bermudo, 1995). In Notch knock out mutants, there are an excess of midline cells and these cells are incorrectly positioned in the dorsal part of the midline, rather than both dorsally and ventrally located. This migration defect is a second effect Notch mutation.

Maternal Notch activity is required for the correct activation of sim (Menne, 1994). Zygotic Notch and neuralized mutants show only low levels of single-minded expression (Martin-Bermudo, 1995).

Snail is required to repress sim expression in mesodermal precursors (Nambu, 1990 and Kasai, 1992).

The initiation of mesoderm differentiation in the Drosophila embryo requires the gene products of twist and snail. In either mutant, the ventral cell invagination during gastrulation is blocked and no mesoderm-derived tissue is formed. One of the functions of Snail is to repress neuroectodermal genes and restrict their expressions to the lateral regions. The derepression of the neuroectodermal genes into the ventral region in snail mutants is a possible cause of defects in gastrulation and in mesoderm differentiation. To investigate such a possibility, a series of snail mutant alleles was analyzed. Different neuroectodermal genes respond differently in various snail mutant backgrounds. Due to the differential response of target genes, one of the mutant alleles, V2, which manifests reduced Snail function, also shows an intermediate phenotype. In V2 embryos, neuroectodermal genes, such as single-minded and rhomboid, are derepressed while ventral invagination proceeds normally. However, the differentiation of these invaginated cells into mesodermal lineage is disrupted. The results suggest that the establishment of mesodermal cell fate requires the proper restriction of neuroectodermal genes, while the ventral cell movement is independent of the expression patterns of these genes. The expression of some ventral genes disappear in snail mutants. Snail function is required for activation of genes such as dGATAb (serpent) and zfh-1. It is proposed that Snail may repress or activate another set of target genes, including folded gastruation, that are required specifically for gastrulation (Hemavathy, 1997).

Notch signal transduction involves the presenilin-dependent intracellular processing of Notch and the nuclear translocation of the intracellular domain of Notch, NICD. NICD associates with Suppressor of Hairless [Su(H)], a DNA binding protein, and Mastermind (Mam), a transcriptional coactivator. In the absence of Notch signaling, Su(H) acts as a transcriptional repressor. Repression by Su(H) is relieved by the activation of Notch. In the Drosophila embryo, this transcriptional switch from repression to activation is important for patterning the expression of the single-minded (sim) gene along the dorsoventral axis. The mechanisms by which Su(H) inhibits the expression of Notch target genes in Drosophila has been investigated. Hairless, an antagonist of Notch signaling, is required to repress the transcription of the sim gene. Hairless forms a DNA-bound complex with Su(H). Furthermore, it directly binds the Drosophila C-terminal Binding Protein (dCtBP), which acts as a transcriptional corepressor. The dCtBP binding motif of Hairless is essential for the function of Hairless in vivo. It is proposed that Hairless mediates transcriptional repression by Su(H) via the recruitment of dCtBP (Morel, 2001).

The sim gene is expressed in a single row of cells abutting the mesoderm in the Drosophila embryo at the cellular blastoderm stage. Sim confers to these cells a mesectodermal identity. Su(H) has a dual function in the regulation of sim expression: (1) Su(H) directly inhibits the expression of the sim gene in the neuroectoderm -- in Su(H) mutant embryos derived from germ-line clones (GLC), both endogenous sim and a sim-lacZ transgene that mimics the expression of sim are ectopically expressed in 2-3 rows of neuroectodermal cells; (2) Su(H) upregulates the expression of sim in the mesectoderm. Transcriptional activation by Su(H) depends on Notch signaling, but repression by Su(H) is independent of Notch activity (Morel, 2001).

Hairless is a nuclear protein that binds Su(H) and antagonizes Notch activity in numerous cell fate decisions. It is, however, unclear how Hairless inhibits transcriptional activation by Su(H). One hypothesis is that Hairless promotes repression by Su(H). Analysis of the sim promoter has allowed for a test of whether Hairless is required for Su(H)-dependent repression. The expression of the sim gene was analyzed in embryos that lack both the maternal and the zygotic contributions of Hairless. In these Hairless GLC embryos, sim-lacZ is ectopically expressed in cells located dorsally to the mesectoderm. This ectopic expression in the neuroectoderm is similar to the one observed in Su(H) mutant embryos. Thus, Hairless is required for the repression of sim in the same cells that also require Su(H) for repression (Morel, 2001).

Hairless binds Su(H) and inhibits transcriptional activation by Su(H). However, because Su(H)-Hairless complexes do not bind DNA in vitro, Hairless has been proposed to inhibit the DNA binding activity of Su(H). In this model, Hairless inhibits Notch signaling by titrating Su(H) and not by repressing Notch target genes. This model does not explain, however, why the loss of Hairless activity leads to the same phenotype as the loss of Su(H) activity, i.e., derepression of sim in neuroectodermal cells. It is therefore envisaged that Hairless and Su(H) act together to repress transcription. This implies that Hairless must bind to a DNA bound form of Su(H). Whether Hairless and Su(H) can form a DNA bound complex has been reexamined in a gel retardation assay. Hairless and a truncated version of Hairless that binds Su(H) (H[1-293]) are shown to supershift a Su(H)-oligonucleotide complex. It is concluded that Hairless associates with DNA via Su(H) (Morel, 2001).

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm (Cowden, 2002).

This study provides further evidence that Notch signaling is essential for the formation of the mesectoderm at the boundary between the mesoderm and neurogenic ectoderm. Two different Notch target genes were examined: m8 expression appears to depend almost exclusively on Notch signaling, whereas sim is a conditional Notch target gene that is activated only in cells containing Dorsal. Evidence is presented that Snail functions as both a repressor and an indirect activator of Notch signaling. In particular, a transient stripe of the Snail repressor creates a domain of Notch signaling in apolar embryos that contain low, uniform levels of Dorsal (Cowden, 2002).

A crucial finding of this study is that a stripe2-snail transgene induces ectopic expression of m8 and sim in both wild-type and Tollrm9/Tollrm10 mutant embryos, suggesting that the Snail repressor is actually playing a positive role in Notch signaling. Importantly, this stimulatory activity depends on the ability of Snail to function as a transcriptional repressor. Mutant forms of the stripe2-snail transgene that contain single amino acid substitutions in the two repression domains (PxDLSxK and PxDLSxR) fail to induce sim and m8 expression in either wild-type or Tollrm9/Tollrm10 mutant embryos. By contrast, a stripe2-snail/hairy transgene that contains the Hairy repression domain continues to activate both sim and m8 in mutant embryos (Cowden, 2002).

The localized Snail repressor restricts Notch signaling to the mesectoderm of early embryos, presumably by directly repressing Notch target genes. Indeed, the sim 5' regulatory region contains a series of high-affinity Snail repressor sites. It is conceivable that Snail restricts Notch signaling in other developmental processes. For example, after its transient expression in the ventral mesoderm of early embryos, snail is reactivated in delaminating neuroblasts at the completion of germ band elongation. At this stage, Notch signaling subdivides the neurogenic ectoderm into neurons and ventral epidermis. Notch is selectively activated in epidermal cells, where it induces the expression of E(spl) repressors that silence Achaete-Scute proneural genes. The localized expression of the Snail repressor in delaminating neuroblasts might help ensure neuronal differentiation by inhibiting Notch-specific target genes. Removal of snail along with two related linked zinc-finger repressors (Worniu and Escargot) leads to a reduction in the number of CNS neuroblasts (Cowden, 2002).

It is proposed that Snail functions as a gradient repressor to restrict Notch signaling. In precellular embryos, the initial snail expression pattern is broad and extends into the future mesectoderm. During cellularization, the pattern is refined and snail expression is lost in the mesectoderm and restricted to the mesoderm. The early, broad snail pattern might create a broad domain of potential Notch signaling by repressing components of the Notch pathway, such as Delta and lethal of scute. After cellularization, Notch signaling is blocked in the presumptive mesoderm by sustained, high levels of the Snail repressor. However, Notch can be activated in the mesectoderm because of the loss of Notch inhibitors repressed by transient expression of the Snail repressor. According to this model, the dynamic snail expression pattern determines both the timing and limits of Notch signaling (Cowden, 2002).

The analysis of Tollrm9/Tollrm10 embryos suggests that Dorsal functions synergistically with Notch signaling to activate sim expression. A stripe2-NotchIC transgene induces strong sim expression in these embryos, even though they contain low levels of Dorsal and lack Twist. However, the same transgene barely activates sim when crossed into embryos that lack both Dorsal and Twist. By contrast, m8 is strongly expressed in these mutants, indicating m8 is primarily activated by Su(H)-NotchIC and does not require Dorsal (Cowden, 2002).

Perhaps the low levels of Dorsal present in the presumptive mesectoderm are not sufficient to activate sim. Instead, activation might rely on protein-protein interactions between Dorsal and the Su(H)-NotchIC complex within the sim 5' cis-regulatory region. sim contains a number of optimal Su(H) recognition sequences; these might help recruit Dorsal to adjacent sites. By contrast, the stripe2-NotchIC transgene appears to be sufficient to activate m8, even though it contains fewer optimal Su(H) binding sites than the sim 5' cis-regulatory region. Perhaps m8 is ‘poised’ for activation by ubiquitous bHLH activators that are maternally expressed and present throughout early embryos (e.g. Daughterless and Scute). Notch signaling might trigger expression upon binding of the Su(H)-NotchIC complex. By relying on ubiquitous bHLH ‘co-factors’, Notch signaling may be sufficient to activate m8 in diverse cellular contexts. Accordingly, the differential regulation of sim and m8 by Notch signaling is combinatorial and depends on the distribution of distinct co-factors (Cowden, 2002).

Drosophila zfh-1 is downregulated in embryos prior to myogenesis. Embryos with zfh-1 loss-of-function mutation show alterations in the number and position of embryonic somatic muscles, suggesting that zfh-1 could have a regulatory role in myogenesis. Zfh-1 is a transcription factor that binds E box sequences and acts as an active transcriptional repressor. When zfh-1 expression is maintained in the embryo beyond its normal temporal pattern of downregulation, the differentiation of somatic but not visceral muscle is blocked. One potential target of zfh-1 in somatic myogenesis could be the myogenic factor mef2. mef2 is known to be regulated by the transcription factor twist, and Zfh-1 is shown to bind to sites in the mef2 upstream regulatory region and inhibit twist transcriptional activation. Even though there is little sequence similarity in the repressor domains of vertebrate ZEB and zfh-1, evidence is presented that Zfh-1 is the functional homolog of ZEB and that the role of these proteins in myogenesis is conserved from Drosophila to mammals (Postigo, 1999).

Among all zfh family members, Zfh-1 and ZEB share the most sequence similarity in the zinc fingers and homeodomain. The zinc fingers of ZEB bind to a subset of E boxes (and E box-like sequences), with highest affinity for the CACCTG site. The similarity in the zinc fingers of ZEB and Zfh-1 suggests that these motifs in Zfh-1 might also be DNA binding domains. Therefore, whether Zfh-1 can bind to the CACCTG site was tested. Both the N- and C-terminal zinc fingers of recombinant Zfh-1 bind to the site in gel retardation assays. This binding is abolished when the site is mutated. Furthermore, as observed for ZEB, Zfh-1 binds to only a subset of E box sequences; it fails to bind the CATTTG E box sequence. Interestingly, the Zfh-1 binding site also matches the high-affinity site recognized by the zinc finger protein Snail, and Zfh-1 binds quite efficiently to various Snail binding sites in the single-minded gene (Zfh-1 binds better than Snail to Sna5ab, the highest-affinity site). These results demonstrate for the first time that Zfh-1 is a DNA binding protein and that it shows DNA binding specificity similar to that of ZEB and Snail (Postigo, 1999).

Both ZEB and snail are transcriptional repressors. To determine whether Zfh-1 has transcriptional activity, a reporter containing the CACCTG binding site 30 bp upstream of an enhancer was transfected in Drosophila Schneider L2 cells. These cells do not express endogenous Zfh-1 or Snail, and thus the presence of the E box site had no effect on promoter activity. However, cotransfection of a Zfh-1 or Snail expression vector results in repression. A similar level of repression by Zfh-1 was observed when the CACCTG sequence was moved 300 bp upstream of the enhancer, demonstrating that Zfh-1 (and ZEB) can repress at long range. In contrast, Snail failed to repress transcription at this long range. Expression of DNA-binding Zfh-1 (DB-Zfh-1), containing only the DNA binding domain of Zfh-1 but not the repression domain, did not repress, suggesting that the protein has separate DNA binding and repressor domains. These results demonstrate that Zfh-1, like Snail and ZEB, functions as an active transcriptional repressor when it binds to E box sequences (Postigo, 1999).

Because of the overlap in DNA binding specificity, Zfh-1 could target the same genes as Snail. One such Snail-regulated gene is single-minded, which is normally restricted to midline cells and a subset of somatic muscle precursor cells. Snail functions to block ectopic expression of single-minded and other nonmesodermal genes in the mesoderm. Zfh-1 not only binds to the Snail sites on the single-minded promoter but also represses the activity of the single-minded promoter in transfection assays in Schneider cells even more efficiently than Snail, consistent with the finding that sites from the single-minded promoter bind to Zfh-1 more efficiently than Snail (Postigo, 1999).

However, Snail also binds other sequences that are not shared with Zfh-1. In the rhomboid promoter, Snail sites are important to block the expression of rhomboid in the ventral regions during embryogenesis. Contrary to what was found for the Snail sites in the single-minded promoter, Zfh-1 showed little or no binding to the four Snail sites of the rhomboid promoter. And, accordingly, Zfh-1 fails to repress the transcriptional activity of the rhomboid promoter. These results demonstrate that Zfh-1 can interact with only a subset of Snail sites (Postigo, 1999).

Involvement of co-repressors Groucho and CtBP in the regulation of single-minded in Drosophila

Dorso-ventral patterning results in the establishment of the two germ layers in the Drosophila embryo, mesoderm and mesectoderm, that are separated by a strip of cells giving rise to the mesectoderm and eventually to the ventral midline. The mesectoderm is specified by the expression of single-minded which is activated through the concerted action of Dorsal and Twist in addition to a Notch signal. In the mesoderm, sim is repressed by Snail together with the co-repressor C-terminal binding protein (CtBP). This study addresses the involvement of the two co-repressors CtBP and Groucho (Gro) in repression of sim in the neuroectoderm. It was shown earlier that sim is restricted in the neuroectoderm with help of Suppressor of Hairless [Su(H)] and Hairless. Using the female sterile technique, germ line clones deficient for Gro, CtBP or Hairless were generated, and sim mRNA was assayed relative to snail mRNA expression. sim repression requires both co-repressors Gro and CtBP to be fully repressed in the neuroectoderm, suggesting that a repression complex is assembled including Su(H) and Hairless as was shown for other Notch target genes before. Moreover, this work implies that Gro is important for the repression of sim specifically within the mesoderm anlagen, indicating that Snail and CtBP are insufficient to entirely silence sim in this germ layer (Nagel, 2007).

Mastermind mutations generate a unique constellation of midline cells within the Drosophila CNS

The Notch pathway functions repeatedly during the development of the central nervous system in metazoan organisms to control cell fate and regulate cell proliferation and asymmetric cell divisions. Within the Drosophila midline cell lineage, which bisects the two symmetrical halves of the central nervous system, Notch is required for initial cell specification and subsequent differentiation of many midline lineages. This study provides the first description of the role of the Notch co-factor, mastermind, in the central nervous system midline of Drosophila. Overall, zygotic mastermind mutations cause an increase in midline cell number and decrease in midline cell diversity. Compared to mutations in other components of the Notch signaling pathway, such as Notch itself and Delta, zygotic mutations in mastermind cause the production of a unique constellation of midline cell types. The major difference is that midline glia form normally in zygotic mastermind mutants, but not in Notch and Delta mutants. Moreover, during late embryogenesis, extra anterior midline glia survive in zygotic mastermind mutants compared to wild type embryos. This is an example of a mutation in a signaling pathway cofactor producing a distinct central nervous system phenotype compared to mutations in major components of the pathway (Zhang, 2011).

Notch has been shown to play multiple developmental roles in the CNS of several organisms. The Drosophila midline, with its easy to identify neural and glial lineages, has provided examples of multiple and reiterative roles of the Notch pathway within a single CNS lineage. In this study, the characterization of mamΔC mutants indicates how a co-factor within a signaling pathway contributes to the development of different midline cell types and adds to understanding of Notch signaling complexity (Zhang, 2011).

Initial activation of sim in the mesectoderm depends on maternal Notch expression, as N55e11 germline clones lack most sim expression and therefore, contain few midline cells. Likewise, mamΔC germline clones also show a reduction in sim expression. Thus, maternal contributions of both mam and Notch appear to act in the same pathway to activate sim early in development. Similarly, many midline neural phenotypes in zygotic mamΔC mutant embryos are largely consistent with those of N55e11 and Dl3, suggesting mam and Notch act together during the development of these neurons. Notch is required for formation of neurons expressing en and may be needed to maintain en expression in midline cells that develop in the posterior compartment of each CNS segment. The results described in this study suggest mam is also required for the formation of the midline neurons that express en and develop into the iVUMs, the MNB and its progeny. While these cells of the posterior compartment were absent, the H cell and mVUM midline neurons were expanded in mamΔC mutants, similar to N55e11 and Dl3 mutants, suggesting that mam function is needed within the Notch signaling pathway to obtain the variety of midline neurons found in wild type embryos (Zhang, 2011).

The major difference observed between zygotic mamΔC and N55e11 mutants was the presence of midline glia in mamΔC, but not N55e11 mutant embryos during mid to late embryogenesis. Not only were AMG present, but additional AMG survived in the mature CNS midline in mamΔC mutants compared to wild type embryos (and N55e11 mutants). The presence of AMG in mamΔC mutants suggests either (1) the mamΔC mutation is hypomorphic, (2) mam is not required within the Notch pathway for midline glial differentiation or (3) maternally deposited mam transcripts are stable and functional during the Notch signaling event needed for midline glial formation. Results with mam deficiency embryos indicated that midline glia formed and persisted in the complete absence of zygotic mam activity, suggesting it is not the hypomorphic nature of the mamΔC allele that allows the midline glia to form. Currently, it is not possible to distinguish between the other two possibilities, although the last hypothesis is favored due to the timing of midline cell divisions. At gastrulation, each segment contains 8 mesectodermal cells, which each divide, resulting in 16 MPs per segment at stage 10. Cells that give rise to AMG and PMG do not divide again, whereas MPs that develop into neurons each divide once at stage 11. Because MPs that give rise to glia undergo their last division earlier than MPs that give rise to neurons, the Notch signaling event needed for midline glial differentiation may occur prior to Notch events that dictate midline neural fates at stage 11. Maternal Mam protein may linger just long enough to allow midline glia to form, but not long enough to function when MPs divide to give rise to midline neurons slightly later. It is thought that this is the reason N55e11 mutants contain more midline cells per segment than wild type (and mamΔC). In N55e11 mutants, MPs that would normally form glia and not divide, instead take on neural fates and do divide. The data are consistent with this hypothesis, but future, additional experiments are required to properly test it (Zhang, 2011).

In addition to this temporal sensitivity, mam may also be sensitive to spatially restricted events within the midline. Existing evidence suggests the 16 MPs fall into 3 equivalence groups at stage 10: the MP1s, MP3s and MP4s. MP1s are in the anterior, MP3s in the middle and MP4s in the posterior of each CNS segment and effects of mamΔC vary according to these positions. The results indicate that neurons derived from the anterior MP1s are sensitive to N55e11, but not mamΔC; the middle MP3s are more sensitive to N55e11 than mamΔC; while the posterior MP4s are equally sensitive to N55e11 and mamΔC. In other words, mamΔC mutants 1) differ with N55e11 mutants in neurons derived from MP1s (MP1 neurons), 2) have similar, less severe effects compared to N55e11 mutants in cells derived from the MP3s (the H cell and H cell sib) and 3) the same effects as N55e11 mutants in cells derived from the posterior MP4s (mVUMs, iVUMS and MNB). These differences may be due to region specific differences in expression of other midline regulators that combine with Notch and/or Mam to control cell fate specification during embryogenesis. Possible candidates include hedgehog and wingless, which are expressed in the midline, affect cell fate and both interact with mam in a Notch-independent manner in other tissues. In any case, clear differences in zygotic mam and Notch mutations within the midline exist and demonstrate that variations in different Notch signaling components can alter the cellular composition of the CNS in unique ways (Zhang, 2011).

Close examination of mamΔC and N55e11 mutants during mid embryogenesis indicates they also differ in sim expression. After stage 10, sim diminishes in N55e11 mutants, but persists in mamΔC mutants. Likewise, midline glia, which are known to require sim expression to differentiate, do not develop in N55e11 mutants, but do develop in mamΔC mutants. The data indicate that all midline lineages that normally express sim are absent in N55e11 mutants, while midline lineages that do not normally express sim are present and expanded in zygotic mutants of N55e11. Therefore, similar to the initiation of sim expression early, the maintenance of sim expression at this later time also appears to require zygotic Notch activity. In contrast, the results suggest sim expression persists in zygotic mamΔC mutants (Zhang, 2011).

In the canonical Notch pathway, Mam normally functions as a co-factor and collaborates with both the NICD and Su(H) to activate target genes. Consistent with this role, overexpression of mam alone does not affect the number of AMG generated at mid embryogenesis, whereas the overexpression of the NICD in wild type embryos increases AMG cell number. Overexpression of the NICD in a mamΔC mutant background still increased the number of AMG during this stage, further supporting the idea that zygotic mam is not needed at this time. During late embryogenesis, mamΔC mutants contained extra AMG. Mutations in mam are known to promote neural tissue at the expense of ectoderm and this may result in the production of additional Spi, which inhibits apoptosis and allows extra midline glia to survive (Zhang, 2011).

Altogether, the data suggest a high level of complexity in the regulation of CNS target genes of Notch. Notch likely interacts with additional cell-lineage specific co-activators other than, or in addition to, Mam in certain cells. In this way, combinatorial interactions between components of Notch signaling and other signaling pathways can lead to different outputs in various cell types, increasing cell diversity and function. These result indicate mamΔC mutants contain AMG and PMG, whereas N55e11 mutants do not. While this report describes major disruptions in mam, less severe mutations, such as small deletions, insertions or polymorphisms could also affect the midline and modify its cellular composition. Because mam mutations have more subtle effects on the midline compared to mutations in Notch or Delta, they may be tolerated more than mutations in major components of the pathway and actually contribute to CNS cellular variation in natural populations. Future experiments are needed to fully explore these functional differences between mam and Notch in the midline, as well as other tissues. Such differences can then be exploited to develop progressively specific research and clinical tools to regulate Notch signaling and the cellular composition of tissues from the different sensitivities (Zhang, 2011).

Targets of Activity

Single-minded regulates its own expression in midline cells (Nambu, 1991) and is required for normal gene expression of slit, Toll, engrailed, and rhomboid (Nambu, 1990). Enhancer trap screening has identified a large number of genes which exhibit various midline expression patterns and may be involved in discrete aspects of midline cell development. These genes are potentially regulated by Single-minded (Crews, 1992).

SIM binding sites have been detected in toll and slit. The site is an E box with consensus sequence GTACGTG. There are five consensus SIM binding sites in the proximal 1.5 kb region of the sim promoter. These provide for sim autoregulation (Wharton, 1994).

The ventral midline provides the site for Spitz expression and processing. The Drosophila EGF receptor (DER) is activated by secreted Spitz to induce different cell fates in the ventral ectoderm. Processing of the precursor transmembrane Spitz to generate the secreted form is shown to be the limiting event. The ectodermal defects in single minded mutant embryos, in which the midline fails to develop, suggests that the ventral midline cells contribute to patterning of the ventral ectoderm. The Rhomboid and Star proteins are also expressed and required in the midline. The ectodermal defects of spitz, rhomboid or Star mutant embryos can be rescued by inducing the expression of the respective normal genes only in the midline cells. Rho and Star thus function non-autonomously, and may be required for the production or processing of the Spitz precursor. Secreted Spitz is the only sim-dependent contribution of the midline to patterning the ectoderm, since the ventral defects observed in sim mutant embryos can be overcome by expression of secreted Spitz in the ectoderm. While ectopic expression of secreted Spitz in the ectoderm or mesoderm gives rise to ventralization of the embryo, increased expression of secreted Spitz in the midline does not lead to alterations in ectoderm patterning. A mechanism for adjustment to variable levels of secreted Spitz emanating from the midline may be provided by Argos, which forms an inhibitory feedback loop for DER activation. The production of secreted Spitz in the midline, may provide a stable source for graded DER activation in the ventral ectoderm (Golembo, 1996).

The gene ventral nervous system defective, also called NK-2, is repressed by snail and therefore not expressed in the mesodermal anlage and repressed by single-minded in mesectodermal cells. In the lateral neuroectodermal and/or dorsal epidermal anlagen ventral nervous system defective /NK-2is not expressed due to repression mediated indirectly by decapentaplegic. Twist activates vnd in the posterior portion of the embryo or is a coactivator with Dorsal (Mellerick, 1995).

Single-minded represses wingless, hedgehog and vnd gene expression in developing midline cells. By doing this sim plays a key role in proper patterning of the neuroetoderm by helping to generate the boundary between mesectoderm and ventral ectoderm. This process likely requires simultaneous function of SIM as both a transcriptional activator (of slit and Toll) and transcriptional repressor within the developing midline cells (Xiao, 1996).

Based on its pattern of expression, eyegone is thought to play a role in salivary gland organogenesis. Salivary gland primordium (SGP) development responds to positional information. On the anteroposterior axis, Sex combs reduced (Scr) specifies PS2. In Scr minus embryos, no salivary glands are formed and eyg expression is lost, except for a small patch of cells present at the PS1/PS2 border. In a teashirt minus mutation, Scr is expanded to both PS2 and PS3 and results in enlarged SGPs. The SGP expression of eyg is duplicated in PS3, although its appearance and fading are delayed slightly. Along the dorsoventral axis, the SGP is restricted dorsally by decapentaplegic (dpp), and ventrally by the spitz group of genes. In dpp minus embryos, eyg expression expands dorsally to form a ring that is interrupted ventrally. In several spitz-group mutant embryos, such as single minded (sim), the SGPs from each side move ventrally, and eyg expression expands ventrally. Expression in the trunk is also disordered, which may be a secondary effect of the disruption of the mesoderm (Jun, 1998).

The jing zinc-finger transcription factor, identified as a downstream target of slbo required for developmental control of border cell migration also plays an essential role in controlling CNS midline and tracheal cell differentiation. The jing locus ('jing' means 'still' in Chinese) was initially identified in a screen for mutations that cause border cell migration defects in mosaic clones (Liu, 2001). Zygotically jing transcripts and protein accumulate from stage 9 in the CNS midline, trachea and in segmental ectodermal stripes. Jing protein localizes to the nuclei of CNS midline and tracheal cells implying a regulatory role during their development. Loss of jing-lacZ expression in homozygous single-minded (sim) mutants and induction of jing-lacZ by ectopic sim expression establish that jing is part of the CNS midline lineage. Embryonic recessive lethal jing mutations display genetic interactions in the embryonic CNS midline and trachea, with mutations in the bHLH-PAS genes sim and trachealess, and their downstream target genes (slit and breathless). Loss- and gain-of-function jing is associated with defects in CNS axon and tracheal tubule patterning. In jing homozygous mutant embryos, reductions in marker gene expression and inappropriate apoptosis in the CNS midline and trachea establish that jing is essential for the proper differentiation and survival of these lineages. These results establish that jing is a key component of CNS midline and tracheal cell development. Given the similarities between Jing and the vertebrate CCAAT-binding protein AEBP2, it is proposed that jing regulates transcriptional mechanisms in Drosophila embryos and promotes cellular differentiation in ectodermal derivatives (Sedaghat, 2002).

The jing expression pattern and gene dose effects in the CNS midline and trachea suggest that jing function may be important for the development of both systems. Therefore, CNS axon and tracheal tubule development was assessed in jing homozygous mutant embryos stained with monoclonal antibodies BP102 and 2A12, respectively. In jing3 homozygous mutant embryos, commissural growth cones are often absent in the midline at stage 12 when compared with wild type. By stage 14, homozygous jing3 mutants show losses of longitudinal connections and reduced commissures compared with wild type. Embryos double mutant for jing and sim display phenotypes similar to those of sim homozygotes. Therefore, the sim embryonic CNS axon phenotype is epistatic to that of jing, implying that jing functions downstream of sim (Sedaghat, 2002).

The GAL4/UAS system was used to determine the effects of overexpressing jing in the CNS midline. Flies containing P[sim-GAL4] were crossed to flies containing P[jing-UAS] and their progeny stained with BP102 to assess CNS axon formation. Expression of one copy of P[jing-UAS] specifically in the CNS midline is sufficient to inhibit commissural and longitudinal axon formation. Therefore, the jing midline overexpression phenotype is similar to that resulting from jing loss of function, and phenotypes of jing and sim double heterozygotes. These results demonstrate that appropriate jing dose is a requirement for proper CNS axon development in the CNS midline. Interestingly, a similar CNS axon phenotype is observed after overexpression of sim in the CNS midline (Sedaghat, 2002).

Based on genetic and phenotypic analyses, a role is proposed for jing downstream of sim and trh during CNS midline and tracheal development, respectively. (1) jing expression is not observed prior to that of either sim or trh in the CNS midline and trachea, respectively. jing expression is detected in the CNS midline during stage 9, which comes after the initiation of sim expression and establishment of midline fates. Jing protein is present in tracheal precursor nuclei, coincident with Trh during stage 10. (2) The CNS axon and tracheal phenotypes of homozygous jing mutations are less severe than those of homozygous sim and trh mutations, respectively. However, it cannot be rule out that maternal Jing may rescue the effects of zygotic jing mutations or that jing functions in a combinatorial fashion and therefore may not display severe phenotypes. (3) jing can be activated by ectopic expression of sim, suggesting that sim may regulate jing. The presence of three E-box ACGTG core sites in the 5' regulatory region of jing suggest that this regulation may be direct. (4) The sim and trh embryonic phenotypes are epistatic to that of jing, as shown by double mutant analysis. (5) jing mutations genetically interact with mutations in bHLH-PAS target genes such as sli and btl. The ventral displacement of midline cells in jing and sli double heterozygotes strongly suggests that jing is required for proper sli regulation (Sedaghat, 2002).

Analysis of genomic DNA sequence (GenBank accession number, AF285778) surrounding two lethal P-element insertions in jing reveals that there are three putative DNA binding sites for Tgo::Sim and Tgo::Trh (CMEs), and one for the HMG SOX protein called Fish-hook (also known as Dichaete, D) (TACAAT) in the 5' regulatory region of jing. This raises the possibility that jing may be a direct transcriptional target of bHLH-PAS heterodimers and SOX proteins including Tgo:Sim, Tgo:Trh or Fish-hook (Sedaghat, 2002).

Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, several cis-regulatory modules have been identified that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, the Gal4/UAS system was used to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16 were identified. Furthermore, these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, it could be shown to regulate rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolishes rst expression; indicating that the regulation of rst by Sim is direct (Apitz, 2005).

Rst has complex and multifaceted functions throughout the development of the fly, which include myogenesis, eye development, as well as axonal pathfinding in the optic lobes. To gain a better understanding of these functions at the levels of gene regulation and signal transduction, a number of tests were designed to identify both the transcriptional activators and their respective targets surrounding the rst locus. In a preceding study (Apitz, 2004), a number of DNA segments upstream of rst were characterized and regulatory regions were discovered that mediate gene expression in myoblasts, midline, and eyes, respectively. In the present study these results were supplemented with an in vivo screen to identify regulators of rst expression using the Gal4/UAS system. Several factors were discovered that are able to induce ectopic Rst expression and to activate reporter gene expression via rst cis-regulatory sequences (Apitz, 2005).

The experimental route taken to identify protein factors involved in the regulation of the rst gene is based on the detection of their potential to induce ectopic Rst expression in vivo. The use of sca-Gal4 as a driver line in this experimental approach is based on the following criteria. sca-Gal4 mediates expression in neuroectodermal cells of the embryo. At embryonic stage 10, these cells can be examined for ectopic Rst expression because no endogenous Rst expression is found at this time in these cells; this allows operators to obtain clear-cut and unequivocal results. The use of alternative Gal4 driver lines did not prove suitable because of the dynamic expression of Rst during all developmental stages, and due to its subcellular localization. For example, when dll-Gal4 is used as a driver, it is difficult to distinguish between ectopic Rst expression induced in the apical tips of cells of the leg discs, and endogenous Rst expression in the overlaying ectodermal cells. Furthermore, the use of sca-Gal4 has the advantage that neuroectodermal cells are not fully differentiated cells. This may more closely resemble the developmental state of the cells in which Rst expression is normally induced endogenously, e.g., in undifferentiated cells of the developing eye disc. However, this approach generally fails to reveal transcription factors that need a coactivator for induction of rst expression, which is not present in the cells of the neuroectoderm at embryonic stage 10. This may explain the failure of Dmef2 to induce rst expression at this stage. It was shown, however, that Dmef2 is able to induce rst expression at later stages by the use of rst-lacZ constructs. The function of the different rst-lacZ constructs has been linked to specific developmental circumstances (Apitz, 2004) and their activation by corresponding factors is consistent with the known roles of these proteins in development (Apitz, 2005).

Single-minded (Sim) is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor that serves as a master regulatory gene of CNS midline development. The CNS midline is derived from two mesectodermal cell rows that intermingle at the ventral midline following gastrulation of the embryo. The ventral midline cells move into the interior of the embryo where they function (among other roles) in the patterning of the CNS by secreting axonal guidance cues. It has been shown that expression of more than 20 genes depends on sim. Because Rst expression in the embryonic midline is lost in sim mutant embryos, rst is added to this list (Apitz, 2005).

Tgo, the coactivator of Sim, is ubiquitously expressed in the embryo. Thus, ectopic expression of target genes can be induced by Sim in transactivation studies. The early onset of strong ectopic expression of Rst in neuroectodermal cells under the transcriptional control of ectopic Sim suggests that rst may be its direct target. Several lines of evidence support this hypothesis. The expression of rst in the embryonic midline sets in shortly after gastrulation in the ventral midline, following accumulation of Sim protein in these cells. In an expression profiling study, rst was assigned to a group of 37 genes that mimic the expression pattern of sim in the embryonic midline. These genes are therefore likely direct targets of Sim/Tgo heterodimers. Since Sim does not act on the F5 fragment in the transactivation assay, the Sim-responsive rst cis-regulatory sequence was mapped to a 2.5-kb region contained within the F6 fragment, i.e., F6d. F6d was shown to contain a regulatory module for expression in the embryonic midline (Apitz, 2004). Two putative central midline elements (CME) are located in this region and both are conserved between D. melanogaster and D. pseudoobscura. It has been shown that heterodimers of Drosophila Sim and human Tgo bind to the CME and that Sim activates midline gene transcription through CME. Site-directed mutagenesis studies described in this paper show that both CME in F6d are required for reporter gene expression in the embryonic midline. Significantly, analysis of polytene chromosomal binding sites of ectopically expressed Sim in salivary gland nuclei revealed that Sim binds to chromatin within chromosomal subdivision 3C, coinciding with the rst locus. These data indicate that rst expression in the embryonic midline is directly regulated by Sim (Apitz, 2005).

Sim is also involved in postembryonic optic lobe development. Although the expression pattern and axonal pathfinding defects of sim and rst point to a possible regulation of rst by Sim in the optic lobes, no evidence was found supporting this hypothesis using simts mutants. This result is corroborated by analysis of rst cis-regulatory elements (Apitz, 2004). F6d, the rst regulatory sequence that mediates reporter gene expression in the embryonic midline and which is activated by ectopic Sim, does not display a corresponding reporter gene expression in the optic lobes. These results are unexpected since they point to a differential set of Sim target genes in postembryonic stages as compared to the embryo. It will be interesting to examine whether other known target genes of Sim are expressed independently of sim in the optic lobes as well, and whether this differential regulation depends on the differential use of alternative cofactors in the embryo and in the optic lobes, respectively. Since Tgo colocalizes with Sim in the optic lobes, one may suspect the involvement of additional uncharacterized cofactors. The existence of tissue-specific cofactors that direct Sim/Tgo activity to the embryonic midline have already been postulated by other authors (Apitz, 2005).

Identification of motifs that are conserved in 12 Drosophila species and regulate midline glia vs. neuron expression

Functional complexity of the central nervous system (CNS) is reflected by the large number and diversity of genes expressed in its many different cell types. Understanding the control of gene expression within cells of the CNS will help reveal how various neurons and glia develop and function. Midline cells of Drosophila differentiate into glial cells and several types of neurons and also serve as a signaling center for surrounding tissues. This study examined regulation of the midline gene, wrapper, required for both neuron-glia interactions and viability of midline glia. A region upstream of wrapper required for midline expression was identified that is highly conserved (87%) between 12 Drosophila species. Site-directed mutagenesis identifies four motifs necessary for midline glial expression: (1) a Single-minded/Tango binding site, (2) a motif resembling a Pointed binding site, (3) a motif resembling a Sox binding site, and (4) a novel motif. An additional highly conserved 27 bp are required to restrict expression to midline glia and exclude it from midline neurons. These results suggest short, highly conserved genomic sequences flanking Drosophila midline genes are indicative of functional regulatory regions and that small changes within these sequences can alter the expression pattern of a gene (Estes, 2008).

To facilitate the identification of sequences responsible for wrapper expression in the midline glia of Drosophila, the genomic region flanking the wrapper transcription unit was examined to determine the degree of conservation between the 12 available Drosophila species. The regions most likely to contain regulatory control elements (motifs) of wrapper are tractable; the genomic regions flanking the transcription unit and the first intron are relatively small. The results of this analysis highlighted a region between -492 and -326 upstream of the transcription start site of wrapper that is highly conserved in all Drosophila 12 species examined, particularly a 70-bp region. To test if these sequences are responsible for the wrapper expression pattern in embryos, this genomic region was amplified within a 884-bp fragment, and then fused it to the green fluorescent protein (GFP) reporter gene within the pHstinger vector, which contains a minimal Hsp70 promoter. This DNA construct (wrapper W:GFP) was injected into D. melanogaster embryos using P element-mediated transformation to generate stable fly lines. Embryos containing this construct express GFP in midline glia beginning at stage 12 of embryogenesis and throughout larval stages. It was confirmed that GFP was expressed in midline glia by staining embryos simultaneously with either (1) wrapper and GFP or (2) sim and GFP. Because wrapper protein is found at the surface of midline glial cells, but the GFP produced by pHstinger localizes to the nucleus, wrapper protein encircles the GFP in these cells. The wrapper W:GFP reporter construct also drives expression in a few additional cells within the lateral CNS and muscles, a pattern that differs from the endogenous wrapper expression pattern. This suggests that the W fragment, although sufficient to drive high levels of expression in midline glia, lacks certain sequences that exclude expression in lateral CNS cells. To confirm the midline expression pattern generated by the reporters, all subsequent experiments were performed by staining embryos with both sim and GFP at stage 16 of embryogenesis. These experiments revealed that GFP generated by the wrapper W:GFP reporter gene was indeed expressed in the midline glia, but not in the cells that develop into midline neurons (Estes, 2008).

Next, to determine the minimal sequences required to provide expression in midline glia, this 884-bp region was divided into several subregions, fused to GFP within the pHstinger vector and tested for the ability to drive midline expression in transgenic embryos. Region E, extending from sequences -756 to -286, is sufficient to drive high levels of GFP expression in midline glia. Moreover, a smaller 166-bp (-492 to -327) G fragment, and an even smaller 119-bp (-492 to -374) internal K fragment, that both include the highly conserved region, are also sufficient to drive GFP expression in midline glia, but the level of expression is reduced compared to that of the E fragment and the intact 884-bp W fragment. None of the other reporter constructs drove GFP expression in the midline. The K fragment is also expressed in a subset of midline neurons, including progeny of the median neuroblast, suggesting that the larger W, E, and G fragments contain a silencer, which is absent from the K fragment and normally represses expression in these midline neurons (Estes, 2008).

Next, to determine if the observed conservation at the sequence level between Drosophila species reflects conservation in function, the corresponding E region from D. virilis was tested to see if it could drive GFP reporter expression in the midline glia of D. melanogaster. The E region is also located upstream of wrapper in D. virilis and is 476 bp in length, while it is 462 bp in melanogaster. The entire E region is 58.4% identical in the two species, and the 70-bp highly conserved section differs by only six nucleotides. The midline expression pattern provided by the D. virilis wrapper E:GFP construct in D. melanogaster flies is indistinguishable from that of the corresponding D. melanogaster E region. These results suggest that the location and function of the regulatory sequences of wrapper have been conserved between D. melanogaster and D. virilis (Estes, 2008).

To determine if previously identified midline transcription factors affect wrapper through these regulatory sequences, the wrapper W:GFP reporter gene was tested in a number of mutant backgrounds. First, the effect of sim mutations on the reporter gene was tested by placing the 884-bp wrapper W:GFP transgene into a simH9 mutant background, a mutation that eliminates Sim protein expression. In this background, GFP expression was abolished in most cells, suggesting that sim expression is required for wrapper transcriptional activation in the midline. A few remaining cells did express GFP and these are likely lateral CNS cells also observed in wild-type embryos containing the wrapper W:GFP reporter (Estes, 2008).

Next, the reporter gene was tested in a spitz (spi) mutant background. Spi is a signaling molecule that plays multiple roles during Drosophila development. Wrapper protein is normally found on the surface of midline glia where it mediates direct contact with the lateral CNS axons that cross the midline and promotes survival of midline glia. In wrapper mutant embryos, this intimate interaction cannot occur and additional midline glia die. The amount of spi signaling provided by lateral CNS axons determines how many midline glia survive in each segment. The spi mutation severely disrupted CNS development so that the sim positive cells remained on the ventral surface of the embryo. Only a few of the sim positive cells also express GFP driven by wrapper regulatory sequences, suggesting these are the remaining midline glia. The cells expressing sim, but not GFP, are likely midline neurons, while cells expressing GFP and not sim are lateral glia, because they also express reversed polarity (repo), a marker of lateral CNS glia. These results indicate spi mutations reduce the number of midline glia in the embryo and also reduce expression of the wrapper W:GFP reporter gene (Estes, 2008).

In addition to sim and tgo, the transcription factors Dichaete (D), a Sox HMG protein, and Dfr, a POU domain protein, regulate genes expressed in midline glia. The D protein directly interacts with the PAS domain of Sim and the POU domain of Dfr and all three genes activate expression of slit in midline glial . The wrapper W:GFP construct was tested in both a D and dfr mutant background. In both cases, the number and behavior of midline cells was altered and they did not migrate to the dorsal region of the ventral nerve cord, as they normally do. While development of midline cells was disrupted in these mutant backgrounds and fewer midline glia were present, robust GFP expression was still observed from the reporter construct in the midline cells that remained, suggesting that (1) D and Dfr do not directly activate wrapper via these regulatory sequences, (2) additional, redundant factors exist that can substitute for them, or (3) they can substitute for one another (Estes, 2008).

In summary, midline cell development was disrupted in sim, spi, D, and dfr mutant backgrounds. The simH9 mutation eliminated midline glia and neurons, while a mutation in spi eliminated most midline glia. As predicted, both sim and spi mutations severely reduced the number of cells expressing GFP driven by the wrapper W:GFP reporter gene. In the D and dfr mutants, the number of midline glia was reduced and the remaining midline glia expressed high levels of GFP (Estes, 2008).

Ectopic sim expression converts neuroectodermal cells into midline cells and activates downstream, midline genes. To test the effect of ectopic sim on wrapper expression, sim was overexpressed using the UAS/GAL4 system and it was found that wrapper was expressed in neuroectodermal cells outside of the midline, but not in all cells that overexpress sim. In the UAS-sim/da-GAL4 embryos, wrapper is activated in cells that correspond to the lateral edges of the CNS and the cells in the anterior of each segment, with gaps in the expression pattern. Next, whether overexpression of the secreted form of spi could expand wrapper to cells outside the midline was tested. Ectopic expression of secreted spi with the da-GAL4 driver also expanded wrapper expression. To determine if it is possible to expand the expression domain of wrapper further, sim was overexpressed together with spi. This caused additional expansion of the wrapper domain into broad stripes within ectodermal cells. In addition, overexpression of either sim or spi causes severe disruption in embryonic development (Estes, 2008).

Next, the ability of sim and spi, either alone or together, to expand expression of the wrapper reporter genes was tested. Expression from both the full-length reporter construct, wrapper W:GFP, and the smaller wrapper G:GFP construct expanded in the UAS-sim/da-GAL4 embryos to a greater extent than the endogenous wrapper gene. The expression pattern provided by the reporter constructs differs from the endogenous wrapper expression pattern, suggesting that either (1) some of the sequences that normally repress wrapper in tissues outside the midline glia may be missing in these wrapper W and G constructs, or (2) ectodermal cells overexpressing sim may undergo cell death and the GFP marker may be more stable in these dying cells compared to wrapper. Overexpression of spi alone also expanded reporter gene expression driven by both the wrapper W:GFP and wrapper G:GFP constructs. The GFP expression domain was expanded to a greater extent in embryos overexpressing sim together with spi compared to those overexpressing either gene alone. Taken together, the results indicate that (1) limiting the wrapper regulatory sequences and (2) increasing the cells that express sim and spi converts the highly specific expression pattern of wrapper from a single strip of CNS cells to a more general pattern throughout the ectoderm of the embryo. In addition, these results suggest that both the sim transcription factor and spi signaling molecule can activate transcription through these sequences derived from the regulatory region of wrapper (Estes, 2008).

To both (1) identify functionally important motifs needed for wrapper expression and (2) determine if all the invariant nucleotides within the conserved 70-bp region of wrapper are essential for the observed midline glial expression pattern, effects of select mutations within the wrapper G region were tested. Previous studies have demonstrated the importance of sim/tgo, D, dfr, and spi for the expression of midline glial genes and, therefore, possible binding sites for these factors were sought. To examine both predicted binding sites, as well as other conserved sequences that may contain binding sites for novel factors, the region was divided into eight motifs that were tested for their effect on midline glia expression (Estes, 2008).

Each of these conserved motifs was tested by changing 2-3 nucleotides in the context of the D. melanogaster G fragment. The altered G fragments were then inserted independently into the pHstinger vector and injected into fly embryos to test their ability to drive midline expression (Estes, 2008).

Despite the high degree of conservation within this region, only four of the eight mutations that were tested (G1, G2, G5, and G7) caused a noticeable reduction in reporter expression. Two of the mutation sets destroyed midline expression of the G reporter construct. The putative Sim/Tgo binding site (G2: CACGT) was needed for midline expression, because changing this sequence to GAAGT eliminated midline glial expression. In addition, another sequence, ATTTTATC (G5), located upstream of the G2, was required for expression of the reporter gene in wild-type embryos and changing this sequence to ATTGGATC eliminated midline glial expression. Two additional sites within the G fragment of wrapper are needed for midline expression: CGGAGAG (G7) and CACAAT (G1). If either of these motifs is altered, midline glial expression is greatly reduced, but not completely eliminated (Estes, 2008). In contrast, the other four sets of mutations had no detectable negative effect on midline glial expression of the reporter gene, even though these sequences are conserved in all 12 Drosophila species. Mutation sets G4, and G8 did cause a low level of reporter gene activation in some midline neurons, suggesting that repressor proteins present in midline neurons may interact with these regions of the wrapper regulatory region. Finally, mutation G3 had no detectable positive or negative effect on expression of the reporter gene, despite being conserved in all 12 Drosophila species. In summary, the various mutations had three different effects on expression driven by the wrapper regulatory sequences: (1) some reduced midline glial expression, (2) some caused the inappropriate activation of the wrapper reporter in midline neurons, and (3) one was conserved, but apparently had no effect on wrapper regulation, in the context of the experiments presented here (Estes, 2008).

Therefore, these experiments suggest that Sim/Tgo heterodimers may directly regulate wrapper gene expression. (1) Activity of the wrapper W:GFP reporter gene is severely reduced in a sim mutant background, suggesting sim is necessary for expression of this transgene and that sim regulates wrapper by activating transcription through these sequences. (2) Midline activity of the wrapper reporter gene is abolished by eliminating the single CME (CACGT) present within this region. (3) wrapper reporter gene expression is expanded in sim overexpression embryos. Future biochemical studies will determine if Sim/Tgo heterodimers directly interact with the wrapper regulatory motif identified in this study (Estes, 2008).

The studies described in this study demonstrate that the wrapper reporter genes are sensitive to levels of spi signaling. Mutations in spi reduce wrapper reporter gene expression and overexpression of the secreted form of Spi, together with Sim expands, not only the expression domain of the endogenous wrapper gene, but the wrapper reporter genes as well. Spi binds the Epidermal Growth Factor Receptor in midline glia, leading to MAPK activation and subsequent activation of the ETS transcription factor, pnt. Therefore, it may be Pnt that directly activates wrapper transcription through the regulatory sequences studied in this study. One of the identified motifs needed for transcriptional activity of wrapper is: CGGAGAG, which loosely conforms to the consensus binding site for ETS transcription factors (C/A)GGA(A/T)(A/G)(C/T). However, further experiments are needed to determine if Pnt directly interacts with these regulatory sequences, as well as the precise mechanism whereby spi signaling regulates wrapper. Taken together with previous studies, these results suggest that the spi signaling pathway may play at least two roles in promoting survival of midline glia: (1) activating wrapper, needed for neuron-glial interactions and (2) phosphorylating, thereby inactivating Head involution defective, which would otherwise cause programmed cell death in midline glia (Estes, 2008).

Many genes expressed in the CNS of metazoan organisms are regulated through synergistic interactions between Sox HMG-containing proteins and POU domain proteins. Recently, many vertebrate genes expressed in the developing CNS have been shown to contain highly conserved noncoding DNA regions enriched for binding sites for three classes of transcription factors: Sox, POU, and homeodomain proteins. Experiments indicated that Sox and POU proteins work together to activate, while homeodomain proteins repress and limit expression of CNS genes. Interestingly, several motifs identified in this study as important for regulation in midline glia of Drosophila resemble binding sites for Sox (G1: CACAAT), POU (G4: ATGCAAAT, G6: ATGCAACA, and G8: ATGCGTGG), and homeodomain proteins (G5: ATTTTATC) (Estes, 2008).

That the wrapper K:GFP, but not the wrapper G:GFP construct is expressed in certain midline neurons, identifies a midline neural silencer in the 43-bp region present in the G fragment, but absent in the K fragment. Within this region, 27 bp are highly conserved in all 12 Drosophila species and two of the three mutations in the G fragment that cause slight activation of reporter gene expression in midline neurons are found within the 43-bp region. All three sites that lead to activation in midline neurons, G4, G6, and G8, conform to a POU domain binding site, suggesting a POU domain protein expressed in midline neurons may bind to one or more of these sites to keep the wrapper gene silent (Estes, 2008).

One POU domain protein, Dfr, binds to the sequence ATGCAAAT in other gene regulatory regions to activate transcription, including those of two genes expressed in midline glia: dfr itself and slit. This sequence is found at site G8 in the wrapper regulatory region, but when changed to ATGCTAGC, caused a low level of activation in midline neurons, rather than reducing expression in midline glia. Although the number of midline glia is reduced in a dfr mutant background, those that remain express a high level of reporter gene expression driven by wrapper sequences and the results suggest dfr is not absolutely required for wrapper reporter gene expression in midline glia (Estes, 2008).

Mutations in the POU domain motifs within the wrapper regulatory sequences suggest a notable difference between the CNS genes studied previously in vertebrates and the midline glial gene studied here. The POU domain binding sites appear to limit expression in midline neurons (rather than activate expression as in vertebrate CNS genes), and it is the Sox and homeodomain binding sites that are needed for activation. This may reflect a key difference in regulatory control of glial vs. neural genes and it is plausible that other midline glial genes excluded from midline neurons will contain silencer elements similar to the one identified in this study, but further experiments are needed to confirm this (Estes, 2008).

Common motifs shared by conserved enhancers of Drosophila midline glial genes

Coding sequences are usually the most highly conserved sectors of DNA, but genomic regions controlling the expression pattern of certain genes can also be conserved across diverse species. In this study, five enhancers were identified capable of activating transcription in the midline glia of Drosophila melanogaster and each contains sequences conserved across at least 11 Drosophila species. In addition, the conserved sequences contain reiterated motifs for binding sites of the known midline transcriptional activators, Single-minded, Tango, Dichaete, and Pointed. To understand the molecular basis for the highly conserved genomic subregions within enhancers of the midline genes, the ability of various motifs to affect midline expression, both individually and in combination, were tested within synthetic reporter constructs. Multiple copies of the binding site for the midline regulators Single-minded and Tango can drive expression in midline cells; however, small changes to the sequences flanking this transcription factor binding site can inactivate expression in midline cells and activate expression in tracheal cells instead. For the midline genes described in this study, the highly conserved sequences appear to juxtapose positive and negative regulatory factors in a configuration that activates genes specifically in the midline glia, while maintaining them inactive in other tissues, including midline neurons and tracheal cells (Fulkerson, 2010).

The results described in this study indicate that the four genes expressed in the midline glia contain enhancers with subregions conserved in 11 or 12 of the sequenced Drosophila genomes. These conserved subregions contain one or more of the four motifs previously identified in the wrapper regulatory region, are highly A/T rich, and needed for robust expression in the midline. These results confirm the importance of several transcription factor-binding sites for midline glial activation. One of these sites, the CME, binds both Sim/Tgo and Trh/Tgo heterodimers and, when multimerized, can drive reporter gene expression in both midline and tracheal cells. Two lines of evidence indicate that the context of the CME determines whether or not it can be utilized to drive expression in these two tissues. (1) The sequences flanking the CMEs are highly conserved in the four genes discussed in this study, Glec, oatp26f, liprinγ and wrapper, suggesting that the location and sequence of other transcription factor-binding sites are constrained. (2) Changing the sequences flanking the CME in the synthetic multimers can eliminate expression in the midline, trachea, or both tissues (Fulkerson, 2010).

A multimerized CME in the context of the 4Toll:GFP reporter was expressed in both the midline and trachea and quite sensitive to slight modifications in flanking sequences. Changing 5-7 nucleotides on either side of the CME within this multimerized construct either substantially elevated expression in the trachea and eliminated it in the midline (T rich:GFP) or eliminated expression in both tissues (Sox:GFP). Additional combinations between the CME and one of the other midline glial motifs restricted expression to the midline (Pnt:GFP) or the trachea (POU:GFP). These results indicate that testing binding sites for two different factors next to one another can disrupt the endogenous ordering and spacing of the sites within the enhancers. Significantly, the Toll:GFP and Pnt:GFP reporters, unlike the intact enhancers described in this study, drive GFP expression in both midline neurons and glia. This midline expression pattern suggests that the synthetic multimers may lack repressor-binding sites that restrict expression to midline glia. Taken together, these results demonstrate the sensitivity of CME function to flanking sequences within the midline enhancers (Fulkerson, 2010).

Existing experimental evidence suggests that unlike most transcription factors, Sim/Tgo heterodimers (as well as Trh/Tgo heterodimers) preferentially binds one sequence over all others: ACGTG, the CME. Within the enhancers described in this study, sites flanking the CME have remained unchanged over evolutionary time due, in part, to similarities between binding sites for Sim and Trh and the molecular consequences of changing nucleotides adjacent to the CME. This conservation may ensure transcription is restricted to the midline glia and repressed in tracheal cells. In addition to the midline enhancers reported in this study, regions conserved among Drosophila species were found within the known midline enhancers. For instance, a 1.0-kb enhancer present in the first intron of slit drives expression in the midline glia and it contains a single CME and a 32-bp sequence conserved in 11 Drosophila species. It is important to note that the number of midline enhancers described in this study is limited and not all the midline glial enhancers are likely to exhibit such a high degree of conservation. For instance, a midline enhancer of the ectoderm3 gene, was identified that exhibits much less conservation among Drosophila species and presently, the basis for the observed variation among enhancers is unknown (Fulkerson, 2010).

The Ets transcriptional activator, pnt, a downstream effector of EGFR signaling, and Drifter, a POU domain protein, are expressed in both embryonic midline glia and tracheal cells. Previous studies have shown that deleting a POU domain-binding site within an enhancer of rhomboid eliminated expression in tracheal cells, but did not affect its midline glial expression. The results described in this study confirm and extend these results and suggest that the location of a POU domain-binding site relative to the CME can play a role in determining if a gene is expressed in the midline glia, the trachea or both. Moreover, swapping the PAS domains between Sim and Trh proteins indicated that additional, midline or tracheal specific cofactors bind to the PAS domains of the individual proteins and likely to determine which genes are expressed in the two different cell types. This may be the reason sequences adjacent to the CME play such a critical and sensitive role in determining which tissues express the various reporter genes described here. To activate the midline genes, Sim may interact with Drifter and Pnt and bind to sequences flanked by different binding sites compared with sequences bound by Trh, Drifter, and Pnt needed to activate tracheal genes. The simplicity of the multimers studied in this paper raise the possibility that different PAS heterodimers may specifically interact with other factors, such as Drifter and Pnt, in a manner that depends on the relative location and/or distance between each binding site, as has been described for nuclear hormone receptor complexes (Fulkerson, 2010).

The results confirm those of Swanson (2010), who found binding sites can be juxtaposed in different ways within enhancers to favor particular short-range interactions, and, in this way, various combinations of transcription factor binding sites (inputs) can result in more than one output. Similarly, the motifs described in this study can be combined in different ways that result in either midline or tracheal expression. The results indicate the proximity of the CME to activators, one another and/or to repressors could contribute to the level of expression observed in the trachea and midline. This study focused on activator sites, but repressor sites are also likely present and restrict expression to certain cell types. Previous studies in Drosophila embryos have revealed the complexity of the transcriptional regulatory 'grammar' and have shown that the transcriptional output from various genes can be determined by the stoichiometry, affinity, spacing, arrangement, and distance between activator and repressor sites (Fulkerson, 2010).

The high degree of conservation within the midline enhancer subregions examined in this study here belies known properties of transcription factors and their recognition sequences, as well as observations made for many early developmental regulators of Drosophila development. Most transcription factors can vary considerably in the sequences they recognize and tend to bind to related sites with different affinities. This property would suggest that enhancers need not be strictly conserved to function, in contrast to what is reported here. The pattern of conserved sequences within these identified enhancers suggests that the transcription factors that bind these regions do so in a conserved order and spacing pattern. These results suggest that Sim and Trh may interact with other proteins to form an 'enhanceosome'-like complex, similar to that observed in the regulation of the interferon-β gene, in which activators and HMG proteins interact to form a specific multiprotein complex, with a defined structure. This model contrasts with the 'information display/billboard' model of enhancer function. In that model, enhancers are bound by a group of independent factors or group of factors that work together to promote or repress transcription in particular cell types. An important distinction between the two models is the arrangement of binding sites within an enhancer. Within an enhanceosome, the arrangement of binding sites relative to each other is constrained, whereas within a billboard enhancer, the relative arrangement of binding sites is rather flexible as long as a sufficient number of binding sites work together, in many possible configurations, to recruit factors for transcriptional activation (Fulkerson, 2010).

Results obtained with the midline glial genes examined in this study suggest that midline enhancers may consist of a nucleating enhanceosome-like region that combines with an 'information display/billboard' constellation of additional binding sites. This is supported by results obtained with the 70-bp conserved region of wrapper. When tested alone, it only marginally drives midline expression, whereas in the context of the 166-bp enhancer, it works quite well. Moreover, the 166-bp region of virilis cannot function on its own, but drives high levels of expression in the midline glia of melanogaster in the context of the larger, 476 bp region. That the 166-bp region from virilis cannot work efficiently in the midline suggests the transcription complex that binds to this region may be slightly different in virilis compared with melanogaster. For each enhancer described in this study, the presence of the conserved region is required to obtain expression in the midline glia (Fulkerson, 2010).

After comparing vertebrate genomes and generating reporter constructs with highly conserved noncoding sequences, Bailey (2006) noticed that many of these direct expression to regions of the CNS. It is possible that enhancers of CNS genes are more conserved compared with other gene sets, such as early developmental regulators of Drosophila that have been studied in detail. This may be due to the highly conserved nature of the transcription factors that regulate gene expression in this tissue, many of which have analogous functions in flies and mammals). Sox-binding sites are present throughout conserved regions of CNS genes and one of the similarities between these conserved CNS genes, the extensively characterized interferon-β enhanceosome and midline glial genes is the importance of HMG proteins. These proteins may bend the DNA, facilitating binding to highly structured, multiprotein complexes. The enhancers described here likely bind PAS and Sox proteins together with other conserved CNS regulators and it may be this combination of transcription factors that contributes to the similarly conserved arrangement of binding sites (Fulkerson, 2010).

Numerous combinations of transcription factor binding sites can be used to drive expression in many tissue types. Despite the conservation found in this study, binding sites for transcription factors do vary considerably, making it, at times, difficult to identify enhancers based on sequence conservation. In certain cases, changes within enhancers can generate diverse phenotypes between Drosophila populations. The continuing challenge is to understand both the forces constraining the enhancer sequences between Drosophila species, as well as how changes in these regions lead to significant modifications in the expression pattern of a gene, which over the long term, leads to variation among Drosophila populations and eventually, Drosophila species. For the midline genes described in this study, selection has stabilized the constellation of binding sites found within enhancers, resulting in their conservation among Drosophila species over approximately 40 million years of evolution (Fulkerson, 2010).

A comparison of midline and tracheal gene regulation during Drosophila development

Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, novel enhancers were identified and analyzed that drive midline and/or tracheal expression, and they were compared to previously characterized enhancers. In addition, expression was tested of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea (Long, 2014).

Several families of transcription factors contain members that bind related, but slightly different DNA recognition sequences. Examples include members of the nuclear receptor family and bHLH proteins. Nuclear receptor homodimers and heterodimers bind DNA response elements consisting of two inverted repeats separated by a trinucleotide spacer. Specificity is determined by interactions between protein loops on the second zinc finger of a particular steroid receptor DNA binding domain and the trinucleotide spacer within the DNA recognition site. Similarly, the recognition sequence of bHLH transcription factors is called the E box and consists of the sequence CANNTG. Specific bHLH heterodimers preferentially bind E boxes containing various internal dinucleotides (represented by the NN within the E box). The bHLH-PAS proteins investigated in this study are a subfamily within the bHLH superfamily of transcription factors. The PAS domain helps stabilize protein-protein interactions with other PAS proteins, as well as with additional co-factors, some of which mediate interactions with the environment. The evolutionary relationship of bHLH and bHLH-PAS proteins is also reflected in the similarity of their DNA recognition sequences. The CME is related to the E box and historically has been considered to consist of a five rather than six base pair consensus. Previous results indicated bHLH-PAS heterodimers strongly prefer the internal two nucleotides of the binding site to be 'CG', while the nucleotide immediately 5' to this core helps to discriminate which heterodimer binds the site. The first crystal structure of a bHLH-PAS heterodimer bound to DNA reveals that the recognition sequence of the human Clock/Bmal bHLH-PAS heterodimer actually consists of seven base pairs, rather than five. This is consistent with results reported in this study that suggest Sim/Tgo and Trh/Tgo heterodimers preferentially bind highly related, but slightly different seven base pair sequences. In addition, experiments with fly Sim and human Tgo, called Aryl hydrocarbon receptor nuclear translocator protein (Arnt), using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach, identify the sequence DDRCGTG (D = A, C or T and R = either purine) as the Sim/Tgo binding site. The current results agree with this, although the consensus sequence this study identified by examining known enhancers, is shifted by one nucleotide (DACGTG). In the midline and tracheal enhancers, sixty-six copies of the CME, ACGTG, were found and forty-eight copies of the related sequence, GCGTG, also identified in the SELEX experiments. Half of these GCGTG sites fit the seven bp consensus TGCGTGR and future experiments are needed to determine their importance within the various enhancers. The results indicated that the CME context favored within midline and tracheal enhancers as well as enhancers active in both tissues, was very similar, yet clearly distinct from binding sites of other bHLH and bHLH-PAS heterodimers. Based on the expression pattern of certain reporter genes examined in this study, the same CME may be bound by Sim/Tgo in the midline and Trh/Tgo in the trachea within certain enhancers. Within other contexts, the CME appears to be discriminated by these different heterodimers, because some enhancers drive expression in only one tissue or the other (Long, 2014).

Results from both endogenous enhancers and the synthetic reporter genes confirm the importance of the proximal sequences in limiting expression to either the midline or trachea. While the proximal context of the CME plays a role, additional sequences clearly combine with the CME to ultimately determine if an enhancer is functional in the midline or trachea. Taken together, these results indicate that proximal motifs combine with additional sequences not only to determine whether or not a gene is expressed in the midline or trachea, but also to determine which cellular subtypes express the gene and when it is activated within a tissue. Future experiments will reveal if 1) changing the sequence, AACGTGC, to TACGTGC within a midline enhancer will cause the enhancer to drive expression in trachea as well and 2) if changing the sequence, TACGTGC, to AACGTGC within an enhancer that drives expression in both the midline and trachea, will restrict expression to only the trachea. Sequences proximal to the CME likely affect the affinity of either Sim/Tgo and/or Trh/Tgo heterodimers to the DNA, but binding sites for additional factors that interact cooperatively to stabilize an entire transcription complex are needed for high levels of expression within a particular cell. Moreover, recent experiments indicate that enhancers containing multiple CMEs are activated earlier in the embryonic midline than enhancers containing only one CME. It has been suggested that Sim/Tgo binding sites may be sufficient for activation in the early embryo, but that binding sites for additional transcription factors must combine with the CME to drive expression within the later, more complex embryo (Long, 2014).

Protein Interactions

bHLH proteins usually recognize the E-box as dimers. This is true for MYC, whose partner is MAX, and for ARNT, whose partner is AHR (Wharton, 1994).

Trachealess (Trh) and Single-minded (Sim) are highly similar Drosophila bHLH/PAS transcription factors. They activate nonoverlapping target genes and induce diverse cell fates. A single Drosophila gene encoding a bHLH/PAS protein homologous to the vertebrate ARNT protein was isolated and may serve as a partner for both Trh and Sim. The Drosophila ARNT (DARNT, officially termed Tango ) protein shows complete identity to the human protein in the basic domain, 95% identity in the HLH region, and 56% identity in the region including PAS A, PAS B and the spacer between them. DARNT is expressed ubiquitously during embryogenesis with elevated levels in the tracheal placodes and pits (Zelzer, 1997).

The Drosophila single-minded and trachealess bHLH-PAS genes control transcription and development of the CNS midline cell lineage and tracheal tubules, respectively. Single-minded and Trachealess activate transcription by forming dimers with the Drosophila Tango protein, which is an ortholog of the mammalian Arnt protein. Both cell culture and in vivo studies show that a DNA enhancer element acts as a binding site for both Single-minded::Tango and Trachealess::Tango heterodimers and functions in controlling CNS midline and tracheal transcription. Isolation and analysis of tango mutants reveal CNS midline and tracheal defects. Gene dosage studies demonstrate in vivo interactions between single-minded::tango and trachealess::tango. These experiments support the existence of an evolutionarily conserved, functionally diverse bHLH-PAS protein regulatory system (Sonnenfeld, 1997).

Trh and Sim complexes recognize similar DNA-binding sites in the embryo. To examine the basis for their distinct target gene specificity, the activity of Trh-Sim chimeric proteins was monitored in embryos. Replacement of the Trh PAS domain by the analogous region of Sim is sufficient to convert it into a functional Sim protein. It is concluded that the basic domains of Trh and Sim bind the same site on the DNA and do not confer specificity. The PAS domain of Sim provides midline specificity. The PAS domain of Trh was replaced with that of SIM. Ubiquitous expression of this protein results in a phenotype identical to the one induced by ectopic Sim expression: wide expansion of midline fates up to the ventral border of the tracheal pits. Expression of genes that are known to be targets of Sim, including breathless, rhomboid and S55 also show an expansion of midline cell fates. The PAS domain thus mediates all the features conferring specificity and the distinct recognition of target genes. The normal expression pattern of additional proteins essential for the activity of the Trh or Sim complexes can be inferred from the induction pattern of target genes and binding-site reporters, triggered by ubiquitous expression of Trh or Sim. It is thought that the capacity of bHLH/PAS heterodimers to associate, through the PAS domain, with additional distinct proteins that bind target-gene DNA, is essential to confer specificity (Zelzer, 1997).

During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent on Single-minded, a basic-helix-loop-helix (bHLH)-PAS transcription factor. Fish-hook/Dichaete, a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression. single-minded, Dichaete, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays reveal genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Dichaete directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Dichaete can form homodimers and also associates with other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes (Ma, 2001).

To address whether the sim, Dichaete, and dfr genes might functionally interact to regulate development of the embryonic CNS midline, whether they exhibit overlapping expression in developing midline cells was examined. This was accomplished using anti-Dichaete and anti-Dfr sera, as well as a P[3.7sim-lacZ] marker that mimics sim midline expression. P[3.7sim-lacZ] embryos were immunostained using anti-ß-gal and either anti-Dichaete or anti-Dfr sera. Prominent overlapping expression was detected between Sim and Dichaete in developing CNS midline cells from stage 8 throughout the remainder of germ band extension. Overlap was also detected in a subset of prospective foregut cells. Similar overlapping expression was also detected between Sim and Dfr. Midline coexpression of Dichaete and Dfr was detected by immunostaining wild-type embryos with anti-Dichaete and anti-Dfr sera. Both genes are expressed together in the CNS midline throughout germ band extension. In germ band-retracted embryos, Dichaete exhibits overlapping expression with Sim and Dfr in the midline glia. Dichaete and Dfr are also detected together in lateral cells of the thoracic ganglia and a subset of ventral epidermal cells. These analyses indicate that sim, Dichaete, and dfr are coexpressed in developing CNS midline cells. The midline expression of these three genes also overlaps that of the slit gene, which is a downstream target of Sim (Ma, 2001).

Both loss-of-function and gain-of-function assays were used to detect genetic interactions between sim, Dichaete, and dfr. Mutants are known to show genetic interactions in CNS midline differentiation and in Slit protein expression. Potential cooperative interactions between sim, Dichaete, and dfr in regulating slit gene transcription were examined through the use of a P[1.0slit-lacZ] marker. This reporter contains a portion of a slit intron that drives lacZ expression mimicking that of the native slit gene in developing midline glia; P[1.0slit-lacZ] expression is first detected in germ band-extended stage 11 embryos and is maintained throughout the remainder of embryogenesis. Dichaete null mutant embryos exhibit a misplacement and loss of midline glia, as detected via anti-ß-gal immunostaining. P[1.0slit-lacZ] is expressed normally in stage 11 Dichaete mutant embryos, but during germ band retraction the number of midline glia becomes reduced from wild type, and many cells are located at aberrant ventral positions within the nerve cord. Similar, although less severe, defects are observed in dfr mutant embryos, where some midline glia are displaced from their normal positions. Notably, ß-gal-expressing midline glia are still detected in both Dichaete and dfr mutants, indicating that unlike Sim, Dichaete and Dfr are not absolutely required for P[1.0slit-lacZ] expression or midline glial development (Ma, 2001).

Because homozygous sim mutants exhibit severe CNS midline defects, it is not informative to analyze the phenotypes of Dichaete-sim or dfr-sim double mutants. Instead, potential interactions between Dichaete and sim were examined via a gain-of-function approach using the Gal4/UAS targeted gene expression system. A P[GMR-Gal4] strain that drives Gal4 expression in and behind the morphogenetic furrow in the developing eye imaginal disc was crossed to P[UAS-Dichaete] and P[UAS-sim] strains. P[GMR-Gal4]/+;P[UAS-Dichaete]/+ animals exhibit a moderate eye roughening with disruption of ommatidia organization and loss of mechanosensory bristles. In contrast, ectopic sim expression results in essentially normal eye morphology. The effects of Dichaete and sim coexpression reveal a nonadditive phenotype; there is a stronger disorganization of ommatidia and mechanosensory bristles than seen in flies expressing Dichaete or sim alone, and there is also a dramatic loss of eye pigmentation. These results indicated that ectopic expression of Dichaete and sim synergistically alters normal eye development, and supports the hypothesis that these genes can interact functionally (Ma, 2001).

Analysis of a 380 bp slit midline regulatory fragment has indicated the presence of a single CNS midline element (CME), through which Sim::Tgo heterodimers act. The CME is located within 300 bp of the distal end (farther from the promoter in the native slit gene) of this fragment. An inverted TTCAAT repeat (TTCAATTTCATTGAA) is located 20 bp proximal to the CME. This sequence resembles a (A/T)(A/T)CAAT consensus binding site for Sox proteins, although binding of Sox proteins to a TTCAAT sequence has not been reported. Because sequences present in an extended 1 kb slit DNA fragment are required for normal levels of slit expression in vivo, additional DNA sequences have been obtained. This analysis indicated that no other CMEs are present in the 1 kb slit DNA fragment. However, two perfect Dfr consensus binding sites, ATGCAAAT and CATAAAT, located within 500 bp of DNA proximal to the CME were identified. These two Dfr binding sites are separated by ~150 bp and flank a consensus Dichaete binding site, TACAAT. These data suggest that Dichaete, Sim, and Dfr may all bind to sites present in the 1 kb slit regulatory DNA fragment. To test this possibility, DNA gel mobility shift assays were performed using the Dichaete HMG domain and full-length Dfr protein on double-stranded oligonucleotide probes corresponding to sequences from the slit 1 kb fragment. The Dichaete HMG domain binds strongly to a 26 mer probe containing the TACAAT site. In contrast, Dichaete does not bind consistently to a 26 mer probe containing both TTCAAT sites, suggesting that Dichaete can distinguish between closely related DNA sequences. Dfr protein binds very strongly to a 33 mer probe that contains the ATGCAAAT site, and less strongly to a 32 mer probe containing the CATAAAT site. Dfr binds the ATGCAAAT site both as an apparent monomer and a dimer, because two distinct bands with reduced mobilities are detected. The 1 kb slit fragment thus may integrate the actions of at least three different types of regulatory proteins, represented by Sim, Dichaete, and Dfr (Ma, 2001).

The ability of Dichaete, Dfr, Sim, and Tgo to directly control slit transcription was examined using transient transcription assays in cultured Drosophila S2 cells. The P[1.0slit-lacZ] construct was used as a reporter with various combinations of plasmids that express Dichaete, Dfr, Sim, or Tgo. Dichaete modestly activates P[1.0slit-lacZ] transcription, indicating that in both yeast and fly cells, Dichaete can function as a direct transcriptional activator. Dfr results in little if any activation of P[1.0slit-lacZ], and Dfr and Dichaete together do not exhibit any increased activation over the levels observed for Dichaete alone. Neither Sim nor Tgo alone is able to activate the P[1.0slit-lacZ] reporter, because only background levels of expression are detected. Furthermore, Sim and Tgo together yield only minimal activation. These results imply that although Sim::Tgo heterodimers strongly activate expression of a P[6XCME-lacZ] reporter (>150 units) that contains six multimerized CMEs, additional factors are required to achieve high levels of reporter expression. Significantly, the combination of either Dichaete and Sim::Tgo or Dfr and Sim::Tgo both result in relatively high levels of activation. Thus, both Dichaete and Dfr strongly enhanced the ability of Sim::Tgo heterodimers to activate slit transcription. Comparable levels of activation are observed when all four proteins are expressed together. Taken together, the DNA binding and transcriptional activation assays provide additional evidence that regulation of slit expression in the midline glia requires functional interactions between Dichaete, Dfr, Sim, and Tgo (Ma, 2001).

Functional interactions between Sim, Dichaete, and Dfr may also regulate the midline expression of other genes, including sim and breathless (btl). Thus, sim has autoregulatory functions, and the combined functions of dfr and Dichaete are also required for sustained midline sim expression. In addition, a 2.8 kb interval in the P[3.7sim-lacZ] transgene used in this study contains six evolutionarily conserved CMEs as well as several consensus Dichaete and Dfr binding sites. btl encodes an FGF receptor homolog whose expression in the CNS midline and tracheal cells has been shown to depend, respectively, on Dfr as well as Sim and Tgo, or Trh and Tgo. A 200 bp btl midline/tracheal regulatory region contains three evolutionarily conserved CMEs. Inspection of this region also revealed the presence of a conserved consensus ATCAAT Dichaete binding site located in a 40 bp interval between CME2 and CME3, as well as a conserved consensus GATAAAT Dfr binding site located 40 bp downstream of CME3. Thus, functional interactions between Sim, Dichaete, and Dfr could be a general mechanism to regulate gene transcription during CNS midline development (Ma, 2001).

Sim is a developmental basic helix-loop-helix (bHLH) transcription factor containing a Per-Arnt-Sim (PAS) region of homology. Sim, in analogy to the structurally related bHLH/PAS dioxin receptor, can stably associated with the molecular chaperone hsp90. In the case of the dioxin receptor, release of hsp90 and derepression of receptor function appear to be regulated by ligand binding and dimerization with Arnt, a non-hsp90-associated bHLH/PAS factor. Dimerization with Arnt very efficiently disrupts Sim-hsp90 interaction, a process that required both the bHLH and PAS dimerization motifs of Arnt. Moreover, hsp90 is also released upon dimerization of Sim with the Drosophila PAS factor Per, whereas the hsp90-associated dioxin receptor fails to interact with Sim. These results indicate that hsp90 may play a role in conditional regulation of Sim function, and that Per and possibly bHLH/PAS partner factors may activate Sim by inducing release of hsp90 during the dimerization process (McGuire, 1995).

single-minded: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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