snail

Targets of Activity

Snail as a repressor

The consensus sequence for SNA binding to randomly selected sequences, 5'G/A A/t G/A A CAGGTG C/t A C 3', with a highly conserved core of 6 bases, CAGGTG, shares no significant homology with known binding sequences of other Drosophila zinc finger proteins. However, the CAGGTG core is identical to the core motif of HLH (helix-loop-helix) binding sites. The strongest SNA binding is obtained with sequences containing this core motif whereas reduced binding is seen for sequences with canonical CANNTG HLH motifs. Interestingly, SNA binding is detected in the promoter region of the snail gene. Transient expression in co-transfection experiments using a SNA binding element (SBE) linked to a heterologous promoter indicates that SNA has the ability to function as a transcription activator (Mauhin, 1993).

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 (sim). snail encodes a protein with a zinc-finger motif. The Snail protein recognizes a 14-base-pair consensus sequence that is found nine times in a 2.8-kilobase sim regulatory region. These results provide evidence for the direct control of sim transcription by Snail (Kasai, 1992).

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

rhomboid (rho) encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis (Ip, 1992). Dorsal acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including Twist, to activate rhomboid in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by Snail (Ip, 1992a).

Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains on either side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc-finger gene escargot (esg), whereas the intermediate column does not. esg expression was examined as a probe to investigate the mechanism of neuroectoderm patterning. The effect of dorsoventral patterning genes on esg expression was studied. decapentaplegic, snail and twist repress esg expression outside the neuroectoderm. The expression of esg in the intermediate column is normally repressed, but is de-repressed when Egfr activity is either elevated or reduced. A neurogenic enhancer of esg was identified, and shown to be separable into a distal region that promotes ubiquitous expression in the neuroectoderm and a proximal region that represses the intermediate expression. It is concluded that decapentaplegic, snail, twist and an activator all act through the distal region to initiate transcription of esg in the neuroectoderm. It is proposed that the combination of opposing gradients of Egfr and its ligand creates a peak of Egfr activity in the intermediate column, where Egfr represses esg transcription through the proximal repressor region. These two kinds of regulation establish the early esg expression that prefigures the neuroectoderm patterning (Yagi, 1997).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. col expression is first detected during the interphase of mitotic cycle 14, when expression of head-gap genes has already resolved from initial broad domains into defined stripes. The stripe of col expression is included in that of btd, overlaps that of ems, and is restricted both dorsally and ventrally to neuroectodermal cells. Examination of dorsal (dl) mutant embryos shows that Dl is required for col repression in the mesodermal plate. The ectopic expression of col observed in twist (twi) and snail (sna) mutant embryos suggests that Dl target genes, rather than Dl itself, are involved. Embryos lacking ems function also show a ventral derepression of col expression. Further, at stage 10, ems mutant embryos show an abnormal pattern of col mRNA accumulation, with a mandibular stripe in addition to intercalary stripe of col-expressing cells. This suggests a second role for ems in regulating col. In btd mutant embryos, there is a complete loss of col expression, whereas there is no change in embryos lacking both slp (slp1 and slp2) genes, consistent with previous data establishing that btd but not slp is required for intercalary en and wg expression (Crozatier, 1999).

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, Notch^IC, in transgenic embryos using the eve stripe2 enhancer. The Notch^IC 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 Toll^rm9/Toll^rm10 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 Toll^rm9/Toll^rm10 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 results obtained in Toll^rm9/Toll^rm10 mutant embryos can be interpreted in the context of this Snail gradient model. Owing to the mutant Toll receptor, these embryos contain low, uniform levels of Dorsal that are insufficient to activate twist or snail. The stripe2-snail transgene produces transient expression of the Snail repressor when compared with the endogenous gene. Consequently, the snail stripe creates an early zone of potential Notch signaling in Toll^rm9/Toll^rm10 by repressing Delta, T3, and other components of the pathway. Perhaps the initially intense expression of the stripe2-snail transgene inhibits the activation of m8 and sim, but these genes are activated as expression from the transgene diminishes. Previous studies lend support to the idea that low levels of Snail can repress some target genes such as T3, while failing to repress others (Cowden, 2002).

This does not mean to imply that repression by a Snail gradient is the sole basis for positioning Notch signaling. Previous studies suggest that expression of neurogenic genes such as neuralized are also important for the restricted expression of sim and m8 within the mesectoderm. Perhaps Neuralized and Snail act separately to establish precise lines of Notch signaling (Cowden, 2002).

Notch, like other signaling pathways, is not dedicated to a particular developmental process. While first identified as an agent of neurogenesis, it has been shown to play a role in the dorsoventral patterning of the wing imaginal disk, and the specification of the R7 photoreceptor cell in the adult eye. This study provides additional evidence that Notch signaling specifies the mesectoderm at the ventral border of the neurogenic ectoderm in the early embryo. The regulation of sim may provide insights into how the Notch signaling cassette can perform so many disparate functions (Cowden, 2002).

The analysis of Toll^rm9/Toll^rm10 embryos suggests that Dorsal functions synergistically with Notch signaling to activate sim expression. A stripe2-Notch^IC 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)-Notch^IC 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)-Notch^IC 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-Notch^IC 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)-Notch^IC 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).

Sequential patterns of vnd, ind, and msh expression respond to distinct thresholds of the Dorsal gradient: Snail excludes vnd expression in the ventral mesoderm and restricts expression to the neuroectoderm

A nuclear concentration gradient of the maternal transcription factor Dorsal establishes three tissues across the dorsal-ventral axis of precellular Drosophila embryos: mesoderm, neuroectoderm, and dorsal ectoderm. Subsequent interactions among Dorsal target genes subdivide the mesoderm and dorsal ectoderm. The subdivision of the neuroectoderm by three conserved homeobox genes, ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) has been investigated. These genes divide the ventral nerve cord into three columns along the dorsal-ventral axis. Sequential patterns of vnd, ind, and msh expression are established prior to gastrulation and evidence is presented that these genes respond to distinct thresholds of the Dorsal gradient. Maintenance of these patterns depends on cross-regulatory interactions, whereby genes expressed in ventral regions repress those expressed in more dorsal regions. This 'ventral dominance' includes regulatory genes that are expressed in the mesectoderm and mesoderm. At least some of these regulatory interactions are direct. For example, the misexpression of vnd in transgenic embryos represses ind and msh, and the addition of Vnd binding sites to a heterologous enhancer is sufficient to mediate repression. The N-terminal domain of Vnd contains a putative eh1 repression domain that binds Groucho in vitro. Mutations in this domain diminish Groucho binding and also attenuate repression in vivo. The significance of ventral dominance is discussed with respect to the patterning of the vertebrate neural tube, and ventral dominance is compared with the previously observed phenomenon of posterior prevalence, which governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos (Cowden, 2003).

The ability of Vnd to repress msh in addition to ind raises the possibility that transcriptional repressors expressed in ventral regions of the embryo can inhibit repressors active in more dorsal regions. Support for this hypothesis came from using the Krüppel enhancer to misexpress both ind and msh along the anterior-posterior axis. Ectopic Ind failed to repress vnd expression, while ectopic Msh did not repress either vnd or ind expression. To determine if 'ventral dominance' is restriced to the neuroectoderm, the mesodermal repressor snail was misexpressed in transgenic embryos using the even-skipped (eve) stripe 2 enhancer. The stripe2-snail transgene creates an ectopic domain of snail along the anterior-posterior axis. This ectopic expression leads to a gap in the sim expression pattern. The transgene also causes a gap in the vnd pattern, confirming the model that Snail excludes vnd expression in the ventral mesoderm and restricts expression to the neuroectoderm. The stripe2-snail transgene also creates a gap in the ind pattern. These results support the ventral dominance model, whereby repressors located in ventral regions inhibit repressors expressed in more dorsal regions. Consistent with this 'directionality' of repression, ectopic expression of Vnd, Ind, or Msh does not repress snail (Cowden, 2003).

Further support for ventral dominance of the Snail repressor was obtained by analyzing mutant embryos derived from CtBP germline clones. CtBP is a maternally deposited corepressor protein essential for snail-mediated repression. Removal of this corepressor results in ventral derepression of sim and vnd into the presumptive mesoderm due to loss of Snail mediated repression. However, this ventral expansion of vnd does not result in a transformation of mesoderm into medial neuroblasts. Instead, the expanded vnd pattern is lost at slightly later stages, and expression becomes restricted to lateral regions, similar to the endogenous expression pattern. This lateral restriction is consistent with the observation that neuroblasts are formed in lateral regions of CtBP^- mutants, and not in ventral regions that normally form the mesoderm. Neuroblast segregation can be visualized using a snail antisense RNA probe, which stains all neuroblasts following gastrulation. Sim may be responsible for the late repression of vnd, because vnd expands into the ventral midline of sim mutant embryos. Repression of vnd by Sim is probably indirect because a Krüppel-sim transgene does not alter vnd expression in the lateral neuroectoderm. Perhaps Sim activates an unknown repressor that ultimately inhibits vnd expression in the midline (Cowden, 2003).

Thus the Dorsal gradient directly subdivides the neuroectoderm into separate dorsal-ventral compartments through the differential regulation of three conserved homeobox genes, vnd, ind, and msh. Maintenance of sequential patterns of gene expression depends on cross-regulatory interactions, whereby repressors expressed in ventral regions inhibit repressors active in more dorsal regions. This ventral dominance is evocative of the posterior prevalence phenomenon that governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos. At least one of the cross-regulatory interactions is direct and evidence was presented that Vnd functions as a sequence-specific transcriptional repressor (Cowden, 2003).

Transcriptional repression of ind by Vnd was predicted from previous genetic studies but lateral repression of msh was somewhat unexpected. Previous studies have shown that ectopic Vnd represses msh expression in the procephalic neuroectoderm, where the vnd and msh expression patterns overlap. This result was extended in the present study using a Krüppel-vnd transgene. It would appear that Vnd represses both ind and msh to specify medial neuroblasts. A similar result was seen using the eve stripe 2 enhancer to misexpress snail. Previous studies have shown that Snail acts as a transcriptional repressor to create the boundary between mesoderm and neuroectoderm. As expected, ectopic snail repressed vnd expression but surprisingly, ind was also repressed. These results suggest that the Dorsal gradient separates domains along the dorsal-ventral axis by activating a series of localized transcriptional repressors. According to this model, repressors located in ventral regions selectively repress those located more dorsally, while dorsal repressors do not inhibit ventral repressors. For example, ectopic Vnd represses ind but not snail, while ectopic Ind fails to repress vnd or snail. According to this model, ectopic Ind should repress msh expression. However, because none of the transgenic Krüppel-ind lines persisted until germband elongation when msh expression is uniform, it was not possible to determine if ectopic Ind repressed msh. Similarly, while ectopic Msh failed to repress snail, vnd, or ind expression, the lack of early target genes that are regulated by Msh prevents any definitive conclusions regarding its role as a transcriptional repressor. Both Ind and Msh contain putative eh1 domains, suggesting that they may function as Groucho dependent repressors and previous work supports such a role for Ind and Msh in the ventral nerve cord (Cowden, 2003).

'Ventral dominance' might govern the patterning of the ventral nerve cord in older embryos, in addition to the prepatterning of the neuroectoderm in pregastrulating embryos. Sim might exclude vnd, ind, and msh expression in the ventral midline. In embryos lacking maternal CtBP products, Snail fails to act as a repressor, allowing the ventral expansion of sim and vnd into the presumptive mesoderm. However, vnd expression is ultimately lost from ventral regions, while sim expression persists. As a result, ventral regions form an expanded mesectoderm, while neuroblasts arise from lateral regions. These observations suggest that Sim excludes vnd expression from ventral regions in CtBP mutants, either directly by acting through a CNS specific enhancer or indirectly by activating an unknown repressor. This putative repressor probably does not rely on the CtBP corepressor, as it is still capable of repressing vnd in CtBP germ line clones. According to a ventral dominance scenario, the misexpression of this unknown repressor should inhibit the expression of vnd, ind, and msh in the ventral midline. One potential target for the indirect repressor could be the EGF pathway. The ventral midline is a well-characterized source of EGF signaling and both vnd and ind rely upon EGF signaling for maintenance of expression. By eliminating EGF activation, this midline repressor could prevent vnd and ind expression (Cowden, 2003).

It is conceivable that the ventral dominance model governing cross-regulatory interactions among Vnd, Ind, Msh, Snail, and possibly sim, also applies to the patterning of the vertebrate neural tube. The vertebrate homolog of vnd, Nkx2.2, is expressed in ventral regions of the neural tube, while the homologs of ind (Gsh) and msh (Msx) are expressed in intermediate and dorsal regions, respectively. These neural tube expression patterns match the dorsal-to-ventral positions of vnd, ind, and msh in the ventral nerve cord of Drosophila. Furthermore, the vertebrate homolog of Vnd, Nkx2.2, also functions as a Groucho-dependent transcriptional repressor. A clear prediction of this study is that the misexpression of Nkx2.2 throughout the vertebrate neural tube should lead to the repression of both Gsh and Msx. In contrast, the misexpression of Gsh should repress Msx, but not Nkx2.2. Thus, a cascade of homologous localized transcriptional repressors could subdivide both the vertebrate and invertebrate CNS (Cowden, 2003).

WntD is a target and an inhibitor of the Dorsal/Twist/Snail network in the gastrulating embryo

The maternal Toll signaling pathway sets up a nuclear gradient of the transcription factor Dorsal in the early Drosophila embryo. Dorsal activates twist and snail, and the Dorsal/Twist/Snail network activates and represses other zygotic genes to form the correct expression patterns along the dorsoventral axis. An essential function of this patterning is to promote ventral cell invagination during mesoderm formation, but how the downstream genes regulate ventral invagination is not yet known. wntD (FlyBase name: Wnt8) is shown to be a member of the Wnt family. The expression of wntD is activated by Dorsal and Twist, but the expression is much reduced in the ventral cells through repression by Snail. Overexpression of WntD in the early embryo inhibits ventral invagination, suggesting that the de-repressed WntD in snail mutant embryos may contribute to inhibiting ventral invagination. The overexpressed WntD inhibits invagination by antagonizing Dorsal nuclear localization, as well as twist and snail expression. Consistent with the early expression of WntD at the poles in wild-type embryos, loss of WntD leads to posterior expansion of nuclear Dorsal and snail expression, demonstrating that physiological levels of WntD can also attenuate Dorsal nuclear localization. The de-repressed WntD in snail mutant embryos contributes to the premature loss of snail expression, probably by inhibiting Dorsal. Thus, these results together demonstrate that WntD is regulated by the Dorsal/Twist/Snail network, and is an inhibitor of Dorsal nuclear localization and function. The closest homologs of Drosophila WntD, vertebrate Wnt8 proteins, regulate mesoderm patterning, neural crest cell induction, neuroectoderm patterning, and axis formation (Hoppler, 1998; Lekven, 2001; Lewis, 2004; Popperl, 1997). These vertebrate Wnt8 proteins may transmit the signal through the canonical pathway, but the exact mechanism remains unclear. So far, the downstream mediators of Drosophila WntD signaling are not known (Ganguly, 2005).

A second study (Gordon, 2005) confirms and extends Ganguly (2005) by inducing a mutation in wntD by homologous replacement. The Gordon study shows that WntD acts as a feedback inhibitor of the NF-kappaB homologue Dorsal, during both embryonic patterning and the innate immune response to infection. wntD expression is under the control of Toll/Dorsal signalling, and increased levels of WntD block Dorsal nuclear accumulation, even in the absence of the IkappaB homologue Cactus. The WntD signal is independent of the common Wnt signalling component Armadillo. By engineering a gene knockout, this study shows that wntD loss-of-function mutants have immune defects and exhibit increased levels of Toll/Dorsal signalling. Furthermore, the wntD mutant phenotype is suppressed by loss of zygotic dorsal (Gordon, 2005).

To identify novel components in the dorsoventral pathway, a microarray assay was carried out using embryos derived from gain-of-function and loss-of-function mutants of the Toll pathway. Among the novel genes identified, the expression and function of wntD was analyzed because the Wnt family of secreted proteins regulates patterning, cell polarity and cell movements. The results show that wntD is activated by Dorsal and Twist but repressed by Snail. Increased expression of WntD in wild-type early embryos inhibits ventral invagination. Thus, wntD is the first Snail target gene shown to have an interfering function in mesoderm invagination. The overexpressed WntD blocks invagination by inhibiting Dorsal nuclear localization. Loss-of-function analyses also show that physiological levels of WntD can attenuate Dorsal nuclear localization and function. Therefore, wntD is a novel downstream gene of the Dorsal/Twist/Snail network and can feed back to inhibit Dorsal (Ganguly, 2005).

The dynamic pattern of wntD expression in the early embryo is a combined result of activation by Dorsal/Twist and repression by Snail. Overexpressed WntD negatively regulates Dorsal nuclear localization, leading to an inhibition of ventral cell invagination. Physiological levels of WntD can also negatively regulate Dorsal, since loss of WntD leads to detectable expansion of both Dorsal nuclear localization and snail expression in the posterior regions. Furthermore, de-repressed WntD expression in the ventral region of snail mutant embryos can also attenuate Dorsal function. However, the loss of WntD could not rescue the invagination defect of the snail mutant embryo, suggesting that in the snail mutant embryo there are other de-repressed genes that can interfere with ventral invagination (Ganguly, 2005).

The wntD loss-of-function phenotype correlates with the expression of wntD at the poles of pre-cellular blastoderms. wntD is also expressed a bit later in the mesectoderm, and weakly in the mesoderm. Because WntD can inhibit Dorsal, one speculation is that WntD in the early mesectoderm may help to establish the sharp snail expression at the mesectoderm-neuroectoderm boundary. However, no changes were detected in the Dorsal protein gradient or snail pattern in the trunk regions of the Df(3R)l26c embryos. It is speculated that the timing of early expression of wntD, which may have additional input from the Torso pathway at the poles, is important for the feedback inhibition of Dorsal. By the time of cellularization, the Dorsal protein gradient is well established. This well-established Dorsal gradient activates the wntD gene in the trunk regions, but the subsequently translated WntD protein may not be capable of exerting a strong negative-feedback effect on the already formed Dorsal gradient. This timing argument is supported by the results of WntD-overexpression experiments. The use of maternal nanos-Gal4 caused a strong inhibition of Dorsal nuclear localization and of ventral invagination, whereas the use of zygotic promoters did not result in a significant phenotype (Ganguly, 2005).

Snail acts as a transcriptional repressor for at least 10 genes in the ventral region where mesoderm arises. In snail mutant embryos, all of these target genes are de-repressed in the ventral cells, concomitant with severe ventral invagination defects. However, no direct evidence has been reported on whether these de-repressed genes interfere with invagination. This study showed for the first time that a target gene of Snail, namely wntD, can block ventral invagination when overexpressed. If de-repressed WntD is solely responsible for inhibiting ventral invagination, it would be expected that, in the snail;Df(3R)l26c double-mutant embryos, ventral invagination would appear again. No rescue of ventral invagination was detected in the double-mutant embryos, suggesting that wntD is not the only de-repressed target gene that inhibits invagination. Nonetheless, the de-repressed WntD can attenuate Dorsal function, and may contribute to the ventral invagination defect (Ganguly, 2005).

Dorsal-ventral pattern of Delta trafficking is established by a Snail-Tom-Neuralized pathway

The intracellular trafficking of the Notch ligand Delta plays an important role in the activation of the Notch pathway. This study addresses Snail-dependent regulation of Delta trafficking during the plasma membrane growth of the mesoderm in the Drosophila embryo. Delta is retained in endocytic vesicles in the mesoderm but expressed on the surface of the adjacent ectoderm. This trafficking pattern requires Neuralized. A protocol based on chromosomal deletion and microarray analysis has led to the identification of tom (see Bearded) as the target of snail regulating Delta trafficking. Snail represses Tom expression in the mesoderm and thereby activates Delta trafficking. Overexpression of Tom abolishes Delta trafficking and signaling to the adjacent mesoectoderm. Loss of Tom produces mesoderm-type Delta trafficking in the entire blastoderm epithelium and an expansion of mesoectoderm gene expression. It is proposed that Tom antagonizes the activity of Neuralized and thus establishes a sharp mesoderm-mesoectoderm boundary of Notch signaling (De Renzis, 2006).

The Neuralized-dependent trafficking of Delta in the signal-sending cell is required for Notch activation in the receiving cell. Therefore, the activity of Neuralized must be tightly controlled between the signal-sending and signal-receiving cell in order to ensure the correct pattern of Delta trafficking and signal polarity. This study has followed the snail-mediated modulation of Delta trafficking during the cellularization of the Drosophila embryo. The concomitant formation of cell membranes and activation of zygotic transcription has offered some unique advantages for the experiments described in this work. Most importantly, the growth of the plasma membrane is timed with the zygotic expression of snail and neuralized and thus allows a precise staging protocol (De Renzis, 2006).

The experiments demonstrate that snail regulates the mesoderm-specific trafficking of Delta by repressing the expression of tom, and presumably of the other brd genes. In the ectoderm, where the brd class genes are expressed and snail is not, Delta and Notch Extra-Cellular Domain (NECD) have a predominantly cell surface localization. Obvious endocytic vesicles containing these proteins do not accumulate in ectodermal cells. Such vesicles do form in the mesoderm where Snail represses tom expression, and in tom^−/− embryos, where the NECD-Delta vesicles extend into the ectoderm. The vesicular trafficking of Delta and NECD characteristic of mesodermal cells requires the ubiquitin ligase Neuralized. Overexpression of Tom recapitulates the loss of function phenotype of neuralized, consistent with the view that Tom may normally function by opposing the role of Neuralized in Delta trafficking. Neuralized expression is dynamic and extends to the lateral region of the embryo, beyond the mesoderm-ectoderm boundary. Thus, on its own it cannot explain the restriction of vesicles to the mesoderm. It is proposed instead that Tom creates a functional boundary for Neuralized by suppressing its activity in the ectoderm. In agreement with this model, in tom^−/− embryos the expression of the mesoectoderm gene sim is extended dorsally (De Renzis, 2006).

The expression of sim is regulated by the maternal Dorsal nuclear gradient that directly or indirectly specifies all ventral cell fates. The mechanisms that precisely position sim expression to one row of cells, however, are not completely understood. sim can respond to the Dorsal gradient and can in principle be expressed in the entire ventral region of the embryo. Recent studies suggest that Notch signaling restricts the expression of sim to the mesoectoderm by relieving Suppressor of Hairless [Su(H)]-mediated repression of sim. Su(H) is uniformly distributed throughout the early embryo and represses sim expression in the ectoderm (De Renzis, 2006).

The precision of sim expression and its one cell diameter reflect the bias that zygotic expression of tom introduces on maternal Notch signaling in that region of the embryo. If the mesoderm cells that do not express Tom are the only cells that retain Neuralized activity and can internalize Delta ligand, they might be the only cells capable of sending signal. The snail repression will allow sim expression outside the mesoderm, and thus only those cells could be effective signal recipients. According to this model, the last snail-expressing cell of the mesoderm becomes the signal-sending cell and its immediate dorsal neighbor becomes the signal-receiving cell; i.e., the mesoectoderm. In the absence of Tom, the dynamic expression of Neuralized, which extends dorsally, would make more cells competent to send Notch signal. Indeed, the dorsal expression of sim is more pronounced at the end of mesoderm invagination at the time when the expression of Neuralized has extended to the neuroectoderm (De Renzis, 2006).

The molecular mechanisms by which Tom functions are most likely related to its interaction with Neuralized; this has been demonstrated in a yeast-two hybrid genomic screening (Giot, 2003). Tom may inhibit the ubiquitin ligase activity of Neuralized or it could compete for the interaction between Delta and Neuralized. Future work will be necessary to discriminate between these two possibilities. It will also be important to test the activity of the other Brd family members in the regulation of Neuralized activity. At least eight bearded-like genes (m2, m4, m6, mα, bob, brd, tom, and ocho) have been identified in the Drosophila genome. An interesting possibility is that different genes in this family may have different effects on Delta trafficking. Any difference may provide important clues into the regulation of the Notch pathway (De Renzis, 2006).

Computational models for neurogenic gene expression in the Drosophila embryo

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. This study applies similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. It is proposed that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo (Zinzen, 2006).

Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo

Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. This study determined the genome-wide occupancy of the dorsoventral (DV) determinants Dorsal, Twist, and Snail in the Drosophila embryo using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip). The in vivo binding of these proteins correlate tightly with the limits of known enhancers. This analysis predicts substantially more target genes than previous estimates, and includes Dpp signaling components and anteroposterior (AP) segmentation determinants. Thus, the ChIP-chip data uncover a much larger than expected regulatory network, which integrates diverse patterning processes during development (Zeitlinger, 2007).

ChIP-chip assays were performed with antibodies directed against Dorsal, Twist, or Snail on Toll^10b mutant embryos, aged 2-4 h. These embryos contain a constitutively activated form of the Toll receptor, which results in high levels of nuclear Dorsal protein and uniform expression of Twist and Snail throughout the embryo. The high levels of Dorsal, Twist, and Snail cause all cells to form derivatives of the mesoderm at the expense of neurogenic and dorsal ectoderm. Thus, these embryos represent a uniform cell type with respect to DV fate (Zeitlinger, 2007).

The whole-genome ChIP-chip experiments reveal several hundred strong binding clusters of Dorsal, Twist, and Snail with up to 40-fold ChIP enrichment, most of which span regions of ~1 kb in length. To identify the binding patterns of bona fide target enhancers of the Dorsal regulatory network, known enhancers were analyzed. The 22 known enhancers fall into three classes: type 1, type 2, and type 3, based on which levels of nuclear Dorsal regulate their expression (Zeitlinger, 2007).

The 10 type 1 enhancers (associated with twi, sna, miR-1, htl, hbr, mes3, CG12177, ady43A, tin, and Phm) are activated by peak levels of Dorsal in the presumptive mesoderm, and are all constitutively activated in Toll^10B mutant embryos. The ChIP-chip experiments identify strong binding peaks (greater than fivefold enrichment) of Dorsal, Twist, and Snail (DTS) within five of the 10 enhancers (twi, sna, miR-1, CG12177 and Phm). Another three enhancers, those associated with htl, tin, and ady43A, show significant but lower (less than fivefold) binding peaks restricted to Twist and Snail (TS) binding. This observation is consistent with earlier studies indicating that these enhancers might be primarily activated by Twist. Hence, eight of the 10 known type 1 enhancers exhibit significant in vivo occupancy by Twist and Snail (Zeitlinger, 2007).

An even greater correspondence between known enhancers and in vivo occupancy is seen for the type 2 [sim, E(spl), vn, rho, vnd and brk] and type 3 enhancers (ths, sog, ind, dpp, zen and tld), which are regulated by intermediate and low levels of the Dorsal gradient, respectively. All 12 enhancers are silenced in Toll^10B mutant embryos due to constitutive expression of the Snail repressor. Remarkably, every enhancer exhibits strong DTS or TS peaks with greater than fivefold enrichment in the ChIP-chip assays. Thus, ChIP-chip assays correctly identified 20 of the 22 known Dorsal target enhancers (Zeitlinger, 2007).

Most known DV enhancers are associated with overlapping binding clusters of Dorsal, Twist, and Snail regardless of whether they mediate activation or repression. Moreover, 17 of the 20 binding clusters at known enhancers display greater than fivefold enrichment of Twist and/or Snail. Using these binding criteria, 428 high-confidence DTS regions and 433 high-confidence TS regions were identified across the genome (Zeitlinger, 2007).

To confirm these regions through independent evidence, sequence analysis on these regions was performed using the known consensus binding motifs of Dorsal, Twist, and Snail. As expected, the identified regions are highly enriched in all three binding motifs. Moreover, a large fraction of the motifs is conserved across the 12 sequenced Drosophila species providing evidence that the discovered regions are functionally important. Finally, when motifs that are enriched in these regions were identified de novo, the known binding motifs can be rediscovered. Hence, the regions identified represent putative target gene enhancers of the DV network (Zeitlinger, 2007).

To show that newly identified regions indeed function as enhancers in vivo, putative enhancers were selected of primary DV genes; i.e., those genes that are expressed as localized stripes across the DV axis. In addition to the 22 known DV enhancers, 47 new putative enhancers were identified , some of which appear to regulate the same gene, were identified. By attaching the genomic sequence to a lacZ reporter and expressing the construct in transgenic embryos, seven of these enhancers were shown to be bona fide DV enhancers and that regulation by multiple enhancers occurs (Zeitlinger, 2007).

The wntD gene is expressed in portions of the presumptive mesoderm where it mediates feedback inhibition of Toll signaling. A cluster of DTS-binding peaks was identified in the 5'-flanking region, and the corresponding genomic DNA fragment mediates lacZ expression in the same region of the mesoderm as the endogenous gene. Similar results were obtained with the DTS-binding cluster located in the 5'-flanking region of mes5/mdr49 (Zeitlinger, 2007).

The vnd locus contains a well-documented intronic enhancer that mediates expression in the neurogenic ectoderm and recapitulates the spatial and temporal expression pattern of the endogenous gene. The ChIP-chip analysis detected this enhancer but also revealed two novel clusters further upstream. When tested for lacZ reporter activity, these novel genomic sequences directed lacZ expression in a pattern resembling that of the endogenous gene over different time periods: One directs early vnd expression in the presumptive ventral neurogenic ectoderm (vNE) while the other directs later expression in the medial column (mc) of the developing nervous system. All three enhancers contain evolutionarily conserved binding sites for Dorsal, Twist, and Snail, suggesting that the enhancers are not redundant but may function to fine-tune the vnd expression pattern. Overlapping enhancer activity was also observed for multiple miR-1 enhancers. Overall, as many as a third of all DV genes have multiple binding clusters, and thus might be subject to similar regulatory control (Zeitlinger, 2007).

Several of the occupied regions are associated with Dpp target genes expressed in the dorsal ectoderm. When the tup and pnr intronic sequences are tested in transgenic embryos, both fragments function as authentic enhancers and direct localized expression in the dorsal ectoderm, comparable to the endogenous tup and pnr expression patterns. These results suggest that the Dorsal patterning network directly regulates the expression of Dpp target genes (see below) (Zeitlinger, 2007).

It was noticed that many of the new DTS/TS clusters are associated with AP genes involved in segmentation. Although classical genetic studies argue that AP and DV patterning of the early embryo are controlled by separate maternal genetic programs, it is conceivable that the expression of AP target genes is modulated by the DV network. Indeed, DV modulation of segmentation gene expression has been observed previously (Zeitlinger, 2007).

The gap gene orthodenticle (otd) is expressed in two stripes across the AP axis in the early embryo. The anterior stripe shows diminished expression on the ventral side. Previous studies identified a 5' enhancer that recapitulates the normal expression pattern, including Dorsal-dependent suppression in ventral regions. ChIP-chip identified a strong DTS cluster within the limits of this enhancer. A similar DV bias in the expression pattern was found for the gap gene tailless (tll) and the pair-rule genes runt and hairy. In each case, the regions identified by ChIP-chip overlap or map close to known regulatory regions and contain several Dorsal-binding motifs (Zeitlinger, 2007).

At the gap gene knirps, a DTS-binding cluster was found in a region distinct from the known Bicoid-dependent enhancer. This newly identified genomic region functions as a bona fide enhancer directing expression in the anteroventral domain like endogenous knirps. Thus, the ChIP-chip analysis identified novel AP regulatory regions modulated by DV activity (Zeitlinger, 2007).

In summary, many segmentation genes contain DTS/TS-binding clusters, and at least some of these regions modulate gene expression across the DV axis, particularly in anterior regions of the embryo. It is concluded that the Dorsal gradient does not only regulate primary DV target genes, but rather appears to fine-tune a large number of genes that do not contribute to DV axis formation themselves, at least based on their known genetic function (Zeitlinger, 2007).

Many DTS/TS-binding clusters are also found at genes encoding signal transduction components. Analysis of the network formed by these pathways suggests that the Dorsal gradient controls the expression of many target genes by multiple regulatory pathways (Zeitlinger, 2007).

Dorsal directly represses Dpp expression in the mesoderm and neuroectoderm, leading to localized Dpp signaling in the dorsal ectoderm. Dpp activates a variety of genes, including tup and pnr. Accurate identification of intronic tup and pnr enhancers suggests that these genes are directly regulated by the Snail repressor, in addition to indirect regulation by the Dorsal gradient via Dpp signaling. zen is another well-known target gene of Dorsal in the dorsal ectoderm, and its product, a homeodomain transcription factor, functions synergistically with Dpp signaling. Target genes of Zen also appear to be subject to additional regulation by the Dorsal gradient. In the dorsal ectoderm, Dorsal may regulate gene expression by two mechanisms: direct repression, and indirect repression via Snail (Zeitlinger, 2007).

Similar network configurations regulate gene expression in the neuroectoderm. High levels of Dorsal repress the expression of rho via Snail in the mesoderm, thereby blocking EGF signaling in Toll^10b mutant embryos. ChIP-chip data suggest that the Dorsal network regulates additional genes encoding EGF signaling components as well as EGF target genes such as pnt, aop/yan, and argos. In the case of Notch signaling, it is known that the Dorsal network represses Notch target genes such as sim in Toll^10B mutant embryos through Snail. The Dorsal network may also regulate Notch signaling more directly, by suppressing genes encoding components of the signaling pathway including Notch itself (Zeitlinger, 2007).

Although repression of neuroectodermal target genes is likely to occur predominantly through Snail, Dorsal also induces the expression of a number of microRNAs in Toll^10b mutant embryos, including miR-1. Some of the neuroectodermal genes repressed by Snail are also predicted targets of these microRNAs. Hence, there may be multiple tiers of repression in the DV system, similar to the activities of the gap repressors in the AP system (Zeitlinger, 2007).

In summary, the present ChIP-chip study revealed an unexpectedly broad distribution of binding peaks for Dorsal, Twist, and Snail in the genome, and suggests extensive integration of the Dorsal regulatory network with additional patterning processes, such as Dpp signaling in the dorsal ectoderm and segmentation across the AP axis. In addition to the observed tight correlation between binding peaks and known enhancers, two lines of evidence suggest that a significant fraction of the newly identified regions is functional: First, the bound regions are highly enriched in evolutionarily conserved Dorsal, Twist, and Snail sequence motifs; and, second, several of the identified enhancers were experimentally confirmed by lacZ reporter gene expression in transgenic embryos. Thus, while genetic studies identified core sets of regulators for each developmental process in Drosophila, gene regulation integrates information more widely from several different systems. It is likely that integration of diverse patterning processes will also apply to mammalian development, including stem cell differentiation (Zeitlinger, 2007).

The Snail repressor inhibits release, not elongation, of paused Pol II in the Drosophila embryo

The development of the precellular Drosophila embryo is characterized by exceptionally rapid transitions in gene activity, with broadly distributed maternal regulatory gradients giving way to precise on/off patterns of gene expression within a one-hour window, between two and three hours after fertilization. Transcriptional repression plays a pivotal role in this process, delineating sharp expression patterns (e.g., pair-rule stripes) within broad domains of gene activation. As many as 20 different sequence-specific repressors have been implicated in this process, yet the mechanisms by which they silence gene expression have remained elusive. This study reports the development of a method for the quantitative visualization of transcriptional repression. The focus of this study was the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm. Elongating Pol II complexes were found to complete transcription after the onset of Snail repression. As a result, moderately sized genes (e.g., the 22 kb sog locus) are fully silenced only after tens of minutes of repression. It is proposed that this 'repression lag' imposes a severe constraint on the regulatory dynamics of embryonic patterning and further suggest that posttranscriptional regulators, like microRNAs, are required to inhibit unwanted transcripts produced during protracted periods of gene silencing (Bothma, 2011).

Snail typically binds to repressor sites located near upstream activation elements within distal enhancers. Repression might result from the passive inhibition of upstream activators, such as the failure of the activators to mediate looping to the core promoter. Alternatively, Snail might alter the chromatin state of the promoter region, resulting in diminished access of the Pol II transcription complex. Such repression mechanisms might cause a lag in gene silencing due to the continued elongation of Pol II complexes that were released from the promoter prior to the onset of repression. As in the case of the delay in the production of mature mRNAs after initiation, the lag in repression would be commensurate with the size of the gene, with large genes taking longer to silence than small genes. This can take a significant amount of time due to the surprisingly slow rate of Pol II elongation, only ∼1 kb/min (Ardehali, 2009; Bothma, 2011 and references therein).

Alternatively, elongating Pol II complexes might be arrested or released from the DNA template due to changes in chromatin structure and/or attenuation of Pol II processivity. Such mechanisms could lead to the immediate silencing of all genes regardless of size. Recent studies have documented rapid changes in the chromatin structure across the entire length of genes, exceeding the rate of Pol II processivity (Petesch, 2008). Certain corepressors in the Drosophila embryo (e.g., Groucho) are thought to mediate repression by a 'spreading' mechanism that modifies chromatin over extensive regions. Indeed, this type of mechanism has been invoked to account for the repression of the pair-rule gene even-skipped (eve) by the gap repressor Knirps. The attenuation of Pol II elongation has been implicated in a variety of processes. For example, Pol II attenuation has been documented for the transcriptional repression of MYC. Moreover, the activation of the HIV genome is regulated by Pol II processivity. In an effort to distinguish these potential mechanisms, the repression dynamics of several Snail target genes were visualized, because they are silenced in the presumptive mesoderm of precellular embryos (Bothma, 2011).

short gastrulation (sog) encodes an inhibitor of BMP/Dpp signaling that restricts peak Dpp signaling to the dorsal midline of cellularizing embryos. The sog locus is ∼22 kb in length and contains three large introns, including a 5′ intron that is ∼10 kb in length and a 3′ intron that is ∼5 kb in length. The use of separate intronic hybridization probes permits independent detection of 5′ and 3′ sequences within nascent sog transcripts. Individual nuclei are then false colored according to the probe combination they contain (Bothma, 2011).

sog exhibits synchronous activation at the onset of cell cycle 13 (cc13), ∼2 hr after fertilization. There is a lag between the time when nascent transcripts are first detected with the 5′ probe and subsequently cross-hybridize with both the 5′ and 3′ intronic probes. This lag is consistent with the established rates of Pol II elongation in flies, ∼1.1–1.5 kb/min. cc13 persists for ∼20 min, and by the completion of this time window, most of the nuclei in ventral and lateral regions exhibit yellow staining, indicating the presence of multiple nascent transcripts containing 5′ and 3′ intronic sequences within each nucleus. There is little or no repression in ventral regions, presumably due to insufficient levels of the Snail repressor prior to cc14 (Bothma, 2011).

As shown previously, nascent transcripts are aborted during mitosis. Consequently, only the 5′ hybridization probe detects nascent sog transcripts at the onset of cc14. Moreover, a small number of nuclei (at the ventral midline) fail to exhibit nascent transcripts with either the 5′ or 3′ probe, suggesting repression by Snail. This repression becomes progressively more pronounced during cc14 (Bothma, 2011).

Within about 10 min of the first detection of nascent sog transcripts at the onset of cc14, most of the nuclei exhibiting sog expression stain yellow, indicating expression of both 5′ (green) and 3′ (red) intronic sequences. During the next several minutes, progressively more nuclei exhibit only 3′ (red) hybridization signals in ventral regions. This transition from yellow to red continues and culminates in a 'red flash' where the majority of the ventral nuclei that contain nascent transcripts express only the 3′ (red) probe. As cc14 continues, there is a progressive loss of staining in the presumptive mesoderm, and eventually, nascent sog transcripts are lost entirely in the presumptive mesoderm (Bothma, 2011).

These results suggest that after its release from the promoter, Pol II continues to elongate along the length of the sog transcription unit, even as Snail actively represses its expression in the mesoderm. The red flash observed during mid-cc14 represents partially processed nascent sog transcripts that have lost the 5′ intron (hence no green signals with the 5′ hybridization probe) but retain 3′ sequences. Previous studies are consistent with sequential processing of nascent transcripts, beginning with the removal of 5′ intronic sequences and concluding with the removal of 3′ introns. As a control, two separate hybridization probes were used to label opposite ends of sog intron 1. As expected, there was no red flash, because both hybridization signals were simultaneously lost when intron 1 was spliced (Bothma, 2011).

There is an ∼20 min lag between the onset of repression at early cc14 and the complete silencing of sog expression in the presumptive mesoderm during mid- to late cc14. To determine whether this repression lag is a common feature of Snail-mediated gene silencing, additional target genes, including ASPP, Delta, canoe, and scabrous (sca) were examined. ASPP encodes a putative inhibitor of apoptosis, whereas Delta encodes the canonical ligand that induces Notch signaling. All four of these genes exhibit repression lag as they are silenced in the presumptive mesoderm of cc14 embryos (Bothma, 2011).

With the notable exception of Delta, the genes examined in this study contain promoter-proximal paused Pol II, as do most developmental patterning genes active in the precellular embryo. Moreover, results from whole-genome Pol II binding assays indicate that these genes maintain promoter-proximal paused Pol II in the presumptive mesoderm as they are actively repressed by Snail. These findings are consistent with the observation that the segmentation gene sloppy paired 1 retains promoter-proximal paused Pol II even after being silenced by the ectopic expression of Runt and Ftz. Thus, the Snail repressor does not appear to affect Pol II recruitment but rather inhibits the release of Pol II from the promoter-proximal regions of paused genes. At every round of de novo transcription, each Pol II complex at the pause site must receive an activation signal for its release into the transcription unit. It is proposed that the Snail repressor interferes with this signal, resulting in the retention of Pol II at the pause site (Bothma, 2011).

It is currently unclear whether repression lag is a general feature of transcriptional silencing. A recent study suggests that the gap repressor Knirps reduces the processivity of Pol II complexes across the eve transcription unit (Li, 2011). Snail and Knirps might employ distinctive modes of transcriptional repression. Snail recruits the short-range corepressor CtBP, whereas Knirps recruits either CtBP or the long-range corepressor Groucho. When bound to certain cis-regulatory elements within the eve locus, Knirps recruits Groucho, which might propagate a repressive chromatin structure. In contrast, Snail-CtBP might interfere with the release of Pol II from the proximal promoter, as discussed above. There is a considerable difference in the lengths of the genes examined in the two studies. The eve transcription unit is only 1.5 kb in length, less than one-tenth the size of sog. In fact, many patterning genes active in the early fly embryo contain small transcription units only a few kilobases in length. Small transcription units offer dual advantages in rapid patterning processes: essentially no lag in activation or repression (Bothma, 2011).

All five Snail target genes examined in this study exhibit Pol II elongation after the onset of repression. The number of transcripts produced during repression lag depends on the Pol II density across the transcription unit at the onset of repression. Whole-genome Pol II binding assays suggest that there are at least several Pol II complexes per kilobase. This estimate is based on comparing the total amount of Pol II within these genes to that present at the promoter of the uninduced hsp70 gene, for which there are accurate measurements. As a point of reference, the Pol II density on induced heat-shock genes is one complex per 75-100 bp, which is comparable to the footprint size, ∼50 bp, of an elongating Pol II complex. Thus, somewhere in the vicinity of ∼50 (or more) sog transcripts may be produced in a diploid cell after the onset of Snail repression. This represents a significant fraction of the steady-state expression of a typical patterning gene (∼200 transcripts per cell (Bothma, 2011).

Repression lag could impinge on a number of patterning processes, such as Notch signaling. The specification of the ventral midline of the central nervous system depends on the activation of Notch signaling in the ventralmost regions of the neurogenic ectoderm. Sca products somehow facilitate the activation of the Notch receptor, and repression lag could potentially disrupt this process by producing high steady-state levels of Sca in the mesoderm where Notch is normally inactive. Similar arguments might apply to the unwanted accumulation of Delta products in the mesoderm. Perhaps microRNAs are required to inhibit these transcripts and thereby facilitate localized activation of Notch signaling. Indeed, miR-1 is expressed in the presumptive mesoderm, at the right time and place to regulate Sca and/or Delta, and is known to be able to target Delta transcripts. Repression lag is potentially quite severe for Hox genes, particularly Antp and Ubx, which contain large transcription units (75–100 kb) that could take over an hour to silence after the onset of repression. It is conceivable that miRNAs encoded by the miR-iab4 gene, which are known to target Antp and Ubx transcripts, might inhibit postrepression transcripts (Bothma, 2011).

The precellular Drosophila embryo possesses a number of inherently elegant features for the detailed visualization of differential gene activity in development. Indeed, such studies were among the first to highlight the importance of transcriptional repression in the delineation of precise on/off patterns of gene expression. This study extends this rich tradition of visualization by providing the first dynamic view of gene silencing. The key feature of this method is the use of sequential 5′ and 3′ intronic probes to distinguish nascent transcripts produced by Pol II complexes shortly after their release from the promoter versus mature Pol II elongation complexes that have already transcribed 5′ intronic sequences. Elongating Pol II complexes have been shown to complete transcription after the onset of Snail repression and, as a result, moderately sized genes are fully silenced only after a significant lag. It is suggested that this repression lag represents a previously unrecognized constraint on the regulatory dynamics of the precellular embryo (Bothma, 2011).

Transcriptional repression via antilooping in the Drosophila embryo

Transcriptional repressors are thought to inhibit gene expression by interfering with the binding or function of RNA Polymerase II, perhaps by promoting local chromatin condensation. This study presents evidence for a distinctive mechanism of repression, whereby sequence-specific repressors prevent the looping of distal enhancers to the promoter. Particular efforts focus on the Snail repressor, which plays a conserved role in promoting epithelial-mesenchyme transitions in both invertebrates and vertebrates, including mesoderm invagination in Drosophila, neural crest migration in vertebrates, and tumorigenesis in mammals. Chromosome conformation capture experiments were used to examine enhancer looping at Snail target genes in wild-type and mutant embryos. These studies suggest that the Snail repressor blocks the formation of fruitful enhancer-promoter interactions when bound to a distal enhancer. This higher-order mechanism of transcriptional repression has broad implications for the control of gene activity in metazoan development (Chopra, 2012).

This study presents evidence that Snail represses gene expression by inhibiting the looping of distal enhancers to the promoter regions of target genes. Such a mechanism is compatible with the recent demonstration that Snail blocks Pol II initiation, but does not interfere with the elongation of RNA polymerases released before the onset of Snail repression. It would appear that Snail blocks enhancer looping, and thereby prevents Pol II release from the promoter region (Chopra, 2012).

Although 3C assays have the potential to produce high background, the use of appropriate controls and the use of different DV mutants provide a critical endogenous control for correlating the loss of specific enhancer-promoter loops and transcriptional repression. There is a loss of looping in two different classes of mutants lacking sog and brk expression. One lacks the Dorsal activator (gd⁷) and the other exhibits constitutive expression of the Snail repressor (Toll^10b) (Chopra, 2012).

In summary, the Snail repressor functions via antilooping, when it is bound to distal enhancers. It is anticipated that this higher-order mechanism of transcriptional repression will prove to be generally used in a variety of developmental processes. For example, gap repressors controlling segmentation, including Kruppel and Knirps, recruit the same corepressor CtBP that is used by Snail. It is possible that CtBP (or Ebi) somehow interferes with the recruitment or function of coactivators [e.g., CBP] or other factors required for the formation of enhancer-promoter loops. Antilooping is a flexible form of repression, in that it need not interfere with the binding or function of Pol II at the core promoter. Such a mechanism might be particularly useful for repressing 'poised' genes (containing paused or stalled Pol II) that are rapidly activated during development (Chopra, 2012).

Common origin of insect trachea and endocrine organs from a segmentally repeated precursor

Segmented organisms have serially repeated structures that become specialized in some segments. The Drosophila corpora allata, prothoracic glands, and trachea are shown to have a homologous origin and can convert into each other. The tracheal epithelial tubes develop from ten trunk placodes, and homologous ectodermal cells in the maxilla and labium form the corpora allata and the prothoracic glands. The early endocrine and trachea gene networks are similar, with STAT and Hox genes inducing their activation. The initial invagination of the trachea and the endocrine primordia is identical, but activation of Snail in the glands induces an epithelial-mesenchymal transition (EMT), after which the corpora allata and prothoracic gland primordia coalesce and migrate dorsally, joining the corpora cardiaca to form the ring gland. It is proposed that the arthropod ectodermal endocrine glands and respiratory organs arose through an extreme process of divergent evolution from a metameric repeated structure (Sanchez-Higueras, 2013).

The endocrine control of molting and metamorphosis in insects is regulated by the corpora allata (ca) and the prothoracic glands (pg), which secrete juvenile hormone and ecdysone, respectively. In Diptera, these glands and the corpora cardiaca (cc) fuse during development to form a tripartite endocrine organ called the ring gland. While the corpora cardiaca is known to originate from the migration of anterior mesodermal cells, the origin of the other two ring gland components is unclear (Sanchez-Higueras, 2013).

The tracheae have a completely different structure consisting of a tubular network of polarized cells. The tracheae are specified in the second thoracic to the eighth abdominal segments (T2-A8) by the activation of trachealess (trh) and ventral veinless (vvl) (Sanchez-Higueras, 2013).

The enhancers controlling trh and vvl in the tracheal primordia have been isolated and shown to be activated by JAK/ STAT signaling. While the trh enhancers are restricted to the tracheal primordia in the T2-A8 segments, the vvl1+2 enhancer is also expressed in cells at homologous positions in the maxilla (Mx), labium (Lb), T1, and A9 segments in a pattern reproducing the early transcription of vvl. The fate of these nontracheal vvl-expressing cells was unknown, but it was shown that ectopic trh expression transforms these cells into tracheae. To identify their fate, vvl1+2-EGFP and mCherry constructs were made (Sanchez-Higueras, 2013).

Although the vvl1+2 enhancer drives expression transiently, the stability of the EGFP and mCherry proteins labels these cells during development. It was observed that while the T1 and A9 patches remained in the surface and integrated with the embryonic epidermis, the patches in the Mx and Lb invaginated just as the tracheal primordia did. Next, the Mx and Lb patches fused, and a group of them underwent an epithelial-mesenchymal transition (EMT) initiating a dorsal migration toward the anterior of the aorta, where they integrate into the ring gland. To find out what controls the EMT, the expression of the snail (sna) gene, a key EMT regulator, was studied. Besides its expression in the mesoderm primordium, it was found that sna is also transcribed in two patches of cells that become the migrating primordium. Using sna bacterial artificial chromosomes (BACs) with different cis-regulatory regions, the enhancer activating sna in the ring gland primordium (sna-rg). A sna-rg-GFP construct labels the subset of Mx and Lb vvl1+2-expressing cells that experience EMT and migrate to form the ring gland. Staining with seven-up (svp) and spalt (sal) (also known as salm) markers, which label the ca and the pg, respectively, showed that the sna-rg-GFP cells form these two endocrine glands. The sna-rg-GFP-expressing cells in the Mx activated svp, and those in the Lb activated sal before they coalesced, indicating that the ca and pg are specified in different segments before they migrate (Sanchez-Higueras, 2013).

To test whether Hox genes, the major regulators of anteroposterior segment differentiation, participate in gland morphogenesis, vvl1+2-GFP embryos were stained, and it was found that the Mx vvl1+2 primordium expressed Deformed (Dfd) and the Lb primordium Sex combs reduced (Scr), while the T1 primordium expressed very low levels of Scr. Dfd mutant embryos lacked the ca, while Scr mutant embryos lacked the pg. Dfd and Scr expression in the gland primordia was transient, suggesting that they control their specification. Consistently, in Dfd, Scr double-mutant embryos, vvl1+2 was not activated in the Mx and Lb patches, and the same was true for vvl transcription. In these mutants, the sna-rg-GFP expression was almost absent, and the ca and pg did not form. In each case, Dfd controlled the expression of the Mx patch and Scr of the Lb patch (Sanchez-Higueras, 2013).

The capacity of different Hox genes to rescue the ring gland defects of Scr, Dfd double mutants was tested. Induction of Dfd with the sal-Gal4 line in these mutants restored the expression of vvl1+2 and sna-rg-GFP in the Mx and the Lb. However, in contrast to the wild-type, both segments formed a ca as all cells express Svp. Similarly, induction of Scr also restored the vvl1+2 and sna-rg-GFP expression, but both primordia formed a pg as they activate Sal and Phantom, an enzyme required for ecdysone synthesis. The capacity of both Dfd and Scr to restore vvl expression, regardless of the segment, led to a test of whether other Hox proteins could have the same function. Induction of Antennapaedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), or Abdominal-B (Abd-B) restored vvl1+2 expression in the Mx and Lb, but these cells formed tubes instead of migratory gland primordia. These cephalic tubes are trachea, as they do not activate sna-rg, they express Trh, and their nuclei accumulate Tango (Tgo), a maternal protein that is only translocated to the nucleus in salivary glands and tracheal cells, indicating that the trunk Hox proteins can restore vvl expression in the Mx and Lb but induce their transformation to trachea (Sanchez-Higueras, 2013).

To investigate whether vvl and trh expression is normally under Hox control in the trunk, focus was placed on Antp, which is expressed at high levels in the tracheal pits. In double-mutant Dfd, Antp embryos, vvl1+2 was maintained in the Lb where Scr was present, while the Mx, T1, and T2 patches were missing. In T3-A8, vvl1+2 expression, although reduced, was present, probably due to the expression of Ubx, Abd-A, and Abd-B in the posterior thorax and abdomen. Thus, Antp regulates vvl expression in the tracheal T2 primordium. Surprisingly, in Dfd, Antp double mutants, Trh and Tgo were maintained in the T2 tracheal pit, indicating that although Hox genes can activate ectopic trh expression, in the tracheal primordia they may be acting redundantly with some other unidentified factor, explaining why the capacity of Hox proteins to specify trachea had not been reported previously (Sanchez-Higueras, 2013).

sna null mutants were studied to determine sna's requirement for ring gland development, but their aberrant gastrulation precluded analyzing specific ring gland defects. To investigate sna function in the gland primordia, the sna mutants were rescued with the sna-squish BAC, which drives normal Sna expression except in the ring gland. These embryos have a normal gastrulation and activate the sna-rg- GFP; however, the gland primordia degenerate and disappear. To block apoptosis, these embryos were made homozygous for the H99 deficiency, which removes three apoptotic inducers. In this situation, the ca and pg primordia invaginated and survived, but they did not undergo EMT. As a result, the gland primordia maintain epithelial polarity, do not migrate, and form small pouches that remain attached to the epidermis. Vvl is required for tracheal migration. In vvl mutant embryos, sna-rg-GFP expression was activated, but the cells degenerated. In vvl mutant embryos also mutant for H99, the primordia underwent EMT and migrated up to the primordia coalescence; however, the later dorsal migration did not progress (Sanchez-Higueras, 2013).

This study has shown that the ca and pg develop from vvl-expressing cephalic cells at positions where other segments form trachea, suggesting that they could be part of a segmentally repeated structure that is modified in each segment by the activity of different Hox proteins. As the cephalic primordia are transformed into trachea by ectopic expression of trunk Hox, tests were performed to see whether the trachea primordia could form gland cells. Ectopic expression of Dfd with arm- Gal4 resulted in the activation of sna-rg-GFP on the ventral side of the tracheal pits. These sna-rg-GFP0-expressing cells also expressed vvl1+2 and Trh and had nuclear Tgo, showing that they conserve tracheal characteristics. These sna-rg-GFP-positive cells did not show EMT and remained associated to the ventral anterior tracheal branch. The strength of ectopic sna-rg-GFP expression increased when ectopic Dfd was induced in trh mutant embryos. However, migratory behaviors in the sna-rg-GFP cells were only observed if Dfd was coexpressed with Sal. Thus, sal is expressed several times in the gland primordia, first at st9-10 repressing trunk Hox expression in the cephalic segments and second from st11 in the prothoracic gland. It is uncertain whether the sal requirement for migration is linked to the first function or whether it represents an additional role (Sanchez-Higueras, 2013).

These results show that the endocrine ectodermal glands and the respiratory trachea develop as serially homologous organs in Drosophila. The identical regulation of vvl in the primordia of trachea and gland by the combined action of the JAK/STAT pathway and Hox proteins could represent the vestiges of an ancestral regulatory network retained to specify these serially repeated structures, while the activation of Sna for gland development and Trh and Tgo for trachea formation could represent network modifications recruited later by specific Hox proteins during the functional specialization of each primordium. This hypothesis or alternative possibilities should be confirmed by analyzing the expression of these gene networks in various arthropod species. The diversification of glands and respiratory organs must have occurred before the split of insects and crustaceans, as there is a correspondence between the endocrine glands in both classes, with the corpora cardiaca corresponding to the pericardial organ, the corpora allata to the mandibular organ, and the prothoracic gland to the Y gland. Despite their divergent morphology, a correspondence between the insect trachea and the crustacean gills can also be made, as both respiratory organs coexpress vvl and trh during their organogenesis. Divergence between endocrine glands and respiratory organs may have occurred when the evolution of the arthropod exoskeleton required solving two simultaneous problems: the need to molt to allow growth, and the need for specialized organs for gas exchange (Sanchez-Higueras, 2013).

Multiple regulatory safeguards confine the expression of the GATA factor serpent to the hemocyte primordium within the Drosophila mesoderm

Serpent (srp) encodes a GATA-factor that controls various aspects of embryogenesis in Drosophila, such as fatbody development, gut differentiation and hematopoiesis. During hematopoiesis, srp expression is required in the embryonic head mesoderm and the larval lymph gland, the two known hematopoietic tissues of Drosophila, to obtain mature hemocytes. srp expression in the hemocyte primordium is known to depend on snail and buttonhead, but the regulatory complexity that defines the primordium has not been addressed yet. This study found that srp is sufficient to transform trunk mesoderm into hemocytes. Two disjoint cis-regulatory modules were identified that direct the early expression in the hemocyte primordium and the late expression in mature hemocytes and lymph gland, respectively. During embryonic hematopoiesis, a combination of snail, buttonhead, empty spiracles and even-skipped confines the mesodermal srp expression to the head region. This restriction to the head mesoderm is crucial as ectopic srp in mesodermal precursors interferes with the development of mesodermal derivates and promotes hemocytes and fatbody development. Thus, several genes work in a combined fashion to restrain early srp expression to the head mesoderm in order to prevent expansion of the hemocyte primordium (Spahn, 2013).

Snail as a gene activator

In mutants of snail or twist, transcription of folded gastrulation is normal in the posterior midgut primordium but almost completely eliminated on the ventral side. Maternal-effect ventralizing mutations that expand the expression of twist and snail also expand the domain of fog transcription. In embryos from torpedoQY mutant mothers, Twist protein expression extends farther laterally, but the ventral furrow is usually split into two narrow ventrolateral invaginations by an unknown patterning mechanism. In this case, fog is transcribed in two separate ventrolateral stripes (Costa, 1994).

Snail positively regulates genes in the mesodermal primordium. Contrary to its postulated role as a repressor of ectodermal genes, snail can also have a positive regulatory function. However, gene C, zfh-1 and DFR1 are still expressed, although at reduced levels in very early snail mutant embryos. Therefore, snail is not essential for their activation, but rather their maintenance at high levels, and this may be due to an indirect function (Casal, 1996).

The fibroblast growth factor (FGF)/receptor system is thought to mediate various developmental events in vertebrates. DFR1 and Breathless proteins, the Drosophila homologs, contain two and five immunoglobulin-like domains, respectively, in the extracellular region, and a split tyrosine kinase domain in the intracellular region. In early embryos, DFR1 mRNA expression, requiring both twist and snail proteins, is specific to mesodermal primordium and invaginated mesodermal cells. At later stages, putative muscle precursor cells and cells in the central nervous system (CNS) express DFR1. Breathless expression occurs in endodermal precursor cells, CNS midline cells and certain ectodermal cells such as those of trachea and salivary duct. FGF-receptor homologs in Drosophila would thus appear essential for generation of mesodermal and endodermal layers, invaginations of various types of cells, and CNS formation (Shishido, 1993).

Both twist and snail are required for the mesodermal activation of the novel zinc-finger homeodomain gene zfh-1. ZFH-1 contains one homeodomain and nine C2H2 zinc fingers. In twist mutants, all the early mesodermal anti-AFH-1 staining posterior to the cephalic furrow is lost, although expression anterior to this furrow, as well as the later expression in the developing CNS is unaffected. A similar result is obtained in snail mutant embryos except that the early mesodermal staining anterior to the the cephalic furrow is also lost. In both mutants, the later sites of presumptive mesodermal zfh-1 expression are also devoid of staining, although this may be due to the failure of mutant embryos to reach later developmental stages (Lai, 1991).

escargot and snail are both required for the expression of vestigial in the wing disc (Fuse, 1996).

Zygotic expression of modifier of variegation modulo depends on the activity of genes which pattern the embryo along dorsoventral and anteroposterior axes and specify diversified morphogenesis. Dorsal and the mesoderm-specific genes twist and snail direct modulo expression in the presumptive mesoderm. The homeotic genes Sex combs reduced and Ultrabithorax positively regulate the gene in the ectoderm of parasegment 2 and abdominal mesoderm (Graba, 1995).

A conserved role for Snail as a potentiator of active transcription

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila approximately 25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. This study demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, 106 genes were identified that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of 'Snail-activated' genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, it was shown that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which this study demonstrates to be essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development (Rembold, 2014).

The function of Snail in distinguishing mesodermal from ectodermal fates has been traditionally seen as a repressor of the ectodermal differentiation program. This study demonstrates that Drosophila Snail can also activate part of the program specific for the mesoderm. The role of Snail in gastrulation is thus dual and involves a balance of repression and activation (Rembold, 2014).

One of the functions of Snail is to enable the formation of the ventral furrow together with Twist. Whereas the target genes of Twist that mediate furrow formation are known, it is completely unclear which genes act downstream from Snail. Only one such gene has been identified so far. The gene bearded, which is repressed in the mesoderm by Snail, is partly responsible for allowing adherens junctions in the mesoderm to be relocalized, but this is not sufficient for furrow formation. Therefore, there must be other genes that fulfill essential functions in gastrulation downstream from Snail. The hypomorphic sna^V2 mutant might give some hints of what genes these might be, since it is still able to make a furrow, although many Snail target genes are misregulated. Stepwise reduction of only the repressive activity of Snail by mutation of one or two corepressor-binding sites results in a stepwise increase in the strength of the gastrulation phenotype. Thus, the repressive activity of Snail is certainly required. However, the 60% of Snail-dependent genes that are not or are only weakly affected in the sna^V2 mutant (i.e., those most likely to be responsible for mediating furrow formation) do not fall into a uniform category; they contain both up-regulated and down-regulated genes. This might be an indication that misregulation of a larger set of both repressed and activated genes leads to the failure in furrow formation. This is also consistent with the fact that simply reducing the level of Snail by half leads to a delay in gastrulation (Rembold, 2014).

In summary, this study revealed a direct activator role for Drosophila Snail, a function that is seemingly conserved from flies to humans and places the Snail family of proteins in the category of dually acting TFs (Rembold, 2014).

A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila

Tribbles activity regulates cell cycle by directly and posttranscriptionally affecting String expression. During early embryonic development, string is transcribed in a spatial pattern controlled by the anterior-posterior and dorsoventral patterning systems. Expression of String mRNA in a given mitotic domain precedes mitosis by a few minutes. By analyzing the exception to this rule found in domain 10 on the ventral side at the embryo, the tribbles mode of regulation was uncovered. Although string is expressed in these cells, they do not divide until they are internalized. This delay depends on the activity of the tribbles gene named after the small, round, fictional organisms (from the television series "Star Trek") that proliferate uncontrollably when they contact water. The tribbles effect is restricted to the ventral furrow, even though TRBL mRNA is also present outside of this domain and the trbl mutation can be rescued by uniform exogeneous expression. This suggests that trbl activity is triggered by an input which is present only in the ventral furrow region. Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination. In embryos mutant for either snail or twist, no ventral furrow forms and cells are shifted to more lateral fates. String mRNA is not present in domain 10 and mitotic patterns in the ventral region of these mutant embryos are difficult to evaluate. String mRNA is restored to wild-type levels in the prospective domain 10 of snail mutants carrying three copies of wild-type twist. In such mutants, the ventral cells are the first ones to divide, indicating that snail is required for the function of the ventral inhibitor. One possibility would be that the persistence of trbl expression in the ventral region requires mesodermal determination and thus wild-type snail activity. However, snail mutants show a normal pattern of trbl expression and maintain trbl expression in the ventral domain. Similarly, in twist homozygous mutants and in embryos homozygous for deficiencies for frs the expression of trbl is not changed. Because snail embryos do not show a ventral mitotic inhibition, even though their trbl expression is normal, it is concluded that some aspect of mesodermal determination mediated by snail is required for Trbl activation (Großhans, 2000).

The Snail protein family regulates neuroblast expression of inscuteable and string, genes involved in asymmetry and cell division in Drosophila

Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of Prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the members of the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string (Ashraf, 2001).

Both snail and worniu have extensive expression in neuroblasts, while that of escargot is transient and sparse. Furthermore, based on genetic analysis, snail and worniu have more important role than escargot in the regulation of CNS development. The expression of snail and worniu in GMCs was carefully examined. In situ hybridization has revealed that worniu RNA, in contrast to its extensive expression in neuroblasts, is present in only a small number of GMCs. Even in later staged embryos, when there should be multiple GMCs surrounding each neuroblast, the staining in no more than one small cell next to each neuroblast could be detected. The limited staining in the GMCs is probably due to the segregation of some RNA from the parental neuroblast. Once the GMC is formed, the active transcription of worniu probably ceases. The protein and RNA expression of snail was also examined. The results showed that there is also very limited expression of snail in GMCs. snail RNA-containing GMCs were rarely detected next to neuroblast. Consistent with RNA expression, antibody staining revealed that the protein is predominantly in the neuroblasts (Ashraf, 2001).

Whether the neuroblast expression of snail and worniu is regulated by proneural genes was examined. Such a result would place the snail family in the well established genetic hierarchy that controls early neuroblast differentiation. The scute^B57 deletion mutant uncovers the three pro-neural genes: achaete, scute and lethal of scute. In this mutant, the expression of worniu in neuroblasts is significantly reduced. Only a few neuroblasts within each segment exhibit staining, and the expression level is substantially lower than in the wild type. The expression of worniu is also regulated by vnd and ind, such that in these mutant embryos the whole ventral and intermediate columns of staining are missing. In the msh^Delta68 mutant, no abnormal expression of worniu was detected. Previous results have shown that the neuroblast expression of snail is slightly affected in achaete-scute and vnd mutants but is not affected in a daughterless mutant. In ind and msh mutants, Snail protein expression was observed in many neuroblasts but the spatial pattern was rather disorganized. In summary, most of the proneural genes tested have profound effects on the expression of worniu, and have detectable but lesser effects on that of snail. The predominant expression of snail and worniu in neuroblasts and their regulation by proneural genes suggests that the snail family genes may have important functions within neuroblasts (Ashraf, 2001).

In mutants containing deletions that uncover escargot, worniu and snail, many early neuroblast markers are normal, but ftz expression in GMCs is abnormal. The regulation of ftz depends on Prospero, a homeodomain protein that controls GMC fate. Prospero protein and mRNA are preferentially segregated to GMCs from the neuroblast through the process of asymmetric division. Genes that are involved in asymmetric segregation of Prospero include inscuteable, miranda and staufen. The expression of these possible Snail family target genes was examined in neuroblasts (Ashraf, 2001).

Mutant embryos collected from deficiency strains that uncover the 35D1 chromosomal region, including the snail family genes were examined. In wild-type embryos, the expression of inscuteable can be detected in delaminating neuroblasts. After delamination, many neuroblasts show localization of the Inscuteable RNA. Embryos homozygous for the region 35D1 osp29 deletion, however, had significantly lower levels of the RNA and the staining was detected in a much smaller number of neuroblasts. Transgenic copies of snail, worniu or escargot efficiently rescues the expression of inscuteable RNA, demonstrating that it is the uncovering of the snail family of genes in the deletion that causes the phenotype. The rescue transgenes are under the control of the 2.8 kb snail promoter, which contains the neuroblast expression element. A 1.6 kb snail promoter construct that contains the mesoderm element but lacks the CNS element could not rescue the defect, demonstrating that expression of the transgenes within neuroblasts is essential for the function (Ashraf, 2001).

The segregation of Prospero protein into GMCs from neuroblasts is a critical event during asymmetric cell division. Since inscuteable plays a role in the segregation of prospero gene products into GMCs, whether there is Prospero protein in GMCs of mutant embryos was examined. Prospero protein staining can be easily detected in many wild type GMC nuclei. The staining is largely absent in the deletion that uncovers the snail family locus; only a few cells with the size of normal GMCs had clear nuclear staining. A band of cells along the midline also had Prospero staining, but these cells probably represent an expansion of the midline. It has been well documented that in all snail mutants there is derepression of the mid-line determinant single-minded in the blastoderm stage embryo (Ashraf, 2001).

To determine whether there are defects within GMCs in addition to the loss of Prospero, the expression of Hunchback, which is present transiently in early neuroblasts and later in many GMCs was examined. In the deletion mutant, the Hunchback protein in GMCs is also absent, while staining in cells surrounding the amnioserosa appeared normal. Transgenes of snail, worniu and escargot rescue the staining of Prospero and Hunchback, indicating that these GMC determinants are downstream of the Snail family. The results also suggest that the regulation of ftz by the Snail family is indirect, probably through an earlier event such as segregation of Prospero from neuroblast to GMC (Ashraf, 2001).

If the misregulation of inscuteable in the deletion mutant is the cause of the loss of Prospero and ftz expression in GMCs, the expression of inscuteable should correct the defects even in the absence of Snail family of proteins. A line carrying an inscuteable transgenic construct driven by the 2.8 kb snail promoter was crossed into the osp29 deletion genetic background. However, the rescue of Prospero expression in GMCs was variable and not nearly as strong as those embryos expressing the snail family transgenes. This suggests that inscuteable may not be the only important target gene of Snail. Another line of evidence supporting the idea of an additional target gene comes from the comparison of the phenotypes in osp29 and inscuteable mutant embryos. In inscuteable mutants, the Prospero crescent is formed but the mitotic spindle rotation is randomized. As a result, the Prospero protein frequently is present both in neuroblasts and GMCs. This phenotype is less severe than the almost total loss of Prospero GMC staining in osp29 deletion mutant. Therefore, it is surmised that in addition to the misregulation of inscuteable, there may be other defects that lead to the more severe phenotype in the deletion mutants (Ashraf, 2001).

One possibility that may explain the severe phenotype in snail family deletion mutants is additional defects in cell division. Neuroblasts are arrested at the G2/M transition at the embryonic cell cycle 14. After delamination, a pulse of string (which encodes a Cdc25 phosphatase homolog) expression in neuroblasts drives the cells to enter mitosis. The expression of string RNA was examined in whole-mount mutant embryos, but the result was ambiguous, owing to the dynamic, high level expression in ectoderm and other tissues, which obscures the signal in the neuroblast cell layer. Therefore tissue sectioning was used in order to better view the expression of string in neuroblasts. The sections clearly showed expression of string RNA in wild-type neuroblasts at stage 9 embryos. There are consistently three to four neuroblasts on each side of the midline that exhibit staining. This neuroblast expression appears very faint in the osp29 mutant embryos, and most sections do not show staining in neuroblasts while expression in ectoderm appears normal. The presence of wor and esg transgenes in the deletion mutant background led to accumulation of string RNA in some neuroblasts, suggesting a positive role for Snail family in regulating string expression (Ashraf, 2001).

If regulation of string is an important downstream event of Snail family of proteins, then cell division of neuroblasts should be affected in the absence of these proteins. The mitotic process was examined by staining for phosphorylated histone H3, which reveals condensed chromosomes. In wild-type embryos, although the neuroblasts do not exhibit highly synchronized mitosis, anti-phosphoH3 staining can be detected in multiple cells. In the osp29 mutant embryos, such staining is consistently reduced. The use of Prospero RNA to mark the neuroblast layer and the use of tissue sectioning has provided further support for the idea that the mutant embryos has reduced mitosis in neuroblasts (Ashraf, 2001).

The severe CNS defects are likely due to a combination of loss of inscuteable and string expression. Similar to the results obtained for inscuteable, transgenic expression of string alone has a weak and variable effect in the rescue of Prospero expression in GMCs. When both inscuteable and string are simultaneously expressed in neuroblasts of osp29 mutants using the UAS-Gal4 system, clear staining of Prospero in many cells resembling GMCs is observed. The staining is particularly apparent alongside the expanded midline, characteristic of mutant embryos with no Snail function in early mesoderm. The results support the idea that both inscuteable and string are relevant targets of the Snail family (Ashraf, 2001).

A clearly demonstrated in vivo function of Snail is transcriptional repression. The repression function is mediated through the recruitment of dCtBP (Drosophila C-terminal binding protein), which acts as a co-repressor for Snail to regulate target genes such as rhomboid, lethal of scute and single-minded. There are two conserved P-DLS-R/K motifs in Snail, as well as in Worniu and Escargot, and they have been shown to be critical for recruiting dCtBP. Mutations of these motifs abolish the repressor function of Snail in the blastoderm. To gain insight into the molecular mechanism of how Snail regulates CNS development, transgenic copies of snail, which had the dCtBP interaction motifs mutated were introduced into the osp29 deletion background. M1 contains the N-terminal motif mutation and M2 contains the C-terminal motif mutation. The expression of inscuteable and ftz was examined. The assay shows that the double mutant (M12) lost most of the ability to rescue, and M1 has lost some ability to rescue. However, M2 functioned quite efficiently, closer to that of the wild-type protein, to rescue inscuteable and ftz expression. These results demonstrate that the dCtBP interaction motifs are essential for the Snail function in the CNS, consistent with the idea that Snail acts as a repressor in neuroblasts to regulate gene expression. Thus, the activation of inscuteable and string by the Snail family may be indirect (Ashraf, 2001).

Protein Interactions

The Dorsal (DL) morphogen gradient initiates the formation of the mesoderm, neuroectoderm, and dorsal ectoderm by setting different limits of regulatory gene expression along the dorsoventral axis in the early Drosophila embryo. Low affinity DL-binding sites restrict target gene expression to the ventralmost regions (presumptive mesoderm), where there are peak levels of DL, while high affinity sites permit expression in ventrolateral regions (mesoderm and mesectoderm) containing intermediate levels of the morphogen. Activation by low levels of DL in lateral regions (the presumptive neuroectoderm) depends on cooperative DNA binding interactions between DL and bHLH proteins. The Snail repressor blocks this interaction and restricts expression to the neuroectoderm (Jiang, 1993).

"Quenching," is a form of gene regulation whereby activators and repressors co-occupy neighboring sites in a target promoter, but the repressor blocks the ability of the activator to contact the transcription complex. The zinc finger repressor, Snail, represses the expression of neuroectodermal regulatory genes in the presumptive mesoderm. SNA can mediate efficient repression when bound 50-100 bp from upstream activator sites. Repression does not depend on proximity of SNA-binding sites to the transcription initiation site. SNA is not a dedicated repressor but, instead, appears to block disparate activators (Gray, 1994).

Krüppel and Snail can mediate either quenching or direct repression of the transcription complex, depending on the location of repressor sites. When located within an upstream enhancer, the repressor locally quenches nearby activators (by preventing them from binding or masking their activation surfaces) and permits other enhancers (acting from a considerable distance from a local repressor) to interact with the transcription complex, thus demonstrating enhancer autonomy. In contrast, when bound to promoter-proximal regions, the repressor functions in a dominant fashion and blocks multiple enhancers. Local quenching and dominant repression require close linkage (less than 100 base pairs) of the repressor within either upstream activators or in a proximal promoter adjacent to the the transcription complex. SNA acts on a 300bp rhomboid neuroectodermal enhancer, acting in a competition mechanism to prevent Dorsal activation, but SNA fails to prevent activation when SNA repressor sites are moved away from the closest activators. Likewise KR can repress DL activation of a rhomboid enhancer by a locally acting quenching mechanism. Snail can also act to repress when bound to promoter-proximal regions, in a dominant fashion that blocks multiple enhancers. The ability of Snail to repress transcription can be uncoupled from DNA binding. Repression is probably caused by short-range inhibition of rather than be direct repression of the transcription complex (Gray, 1996). It is conceivable that KNI, and other short-range repressors such as Krüppel and Snail recruit 'corepressors', which cause local changes in chromatin structure (e.g. positioning a nucleosome) or in some other way interfere with access to the DNA by activators or basal transcription factors. Alternatively, short-range repressors do not interact with neighboring activators, but instead might 'hitchhike' with neighboring activators, looping to contact the basal promoter, and then inhibit components of the transcription complex (Arnosti, 1996).

Human CtBP attenuates transcriptional activation and tumorigenesis mediated by the adenovirus E1A protein. CtBp is known to interact with a conserved sequence in the adenovirus E1A protein, Pro-X-Asp-Leu-Ser-X-Lys (P-DLS-K). Mutations in this sequence eliminate E1a-CtBP interactions so that CtBP no longer inhibits E1A mediated transcriptional activation and tumorigenesis in mammalian cell cultures. The P-DLS-K sequence is present in the repression domains of two unrelated short-range repressors, Knirps and Snail; the latter protein also contains the related sequence P-DLS-R. The P-DLS-K sequence is essential for the interaction of these proteins with Drosophila CtBP (dCtBP). A P-element-induced mutation in dCtBP exhibits gene-dosage interactions with a null mutation in knirps, which is consistent with the occurrence of Knirps-dCtBP interactions in vivo. Mutation of the D-DLS-K motif in Knirps, results in a Knirps protein that cannot mediate repression. These observations suggest that CtBP and dCtBP are engaged in an evolutionarily conserved mechanism of transcriptional repression, which is used in both Drosophila and mammals. Mammalian and Drosophila CtBP may mediate repression through the enzymic modification of chromatin because both proteins are related to D-isomer 2-hydroxy acid dehydrogenases. Despite this rather unexpected homology, immunolocalization assays indicate that the Drosophila CtBP protein accumulates in nuclei. Perhaps CtBP and dCtBP cause local changes in chromatin structure by introducing subtle changes in core histones. Alternatively, it is possible that CtBPs are components of an enzymatic cascade that modulates the activities of histone deacetylases or other co-repressor proteins (Nibu, 1998a).

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Two of the repressors, Hairy and Dorsal, are known to recruit a common co-repressor protein, Groucho. Evidence is presented that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, all of which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H (also termed P-DLS-R). This motif is essential for the repression activity of these proteins in transgenic embryos. It is proposed that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression (Nibu, 1998b).

A Gal4-Knirps fusion protein containing the C-terminal third of the Knirps protein (amino acid residues 255-429) has been shown to be able to repress a modified eve stripe 2-lacZ reporter gene in transgenic embryos. The fusion protein contains the Knirps P-DLS-K motif, and mutations in this sequence (PMDLSMK to AAAASMK) inactivate its repression activity. These results suggest that dCtBP is an important component of Knirps-mediated repression (Nibu, 1998b).

Similar assays were used to assess the significance of the P-DLS-K and P-DLS-R motifs in the Snail repressor. The eve stripe 2 enhancer was used to misexpress snail in transgenic embryos. snail is normally expressed in ventral regions where it helps establish the limits of the presumptive mesoderm by repressing various target genes such as rhomboid. The ectopic snail stripe results in an abnormal rhomboid pattern that contains a gap in the vicinity of eve stripe 2. This observation suggests that ectopic Snail products bind to the endogenous rhomboid NEE and repress its transcription. Point mutations in the P-DLS-K and P-DLS-R motifs eliminate the repression activity of an otherwise normal stripe 2-snail transgene. The mutant Snail mRNA is expressed at levels comparable with the wild-type RNA. Additional studies indicate that mutations in the P-DLS-K motif alone, with P-DLS-R intact, result in only weak repression of the rhomboid pattern.

The mechanism by which dCtBP mediates transcriptional repression is unknown. However, the current study provides evidence against a previously proposed mechanism for Krüppel (Sauer, 1995). Krüppel activity is shown to be lost in dCtBP mutants, and the C-terminal region of the protein contains an essential P-DLS-H repression motif. Moreover, preliminary studies suggest that ectopic expression of the native Krüppel protein causes patterning defects in early embryos, which are reversed when the P-DLS-H motif is mutagenized. These results strongly suggest that Krüppel-mediated repression depends on the recruitment of the dCtBP co-repressor. The earlier study provided evidence that repression depends on the direct interactions of Krüppel with the beta-subunit of the TFIIE general transcription factor (Sauer, 1995). It is conceivable that this mechanism of repression is employed in other tissues at later stages in the Drosophila life cycle, although it is noted that a recent study provides strong evidence that a mammalian Krüppel-like protein also employs a CtBP co-repressor (Nibu, 1998b and references).

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. The structures of synthetic peptides identical to the CtBP binding sites on these proteins have been investigated using NMR spectroscopy. Peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity [K(i) of 1.04, 1.34 and 0.52 microM, respectively]. However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides are markedly reduced [K(i) of 332 and 56 microM, respectively] whilst the Hairy peptide binds much more strongly [K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP]. In addition peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. It is concluded that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data constitute evidence for a multiplicity of CtBPs and partner proteins (Molloy, 2001).

Drosophila Ebi mediates Snail-dependent transcriptional repression through HDAC3-induced histone deacetylation

The Drosophila Snail protein is a transcriptional repressor that is necessary for mesoderm formation. This study identified the Ebi protein as an essential Snail co-repressor. In ebi mutant embryos, Snail target genes are derepressed in the presumptive mesoderm. Ebi and Snail interact both genetically and physically. A Snail domain was identified that is sufficient for Ebi binding and functions independently of another Snail co-repressor, Drosophila CtBP. This Ebi interaction domain is conserved among all insect Snail-related proteins, is a potent repression domain and is required for Snail function in transgenic embryos. In mammalian cells, the Ebi homologue TBL1 is part of the NCoR/SMRT-HDAC3 (histone deacetylase 3) co-repressor complex. It was found that Ebi interacts with Drosophila HDAC3, and that HDAC3 knockdown or addition of a HDAC inhibitor impairs Snail-mediated repression in cells. In the early embryo, Ebi is recruited to a Snail target gene in a Snail-dependent manner, which coincides with histone hypoacetylation. These results demonstrate that Snail requires the combined activities of Ebi and CtBP, and indicate that histone deacetylation is a repression mechanism in early Drosophila development (Qi, 2008).

Previous studies have suggested that CtBP mediates transcriptional repression by Snail in the early embryo. However, disruption of Snail repressor activity in ebi mutant embryos cannot be due to an indirect effect on CtBP, based on several observations. Comparable CtBP protein levels were detected in ebi mutant and wt embryos using a CtBP-specific antibody. In addition, the ebi mutation did not affect the function of the Kr repressor, which also requires CtBP, on the NEE-lacZ reporter gene. Furthermore, the segment polarity gene engrailed that is indirectly regulated by CtBP-dependent repressors such as Kr and Knirps is normally expressed in ebi mutant embryos, indicating that CtBP activity is not disrupted by the ebi mutation (Qi, 2008).

Moreover, the Ebi interaction motif (Sna 1-40) that does not bind to CtBP in vitro still has repression activity in S2 cells and in transgenic embryos, suggesting that Ebi functions directly as a cofactor for Snail through a physical association. Removing this motif from the Snail protein abolishes its repression activity. This result is consistent with data that used snail transgenes to rescue snail^−/− embryos. It was found that Snail lacking amino acids 6-25 fails to repress sim expression in snail^−/− embryos. It was proposed that this part of Snail might be involved in nuclear localization of the protein. However, this study demonstrated that the mutant protein is normally localized to nuclei in S2 cells and embryos. This suggests that mutant Snail loses the ability to repress because it is unable to interact with Ebi. Taken together, it is concluded that Ebi specifically regulates Snail-mediated repression through a new, CtBP-independent pathway (Qi, 2008).

This study suggests that Snail mediates repression through two pathways, a CtBP-dependent and an Ebi-dependent pathway. Several repression activities in one protein could contribute qualitatively or quantitatively to repression. In some cases, different target genes are repressed through distinct co-repressors. By contrast, the CtBP-dependent and -independent repression activities in Knirps and Hairless exerts an effect quantitatively. The current experiments show that in the presumptive mesoderm, repression of several Snail target genes requires both CtBP and Ebi, that Snail recruits both CtBP and Ebi to the same rho enhancer and that CtBP and Ebi can interact simultaneously with Snail. Deletion of either the Ebi or CtBP interaction motifs impairs Snail function in transgenic mis-expression and rescue assays. Furthermore, derepression of Snail target genes is not complete in either ebi or CtBP mutant embryos. In ebi mutant embryos, and snail mutant embryos rescued with Snail lacking amino acids 6-25, gene repression is impaired but ventral furrow formation and mesoderm invagination normal, which is similar to the situation in the snail hypomorphic allele V2. Taken together, these results strongly suggest that Snail requires both Ebi and CtBP for full repressor activity (Qi, 2008).

By what mechanism does Ebi contribute to repression? Previous studies have shown that Ebi and its mammalian homologue TBL1 can function through two different complexes, the NCoR-SMRT-HDAC3 complex and a Sina E3 ubiquitin ligase complex. Tests were performed to see if ubiquitin-dependent protein degradation is involved in Snail-mediated repression by adding a proteasome inhibitor to Tet-Sna-expressing cells. No change in luciferase activity was observed in response to this drug. This indicates that proteasomal degradation is not necessary for Snail repressor activity, which is also supported by the lack of rho derepression in sina germline clone mutant embryos (Qi, 2008).

It is well established that histone deacetylation correlates with transcription repression. Local deacetylation of histones by HDAC3 results in repression of gene transcription. HDAC3 was purified as a core subunit of the NCoR-TBL1 (Ebi) complex in mammalian cells, suggesting that histone deacetylation is functionally linked to the activity of this complex. Although the composition of a similar complex in Drosophila has not been determined, a physical association and functional connection between Ebi and SMRTER have been reported. In this study, it was found that HDAC3 and Ebi associate and that both are required for Snail repression domain function in S2 cells, as determined by RNAi and inhibition of HDAC activity. The observation that Sna 1-245 and 1-245Δ5-25 are resistant to TSA treatment implies that these proteins can repress by an Ebi-independent mechanism. Taken together, these results suggest that Ebi-dependent repression requires histone deacetylation, whereas CtBP-dependent repression does not in this assay (Qi, 2008).

In contrast to the situation in embryos where the Ebi interaction domain and the CtBP interaction domain in Snail cooperate, in the cell culture assay these domains (1-40 and 1-245Δ5-25) can repress transcription independently of one another. It is surprising, therefore, that Ebi or HDAC3 RNAi weakly relieved repression by Sna 1-245 containing both repression domains, and produce stronger effects together with CtBP RNAi. This indicates that repression in the cell culture assay may involve further components (Qi, 2008).

The role of SMRTER in this process remains to be determined. Unfortunately, SMRTER knock down by RNAi results in cell cycle arrest and is cell lethal, precluding an investigation of its function in Snail-mediated repression. However, it has been shown that mammalian NCoR and SMRT contain a deacetylase-activating domain (DAD) that is essential for catalytic activity of HDAC3. The DAD is evolutionarily conserved and present also in SMRTER. Presumably, Drosophila HDAC3 also requires SMRTER binding for activation of its enzymatic activity. Moreover, the association between TBL1 and HDAC3 in mammalian cells is bridged by SMRT. For these reasons, it is likely that the Ebi-HDAC3 complex also includes SMRTER. Reciprocal BLAST searches also reveal a homologue of the SMRT-NCoR complex core component GPS2 in Drosophila. It appears that the composition and dependence on histone deacetylation by HDAC3 for SMRT-NCoR complex function has been evolutionarily conserved (Qi, 2008).

Ever since the discovery that Rpd3 is a HDAC over 10 years ago, a strong link between histone hypoacetylation and transcriptional repression has been established. In Drosophila, five HDACs of the class I and II type, and five Sir2-like HDACs are present. However, it is not known whether regulation of histone acetylation contributes to transcriptional control during the rapid nuclear divisions in early Drosophila embryogenesis. Although Rpd3 or the Mi-2-Rpd3 complex has been implicated in repression by the Even-skipped, Runt, Knirps, Tramtrack and Hunchback repressor proteins, and as part of Groucho and Atrophin co-repressor complexes, a direct role of histone deacetylation in repression has not been established in these instances. A recent report has invoked regulation of transcription elongation in repression by the pair-rule proteins Runt and Fushi-tarazu in early embryos. In this case, no change in histone acetylation was observed on the target gene in transcriptionally active cells compared with inactive cells. By contrast, this study demonstrates that H3 becomes hypoacetylated at a Snail-regulated enhancer in the presence of Snail, and suggests that histone deacetylation participates in Snail-mediated repression based on a cell culture assay. This is the first evidence that histone deacetylation may be involved in cell-fate specification during Drosophila embryo development (Qi, 2008).

Vertebrate Snail proteins contain a different conserved motif, the SNAG domain in their very N termini. The Snail SNAG domain is necessary to recruit a Sin3A-HDAC1/HDAC2 co-repressor complex to the E-cadherin promoter, which is sensitive to the HDAC inhibitor TSA. This indicates that both vertebrate and insect Snail proteins rely on histone deacetylation for their repressor function, but that they recruit different co-repressor complexes. Whereas vertebrate Snail depends on Sin3A-HDAC1/HDAC2, insect Snail proteins require an Ebi-HDAC3 complex for maximal activity (Qi, 2008).

Gene length may contribute to graded transcriptional responses in the Drosophila embryo

An important question in developmental biology is how relatively shallow gradients of morphogens can reliably establish a series of distinct transcriptional readouts. Current models emphasize interactions between transcription factors binding in distinct modes to cis-acting sequences of target genes. Another recent idea is that the cis-acting interactions may amplify preexisting biases or prepatterns to establish robust transcriptional responses. This study examined the possible contribution of one such source of prepattern, namely gene length. Quantitative imaging tools were developed to measure gene expression levels for several loci at a time on a single-cell basis, and these quantitative imaging tools were applied to dissect the establishment of a gene expression border separating the mesoderm and neuroectoderm in the early Drosophila embryo. First, the formation of a transient ventral-to-dorsal gradient of the Snail (Sna) repressor was characterized, and then the relationship was examined between this gradient and repression of neural target genes in the mesoderm. It was found that neural genes are repressed in a nested pattern within a zone of the mesoderm abutting the neuroectoderm, where Sna levels are graded. While several factors may contribute to the transient graded response to the Sna gradient, this analysis suggests that gene length may play an important, albeit transient, role in establishing these distinct transcriptional responses. One prediction of the gene-length-dependent transcriptional patterning model is that the co-regulated genes knirps (a short gene) and knirps-related (a long gene) should be transiently expressed in domains of differing widths, which was confirmed experimentally. These findings suggest that gene length may contribute to establishing graded responses to morphogen gradients by providing transient prepatterns that are subsequently amplified and stabilized by traditional cis-regulatory inte

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