Promoter Structure

Two cis-acting control elements, CD1 and CD2, control early expression. CD2 is located at -1000 to -3000 from start of transcription, and CD1 is at -3000 to -4000. The initial expression of Krüppel occurs in a precisely bounded central region of the Drosophila blastoderm embryo. The spatial limits of the Kr expression domain are controlled by the morphogenetic activities of the anterior organizer gene bicoid and the anterior gap gene hunchback . A 730 bp KR control element, (CD1) drives gene expression in the endogenous KR central domain. This cis-acting element, Kr730, is composed of BCD and HB responsive sequences. They map into regions of multiple HB and BCD protein in vitro binding sites. A 142 bp core fragment containing one low affinity HB and five medium to strong BCD protein binding sites drives gene expression in a Kr-like location in the center of the embryo. This fragment represents a target for the redundant activator/repressor system provided by the anterior morphogens BCD and HB (Hoch, 1991).

The proximal promoter of Krüppel consists of a 44-base pair fragment containing the RNA start sites with significant promoter activity. This minimal promoter is flanked both upstream and downstream by binding sites for the GAGA factor. The GAGA factor is the predominant sequence-specific DNA binding factor that interacts with the KR promoter region, and the purified protein activates Kr transcription in vitro. However, strong transcriptional activation of Kr as well as of Ultrabithorax, another GAGA factor-responsive gene, requires the presence of a DNA binding transcriptional repressor. The GAGA factor is able to relieve this repression in a binding site-dependent manner, and, thus, these data suggest that the GAGA factor functions as an antirepressor, rather than an activator, of the Kr gene (Kerrigan, 1991).

High hunchback activity represses Krüppel in the anterior (Schulz, 1994).

Knirps regulates Krüppel by acting as a competitive repressor of Bicoid-mediated activation of Krüppel. Knirps binds at a single site in a 730bp regulatory region located 3.4 kb upstream of Krüppel. The Krüppel site (Kr730) contains a single strong KNI binding site, a 16 base pair element. The KNI binding site overlaps with one BCD binding site (Hoch, 1992 and references).

After the blastoderm stage Kr is under the control of at least 10 different regulatory units, sometime overlapping, up to10kb upstream of the gene. These different regulatory units drive expression in the Malpighian tubules, amnioserosa cells, muscle precursor cells, the developing nervous system, midline precursor cells and the larval eye (Bolwig's organ) (Hoch, 1990).

Knirps regulates krüppel by acting as a competitive repressor of Bicoid-mediated activation of Krüppel. Knirps binds at a single site in a 730bp regulatory region located 3.4 kb upstream of krüppel. The Krüppel site (Kr730) contains a single strong KNI binding site, a 16 base pair element. The KNI binding site overlaps with one BCD binding site (Hoch, 1992 and references).

Early developmental patterning of the Drosophila embryo is driven by the activities of a diverse set of maternally and zygotically derived transcription factors, including repressors encoded by gap genes such as Krüppel, knirps, and giant and the mesoderm-specific snail gene. At a molecular level, the mechanism of repression by gap transcription factors is not well understood. Initial characterization of these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better understand the molecular mechanism of short-range repression, the properties of the Giant gap protein have been investigated. The ability of endogenous Giant to repress when bound close to the transcriptional initiation site was tested. Tandem 'CD1' Giant binding sites derived from the Krüppel (Kr) promoter were inserted 5' of a transposase basal promoter (55 bp from the initiation of transcription of the lacZ reporter gene). Strong repression of the lacZ gene is detected in embryos bearing this transgene; expression of the lacZ gene in both anterior and posterior Giant regions is strongly attenuated. The repression of lacZ in anterior and posterior regions is relieved when the transgene is assayed in gt mutant embryos, confirming that the Giant protein is mediating the repression. Giant effectively represses a heterologous promoter when binding sites are located at -55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression is diminished (Hewitt, 1999).

It is probable that more distally located repressor sites (gt-110 and gt-160, located respectively 110 and 160 bp from the start of transcription) may be effective only at higher concentrations of Giant because the binding sites may not be filled effectively. A partially filled site may still be effective at close range because when Giant is close to the promoter, chance interactions between Giant and its target will occur more frequently, obviating the need for saturation of the binding site by Giant. At a greater distance, these chance interactions would be less frequent, leading to weak repression. Partial filling of binding sites may simply be an indication that the levels of Giant protein are below the Kd for the binding site, so the site is empty for a fraction of the time. Alternatively, even identical sites may not bind Giant protein equally well; Giant may bind cooperatively to its cognate sites with a target protein, so that moving the sites farther from the target may break repressor-target cooperative interactions. Raising the level of Giant protein (moving up the gradient) would suffice to give greater occupancy of Giant sites, and reestablish repression. Quantitation of relative Giant protein levels in embryos suggests that differences in repressor protein levels of less than two fold is sufficient to switch a gene from an active to an inactive state. Similar cooperative effects have been reported for activators, including the Bicoid activator. If Giant is located close to the promoter, chance interactions between Giant and its target will occur more frequently because of the short diffusion distance, obviating the need for saturation of the binding site by Giant. The mechanism whereby endogenous promoters might differentially respond to different concentrations of repressors is not known, but the results obtained from this study suggest that exact placement of short-range repressors with respect to other promoter elements and the number of binding sites might suffice to endow a promoter with high or low sensitivity. Additional factors, such as binding site affinity, may also contribute to differential promoter sensitivity toward repression. For example, a binding site located within the eve stripe 2 enhancer was found to bind Giant protein less well than a site in the Kr promoter: consistent with this finding, eve has been found to be less sensitive to Giant repression than Kr. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient (Hewitt, 1999 and references).

A chimeric Gal4-Giant protein lacking the basic leucine zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain. The three Giant sites in the even-skipped promoter cover large regions and do not closely resemble the compact sites from the Kr promoter. Giant is likely to interact as a homodimer with the Kr promoter, and as a heterodimer with the complex eve stripe 2 enhancer sites, using an as-yet unidentified partner basic leucine zipper protein. Such a gene product may be encoded by a locus identified on the left arm of chromosome 2, mutations in which can cause a gt-like phenotype. Consistent with the possibility that Giant protein repression may involve other factors, recent experiments suggest that eve might be co-regulated by a Giant partner protein localized to the anterior portion of the embryo (Wu, 1998). However, the activity of the Gal4-Giant protein on a variety of activators shows Giant can act as a homodimer on reporter genes (the Gal4 DNA binding domain binds to its cognate site as a dimer). If Giant binds to DNA as a heterodimer in the embryo, the partner protein may serve to modulate DNA binding rather than to effect transcriptional repression (Hewitt, 1999 and references).

The analysis of transgenes with successively greater repressor-promoter spacing demonstrates that short-range repression can act on more than an all-or-nothing basis, however, the activity of these repressors is sharply attenuated over relatively short distances. It has been suggested that short-range repression may directly target adjacent transcription factors in a process akin to quenching; in this case, Giant might alternately quench transcription activators or the basal machinery, depending on the location of the binding site. Such lack of specificity may be consistent with quenching acting through chromatin modification on an extremely local level, as has been reported for Ume6-mediated repression in yeast. Alternatively, repression by direct targeting of the basal transcription machinery is possible, as has been reported for the Krüppel and Even-Skipped proteins. Cofactors such as dCtBP are implicated in the activity of some short-range repressors, such as Knirps, Krüppel and Snail, but apparently not Giant. Further molecular characterization of Giant repression activity will be necessary to distinguish between these alternatives (Hewitt, 1999 and references).

The Giant protein is a short-range transcriptional repressor that refines the expression pattern of gap and pair-rule genes in the Drosophila blastoderm embryo. Short-range repressors including Knirps, Krüppel, and Snail utilize the CtBP cofactor for repression, but it is not known whether a functional interaction with CtBP is a general property of all short-range repressors. Giant repression activity was studied in a CtBP mutant and it has been found that this cofactor is required for Giant repression of some, but not all, genes. While targets of Giant such as the even-skipped stripe 2 enhancer and a synthetic lacZ reporter show clear derepression in the CtBP mutant, another Giant target, the hunchback gene, is expressed normally. A more complex situation is seen with regulation of the Krüppel gene, in which one enhancer is repressed by Giant in a CtBP-dependent manner, while another is repressed in a CtBP-independent manner. These results demonstrate that Giant can repress both via CtBP-dependent and CtBP-independent pathways, and that promoter context is critical for determining giant-CtBP functional interaction. To initiate mechanistic studies of the Giant repression activity, a minimal repression domain within Giant has been identified that encompasses residues 89-205, including an evolutionarily conserved region bearing a putative CtBP binding motif (Stunk, 2001).

What characteristics of a regulatory region dictate CtBP-dependent or CtBP-independent repression? In considering which features of a gene determine CtBP-dependence or -independence, the structure of the basal promoter cannot be the deciding factor, for the same Kr promoter is regulated by distinct elements, some that exhibit CtBP-dependence and some that show CtBP-independence. Similarly, the eve gene is repressed by Knirps via CtBP-dependent and CtBP-independent regulatory elements. While the eve enhancers in question are kilobases apart, the Kr regulatory elements driving anterior and central domain (CD) expression are closely intertwined, and appear to share at least some of the same activator binding sites, suggesting that subtle differences in enhancer architecture or differences in levels of regulatory proteins interacting with those elements may dictate CtBP dependence. The Giant binding site in the Kr CD2 enhancer site was shown to be of higher affinity than the gt1 site in the eve stripe 2 enhancer. Thus, there may be a correlation between Giant binding site affinity and the requirement for CtBP, with elements containing Giant sites of lower affinity showing CtBP-dependence. A consensus has been derived for the Giant protein by aligning binding sites for Giant from eve, Kr, and the recently identified abdA iab-2 enhancer site. The consensus features an extended half-site inverted repeat TNTTAC, consistent with the dimeric nature of basic zipper proteins, and a central ACGT core common to recognition motifs for many basic zipper proteins. The higher affinity sequences from the CtBP-independent Kr CD element are closer to the consensus than those of the CtBP-dependent eve stripe 2 enhancer. Weaker sites may only be partially occupied, resulting in an overall lower level of Giant mediated repression. A loss of CtBP might further depress repression activity below a critical threshold, leading to the derepression observed. Repression of the lacZ reporter containing the giant CD1 site from Kr is CtBP-dependent, a result that contrasts with the CtBP independence of the CD itself, but this particular site may not be optimal, since it contains two mismatches. Full Giant activity may also be mediated on the native CD element through the additional high-affinity CD2 site (Stunk, 2001).

Drosophila Brakeless interacts with Atrophin and is required for Tailless-mediated transcriptional repression in early embryos: Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. Mutations were isolated in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, an expanded expression pattern was noted of the Krüppel (Kr) and knirps (kni) genes. Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. Both Drosophila and human Brakeless and Atrophin interact in vitro, and it is proposed that they act together as a co-repressor complex in many developmental contexts. The possibility is discussed that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA) (Haecker, 2007).

Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo

Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. This study presents evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. Different models are presented for 'enhancer synergy,' whereby two enhancers with overlapping activities produce authentic patterns of gene expression (Perry, 2011).

Candidate gap enhancers were identified using ChIP-chip data. Specifically, clustered binding sites for maternal and gap proteins were identified within 100 kb of every gap gene. This survey identified each of the known enhancers, as well as putative shadow enhancers. For example, a potential distal shadow enhancer was identified for hb, located 4.5-kb upstream of the proximal transcription start site (designated 'P2' in earlier literature) and upstream of the later-acting distal promoter (designated 'P1') (Perry, 2011).

A 400-bp genomic DNA fragment from this newly identified region was attached to a lacZ reporter gene and expressed in transgenic embryos. The resulting hb/lacZ fusion gene exhibits localized expression in anterior regions of the embryo similar to that seen for the endogenous gene and 'classic' enhancer identified over 20 y ago. The classic proximal and distal shadow enhancers exhibit similar responses to increasing Bicoid copy number (Perry, 2011).

ChIP-chip data also identified potential pairs of enhancers for Kr and kni. There are two distinct clusters of transcription factor binding sites upstream of Kr. The previously identified Kr 'CD2' enhancer contains the proximal enhancer but also part of the distal binding cluster. Subsequent lacZ fusion assays identified each ChIP-chip peak and underlying binding sites as separable proximal and distal enhancers. Similarly, more refined limits were determined for the kni intronic enhancer, in addition to the previously identified 5' distal enhancer. Both the distal Kr enhancer and the intronic kni enhancer produce somewhat broader patterns of expression than the endogenous gene. Additional gap enhancers were also identified for giant, including an additional distal enhancer located ~35-kb downstream within a neighboring gene (Perry, 2011).

The survey of gap and maternal binding clusters was extended to include the so-called 'head' and 'terminal' gap genes, critical for the differentiation of head structures and the nonsegmented termini of early embryos. Additional enhancers were identified for empty-spiracles (ems), huckebein (hkb), and forkhead (fkh). More refined limits were also determined for the previously identified ocelliless/orthodenticle (oc/otd) intronic enhancer. For simplicity, the two enhancers regulating a given gap gene will be identified as proximal and distal, based on their relative locations to the transcription start site (Perry, 2011).

BAC recombineering, phiC31-targeted genome integration, and quantitative in situ hybridization assays were used to determine the contributions of the proximal and distal enhancers to the hb expression pattern. BACs containing ~20 kb of genomic DNA encompassing the hb gene and flanking sequences were integrated into the same position in the Drosophila genome. The hb transcription unit was replaced with the yellow gene, which permits quantitative detection of nascent transcripts using an intronic hybridization probe. The modified BAC retains the complete hb 5' and 3' UTRs. Additional BACs were created by inactivating the proximal or distal enhancers by substituting critical regulatory elements with 'random' DNA sequences (Perry, 2011).

BAC transgenes lacking either the distal or proximal enhancer continue to produce localized patterns of transcription in anterior regions of transgenic embryos in response to the Bicoid gradient. However, the patterns are not as faithful compared with the BAC transgene containing both enhancers. Embryos were double-labeled to detect both yellow and hb nascent transcripts. During nuclear cleavage cycle (cc) 13, a substantial fraction of nuclei (14%) expressing hb nascent transcripts lack yellow transcription upon removal of the shadow enhancer. An even higher fraction of nuclei (24%) lack yellow transcription when the proximal enhancer is removed. Control transgenic embryos containing both enhancers exhibit more uniform patterns of transcription, whereby only an average of ~3% of nuclei fail to match the endogenous pattern of transcription (Perry, 2011).

The pairwise Wilcoxon rank sum test (also called the Mann-Whitney u test) was used to determine the significance of the apparent variation in gene expression resulting from the removal of either the proximal or distal enhancer. Control embryos containing the hb BAC transgene with both enhancers exhibit some variation in the number of nuclei that lack yellow nascent transcripts. Despite this variation, the statistical analyses indicate that the loss of either the proximal or distal enhancer results in a significant change in yellow transcription patterns compared with the control BAC transgene (Perry, 2011).

The preceding analyses suggest that multiple enhancers produce more uniform patterns of de novo transcription than individual proximal or distal enhancers. Additional studies were done to determine whether multiple enhancers also help produce authentic spatial limits of transcription (Perry, 2011).

The expression of hb normally diminishes at the anterior pole of cc13 to 14 embryos. This loss in expression has been attributed to attenuation of Bcd activity by Torso RTK signaling. However, the proximal enhancer fails to recapitulate this loss. In contrast, the distal enhancer is inactive at the anterior pole, and the two enhancers together produce a pattern that is similar to endogenous expression, including reduced expression at the pole (Perry, 2011).

To examine the relative contributions of the proximal and distal enhancers in this repression, yellow nascent transcripts were measured in transgenic embryos expressing BAC reporter genes containing one or both hb enhancers. Particular efforts focused on the early phases of cc14, when repression of endogenous hb transcripts is clearly evident. For the transgene lacking the proximal, classic enhancer, but containing the newly identified distal enhancer, a median of 6% (std 6%) of nuclei exhibit expression of yellow nascent transcripts but lack expression of the endogenous gene. In contrast, a median of 24% (std 11%) of nuclei displays a similar discordance upon removal of the distal enhancer. In control embryos, 16% (std 11%) of nuclei express yellow but lack hb nascent transcripts. It should be noted that the BAC transgene lacking the proximal enhancer exhibits 'super-repression' because of reduced activation at the anterior pole (Perry, 2011).

Kr/lacZ and kni/lacZ fusion genes containing either one or two enhancers were inserted into the same position in the Drosophila genome. Transgenic embryos were double-labeled to detect the expression of the transgene (lacZ) as well as the endogenous gap gene (Perry, 2011).

The kni proximal (intronic) enhancer alone produces an abnormally broad pattern of expression, especially in posterior regions. In contrast, the kni distal (5') enhancer produces erratic lacZ activation within nearly normal spatial limits. An essentially normal pattern of lacZ transcription is observed when both enhancers are combined in a common transgene (intronic enhancer 5' and distal enhancer 3' of lacZ). It appears that lacZ transcription is slightly broader than the endogenous pattern, but considerably narrower than the pattern observed for the intronic enhancer alone, and not statistically different from the expression limits of the distal enhancer alone. There is no significant narrowing of the Kr/lacZ expression pattern when both the distal and proximal enhancers are combined within the same transgene. Perhaps additional Kr regulatory elements are required for the type of narrowing observed for the kni intronic enhancer. Alternately, all of these transgenes use the eve basal promoter and it is possible that promoter-specific interactions are important for establishing the normal limits of the Kr expression pattern (Perry, 2011).

As discussed earlier, long-range repressors bound to the distal hb enhancer might inhibit the activities of the proximal enhancer at the anterior pole of precellular embryos. The distal kni enhancer might function in a similar manner to sharpen the expression limits of the intronic enhancer. The spatial limits of gap gene-expression patterns have been shown to depend on cross-repressive interactions. The kni intronic enhancer might lack critical gap repression elements because it produces an abnormally broad expression pattern. Indeed, whole-genome ChIP assays identify more putative Tailless binding sites in the distal vs. intronic enhancer. These Tailless repression elements might function in a dominant fashion to restrict the limits of the intronic enhancer (Perry, 2011).

The modest anterior expansion of the expression pattern driven by the kni intronic enhancer is more difficult to explain because this boundary is probably formed by the Hb repressor, which is not known to function in a long-range and dominant manner. If the action of short-range repressors is also affected by stochastic processes (e.g., binding of the repressor to enhancer or looping of a bound enhancer to promoter), perhaps having two enhancers might improve the chances of maintaining proper repression (Perry, 2011).

This study has presented evidence that the robust and tightly defined patterns of gap gene expression do not arise from the unique action of individual enhancers. Rather, these patterns depend on multiple and separable enhancers with similar, but slightly distinct regulatory activities. This enhancer synergy produces more homogeneous patterns of transcriptional activity, as well as more faithful spatial limits of expression (Perry, 2011).

The enhancer synergy documented in this study is somewhat distinct from the proposed role of the shadow enhancer regulating snail expression in the presumptive mesoderm. The dual regulation of snail by the proximal and distal (shadow) enhancers was shown to ensure homogenous and reproducible expression in embryo after embryo in large populations of embryos, even when they are subject to increases in temperature. In contrast, dual regulation of hb expression by proximal and distal enhancers appears to ensure homogenous activation in response to limiting amounts of the Bicoid gradient. They are used as an obligatory patterning mechanism rather than buffering environmental changes. Despite these apparent differences, it is possible that dominant repression is also used as a mechanism of synergy for the regulation of snail expression. The distal enhancer contains repressor elements (e.g., Huckebein) that inhibit the expression of the proximal enhancer at the termini (Perry, 2011).

Different mechanisms can be envisioned to account for enhancer synergy. Perhaps the simplest is that there are fewer inactive nuclei within a given gap expression domain because of the diminished failure rate of successful enhancer-promoter interactions with two enhancers rather than one. If the rates at which enhancers fail to activate transcription are completely independent, then one would expect the combined action of two enhancers to yield a multiplicative reduction in how often a given cell fails to express the gene within a given window of time. This sort of synergy does not require any direct physical or cooperative interactions between the enhancers. Nonetheless, the effect can be significant (as seen for hb). For example, two enhancers, each with a 10% uncorrelated failure rate, may together be expected to have a 1% failure rate, a 10-fold reduction. For genes that produce strong bursts of mRNA expression, this change in frequency of transcription may have a dramatic effect on the variation of total mRNA levels (Perry, 2011).

A second but critical potential mechanism of enhancer synergy concerns long-range, dominant repression. Repressors (such as Tailless) bound to one enhancer are sufficient to restrict the spatial limits of the other enhancer. There is no need for long-range repressor elements to appear in both enhancers to achieve normal spatial limits of gene expression. It has been suggested that long-range repressors, such as Hairy, mediate the assembly of positioned nucleosomes at the core promoter. Such repressive nucleosomes should block productive enhancer-promoter interactions, even for enhancers lacking repressor sites (Perry, 2011).

Regardless of the detailed molecular mechanisms, the combined action of multiple enhancers helps explain why an individual enhancer sometimes fails to recapitulate an authentic expression pattern when taken from its native context. Enhancers that produce abnormal patterns of expression (e.g., kni intronic enhancer) can nonetheless contribute to homogeneous and robust patterns of gene expression in conjunction with the additional enhancers contained within the endogenous locus (Perry, 2011).

Transcriptional Regulation

HB is autonomously capable of activating the target gene Krüppel at low concentrations and repressing it at high concentrations (Schulz, 1994).

Bicoid and Hunchback activate Krüppel expression (Hoch, 1991), while Knirps and Tailless repress Krüppel in the posterior end of the embryo (Hoch, 1992). However, Krüppel is activated by Tailless, Huckebein and Forkhead in the posterior Malpighian tubules domain (Gaul, 1990 and Schulz, 1994). Kruppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. The interactions of Kruppel with other terminal genes has been studied. The gap genes tailless and huckebein, which repress Kruppel in the central segmentation domain, activate Kruppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Kruppel expression is achieved by the interposition of forkhead, which is not involved in the control of the central domain. In addition, Kruppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Kruppel in the Malpighian tubules differ strikingly from its role in segmentation (Gaul, 1990). Kr activity determines the neural fate of tip cells by acting as a direct downstream target of proneural basic helix-loop-helix (bHLH) proteins that are restricted in response to Notch signalling. Forkhead interacts with a 400 bp cis active element of Krüppel directing Kr expression in Malpighian tubules. There are two FKH binding sites. This element also contains functional binding sites for the restricted proneural bHLH factors (Hoch, 1998).

giant, ectopically expressed, represses the expression of both the Krüppel and knirps segmentation gap genes. An analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. The apparent positive regulation of knirps by Krüppel is mediated by the negative effect of Krüppel on giant and a negative effect of Giant on knirps (Capovilla, 1992).

hindsight may regulate Krüppel. Krüppel, which accumulates in wild-type embryos in the nuclei of amnioserosal cells, is absent from most but not all of these cells in stage 11 hnt mutants (Yip, 1997).

As the germ band shortens in Drosophila melanogaster embryos, cell shape changes cause segments to narrow anteroposteriorly and to lengthen dorsoventrally. One of the genes required for this retraction process is the hindsight (hnt) gene. hnt encodes a nuclear Zinc-finger protein that is expressed in the extraembryonic amnioserosa and the endodermal midgut prior to and during germ band retraction. Through analysis of hnt genetic mosaic embryos, it has been shown that hnt activity in the amnioserosa, particularly in those cells that are adjacent to the epidermis, is necessary for germ band retraction. In hnt mutant embryos the amnioserosa undergoes premature cell death. Prevention of premature apoptosis in hnt mutants does not rescue retraction. Thus, failure of this process is not an indirect consequence of premature amnioserosal apoptosis; instead, hnt must function in a pathway that controls germ band retraction (Lamka, 1999).

The Kruppel gene is activated by hnt in the amnioserosa while the Drosophila insulin receptor (INR) functions downstream of hnt in the germ band. Kr protein is first detected in the amnioserosa of wild-type embryos during germ band extension; however, in hnt mutants, Kr is clearly absent from most amnioserosal cells by stage 11. Loss of Kr in hnt mutants is not a result of premature apoptosis of the amnioserosa. Rather the Kr gene resides downstram of Hnt in the hnt genetic hierarchy in the amniserosa. A specific role for Kr in germ band retraction remains to be defined. Using a heat shock insulin receptor transgene to overexpress the wild-type insulin receptor, nearly complete rescue of germ band retraction is observed in hnt mutants. The extent of rescue depends on the strengtth of the hnt used. The fact that high levels of the Inr can rescue germ band retraction in hnt mutants is consistent with the possibility that the Inr functions downstream of hnt in a germ band retraction pathway. Evidence against a physical model in which the amnioserosa 'pushes' the germ band during retraction is presented. Rather, it is likely that the amnioserosa functions in production, activation, or presentation of a diffusible signal required for retraction (Lamka, 1999).

Mutations in several Polycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting that Pc group genes may regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other Pc group genes show gap, pair rule, and segment polarity segmentation defects. Posterior sex combs and polyhomeotic interact with Krüppel (McKeon, 1994).

Genetic experiments and a targeted misexpression approach have been combined to examine the role of the gap gene giant (gt) in patterning anterior regions of the Drosophila embryo. The results suggest that gt functions in the repression of three target genes, the gap genes Kruppel (Kr) and hunchback (hb), and the pair-rule gene even-skipped (eve). The anterior border of Kr, which lies 4-5 nucleus diameters posterior to nuclei that express GT mRNA, is set by a threshold repression mechanism involving very low levels of Gt protein. The gap gene Kr is activated in a broad central region of precellular embryos. Midway through cleavage cycle 14, this domain extends from 41-59% egg length. The initial positioning of the anterior border of this domain is thought to be controlled by repression involving a combination of maternal and zygotic hunchback transcripts. To test whether gt is also involved in setting or maintaining this border, the Kr expression pattern was analyzed in embryos containing the st2-gt transgene, a modified version of the 480 bp eve stripe 2 enhancer. These embryos show no changes in the initial positioning of the Kr expression domain early in cleavage cycle 14, but slightly later there is a dramatic retraction of the anterior Kr border. The delay in the observed repressive effect on the Kr anterior border is probably due to the fact that the Kr domain is expressed earlier than the st2-gt transgene. Higher levels of ectopic gt result in a more severe retraction, suggesting that Kr transcription is very sensitive to repression by gt. To test whether gt affects Kr expression during normal development, Kr expression was examined in embryos that carry a strong hypomorphic gt allele. The initial Kr expression pattern was correctly established in these gt hypomorphic embryos. However, slightly later, a significant anterior expansion (from 59% to 65% egg length) is observed, suggesting that gt-mediated repression is essential for maintaining the position of the anterior border of the Kr domain (Wu, 1998).

giant activity is required, but not sufficient, for the formation of the anterior border of eve stripe 2, which lies adjacent to nuclei that express GT mRNA. It is proposed that gt's role in forming this border is to potentiate repressive interaction(s) mediated by other factor(s) that are also localized to anterior regions of the early embryo. It is not clear whether gt is sufficient for repression of the in vivo eve stripe 2 response. To test this, eve expression was examined in embryos containing the st2-gt transgene, which extends the gt domain so that it overlaps the position of eve stripe 2. Surprisingly, the ectopic gt causes only a weak transient reduction of the stripe early in cycle 14. Later the stripe recovers to full strength, but expands toward the posterior by about two nucleus diameters. Double in situ hybridization experiments show that the timing and the extent of the expansion correlates well with the retraction of the Kr domain, suggesting that the expansion of eve stripe 2 is indirectly caused by relief from Kr repression. Doubling the ectopic gt expression levels still does not cause a significantly stronger repression, suggesting that eve stripe 2 is quite insensitive to gt repression. To test whether the effects of ectopic gt on eve stripe 2 are controlled by the early or late regulatory elements, the expression of lacZ reporter genes was examined in embryos containing st2-gt transgenes. It is likely that the posterior expansion of endogenous eve stripe 2 caused by the st2-gt transgene is mediated through the early acting enhancer (Wu, 1998).

The recalcitrance of the eve stripe 2 response to ectopic gt expression led to a reexamination of the eve expression pattern in gt mutant embryos. Early in cycle 14, these mutants show a derepression in the interstripe region between stripes 1 and 2. However, later in cycle 14, gt mutants show a dramatic reduction in stripe 2 expression levels, suggesting a role for gt in maintaining the stripe. Since Kr has been previously implicated as the repressor that forms the stripe 2 posterior border, it is possible that the stripe 2 reduction in gt mutants is indirectly caused by Kr, which expands anteriorly to completely overlap the diminishing stripe. The repression of eve stripe 2 observed in gt mutants can be relieved by reducing Kr levels. These results suggest that a major function of the anterior gt domain is to prevent Kr from expanding anteriorly, thus permitting the expression of eve stripe 2. Furthermore, since gt repression maintains the position of the anterior Kr border in wild-type embryos, it indirectly defines the position of the posterior border of eve stripe 2 (Wu, 1998).

In principle, the preceding experiments support the hypothesis that gt acts as a concentration-dependent repressor to set the anterior borders of the Kr and eve stripe 2 expression domains in different positions. Ectopic gt is an effective repressor of Kr, but has little effect on the activation of eve stripe 2. In situ hybridization experiments indicate that endogenous gt levels are significantly higher than the ectopic gt driven by even the strongest st2-gt transgenic lines. Perhaps these higher endogenous levels are required for effectively setting the anterior border of eve stripe 2. If this is the case, the early expansion of eve stripe 2 toward stripe 1 detected in gt mutants should not be affected in embryos in which the endogenous gt gene is replaced by the st2-gt misexpression domain. To test this, eve expression was examined in gt mutants that contained the st2-gt5 transgene. Surprisingly, a sharp anterior eve stripe 2 border is formed in these embryos, with a clear interstripe between eve stripes 1 and 2. Furthermore, the st2-gt domain rescues eve stripe 2 to full strength, with a posterior expansion that is probably due to repression of the anterior Kr border. The relatively low levels of ectopic gt driven by the st2-gt construct overlap the endogenous gt domain and extend 4-5 nucleus diameters posteriorly. The fact that a sharp anterior eve stripe 2 border is formed in embryos containing only this domain argues against a simple concentration-dependent mechanism for setting this border. Rather, it is proposed that other factor(s) are involved along with gt in defining the anterior border of eve stripe 2 in vivo. Thus, gt may act as a potentiator of repression mediated by these localized factors. Since gt encodes a putative leucine zipper (b-ZIP) protein, one possibility is that this activity is also a b-ZIP protein that can heterodimerize with gt as part of an effective repressor complex. Repressive function in the absence of gt would be provided by a homodimer of this protein. Alternatively, since the gt site deletions tested in previous experiments removed relatively long sequences (14-43 bp), it is possible that these deletions may have removed or interrupted binding sites for other protein(s) (Wu, 1998).

gt is required for repression of zygotic hb expression in more anterior regions of the embryo. Zygotic expression of hb is initially activated by the bcd and maternal hb gradients in a broad domain that spans the anterior half of the embryo. This expression is then rapidly refined during nuclear division cycle 14, leaving a secondary pattern that includes a variable head domain, a stripe at the position of parasegment 4 (PS4), and a posterior stripe. The PS4 stripe overlaps the anterior border of the Kr domain. By examining hb expression in gt mutants, significant changes in this secondary pattern were detected. Initially, hb expression at the position of PS4 is greatly reduced, possibly because of the anterior expansion of the Kr domain in gt mutants. High levels of hb expression persist in more anterior regions of gt mutant embryos. The persistent hb expression domain appears very similar in shape to the normal gt domain, suggesting that gt may act as a repressor to clear hb expression from this part of the embryo during wild-type development. To test whether endogenous gt levels were required for this repression, hb expression was examined in gt mutants that also contained the st2-gt transgene. hb expression is repressed normally by a single copy of the st2-gt5 transgene, suggesting that relatively low levels of ectopic gt can replace this function of the endogenous gene. Since gt seems to be involved in repression of hb in anterior regions, it is possible that this repression is important for setting the anterior border of the hb PS4 stripe during wild-type development. To test this, hb expression was examined in embryos containing the st2-gt transgene. The position of the anterior border of the hb PS4 stripe appears unchanged in these embryos, suggesting that the levels of ectopic gt tested here are not sufficient to repress hb PS4 expression. However, a slight posterior expansion of this stripe could be detected in embryos with high levels of misexpression, which is probably caused by the retraction of the Kr domain. This supports the hypothesis that Kr activity is important for setting the posterior PS4 stripe border, and further demonstrates the importance of gt-mediated restriction of Kr expression to central regions of the embryo (Wu, 1998).

Localized gene expression patterns are critical for establishing body plans in all multicellular animals. In Drosophila, the gap gene hunchback is expressed in a dynamic pattern in anterior regions of the embryo. Hb protein is first detected as a shallow maternal gradient that prevents expression of posterior gap genes in anterior regions. HB mRNA is also expressed zygotically, first as a broad anterior domain controlled by the Bicoid morphogen, and then in a stripe at the position of parasegment 4 (PS4). The PS4-hb stripe changes the profile of the anterior Hb gradient by generating a localized peak of protein that persists until after the broad domain has started to decline. This peak is required specifically for the formation of the mesothoracic (T2) segment. At the molecular level, the PS4-hb stripe is critical for activation of the homeotic gene Antennapedia, but does not affect a gradient of Hb repressive activity formed by the combination of maternal and Bcd-dependent Hb. The repressive gradient is critical for establishing the positions of several target genes, including the gap genes Kruppel, knirps, and giant, and the homeotic gene Ultrabithorax. Different Hb concentrations are sufficient for repression of gt, kni, and Ubx, but a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr. These results suggest that the individual phases of hb transcription, which overlap temporally and spatially, contribute specific patterning functions in early embryogenesis (Wu, 2001).

Primary zygotic expression of HB mRNA (P2-hb) covers much of the anterior half of wild-type embryos early in nuclear cleavage cycle 14. This pattern is soon transformed into the secondary pattern, which includes the PS4-hb stripe. This stripe appears just before midcycle 14, and persists until the onset of gastrulation. The distribution of Hb protein is similar to the mRNA profile, but the protein seems to degrade more slowly. Thus, in midcycle-14 embryos, the Hb pattern consists of a broad anterior domain, with a peak at the position of PS4. Later, when cellularization is complete, Hb in anterior regions degrades further, leaving a clear stripe at PS4 (Wu, 2001).

To specifically remove this stripe, a misexpression transgene (st2-kni) was used that directs barely detectable levels of the gap gene kni at the position of PS3. Ectopic kni expression completely abolishes the PS4-hb stripe, and the peak of Hb protein observed in wild-type embryos. By the end of cellularization, no protein is detectable at the PS4 position of st2-kni embryos. The relative levels of Hb were further quantified at midcycle 14 in wild-type and st2-kni embryos. The PS4 position was determined by simultaneous fluorescence in situ hybridization with an RNA probe directed against fushi tarazu, a pair-rule gene expressed in stripes that correspond to the even-numbered parasegments (ftz stripe 2 corresponds to PS4). In st2-kni embryos, nuclei at the center of ftz stripe 2 contain only about 50% of the Hb normally present at this position. Thus, in wild-type embryos, the PS4-hb stripe alters the profile of the anterior Hb gradient by creating a peak of protein concentration at midcycle 14, and also ensures the perdurance of high Hb levels at this position until the end of cellularization (Wu, 2001).

In contrast to previous hb rescue experiments, the st2-kni transgene removes the PS4-hb stripe without changing the normal maternal and P2-hb gradients. Embryos carrying this transgene were examined for morphological defects later in development. More than 75% of these embryos die by the end of embryogenesis and show deletions of the T2 denticle band, which is derived from cells in PS4. While approximately 50% show a complete T2 deletion in dorsal and ventral regions, the phenotype is more severe on the ventral side, with ~90% completely lacking the T2 ventral denticle band. The cause of the phenotypic difference between dorsal and ventral regions is not clear. These results indicate that high levels of Hb at the position of PS4 are critical for T2 development (Wu, 2001).

To investigate the role of PS4-hb in T2 development, the expression patterns of segmentation and homeotic genes was examined in st2-kni embryos, and those fully rescued by the st2DeltaK-hb-1 transgene (a transgenic line that directs high levels of hb in a wide stripe that overlaps the PS4 position). No changes in the expression patterns of the gap genes Kr, kni, or gt were detected in st2-kni embryos. This suggests that Hb-mediated repression of these genes is not dependent on the PS4-hb stripe. However, several genes normally expressed in PS4 were significantly altered. One such gene is ftz. In st2-kni embryos, activation of ftz stripe 2 is delayed, and reduced in intensity. To test whether this reduction affects ftz function, expression of the ftz target gene engrailed was examined. en stripe 4, which is normally activated by ftz stripe 2, is also significantly reduced in st2-kni embryos (Wu, 2001).

The observed reductions in ftz stripe 2 and en stripe 4 could lead to the T2 deletion caused by the removal of the PS4-hb stripe. Surprisingly, however, addition of the st2DeltaK-hb-1 transgene, which mediates complete rescue of morphological defects in st2-kni embryos, does not detectably alter the level of ftz stripe 2 or en stripe 4. The failure to rescue these stripes may be due to the ectopic Kni in this region. However, since these embryos are rescued to adulthood, the reduced ftz and en stripes must be capable of supporting the establishment of the segment (Wu, 2001).

Previous experiments have implicated Hb as a repressor of the gap genes Kr, kni, and gt, but the expression domains of these genes are not affected by the removal of the PS4-hb stripe. Another gene controlled by Hb-mediated repression is the homeotic gene Ultrabithorax, which is strongly expressed as a stripe at the position of PS6 in late-blastoderm embryos. Since the PS4-hb stripe is also expressed at this stage, whether it is required for setting the anterior Ubx expression border was tested. Like the gap gene targets (Kr, kni, and gt), Ubx expression is undisturbed in embryos specifically lacking the PS4-hb stripe. Thus, the peak of protein provided by this stripe is not required for repression of any of these four genes (Wu, 2001).

In wild-type embryos, the anterior borders of Kr, kni, gt, and Ubx are located at different positions along the anterior-posterior axis, suggesting that they respond to different thresholds of Hb concentration. The position of the gt border, which lies most posteriorly, is established by the maternal Hb gradient. There is very little change in the position of this border in zygotic hb mutants, suggesting that the maternal gradient is sufficient for gt repression. The anterior kni border, which lies six to eight nuclear diameters from nuclei that produce zygotic HB mRNA, is also initially established by the maternal gradient. In mutants lacking zygotic hb, this border is correctly positioned early in cycle 14, but there is an anterior expansion at midcycle 14, possibly due to degradation of the maternal gradient. Whether this expansion is sensitive to ectopic Hb driven by the st2DeltaK-hb construct was tested. These experiments show a dose-dependent repression of kni back to its original position. Since only one copy of the transgene causes a strong repression, kni is sensitive to very low levels of Hb. The homeotic gene Ubx, which is normally expressed about four nuclear diameters from the posterior edge of the hb domain, also expands anteriorly in zygotic hb mutants. Addition of the st2DeltaK-hb transgene causes a dose-dependent repression of this gene as well. In this case, two copies of the transgene are significantly more effective than one, suggesting that Ubx repression requires a higher level of Hb than kni (Wu, 2001).

Finally, the gap gene Kr, whose anterior border overlaps the hb expression domain in wild-type embryos, was tested. This gene expands anteriorly in zygotic hb mutants, but cannot be repressed even by two copies of the st2DeltaK-hb transgene. This suggests that a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr (Wu, 2001).

In presumptive cephalic regions, zygotic hb mutants lack elements of the labial segment, including the H-piece and labial sense organs, and other head structures are severely disorganized, probably due to a disruption of head involution. Introduction of the st2DeltaK-hb-1 transgene causes a very weak rescue of these defects, compared to the mostly wild-type head structures found in hb mutants rescued by P2-hb transgenes. Since the st2DeltaK-hb transgene is activated at a later stage than P2-hb, perhaps high levels of Hb present earlier in anterior regions are required for the correct formation of these structures. However, the expression patterns of the head gap genes orthodenticle, button-head, and empty-spiracles are not affected in zygotic hb mutants, raising the possibility that Hb may function indirectly by preventing the expression of repressors of head development. A candidate for such a repressor is Kr, which cannot be repressed by even two copies of the st2DeltaK-hb-1 transgene. Perhaps the expanded Kr domain causes the remaining head defects in hb mutants that are partially rescued by the st2DeltaK-hb transgene (Wu, 2001).

To test this, one copy of the Kr gene was genetically removed from these embryos. This manipulation causes a dramatic rescue of most head structures, and a few embryos (~10%) display anterior phenotypes that are indistinguishable from wild type. This suggests that the expansion of Kr into presumptive head regions prevents normal head development, and that an important function of P2-hb is to restrict Kr to more posterior regions. However, the larval heads of Kr/+;hb/hb embryos still exhibit many morphological defects, suggesting that Hb also patterns the head by mechanisms that are independent of its role in Kr repression (Wu, 2001).

To understand how loss of Grunge activity affects segmentation, the expression of hunchback (hb), Krüppel (Kr), knirps (kni) and fushi tarazu (ftz) was analyzed in embryos derived from Gug35 germline clones fertilized by Gug35 sperm. In wild-type embryos, the expression of these segmentation gene products localizes to discrete domains in the early embryo. In almost all of the expression domains, loss of Gug activity increases the number of cells expressing these segmentation genes, suggesting that Gug plays a role in their repression. Later the expression of ftz displays a more complex defective pattern with some stripes being broader, and others narrower, than wild type (Erkner, 2002).

Loss of Gug activity severely affects the process of segmentation and the expression of segmentation genes when missing from the female germ line. At the blastoderm stage, most of the expression domains of hb, Kr, kni and ftz genes are expanded compared with wild type. These observations indicate that maternal production of Gug is crucial for the repression of these genes to precise domains in the early embryo. Gap proteins, including Hb, Kr and Kni are known to be required to restrict each others domains of expression. It will be interesting to test if Gug acts with these proteins for these repression activities (Erkner, 2002).

Cooperative interactions by DNA-binding proteins have been implicated in cell-fate decisions in a variety of organisms. To date, however, there are few examples in which the importance of such interactions has been explicitly tested in vivo. This study tests the importance of cooperative DNA binding by the Bicoid protein in establishing a pattern along the anterior-posterior axis of the early Drosophila embryo. bicoid mutants specifically defective in cooperative DNA binding fail to direct proper development of the head and thorax, leading to embryonic lethality. The mutants do not faithfully stimulate transcription of downstream target genes such as hunchback (hb), giant, and Krüppel. Quantitative analysis of gene expression in vivo indicates that bcd cooperativity mutants are unable to accurately direct the extent to which hb is expressed along the anterior-posterior axis; they display a reduced ability to generate sharp on/off transitions for hb gene expression. These failures in precise transcriptional control demonstrate the importance of cooperative DNA binding for embryonic patterning in vivo (Lebrecht, 2005).

A modifier screen in the eye reveals control genes for Kruppel activity in the Drosophila embryo

Irregular facets (If) is a dominant mutation of Drosophila that results in small eyes with fused ommatidia. Previous results showed that the gene Krüppel (Kr), which is best known for its early segmentation function, is expressed ectopically in If mutant eye discs. However, it was not known whether ectopic Kr activity is either the cause or the result of the If mutation. This study shows that If is a gain-of-function allele of Kr. The If mutation was used in a genetic screen to identify dominant enhancers and suppressors of Kr activity on the third chromosome. Of 30 identified Kr-interacting loci, two were cloned, and whether they also represent components of a natural Kr-dependent developmental pathway of the embryo was tested. The two genes, eyelid (eld) and extramacrochaetae (emc), which encode a Bright family-type DNA binding protein and a helix-loop-helix factor, respectively, are necessary to achieve the singling-out of a unique Kr-expressing cell during the development of the Malpighian tubules, the excretory organs of the fly. The results indicate that the Kr gain-of-function mutation If provides a tool to identify genes that are active during eye development and that a number of them function also in the control of Kr-dependent developmental processes (Carrera, 1998).

Kr expression defines the Malpighian tubule anlage at late blastoderm stage and becomes restricted to a ring of cells at the midgut/hindgut boundary from where Kr-expressing Malpighian tubule precursors evert. Previous studies have shown that the specification of Malpighian tubule fate and the segregation of the cells depend on Kr expression in the Malpighian tubule anlage. In Kr-deficient embryos, the respective cells become part of the hindgut epithelium (Carrera, 1998).

Once the tubules evert, Kr expression becomes restricted to a single cell, termed the "tip mother cell". The singling-out process of this cell from an equivalence group of Malpighian tubule precursors involves the activated Notch pathway, which restricts the proneural bHLH proteins encoded by the achaete-scute-complex (ASC) genes to the tip mother cell. In this cell, the ASC proteins act in concert with bHLH protein encoded by daughterless (da) to maintain Kr expression. The tip mother cell divides once, and the daughters give rise to the tip cell, which controls proliferation during tubule elongation and differentiates neuronal characteristics, and an excretory cell, termed "satellite cell". The satellite cell loses Kr expression in a Notch-dependent manner, whereas Kr expression is maintained in the tip cell until the end of embryogenesis (Carrera, 1998).

emc expression accompanies Malpighian tubule development in a manner similar to Kr expression. However, once the tip cell is formed, the patterns of expression become complementary, meaning that emc expression continues in all cells of the elongating Malpighian tubules except in the tip cell. To test whether the complementary patterns of Kr and emc expression reflect a regulatory effect of emc on Kr, as indicated during eye development in the If mutant, Kr expression was examined in the Malpighian tubules of emc mutant embryos. Multiple Kr-expressing cells are seen in emc mutant Malpighian tubules. This finding is consistent with the previous finding that emc mutant embryos develop multiple tip cells and that each of them continues to express achaete. Virtually the same observations have been made with Notch mutants, and Notch acts toward restricting the activity of the proneural bHLH proteins, which are required to maintain Kr expression first in the tip mother cell and subsequently in the tip cell. However, although the activated Notch pathway acts through transcriptional repression of the ASC genes, emc protein antagonizes proneural bHLH activities by sequestering the proteins as heterodimers that are incapable of binding to DNA. The results are therefore consistent with the proposal that emc functions in the control of Kr expression by antagonizing proneural bHLH activities that are required to maintain Kr expression in the tip mother cell (Carrera, 1998).

The Eld protein shows a nuclear localization, consistent with its suspected function as a transcription factor. It appears to act in multiple signaling pathways because it antagonizes wingless activity, suppresses Ras1 activity in the eye, and blocks Notch-dependent neuronal differentiation. During Malpighian tubule development, eld is expressed in a restriced area of the everting precursors that corresponds to the equivalence group of cells expressing the proneural genes (Carrera, 1998).

eld mutant embryos exert a distinct phenotype during Malpighian tubule development that is linked to Kr activity. Whereas the anlage and the four tubules evert normally, each tubule develops two instead of the normal one tip cell. Tip cell development is under the control of Kr activity, so it was next asked whether and when Kr expression is altered in eld mutant embryos. In correspondence with the mutant phenotype, the initial expression of Kr, including its restriction to the tip mother cell, appears to be normal. However, once the tip mother cell has undergone division, two instead of only one of the daughter cells maintain Kr expression. This indicates that eld activity is necessary to prevent Kr expression in the sibling of the tip cell and allows for its differentiation into a satellite cell. Thus, although emc is necessary for the restriction of Kr to the tip mother cell, eld functions specifically at the subsequent step during Malpighian tubule development where an alternative and Kr-dependent cell fate decision is taken between the daughters of the tip mother cell (Carrera, 1998).

Notch signaling is required first for the selection of the tip mother cell and subsequently for the distinction between its daughters to either develop a tip cell or a satellite cell. Consistently, in Notch mutant embryos, all cells of the proneural equivalence group develop first into tip mother cells; these cells divide and subsequently develop into the multiple tip cells that continue Kr expression. In contrast, only two tip cells were found in eld mutants. This finding implies that, if eld acts in a Notch-dependent manner and/or mediates Notch signaling, its activity is required only for the second of the two Notch-dependent differentiation steps during Malpighian tubule development. Thus, eld participates as an optional component in the Notch-signaling pathway and is needed to prevent, directly or indirectly, the maintenance of Kr expression in the satellite cell that would otherwise develop into a second tip cell (Carrera, 1998).

The results of this study demonstrate that gene activities that were identified via an artificial experimental situation, namely the ectopic expression of Kr in the developing eye disc, can lead to the identification of integral components of a Kr-dependent developmental pathway during embryogenesis. In the eye imaginal disc, emc suppresses Kr activity whereas eld has an opposite effect, but both act during embryonic Malpighian tubule development as negative regulators of Kr. No explanation is available for this phenomenon. It could mean, in negative terms, that the Kr misexpression screen turned up dosage-sensitive genes affecting cell fate that were several steps downstream from Kr activity and thus have no direct interaction with Kr. Thus, each gene identified in the modifier screen represents a candidate gene that needs to be evaluated critically through additional criteria as outlined here for eld and emc. The additional screening is essential to distinguish between direct Kr interactors and genes that mediate different read-outs of the Kr pathway in cells that have a different organ or tissue competence. However, in view of the fragmentary information concerning the spatial and temporal control of postblastodermal Kr expression and in view of the fact that the few Kr target genes of Kr were identified by molecular approaches, this experimental strategy to assess components of a Kr-dependent regulatory circuitry seems a valid one (Carrera, 1998).

Prospero, targeting Kruppel, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions, muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search were performed for direct Ladybird targets. The data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, bric-a-brac 2 gene was identified as a new component of identity code and inflated encoding αPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells (Junion, 2007).

Uncovering how the cell fate-specifying genes exert their functions and determine unique properties of cells in a tissue is central to understanding the basic rules governing normal and pathological development. To approach the cell fate determination process at a whole genome level a search was performed for transcriptional targets of the homeobox transcription factor Lb known to be evolutionarily conserved and required for specification of a subset of cardiac and muscular precursors. To this end the targeted expression profiling and the novel ChIP-on-chip method ChEST were combined. The data revealed an unexpectedly complex gene network operating downstream from lb, which appears to act not only by regulating components of the cell identity code but also as a modulator of pan-muscular gene expression at fiber-type level. Of note, the role of Drosophila lb in regulating segment border muscle (SBM) founder motility appears reminiscent of the role of its vertebrate ortholog Lbx1, known to control the migration of leg myoblasts (Vasyutina, 2005) in mouse embryos (Junion, 2007).

Earlier genetic studies revealed that within the same competence domain the cell fate specifying factors acted as repressors to down-regulate genes determining the identity of neighboring cells. Consistent with this finding, lb was found to repress msh and kruppel (kr) during diversification of lateral muscle precursors and even skipped (eve) within the heart primordium. This study found that additional identity code components are regulated negatively by lb. In the lateral muscle domain lb acts as a repressor of the MyoD ortholog nau and the NK homeobox gene slou, both known to be required for the specification of a subset of somatic muscles. This suggests that a particularly complex network of transcription factors (Ap, Msh, Kr, Nau, Slou) controls the specification of individual muscle fates in the lateral domain. Interestingly, none of these factors is coexpressed with lb in the SBM, which appears to be a functionally distinct muscle requiring a specific developmental program. Besides factors with well-documented roles in diversification of muscle fibers, the global approach identified a few novel potential players in the muscle identity network. Among them those expressed in somatic muscle precursors including the Pdp1 gene encoding Par domain factor and the CG32611 gene containing a zinc finger motif (Junion, 2007).

Interestingly, in the cardiac domain the data demonstrate that lb is able to positively regulate the expression of tin and the effector of RTK pathway pointed (pnt), both involved in cardiac cell fate specification. These findings are consistent with earlier observations that the forced lb expression leads to the ectopic tin-positive cells within the dorsal vessel. Also, during early cardiogenesis lb directly represses bric a brac 2 (bab2), which emerges as a novel component of the genetic cascade controlling the diversification of cardiac cells. Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (Junion, 2007).

The data indicate that lb exerts its muscle identity functions via regulation of pan-muscular genes that control cell movements, cell shapes and cell-cell interactions including myoblast fusion, myotube growth, and attachment events (Junion, 2007).

Establishment of cell fate during early Drosophila embryogenesis requires transcriptional Mediator subunit dMED31

During early Drosophila embryogenesis, formation of the anterior-posterior (A/P) axis depends on spatial gradients of maternal morphogens. It is well recognized that positional information is transmitted from these morphogens to the gap genes. However, how this information is being transmitted is largely unknown. The transcriptional Mediator complex is involved in the fine tuning of the signaling between chromatin status, transcription factors and the RNA polymerase II transcription machinery. This study found that a mutation in the conserved subunit of the Mediator complex, dMED31, hampers embryogenesis prior to gastrulation and leads to aberrant expression of the gap genes knirps and Krüppel and the pair-rule genes fushi tarazu and even-skipped along the A/P axis. Expression of the maternal morphogens dorsal and hunchback was not affected in dMED31 mutants. mRNA expression of dMED31 exactly peaks between the highest expression levels of the maternal genes and the gap genes. Together, these results point to a role for dMED31 in guiding maternal morphogen directed zygotic gap gene expression and provide the first in vivo evidence for a role of the Mediator complex in the establishment of cell fate during the cellular blastoderm stage of Drosophila (Bosveld, 2008).

Proper fine tuning of the eukaryotic transcriptome depends on numerous cis and trans acting factors that modulate the chromatin environment of genes and influence the RNA polymerase II (RNAPII) transcription machinery. The Mediator complex is a core processor in the signaling between RNAPII and transcription factors. This complex is an evolutionary conserved protein assembly of 25-30 subunits (Bosveld, 2008).

Support for a specific role during development of Mediator subunits is provided by several studies in Drosophila. These studies describe mostly functions of Mediator subunits during late developmental stages, but a role of the subunits during early embryonic development is largely unknown. The Mediator consists of more then 25 subunits, pointing to a multifaceted role of this complex during metazoa development. Understanding this complexity starts with the identification of the function of each subunit (Bosveld, 2008).

Drosophila MED31 was identified by bioinformatics analysis (Boube, 2000) and its presence in the Mediator complex was confirmed in purified complexes from embryos and cells (Park, 2001; Gu, 2002). In a pull down assay, the Mediator (containing dMED31) complex binds to the transcription factors Bicoid (Bcd), Krüppel (Kr), Fushi tarazu (Ftz), Dorsal (Dl) and HSF, but not Twist (Twi), Hunchback (Hb) and Even-skipped (Eve) (Park, 2001). Moreover, the Mediator complex is required for in vitro transcription from developmentally important promoters regulated by these transcription factors. Despite these in silico and in vitro results, to date the functional role of MED31 in eumetazoa remains elusive. This study reports the identification of the highly conserved Drosophila Mediator subunit dMED31 as a novel maternal-effect gene necessary for proper segment specification during early embryogenesis. dMED31 mutant females have fecundity defects and embryos deposited by homozygous mothers display severe defects along the anterior-posterior (A/P) axis when gastrulation is initiated. Whereas expression of maternal morphogens is not affected, alterations in gap and pair-rule gene expression during the proceeding blastoderm stage correlate with these defects observed in dMED31 mutant embryos. Remarkably, a small percentage of the progeny of homozygous mutant females escape from embryonic death and develop into adults. These escapers have defects in their abdominal segmentation pattern, a phenotype enhanced by mutations in dMED13. These findings provide the first in vivo evidence for a specific role of dMED31 in establishing cell fate in the cellular blastoderm and point to a role for the Mediator in guiding maternal morphogen directed zygotic gap gene expression (Bosveld, 2008).

These findings identify a component of the conserved eukaryotic transcriptional Mediator complex, dMED31, that is required for normal initiation of zygotic gene expression during the blastoderm stage of Drosophila embryogenesis. Female flies that carry a mutation in the dMED31 gene suffer from fecundity defects and the embryos deposited by these females display abnormal embryogenesis due to aberrant cell migration events upon gastrulation. Impaired embryogenesis coincided with changes in kni, Kr, ftz and eve expression along the A/P axis. Furthermore, adult flies derived from embryos that escaped from embryonic death displayed severe defects in their abdominal segmentation. Because mRNA production was hampered in dMED311/1, these abdominal defects were likely the result of abnormal maternal and zygotic dMED31 mRNA production. A mutation in the Mediator subunit dMED13 also caused segmentation defects and this mutant enhanced the dMED31 mutant maternal effect phenotype. Therefore, these data indicate that the Mediator complex directs zygotic gene expression upon egg deposition to establish cell fate in the embryonic blastoderm (Bosveld, 2008).

In order to accomplish cell fate determination, cells gain a transcriptional poised state during early embryogenesis that is maintained throughout development and requires many cis and trans acting factors that modulate the chromatin environment of the genes involved. In Drosophila, cell identity along the A/P is established in the blastoderm stage when the pair-rule genes are expressed. A/P polarity is controlled by the maternal morphogenes cad, nos, bcd and hb whose activity results in the spatio-temporal expression of the gap genes gt, kni, tll and Kr. These gap genes are the first genes expressed along the A/P axis and encode transcription factors that in turn govern patterned expression of the pair-rule genes. Pair-rule gene expression occurs in distinct stripes and is accompanied by cellularization. Thus when cellularization takes place, large clusters of cells gain an imprint that defines the primordial segments. Cell identity is fine tuned when expression of the segment polarity and Hox genes is activated. Although this cascade of maternal, gap, pair-rule and segment polarity genes is well studied, much remains unknown how the maternal morphogens regulate RNAPII activity at their cognate promoters in order to establish regional domains of gap gene expression (Bosveld, 2008).

Because segmentation defects in escaper flies derived from dMED311/1 mothers were restricted to the abdomen, it is possible that the bithorax complex (BX-C) is abnormally expressed. This complex contains the homeotic genes Ultrabithorax, abdominal A and Abdominal B, which control the identity of the posterior two-thirds of the fly. Mutations in hb, Kr, tll and kni affect expression of BX-C and result in homeotic transformation. No complete homeotic transformations of entire parasegments were observed, suggesting an indirect effect of dMED311 on Hox activation. Since segment identity is established during early embryogenesis, this implies that only groups of cells and not whole primordial segments gained abnormal imprinting. Regional errors in cellular imprinting are supported by the variety of the abdominal defects observed in adult flies. Moreover, defects in embryogenesis were accompanied by cell loss at the embryonic poles and aberrant migratory behavior of cells upon gastrulation, processes which occur prior to the activation of the segment polarity and the Hox genes. Finally, early developmental defects coincided with abnormal expression of the gap genes kni and Kr and subsequently the pair-rule genes ftz and eve. These genes are expressed prior to Hox gene expression and are required for activation/repression of the Hox cluster. Although it is possible that the abdominal region is preferentially sensitive for a mutation in dMED31, it is more likely that random defects during formation of the abdomen are tolerated, whereas defects in other regions of the embryo are incompatible with adult viability and these adults never eclose (Bosveld, 2008).

Several intriguing questions remain: why is the embryonic phenotype so variable (>90% of the mutant embryos die, while a small percentage of embryos is able to reach the adult stage), why are mainly embryos affected by a mutation in dMED31 and what is the primary embryonic defect caused by a mutation in dMED31? Answers to these questions can be derived from studies of the Mediator in yeast in combination with the current data. The budding yeast MED31 protein is part of the Mediator transcription initiation complex. Although a mutation in yeast MED31 affects gene expression, mutants display no sensitivity to transcriptional inhibition by 6-azauracil and MED31 is not essential for growth. However, yeast MED31 mutants have a synthetic growth defective phenotype when combined with mutations in genes encoding for the two largest subunits of RNAPII (RPB1, RPB2) and the transcription initiation factors TFIIB and TFIIS. As in yeast, depletion of dMED31 in Drosophila SL2 cells by RNAi does not interfere with the Mediator composition and no growth alterations have been reported. Thus, Drosophila MED31, like yeast MED31, might not be essential for RNAPII activity per se, but could be an auxiliary factor involved in the signaling between specific transcription factors and the RNAPII machinery. Together, these findings and the current data suggest that dMED31 is not required for transcription in general, but is merely required for the fine tuning of transcription of specific genes (Bosveld, 2008).

Based on studies in yeast, it has been proposed that the Mediator functions as a platform that allows rapid regulation of transcription at (re)initiation. Fast regulation and (re)initiation of transcription might be key during the interphase periods of the final syncytial cell cycles when zygotic transcription is initiated, while such large scale, strict and 'fast' control over transcription would not be essential during subsequent stages of development and thus may explain why dMED31 function is essential during early embryogenesis. The observation that a small percentage of embryos derived from dMED311/1 mothers is able to develop into an adult, while the majority of the embryos displayed severe defects during embryogenesis, might also be attributed to such auxiliary function(s) of the dMED31 protein. Minor differences in dMED31 protein levels, due to the hypomorphic dMED311 allele, may result in subtle changes in the expression of the gap and pair-rule genes and allow embryos to progress throughout embryogenesis, but with the formation of segmentation defects. On the other hand, in the majority of embryos, more severe changes in gap and pair-rule patterning occur, which results in embryonic death (Bosveld, 2008).

In summary, this study demonstrated that dMED31 is essential to establish regional domains of expression of cell fate determinants kni, Kr, ftz and eve. mRNA expression of dMED31 peaks exactly between maternal morphogen and gap gene expression and it has been demonstrated that the Mediator complex is able to bind to several maternal transcription factors. Together this indicates that the Mediator complex constitutes an interface between the maternal morphogens and the RNAPII machinery to guide zygotic gene expression of cell fate determinants that specify primordial segment identity. These findings provide the first in vivo evidence for a role of the Mediator complex in establishing cell fate during early embryogenesis and since MED31 resembles one of the most conserved subunits within the Mediator complex this protein could serve a crucial role in the control of RNAPII activity during early developmental processes in all higher eukaryotes (Bosveld, 2008).

STAT is an essential activator of the zygotic genome in the early Drosophila embryo

In many organisms, transcription of the zygotic genome begins during the maternal-to-zygotic transition (MZT), which is characterized by a dramatic increase in global transcriptional activities and coincides with embryonic stem cell differentiation. In Drosophila, it has been shown that maternal morphogen gradients and ubiquitously distributed general transcription factors may cooperate to upregulate zygotic genes that are essential for pattern formation in the early embryo. This study shows that Drosophila STAT (STAT92E) functions as a general transcription factor that, together with the transcription factor Zelda, induces transcription of a large number of early-transcribed zygotic genes during the MZT. STAT92E is present in the early embryo as a maternal product and is active around the MZT. DNA-binding motifs for STAT and Zelda are highly enriched in promoters of early zygotic genes but not in housekeeping genes. Loss of Stat92E in the early embryo, similarly to loss of zelda, preferentially down-regulates early zygotic genes important for pattern formation. STAT92E and Zelda synergistically regulate transcription. It is concluded that STAT92E, in conjunction with Zelda, plays an important role in transcription of the zygotic genome at the onset of embryonic development (Tsurumi, 2011).

This study describes a bioinformatics approach to investigating the mechanisms controlling transcription of the zygotic genome that occurs during the MZT; STAT92E was identified as an important general transcription factor essential for up-regulation of a large number of early 'zygotic genes'. The role of STAT92E was described in controlling transcription of a few representative early zygotic genes, such as dpp, Kr, and tll, that are important for pattern formation and/or cell fate specification in the early embryo. These studies suggest that STAT92E cooperates with Zelda to control transcription of many 'zygotic genes' expressed during the MZT. While STAT mainly regulates transcription levels, but not spatial patterns, of dpp, tll, and Kr, and possibly also other 'zygotic genes', Zelda is essential for both levels and expression patterns of these genes (Tsurumi, 2011).

The transcriptional network that controls the onset of zygotic gene expression during the MZT has remained incompletely understood. It has been proposed that transcription of the zygotic genome depends on the combined input from maternally derived morphogens and general transcription factors. The former are distributed in broad gradients in the early embryo and directly control positional information (e.g., Bicoid, Caudal, and Dorsal), whereas the latter are presumably uniformly distributed regulators that augment the upregulation of a large number of zygotic genes. Other than Zelda, which plays a key role as a general regulator of early zygotic expression, the identities of these general transcriptional activators have remained largely elusive. It has been shown that combining Dorsal with Zelda- or STAT-binding sites supports transcription in a broad domain in the embryo. The demonstration of STAT92E as another general transcription factor sheds light on the components and mechanisms of the controlling network in the early embryo. Moreover, STAT92E and Zelda may cooperate to synergistically regulate zygotic genes. The results thus validate the bioinformatics approach as useful in identifying ubiquitously expressed transcription factors that may play redundant roles with other factors and thus might otherwise be difficult to identify (Tsurumi, 2011).

The conclusion that STAT92E is important for the levels but not the spatial domains of target gene expression in the early embryo is consistent with several previous reports. It has been shown that in Stat92E or hop mutant embryos, expression of eve stripes 3 and 5 are significantly reduced but not completely abolished. In addition, JAK/STAT activation is required for the maintenance of high levels, but not initiation, of Sxl expression during the MZT. Moreover, it has previously been shown that STAT92E is particularly important for TorsoGOF-induced ectopic tll expression but not essential for the spatial domains of tll expression in wild-type embryos under normal conditions. On the other hand, Zelda may be important for both levels and spatial patterns of gene expression. This idea is consistent with the finding that Zelda-binding sites are enriched in both promoter and promoter-distal enhancers regions, whereas STAT-binding sites are enriched in promoter regions only. It has been reported that pausing of RNA polymerase II is prominently detected at promoters of highly regulated genes, but not in those of housekeeping genes. In light of these results that STAT and Zelda sites are highly enriched in the early zygotic gene promoters, it is suggested that these transcription factors might contribute to chromatin remodeling that favors RNA polymerase II pausing at these promoters (Tsurumi, 2011).

Finally, the MZT marks the transition from a totipotent state to that of differentiation of the early embryo. As a general transcription factor at this transition, STAT, together with additional factors (such as Zelda), is important for embryonic stem cell differentiation. Further investigation is required to understand the molecular mechanism by which STAT and Zelda cooperate in controlling zygotic transcription in the early Drosophila embryo. Moreover, it would be interesting to investigate whether STAT plays similar roles in embryonic stem cell differentiation in other animals (Tsurumi, 2011).

The Drosophila gap gene network is composed of two parallel toggle switches

Drosophila gap genes provide the first response to maternal gradients in the early fly embryo. Gap genes are expressed in a series of broad bands across the embryo during first hours of development. The gene network controlling the gap gene expression patterns includes inputs from maternal gradients and mutual repression between the gap genes themselves. In this study a modular design is proposed for the gap gene network, involving two relatively independent network domains. The core of each network domain includes a toggle switch corresponding to a pair of mutually repressive gap genes, operated in space by maternal inputs. The toggle switches present in the gap network are evocative of the phage lambda switch, but they are operated positionally (in space) by the maternal gradients, so the synthesis rates for the competing components change along the embryo anterior-posterior axis. Dynamic model, constructed based on the proposed principle, with elements of fractional site occupancy, required 5-7 parameters to fit quantitative spatial expression data for gap gradients. The identified model solutions (parameter combinations) reproduced major dynamic features of the gap gradient system and explained gap expression in a variety of segmentation mutants (Papatsenko, 2011).

Fertilized eggs of Drosophila contain several spatially distributed maternal determinants - morphogen gradients, initiating spatial patterning of the embryo. One of the first steps of Drosophila embryogenesis is the formation of several broad gap gene expression patterns within first 2 hrs of development. Gap genes are regulated by the maternal gradients, so their expression appears to be hardwired to the spatial (positional) cues provided by the maternal gradients; in addition, gap genes are involved into mutual repression. How the maternal positional cues and the mutual repression contribute to the formation of the gap stripes has been a subject of active discussion (Papatsenko, 2011).

Accumulated genetics evidence and results of quantitative modeling suggest the occurrence of maternal positional cues (position-specific activation potentials), contributing to spatial expression of four trunk gap genes: knirps (kni), Kruppel (Kr), hunchback (hb) and giant (gt). Existing data suggest that the central Knirps domain stripe is largely the result of activation by Bicoid (Bcd) and repression by Hunchback. Central domain Kruppel stripe is the result of both activation and repression from Hunchback, which acts as a dual transcriptional regulator on Kr. Hunchback is one of the most intriguing among the segmentation genes. Maternal hb mRNA is deposited uniformly, but its translation is limited to the anterior, zygotic anterior expression of hb is under control of Bcd and Hb itself. Zygotic posterior expression of Hunchback (not included in the current model) is under the control of the terminal torso signaling system. Giant is activated by opposing gradients of Bicoid and Caudal and initially expressed in a broad domain, which refines later into anterior and posterior stripes. This late pattern appears to be the consequence of Kruppel repression (Papatsenko, 2011).

Predicting functional properties of a gene network combining even a dozen genes may be a difficult task. To facilitate the functional exploration, gene regulatory networks are often split into network domains or smaller units, network motifs with known or predictable properties. The network motif based models can explain dynamics of developmental gradients and even evolution of gradient systems and underlying gene regulatory networks. The gene network leading to the formation of spatial gap gene expression patterns is an example, where simple logic appeared to be far behind the system's complexity. Gap genes provide first response to maternal gradients in the early fly embryo and form a series of broad stripes of gene expression in the first hours of the embryo development. While the system has been extensively studied in the past two decades both in vivo and in silico a simple and comprehensive model explaining function of the entire network has been missing (Papatsenko, 2011).

In the current study, a modular design has been proposed for the gap gene network; the network has been represented as two similar parallel modules (or two sub networks). Each module involved three network motifs, two for maternal inputs (one for one gap gene) and a toggle switch describing mutual repression in the pair of the gap genes. Formally, the toggle switches present in the gap gene network are evocative of the bistable phage lambda switch; however, they are operated by maternal inputs and their steady state solutions depend on spatial position in embryo, not environmental variables. The proposed modular design accommodated 5-7 realistic parameters and reproduced major known features of the gap gene network (Papatsenko, 2011).

Krüppel expression levels are maintained through compensatory evolution of shadow enhancers

Many developmental genes are controlled by shadow enhancers-pairs of enhancers that drive overlapping expression patterns. This study hypothesized that compensatory evolution can maintain the total expression of a gene, while individual shadow enhancers diverge between species. This study analyzed expression driven by orthologous pairs of shadow enhancers from Drosophila melanogaster, Drosophila yakuba, and Drosophila pseudoobscura that control expression of Krüppel, a transcription factor that patterns the anterior-posterior axis of blastoderm embryos. The expression driven by the pair of enhancers was conserved between these three species, but expression levels driven by the individual enhancers was not. Using sequence analysis and experimental perturbation, it was shown that each shadow enhancer is regulated by different transcription factors. These results support the hypothesis that compensatory evolution can occur between shadow enhancers, which has implications for mechanistic and evolutionary studies of gene regulation (Wunderlich, 2015)

It was first verified that the level and position of mRNA expression driven by the pair of shadow enhancers are quantitatively conserved between D. mel, D. yak, and D. pse. The endogenous Kr pattern in the three species is spatially conserved with respect to the expression pattern of its regulators. Compared to other TFs involved in embryonic patterning, RNA sequencing (RNA-seq) measurements show that the overall levels of Kr expression are among the most strongly conserved between the three species. Shadow enhancer function was directly measured using transgenic reporter lines in D. mel, where the trans environment, promoter, and other variables affecting expression are identical. By normalizing levels of mRNA with a co-stain, both expression patterns and levels between reporter constructs were compared (Wunderlich, 2015).

It was found that the pair of Kr shadow enhancers from D. mel, D. yak, and D. pse drive indistinguishable gene expression levels and highly similar expression patterns. The median expression levels are not statistically different between reporter lines. The boundaries of the expression patterns are similar, but the D. mel reporter line drives a pattern that is shifted to the anterior by 1% or approximately one cell width (Wunderlich, 2015).

If compensatory evolution occurs between a pair of shadow enhancers, expression driven by each individual enhancer may differ between species. Therefore, the mRNA expression driven by all six individual enhancers constituting the three orthologous Kr pairs was measured. In this setting, the signature of compensatory evolution is that the function of the combined construct is conserved while the function of individual enhancers may diverge. For this reason, focus was placed on expression level, which is conserved in the combined constructs, and not the spatial pattern, which differs slightly between species. However, it is noted that the differences in spatial expression patterns are more dramatic in constructs driven by individual enhancers compared to the combined constructs, suggesting that compensatory evolution may also stabilize the spatial pattern (Wunderlich, 2015).

The expression levels driven by isolated enhancers differ between species. The D. pse proximal enhancer drives higher levels of mRNA expression than the D. mel and D. yak proximal enhancers. In contrast, the D. mel and D. yak distal enhancers drive higher levels of mRNA expression than the D. pse distal enhancer. These differences are consistent with the phylogenetic distances between the species, which are ~25 million years between D. mel and D. pse and ~10 million years between D. mel and D. yak. Thus, while the expression level driven by the pair of Kr shadow enhancers is conserved between these three species, the levels of mRNA expression driven by each individual enhancer are not, supporting the hypothesis that the shadow enhancers are subject to compensatory evolution (Wunderlich, 2015).

Chimeric constructs were made that combine the proximal and distal enhancers from different species. It was hypothesized that these chimeras would not drive the same expression levels as constructs containing pairs of shadow enhancers from a single species. Because the endogenous sequence between the enhancers is unalignable between D. mel and D. pse, this sequence was replaced with an artificial spacer from the lambda phage genome. It was confirmed that the single-species constructs with a lambda spacer drive the same expression levels as those with endogenous spacers. Chimeras with combinations of the D. mel and D. pse enhancers were generated, and these chimeras were each found to drive significantly different levels of gene expression; they also drive significantly different levels of gene expression than the single-species constructs, with the exception of chimera 1 and the D. mel lambda spacer construct. Together, these results suggest that compensatory evolution acts to maintain overall Kr expression levels and, more broadly, that selection simultaneously acts on all the regulatory DNA controlling a gene, in line with previous work (Wunderlich, 2015).

To uncover which DNA sequence changes are responsible for compensatory evolution of expression level, the binding site content of each enhancer was examined. Focused was placed on activators because, at this stage in development, the spatial pattern of Kr's expression is set by repressors while the level of mRNA is set by activators. Genetic studies have shown that Kr is activated by bicoid (bcd), Stat92E, Zelda (Zld), and Hunchback (Hb). To map these genetic interactions to the individual shadow enhancers, TFs whose binding sites are overrepresented in each enhancer sequence were examined compared to the genomic background. Bcd and Zld sites were overrepresented in the D. mel and D. pse distal enhancers, and Stat92E sites were overrepresented in the D. mel and D. pse proximal enhancers. Hb sites were overrepresented in the proximal and distal enhancers from both species. Therefore, it is hypothesized that the proximal and distal enhancers are activated by different TFs (Wunderlich, 2015).

To test this hypothesis for Bcd and Stat92E, the activity of each enhancer reporter line was measure in embryos depleted for each gene separately. In support of the hypothesis, the proximal enhancers drive very low expression levels in Stat92E RNAi embryos. In bcd RNAi embryos, the distal enhancers drive weaker expression than the proximal enhancers (Wunderlich, 2015).

To test the role of zld in regulating these two enhancers, focus was placed on the onset of expression, because zld regulates timing of transcription in blastoderm embryos. Therefore, it was hypothesized that the zld-sensitive distal enhancers drive transcription earlier than proximal enhancers. The fraction of embryos expressing lacZ was measured during stage 4, the stage of development immediately preceding the blastoderm stage. The D. mel distal enhancer reporter line drove robust lacZ expression in all stage-4 embryos, compared with none from the D. mel proximal line. The same trend held true for the D. pse distal and proximal reporter lines (Wunderlich, 2015).

To test how Hb regulates the Kr shadow enhancers, the D. mel enhancers were measured in embryos with ventrally mis-expressed hb. In the ventral region, the proximal enhancer's expression pattern expands to the posterior, while the distal enhancer's pattern retreats, suggesting that Hb activates the proximal enhancer and represses the distal. This is consistent with genetic evidence that Hb both activates and represses Kr (Wunderlich, 2015).

Because these experiments are trans-perturbations, whether each TF is directly acting on each enhancer cannot be assessed; testing direct interaction would require mutation of the corresponding binding sites in each enhancer. Though possible, this can be challenging. The hypothesis is favored that these interactions are direct because of the binding site analysis and existing chromatin immunoprecipitation sequencing (ChIP-seq) data, which shows differential binding of Bcd, Zld, and Hb activators to the Kr shadow enhancers. However, even if these interactions are not direct, the different responses of the proximal and distal enhancers to perturbation show that these enhancers use different regulatory logic, contradicting the initial hypothesis that shadow enhancers bind to the same set of TFs. The emerging picture is that shadow enhancers each build the same pattern in different ways. Previous studies have shown that the Snail shadow enhancers respond differently to a single repressor, and in the eve locus, Hb activates one shadow enhancer for stripe 7 and represses the other. This study finds an even more dramatic difference in the regulatory logic of the Kr shadow enhancers: each is activated by a non-overlapping set of TFs (Wunderlich, 2015).

The motivation for identifying the activators of the two Kr enhancers was to uncover the changes in regulatory DNA that lead to compensation in expression levels. However, because the pair uses different TFs, this is impossible in a small dataset. If both enhancers were controlled by the same TF, it would be possible to look for anti-correlation in TF binding sites, such as gain of Bcd sites in one enhancer with compensatory loss in the other. However, because the two enhancers are controlled by different sets of activators, a large number of sequence differences could explain expression-level changes between species, e.g., changes in the number, strength or arrangement of any of the activator binding sites. This is insufficiently constrained by measurements of three pairs of orthologous shadow enhancers. However, with measurements from a much larger set of orthologs, it may be possible to discern the sequence changes underlying the compensatory evolution of expression levels (Wunderlich, 2015).

This study hasassessed compensatory evolution between shadow enhancers using reporter constructs that contain the eve basal promoter (Experimental Procedures), which come with inherent strengths and weaknesses. The reporter constructs are highly controlled: when integrated in a site-specific manner, any significant differences between transgenic reporter lines can be ascribed to the differences in the enhancer sequences driving the reporter. A previous study showed the power of this approach in a study of compensatory evolution within the eve stripe 2 enhancer. However, in the intact animal, promoters, UTRs, and other factors also affect expression; therefore, reporter constructs do not fully recapitulate evolution in the natural setting. The advent of genome editing tools will make experiments in the intact locus more feasible, but interpretation of these experiments will be complicated by the feedback mechanisms present in many transcriptional circuits, which will make it hard to discern the direct effects of changes in regulatory DNA (Wunderlich, 2015).

The observation of compensatory evolution within enhancers led to the influential idea that stabilizing selection acts on entire enhancers rather than on individual TF binding sites. This allows for flexible constraints on TF binding site organization within an enhancer). The results support the hypothesis that there is also stabilizing selection between shadow enhancers and, therefore, flexibility in how expression is controlled by multiple enhancers in a locus. The fact that there are multiple ways to get the same gene expression pattern, with flexibility both in individual pieces of regulatory DNA and in how information is allocated between them, leads to a vast neutral sequence space of regulatory DNA. This genetic variation is the substrate upon which evolution can act. Within a species, this large sequence space allows the species to be mutationally close to a wide range of phenotypes, allowing for a large number of evolutionary paths to novelty (Wunderlich, 2015).

The list of genes controlled by shadow enhancers, super enhancers, and other large constellations of regulatory DNA is rapidly growing, and these genes are often involved in key developmental programs or disease progression. Deciphering how these pieces of regulatory DNA work together to control expression -- and, by extension, how they are constrained during evolution -- is an important step toward decoding transcriptional regulation in animals (Wunderlich, 2015).

Analysis of functional importance of binding sites in the Drosophila gap gene network model

The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. This study elaborates a two-layer model for the Drosophila gap gene network and includes in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. The new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. The functional importance of binding sites was quantified by calculating their impact on gene expression in the model, and how these impacts correlate across all sites were calculated under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains (Kozlov, 2015).

Krüppel: Biological Overview | Evolutionary Homologs | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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