bicoid


REGULATION

Targets of Activity (part 1/2)

The anterior-posterior (A-P) and dorsal-ventral (D-V) axes of the early Drosophila embryo are established by two key maternal morphogens: BCD and Dorsal, respectively. The BCD protein is expressed in a broad concentration gradient along the A-P axis, with peak levels present at the anterior pole, while DL is expressed in a gradient along the D-V axis with peak levels along the ventral surface. The two morphogens are unrelated and their gradients are formed by distinct processes. Nonetheless, they generate sharp on/off stripes of target gene expression using similar mechanisms. Both morphogens act to induce overlapping patterns of other genes in the early embryo, both transcriptional activators and repressors. The activators and repressors bind to closely linked sites within short (300 to 500 bp) target promoter elements that act like on/off switches. The activators act in concert with the morphogen to define a broad region where target genes can be initiated. Borders of target gene expression are established by the repressors, resulting in the formation of stripes (Ip, 1992).

How are morphogenetic gradients interpreted in terms of embryonic gene transcription patterns within a syncytium such as the Drosophila blastoderm? A hypothetical model (Kerszberg, 1994) postulates a morphogen which is itself a spatially distributed transcription factor M or which generates a distribution of such a morphogenic factor. This model also postulates an additional, zygotically transcribed "vernier" factor V. M and V form all possible dimers: MM, MV, and VV. These are differentially translocated to the nuclei and bind with various affinities to responsive elements in the V promoter, thereby contributing to activation/inactivation of V transcription. A hypothetical model is presented in which the different dimers serve to activate the vernier factor in various distributions through the embryo. Interpretations in terms of Drosophila genes bicoid and hunchback are proposed (Kerszberg, 1994).

Described here are experiments to compare the activities of two Drosophila homeodomain proteins, Bicoid (Bcd) and an altered-specificity mutant of Fushi tarazu, Ftz(Q50K). Although the homeodomains of these proteins share a virtually indistinguishable ability to recognize a consensus Bcd site, only Bcd can activate transcription from natural enhancer elements when assayed in both yeast and Drosophila Schneider S2 cells. Analysis of chimeric proteins suggests that both the homeodomain of Bcd and sequences outside the homeodomain contribute to its ability to recognize natural enhancer elements. Unlike the Bcd homeodomain, the Ftz(Q50K) homeodomain fails to recognize nonconsensus sites found in natural enhancer elements. The defect of a chimeric protein containing the homeodomain of Ftz(Q50K) in place of that of Bcd can be preferentially restored by converting the nonconsensus sites in natural enhancer elements to consensus sites. These experiments suggest that the biological specificity of Bcd is determined by combinatorial contributions of two important mechanisms: the nonconsensus site recognition function conferred by the homeodomain and the cooperativity function conferred primarily by sequences outside the homeodomain. A systematic comparison of different assay methods and enhancer elements further suggests a fluid nature of the requirements for these two Bcd functions in target selection (Zhao, 2000).

The two K50 homeodomain proteins, Bcd and Ftz(Q50K), which have similar affinities to a consensus TAATCC site, exhibit distinct abilities in mediating transcriptional activation from natural enhancer elements. This observation exemplifies a puzzle underlying target selection by homeodomain proteins: why do homeodomain proteins behave differently in vivo while sharing similar or identical DNA binding specificities? It is suggested that the recognition of nonconsensus sites represents an essential biochemical function that helps define biological specificity. This idea is supported by experiments demonstrating that the activity of LexA-Bcd-Ftz(Q50K)HD, which contains the Ftz(Q50K) homeodomain and fails to bind to nonconsensus sites, can be preferentially restored by converting the natural nonconsensus sites to consensus sites. Nonconsensus sites are also found in the hb enhancer elements from other fly species. Previous studies have shown that efficient activation by homeodomain proteins requires a minimal number of recognition sites, reflecting their intrinsically weak properties. Thus, nonconsensus sites found in natural enhancers, depending on their architectures (e.g., number and type of sites), are expected to either merely modulate transcription levels or act as specificity-defining elements (Zhao, 2000).

Because of their critical role in mediating Bcd function, it is important to understand how nonconsensus sites are recognized by the Bcd homeodomain. Chemical-footprint experiments with the consensus site A1 and the nonconsensus site X1 suggest that the Bcd homeodomain can establish different sets of contacts with different recognition sequences. The experiments further suggest that Arg 54 of the Bcd homeodomain makes a base-specific contact with the fourth-position guanine (i.e.,TAAGCT, shown underlined) unique to X1. In the Ftz(Q50K) homeodomain, the 54th position contains methionine. However, an arginine residue artificially introduced in the 54th position of Ftz(Q50K) fails to confer an X1 recognition ability on the protein. It is suggested that both the homeodomain framework and specific residues play important roles in nonconsensus-site recognition. In this context, it is interesting to note that complexes containing Ftz(Q50K) and Bcd homeodomains exhibit slightly different mobilities in electrophoresis. The analysis of several other natural K50 homeodomains further reveals that the ability to recognize all tested nonconsensus sites is unique to the Bcd homeodomain. It is proposed that the nonconsensus site recognition function of the Bcd homeodomain is a noncoincidental property that defines a unique biological specificity for Bcd (Zhao, 2000).

The present study also further underscores the importance of protein-protein interaction between Bcd molecules in natural-target selection. Such a protein interaction function, which is conferred by Bcd sequences outside its homeodomain, is responsible primarily for its cooperative DNA binding activity. Interestingly, the hb and kni enhancer elements used in this study exhibit different requirements for the protein interaction function. In particular, Ftz-BcdHD, which contains the Bcd homeodomain in the framework of Ftz, can efficiently activate transcription from the hb enhancer element while it is virtually inactive on the kni enhancer element. It is proposed that a residual cooperativity function conferred by the Bcd homeodomain, while insufficient on the kni enhancer element, contributes to the chimeric protein's ability to recognize the hb enhancer element. It is noted that the hb and kni enhancer elements have architectural differences in both Bcd site composition and alignments. The hb enhancer element contains three dispersed perfect TAATCC consensus sites, in addition to at least three centrally located, tightly linked nonconsensus sites. In contrast, the kni enhancer element contains symmetrically arranged and tightly linked sites that do not match the TAATCC consensus. Exactly how these architectural features determine the different requirements for Bcd functions remains to be determined (Zhao, 2000).

The results suggest that both the cooperativity and nonconsensus site recognition functions of Bcd contribute combinatorially to target selection. Interestingly, the degree of reliance on these two functions can be influenced not only by enhancer architecture but also by the host factor(s). In particular, Bcd-Ftz(Q50K)HD-VP16 can activate transcription from the kni enhancer elements in Schneider cells but not in yeast. This difference is unlikely to be due to the reporter gene status, because this protein fails to activate the kni-lacZ reporter gene in yeast regardless of whether it is integrated or carried on a replicating plasmid. It is possible that a factor(s) present in Schneider cells but absent from yeast can influence the activity of this derivative on the kni enhancer element (but not on the hb enhancer element). Although a cofactor for Bcd has also been proposed previously, its identity remains elusive; interestingly, a recent study suggests that Bcd activity can be potentiated modestly by the Drosophila protein Chip. A systematic comparison of different assay systems also reveals that, in many instances, dependence on Bcd functions is reduced on reporter genes carried on plasmids, presumably because they are more accessible to activators than are integrated reporters. For example, Ftz-BcdHD-VP16 shows a higher relative activity on plasmid reporters containing the hb, kni, and kni(6A) enhancer elements than on integrated reporters. Similarly, Bcd-Ftz(Q50K)HD-VP16 has a higher relative activity on hb(6A)-lacZ and kni(6A)-lacZ plasmid reporters than on the integrated reporters. Together, these results illustrate a fluid nature of the requirements for Bcd functions in target selection, a process reflective of an efficient interaction between the activator and specific enhancers in physiological environments (Zhao, 2000).

Extensive studies of Q50 homeodomain proteins have produced two contrasting models to explain how their biological specificities are achieved. Both models center on the existence of cofactors, but the roles of these cofactors differ. The first model, referred to as the coselector model, suggests that cofactors selectively interact with different homeodomain proteins to enhance their DNA binding specificities. The second model, referred to as the widespread-binding model, proposes that, although most Q50 homeodomain proteins recognize similar or identical targets in vivo, cofactors can modulate the regulatory activities of these DNA-bound proteins. The latter model is supported by in vivo cross-linking experiments and a recent finding that a Ubx derivative with a strong activation function gains a novel biological specificity. Although the present studies focus on the K50 homeodomain protein Bcd, nonconsensus site recognition most likely also plays an important role, to various extents, in target selection by all homeodomain proteins (Zhao, 2000 and references therein).

Organization of developmental enhancers in the Drosophila embryo

Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. This study investigated the regulatory 'grammar' of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid-Bicoid, Hunchback-Hunchback, Bicoid-Dorsal, Bicoid-Caudal and Dorsal-Twist. Direct evidence is presented for the importance of Bicoid-Dorsal linkage in the integration of the anterior-posterior and dorsal-ventral patterning systems. Hunchback-Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. Evidence is presented that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. It is concluded that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo (Papatsenko, 2009).

Nearly 100 characterized enhancers and ~30 associated binding motifs control the patterning of the early Drosophila embryo, probably the best understood developmental process. These enhancers and sequence-specific TFs regulate the expression of ~50 genes controlling AP and DV patterning, including segmentation and gastrulation. The known TFs controlling embryogenesis represent less than ~10% of all TFs in the Drosophila genome. Thus, this analysis of regulatory grammar was restricted to the ~100 AP and DV enhancers and their ~30 TF inputs (31) (Papatsenko, 2009).

The recent completion of whole-genome sequence assemblies for 12 divergent Drosophila species has created an unprecedented opportunity for analyzing enhancer evolution. In this study 96 selected enhancer sequences from D. melanogaster were mapped to all 12 Drosophila genomes, using the UCSC Browser. The resulting collection combined 1420 kb of genomic sequence data in 1127 sequences, representing 60 enhancers in 23 AP genes and 36 enhancers in 31 DV genes. The entire collection of sequences and binding motifs is available at the Berkeley on-line resource (Papatsenko, 2009).

Inspection of aligned enhancer sequences among all 12 Drosophila species revealed strong conservation within the D. melanogaster subgroup (D. melanogaster, D. simulans, D. seichellia, D. yakuba and D. erecta) and also within the D. obscura group (D. pseudoobscura and D. persimilis). In order to focus on evolutionary changes in these enhancers the seven most divergent Drosophilids were analyzed: D. melanogaster, D. ananassae, D. pseudoobscura, D. willistoni, D. mojavensis, D. virilis and D. grimshawi. The remaining five species contain conservation patterns that are similar to those present in D. melanogaster or D. pseudoobscura (Papatsenko, 2009).

Short-range TF-binding linkages (0-80 bp) were examined in the collection of 96 enhancers from seven species for homo- and heterotypic pairs of binding motifs. Binding sites for the 30 most reliable TF motifs (see the Berkeley online resource) were mapped in enhancers using position weight matrices with match probability cutoff values set to ~2E-04. Distance histograms were generated for distances smaller than 80 bp, measured between the putative centers of each pair of neighboring site matches. Periodic signals were identified in the distance histograms using Fourier analysis, and statistical significance was estimated by bootstrapping positions of site matches in each enhancer sequence (Papatsenko, 2009).

Fourier analysis has identified helical phasing (~11 bp spacing) for several different homotypic activator-activator motif pairs. Such periodic signals were found in the distributions of Bcd-binding sites. Weaker helical-phasing signals were also identified for Caudal (Cad) and Dl-binding sites. Periodic signals close to two DNA turns (~20-22 bp) were found for Twi, Hb and Kruppel. Such helical phasing raises the possibility of direct protein-protein interactions (Papatsenko, 2009).

A weaker, ~11.4-bp periodic signal was detected in the distribution of heterotypic activator-activator site pairs, including Dl-Twi and Bcd-Cad. In contrast, there is a significant reduction in helical phasing signatures for activator-repressor motif pairs, and in fact, an over-representation of site pairs with 'anti-helical' spacing (15.2 bp). A similar 15.2 bp anti-helical signal was detected in distributions of all possible pair-wise combinations of the 30 binding motifs examined in this study. Thus, it would appear that any two randomly chosen binding sites are more likely to occupy the opposite sides of the DNA duplex as compared with helical phasing. This observation raises the possibility that most TFs function either additively or antagonistically to one another and just a special subset of TFs function in a synergistic fashion as reflected by helical phasing of the associated binding sites (Papatsenko, 2009).

The preceding analysis considered 'short-range' organizational constraints, involving linked binding sites separated by <25-30 bp. The possibility of 'long-range' constraints were also considered. The 96 enhancers under study possess characteristic 'unit lengths' of ~500 bp to 1.5 kb (300 bp minimum). The minimal/maximal sizes of the functional enhancers and the 'optimal' site densities can be determined by the amount of encoded information (pattern complexity), mechanisms of TF-DNA recognition such as lateral diffusion, or structural chromatin features like nucleosome positioning (Papatsenko, 2009).

Differential distance histograms reveal an over-representation of short-range linkages (<50 bp), but a depletion in mid-range distances (100-500 bp). These observations raise the possibility that TFs are distributed in a non-uniform manner across the length of the enhancer. That is, there may be sub-clusters, or 'hotspots', of binding sites within a typical enhancer. Such hotspots are observed in the prototypic eve stripe 2 enhancer, whereby 8 of the 12 critical binding sites are observed within two ~50-bp fragments located at either end of the minimal 480 bp enhancer. Homotypic motifs display the greatest propensity for such sub-clustering. Homotypic clusters (38) usually contain 3-5-binding sites distributed over 50-100 bp. Heterotypic activator-activator motif pairs also demonstrate sub-clustering, but these clusters are smaller (<25-30 bp) and usually contain just a pair of heterotypic sites. Heterotypic activator-repressor pairs show moderate enrichment over a distance of 50-70 bp, which is in agreement with the well-documented phenomenon of 'short-range repression'. Depletion of mid-range spacing constraints (around ~200 bp) is especially striking in the case of heterotypic motif pairs. Thus, activator synergy is like short-range repression: it appears to depend on closely linked binding sites (Papatsenko, 2009).

A possible explanation for this depletion of mid-range spacing is the occurrence of positioned nucleosomes, which might separate functionally distinct regions within an enhancer, and also separate neighboring enhancers. To test this hypothesis, nucleosome formation potential was compared with the distributions of TF-binding motifs in enhancers using the 'Recon' program. Three of the four eve enhancers that were examined (eve 1+5, eve 2 and eve 4+6) display a clear negative correlation between potential nucleosome formation and the distribution of TF-binding sites. This observation is consistent with the depletion of nucleosomes near TF-binding sites in vertebrates. This anti-correlation is especially striking in the case of the bipartite eve stripe 1+5 enhancer, where two enhancer regions (stripe 1 and stripe 5) are separated by a 400 bp 'spacer' DNA (in positions 600-1000), which might promote positioning of two nucleosomes and associated linker sequences (Papatsenko, 2009).

To investigate nucleosome positioning further, nucleosome-forming potential was measured in two sets of sequences, previously identified based on clustering of Dl sites and tested in vivo for enhancer activity. One set of sequences functioned as bona fide enhancers and produced localized patterns of gene expression across the DV axis of early embryos. The other set produced no expression in transgenic embryos, despite the presence of the same quality Dl-binding site clusters. The nucleosome-forming potential of the enhancers (true positives) was lower than that of the non-functional sequences (false-positives). These observations raise the possibility that the false Dl-binding clusters fail to function due to the formation of inactive nucleosomal structures (Papatsenko, 2009).

All 465 possible pairwise motif combinations for the 30 relevant binding motifs were tested for conservation in divergent drosophilids. Only linked binding sites, separated by a distance with small variations (max. distance bin = five bases) were considered. In the case of motif pairs, statistical significance was evaluated by bootstrapping columns in the binding motif alignments, thus preserving patterns of conservation. Pairs of homotypic motifs strongly prevailed in this type of analysis (28% of total pairs versus 6.5% expected), suggesting that homotypic interactions are important and pervasive in embryonic patterning. The strongest linkages were found for Bcd, Cad and Hb homotypic pairs. Each of these pairs was shared by five to six different enhancers and conserved in four to seven species. Among the identified heterotypic motif pairs, the most interesting were Bcd-Dl, Bcd-Cad and Dl-Twi (Papatsenko, 2009).

To identify cases of binding site pairs organized in a more flexible fashion, significant motif combinations were extracted using large distance bins or large distance variations. Along with the previously identified motif pairs, this analysis revealed several additional combinations, mainly involving the 'TAG-team' sequence motif, which is recognized by Zelda, a ubiquitous zinc finger TF. Zelda participates in the activation of the early zygotic genome and regulates a wide range of critical patterning genes. Indeed, significant combinations were identified for the TAG motif and Bcd, Dl and Hb. However, all of these TAG-X combinations exhibit spacing variability in different Drosophilids (Papatsenko, 2009).

It is conceivable that these results represent an underestimate of significantly linked motif combinations since very conservative cutoff values were used for statistical evaluation. A database of shared and/or conserved motif pairs, including those below the selected significance cutoff P = 0.03 is available from the Berkeley online resource (Papatsenko, 2009).

Conserved Bcd-Dl-binding site pairs were identified in the enhancers of several AP- and DV-patterning genes, including sal (AP), brk and sog (DV). The sites were found at similar distances, in the same orientation and were conserved in all seven species. It was suggested that the Bcd sites in the brk enhancer might augment gene expression in anterior regions, but this possibility was not directly tested. In wild-type embryos, both brk and sog exhibit significantly broader patterns of gene expression in anterior regions. This expanded pattern is lost in bcd mutants (Papatsenko, 2009).

Highly conserved Hb tandem repeats were detected in the regulatory regions of pair-rule genes, in the gap gene Kruppel, and in the Notch-signaling gene nubbin. Most of the homotypic Hb-Hb site pairs fall into two major groups, separated by either 6-8 or 13-15 bases. Some of the pair-rule enhancers selectively conserve either the 'short' or 'long' arrangement. For example, the eve stripe 4 + 6 enhancer contains two short Hb elements, while the stripe 3 + 7 enhancer contains a single long element. The odd 3 + 6 enhancer contains both short and long elements with various degrees of conservation. The hairy stripe 2,6,7 enhancer contains a single short element. Among the known gap genes, the long and short Hb elements were widely present in the enhancers of Kruppel, and in the blastoderm enhancer of nubbin, but not in any of the known knirps enhancers. It is conceivable that the distinct Hb site arrangements are important for the differential regulation of pair-rule genes by the Hb gradient (Papatsenko, 2009).

In conclusion, the systematic analysis of TF-binding sites in AP and DV patterning enhancers suggests a much higher degree of grammar, or fixed arrangements of binding sites, than is commonly believed. Developmental enhancers are thought to be highly flexible, with randomly distributed binding sites sufficing for the integration of multiple TFs. The results suggest that a large number of enhancers contain conserved short-range arrangements of pairs of binding sites. For instance, virtually all of the enhancers that respond to intermediate and low levels of the Dl gradient contain conserved arrangements of Dl-binding sites along with recognition sequences for other critical DV determinants, such as Twist and Zelda. Cooperating pairs of Bcd sites are found in enhancers responding to low Bcd concentrations, such as Knirps. Finally, distinctive arrangements of Hb-binding sites might influence whether the associated target genes are activated or repressed by high or low levels of the Hb gradient (Papatsenko, 2009).

Precision of hunchback expression in the Drosophila embryo

Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to understand this process have focused on the classical Bicoid target enhancer located immediately upstream of the P2 promoter. However, hb is also regulated by a recently identified distal shadow enhancer as well as a neglected 'stripe' enhancer, which mediates expression in both central and posterior regions of cellularizing embryos. This study employed BAC transgenesis and quantitative imaging methods to investigate the individual contributions of these different enhancers to the dynamic hb expression pattern. These studies reveal that the stripe enhancer is crucial for establishing the definitive border of the anterior Hb expression pattern, just beyond the initial border delineated by Bicoid. Removal of this enhancer impairs dynamic expansion of hb expression and results in variable cuticular defects in the mesothorax (T2) due to abnormal patterns of segmentation gene expression. The stripe enhancer is subject to extensive regulation by gap repressors, including Kruppel, Knirps, and Hb itself. It is proposed that this repression helps ensure precision of the anterior Hb border in response to variations in the Bicoid gradient (Perry, 2012).

hunchback (hb) is the premier gap gene of the segmentation regulatory network. It coordinates the expression of other gap genes, including Kruppel (Kr), knirps (kni), and giant (gt) in central and posterior regions of cellularizing embryos. The gap genes encode transcriptional repressors that delineate the borders of pair-rule stripes of gene expression. hb is activated in the anterior half of the precellular embryo, within 20-30 min after the establishment of the Bicoid gradient during nuclear cleavage cycles 9 and 10 (~90 min following fertilization). This initial hb mRNA transcription pattern exhibits a reasonably sharp on/off border within the presumptive thorax. This border depends on cooperative interactions of Bicoid monomers bound to linked sites in the proximal ('classical') enhancer. However, past studies and recent computational modeling suggest that Bicoid cooperativity is not sufficient to account for this precision in hb expression (Perry, 2012).

The hb locus contains two promoters, P2 and P1, and three enhancers. The 'classical' proximal enhance and distal shadow enhancer mediate activation in response to the Bicoid gradient. Expression is also regulated by a third enhancer, the 'stripe' enhancer, which is located over 5 kb upstream of P2. Each of these enhancers was separately attached to a lacZ reporter gene and expressed in transgenic embryos. As shown previously, the Bicoid target enhancers mediate expression in anterior regions of nuclear cleavage cycle (cc) 12-13 embryos, whereas the stripe enhancer mediates two stripes of gene expression at later stages, during cc14. The anterior stripe is located immediately posterior to the initial hb border established by the proximal and distal Bicoid target enhancers (Perry, 2012).

BAC transgenesis was used to determine the contribution of the stripe enhancer to the complex hb expression pattern. For some of the experiments, the hb transcription unit was replaced with the yellow (y) reporter gene, which contains a large intron permitting quantitative detection of nascent transcripts. The resulting BAC mimics the endogenous expression pattern, including augmented expression at the Hb border. However, removal of the stripe enhancer from an otherwise intact y-BAC transgene leads to diminished expression at this border and in posterior regions (Perry, 2012).

The functional impact of removing the stripe enhancer was investigated by genetic complementation assays. A BAC transgene containing 44 kb of genomic DNA encompassing the entire hb locus and flanking regulatory DNAs fully complements deficiency homozygotes carrying a newly created deletion that cleanly removes the hb transcription unit. The resulting adults are fully viable, fertile, and indistinguishable from normal strains. Embryos obtained from these adults exhibit a normal Hb protein gradient, including a sharp border located between eve stripes 2 and 3 (Perry, 2012).

The Hb BAC transgene lacking the stripe enhancer fails to complement hb/hb mutant embryos due to the absence of the posterior hb expression pattern, which results in the fusion of the seventh and eighth abdominal segments. In addition, the anterior Hb domain lacks the sharp 'stripe' at its posterior limit, resulting in an anterior expansion of Even-skipped (Eve) stripe 3 because the Hb repressor directly specifies this border. There is also a corresponding shift in the position of Engrailed (En) stripe 5, which is regulated by Eve stripe 3. The narrowing of En stripes 4 and 5, due to the anterior shift of stripe 5, correlates with patterning defects in the mesothorax (Perry, 2012).

Quantitative measurements indicate significant alterations of the anterior Hb expression pattern upon removal of the stripe enhancer. There is an anterior shift at the midpoint of the mature pattern, spanning two to three cell diameters. This boundary normally occurs at 47.2% egg length (EL; measured from the anterior pole). In contrast, removal of the stripe enhancer shifts the boundary to 45.6% EL. The border also exhibits a significant diminishment in slope. Normally, there is a decrease in Hb protein concentration of 20% over 1% EL. Removal of the stripe enhancer diminishes this drop in concentration, with a reduction of just 10% over 1% EL. The most obvious qualitative change in the distribution of Hb protein is seen in regions where there are rapidly diminishing levels of the Bicoid gradient. Normally, the transition from maximum to minimal Hb levels occurs over a region of 10% EL (43%-53% EL). Removal of the stripe enhancer causes a significant expansion of this transition, to 26% EL (27%-53% EL). It is therefore concluded that the stripe enhancer is essential for shaping the definitive Hb border (Perry, 2012).

The preceding studies suggest that the proximal and distal Bicoid target enhancers are not sufficient to establish the definitive Hb border at the onset of segmentation during cc14. Instead, the initial border undergoes a dynamic posterior expansion encompassing several cell diameters due to the action of the stripe enhancer. This enhancer is similar to the eve stripe 3+7 enhancer. Both enhancers mediate two stripes, one in central regions and the other in the posterior abdomen, and the two sets of stripes extensively overlap. Previous studies provide a comprehensive model for the specification of eve stripes 3 and 7, whereby the Hb repressor establishes the anterior border of stripe 3 and the posterior border of stripe 7 while the Kni repressor establishes the posterior border of stripe 3 and anterior border of stripe 7. Whole-genome chromatin immunoprecipitation (ChIP) binding assays and binding site analysis identify numerous Hb and Kni binding sites in the hb stripe enhancer, along with several Kr sites (Perry, 2012).

Site-directed mutagenesis was used to examine the function of gap binding sites in the hb stripe enhancer. Since the full-length, 1.4 kb enhancer contains too many binding sites for systematic mutagenesis, a 718 bp DNA fragment was identified that mediates weak but consistent expression of both stripes, particularly the posterior stripe. Mutagenesis of all ten Hb binding sites in this minimal enhancer resulted in a striking anterior expansion of the expression pattern. This observation suggests that the Hb repressor establishes the anterior border of the central stripe, as seen for eve stripe 3. There is no significant change in the posterior border of the central stripe or the anterior border of the posterior stripe, and repression persists in the presumptive abdomen (Perry, 2012).

Mutagenesis of the Kni binding sites resulted in expanded expression in the presumptive abdomen, similar to that seen for the eve 3+7 enhancer. More extensive depression was observed upon mutagenesis of both the Kni and Kr binding sites. These results suggest that the Kr and Kni repressors establish the posterior border of the central Hb stripe and the anterior border of the posterior stripe. This depressed pattern is virtually identical to the late hb expression pattern observed in Kr1;kni10 double mutants. The reliance on Kr could explain why the Hb central stripe is shifted anterior of eve stripe 3, which is regulated solely by Kni (Perry, 2012).

The dynamic regulation of the zygotic Hb expression pattern can be explained by the combinatorial action of the proximal, shadow, and stripe enhancers. The proximal and distal shadow enhancers mediate activation of hb transcription in response to the Bicoid gradient in anterior regions of cc10-13 embryos. The initial border of hb transcription is rather sharp, but the protein that is synthesized from this early pattern is distributed in a broad and shallow gradient, extending from 30% to 50% EL. During cc14 the stripe enhancer mediates transcription in a domain that extends just beyond the initial hb border. Gap repressors, including Hb itself, restrict this second wave of zygotic hb transcription to the region when there are rapidly diminishing levels of the Bicoid gradient, in a stripe that encompasses 44%-47% EL. The protein produced from the stripe enhancer is distributed in a sharp and steep gradient in the anterior thorax. It has been previously suggested that the steep Hb protein gradient is a direct readout of the broad Bicoid gradient. However, the current studies indicate that this is not the case. It is the combination of the Bicoid target enhancers and the hb stripe enhancer that produces the definitive pattern (Perry, 2012).

It has been proposed that Hb positive autofeedback is an important feature of the dynamic expression pattern. However, the mutagenesis of the hb stripe enhancer is consistent with past studies suggesting that Hb primarily functions as a repressor. The only clear-cut example of positive regulation is seen for the eve stripe 2 enhancer. Mutagenesis of the lone Hb-3 binding site results in diminished expression from a minimal enhancer. It was suggested that Hb somehow facilitates neighboring Bicoid activator sites, and attempts were made to determine whether a similar mechanism might apply to the proximal Bicoid target enhancer. The two Hb binding sites contained in this enhancer were mutagenized, but the resulting fusion gene mediates an expression pattern that is indistinguishable from the normal enhancer). It is therefore likely that the reduction of the central hb stripe in hb/hb embryos is the indirect consequence of expanded expression of other gap repressors, particularly Kr and Kni (Perry, 2012).

The hb stripe enhancer mediates expression in a central domain spanning 44%-47% EL, which coincides with the region exhibiting population variation in the distribution of the Bicoid gradient. Despite this variability, the definitive Hb border was shown to be relatively constant among different embryos. Previous studies suggest that the Kr and Kni repressors function in a partially redundant fashion to ensure the reliability of this border. This paper has presented evidence for direct interactions of these repressors with the hb stripe enhancer, and suggest that a major function of the enhancer is to 'dampen' the variable Bicoid gradient. Indeed, removal of this enhancer from an otherwise normal Hb BAC transgene results in variable patterning defects in the mesothorax, possibly reflecting increased noise in the Hb border (Perry, 2012).

Regulation of pair-rule genes by Bicoid

Transient over-expression of runt under the control of a Drosophila heat-shock promoter caused stripe-specific defects in the expression patterns of the pair-rule genes hairy and even-skipped, but had a more uniform effect on the secondary pair-rule gene fushi tarazu. The expression of the gap segmentation genes upstream of runt in the segmentation hierarchy is also altered in heat shock/runt embryos. A subset of these effects have been interpreted as due to an antagonistic effect of runt on transcriptional activation by Bicoid (Tsai, 1994).

A 480 bp region of the eve promoter is both necessary and sufficient to direct a stripe of eve expression within the limits of the endogenous Eve stripe 2. The maternal morphogen Bicoid and the gap proteins Hunchback, Krüppel and Giant all bind with high affinity to closely linked sites within this small promoter element. Activation appears to depend on cooperative interactions among BCD and HB proteins, since the disruption of single binding sites causes catastrophic reductions in expression. Forming the posterior border of the stripe involves a delicate balance between limiting amounts of the BCD activator and the KR repressor (Small, 1992).

The expression of the pair-rule gene hairy (h) in seven evenly spaced stripes along the longitudinal axis of the Drosophila blastoderm embryo is mediated by a modular array of separate stripe enhancer elements. The minimal enhancer element, which generates reporter gene expression in place of the most posterior h stripe 7 (h7-element), contains a dense array of binding sites for factors providing the trans-acting control of h stripe 7 expression as revealed by genetic analyses. The stripe seven enhancer is found in a minimal 932 bp region from a 1.5 kb DNA fragment of the h upstream region. The h7-element mediates position-dependent gene expression by sensing region-specific combinations and concentrations of both the maternal homeodomain transcriptional activators, Caudal and Bicoid, and of transcriptional repressors encoded by locally expressed zygotic gap genes. Zygotic caudal expression is not required for activation. Caudal and Bicoid, which form complementing concentration gradients along the longitudinal axis of the embryo, function as redundant activators, indicating that the anterior determinant Bicoid is able to activate gene expression in the most posterior region of the embryo. The spatial limits of the h stripe-7 domain are brought about by the local activities of repressors that prevent activation. The spatial limit of h7 is significantly altered in the gap mutants tailless, knirps and kruppel, but not in embryos lacking either hunchback, giant or huckebein. There are seven binding sites for Bcd, twenty-three for caudal, five for Kruppel, fourteen for Knirps, eight for Hunchback and five for Tailless. In the absence of both cad and bcd, activation still occurs. Thus, a third activator, likely to be Kr, must function in such embryos. It is thought that Kr acts as both a repressor and an activator within the h7 element depending on its concentration. The posterior border is set in response to Tll activity under the control of the terminal maternal organizer system. The anterior border of the expression domain is due to repression in response to Kni. The results suggest that the gradients of Bicoid and Caudal combine their activities to activate segmentation genes along the entire axis of the embryo (La Rosee, 1997).

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). Since these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. A well-conserved sequence repeat, (GTTT)4 has been identified that is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)4-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers (Andrioli, 2002).

The eve2Delta(GTTT)4-lacZ transgene is repressed at the anterior tip, even in gt mutants, suggesting that yet another mechanism prevents activation in this region. This mechanism could work through another localized repressor activity, or by modifying Bcd, the major activator of eve 2. Consistent with the latter possibility, it has been previously shown that Bcd-dependent activation of hb and orthodenticle (otd) is downregulated by the Tor phosphorylation cascade at the anterior tip. To test whether tor controls the ability of Bcd to activate eve 2, the eve2Delta(GTTT)4-lacZ transgene was crossed into embryos lacking tor activity. This causes a significant derepression at the anterior tip, suggesting that tor-mediated modification of bcd activity is important for preventing activation in this region. A similar derepression is not detected with the wild-type eve2-lacZ transgene in tor mutants, suggesting that Tor-mediated repression is dependent on the (GTTT)4-binding activity. In summary, these results suggest that multiple activities are required for anterior repression of eve 2, and that three different mechanisms prevent activation in different anterior regions (Andrioli, 2002).

Ancestral resurrection of the Drosophila S2E enhancer reveals accessible evolutionary paths through compensatory change

Upstream regulatory sequences that control gene expression evolve rapidly, yet the expression patterns and functions of most genes are typically conserved. In order to address this paradox, this study has reconstructed computationally and resurrected in vivo the cis-regulatory regions of the ancestral Drosophila eve stripe 2 element and evaluated its evolution using a mathematical model of promoter function. A feed-forward transcriptional model predicts gene expression patterns directly from enhancer sequence. This functional model was used along with phylogenetics to generate a set of possible ancestral eve stripe 2 sequences for the common ancestors of 1) Drosophila simulans and D. sechellia, 2) D. melanogaster, D. simulans, D. sechellia, and 3) D. erecta and D. yakuba. These ancestral sequences were synthesized and resurrected in vivo. Using a combination of quantitative and computational analysis, clear support was foumd for functional compensation between the binding sites for Bicoid, Giant, and Kruppel over the course of 40-60 million years of Drosophila evolution. This compensation is driven by a coupling interaction between Bicoid activation and repression at the anterior and posterior border necessary for proper placement of the anterior stripe 2 border. A multiplicity of mechanisms for binding site turnover exemplified by Bicoid, Giant, and Kruppel sites, explains how rapid sequence change may occur while maintaining the function of the cis-regulatory element (Martinez, 2014).

Regulation of other genes by Bicoid

The activation of Deformed is dependent on combinatorial input from at least three levels of the early hierarchy. The simplest activation code sufficient to establish Deformed expression consists of a moderate level of expression from the coordinate gene bicoid, in combination with expression from both the gap gene hunchback, and the pair-rule gene even-skipped (Jack, 1990).

Early polyhomeotic expression is under the control of bicoid and engrailed as activators, and oskar, acting as an inhibitor (Fauvarque, 1995).

The three maternal systems (anterioposterior (bicoid); terminal (torso); dorsoventral (dorsal) control the early expression of Goosecoid. The GSC stripe never appears in bicoid mutants, the stripe is shifted anteriorly in torso mutants and the ventral repression of the stripe is abolished in dorsal mutants (Goriely, 1996).

Little is known about the range of DNA sequences bound by transcription factors in vivo. Using a sensitive UV cross-linking technique, it has been shown that three classes of homeoprotein bind at significant levels to the majority of genes in Drosophila embryos. The three classes, represented by Even skipped, Bicoid and Paired, bind with specificities different from one another; however, their levels of binding on any single DNA fragment differ by no more than 5- to 10-fold. On actively transcribed genes, there is a good correlation between the in vivo DNA-binding specificity of each class and its in vitro DNA-binding specificity. In contrast, no such correlation is seen on inactive or weakly transcribed genes. These genes are bound poorly in vivo, even though they contain many high affinity homeoprotein-binding sites (Carr, 1999).

The amino acid at position 50 of the homeodomain makes specific contacts with the two bases 5' of the ATTA core recognition sequence. All of the selector homeoproteins have a glutamine at this position, whereas Bicoid has a lysine and Paired has a serine. These different residues give Bicoid and Paired unique preferences for variants of the NNATTA consensus sequence. For example, Bicoid binds in vitro >10 times more strongly than the selector homeoproteins to the sequence GGATTA but binds at least 10 times more weakly than the selector homeoproteins to the sequence CCATTA. In addition, Paired contains a second DNA-binding domain, the paired domain. This domain recognizes an entirely different 10-14 bp sequence, which is found adjacent to homeodomain recognition sites in Paired target elements. How are the distinct in vitro preferences of these three classes of homeoprotein related to their DNA binding in vivo? The results indicate that, in embryos, Paired and Bicoid bind most strongly to known target elements within a promoter and that, like the selector homeoproteins, they may also bind at significant levels to the majority of genes (Carr, 1999 and references).

Because Paired and Bicoid are expressed at similarly high levels, it was of interest to determine if they would bind to a wide array of genes in embryos. Consequently, a quantitation was carried out of the mean cross-linking per kb of DNA of Paired and Bicoid to the same series of DNA fragments used in previous studies of Eve and Ftz. Paired and Bicoid cross-link at levels above the limit of detection of the assay to almost all gene fragments tested; only the interactions of Bicoid with Adh and of Paired with rosy and the hsp70 transcription unit are too weak to be detected in the assay. Thus, like Eve and Ftz, Paired and Bicoid may bind at appreciable levels to most genes in Drosophila (Carr, 1999).

It is suggested that the major factor affecting DNA binding in vivo is the inhibition of binding at some gene loci by chromatin structure. Cooperative interactions with other transcription factors (cofactors) are thought to play only a minor role by increasing DNA binding at a limited number of lower affinity sites within genes. At the stage of embryogenesis examined in the UV cross-linking experiments, the Adh gene is not transcribed and the rosy gene is inactive in most cells. These two genes are bound most weakly in vivo by Eve, Ftz, Bicoid and Paired, even though these two genes are bound relatively well in vitro. The chromatin structure of transcriptionally inactive genes is thought to inhibit DNA binding by certain classes of transcription factor. Therefore, closed chromatin structure could explain the reduced binding to Adh and rosy. The Ubx gene is only weakly transcribed at cellular blastoderm, and the hsp70 fragment examined is only open to transcription factor binding over part of its length in vivo. Thus, partially open chromatin structure may explain the intermediate levels of UV cross-linking to Ubx and hsp70 in vivo. The eve, ftz and hunchback genes are all highly transcribed. Thus, their chromatin structure may be fully permissive for homeoprotein binding, and this could explain why they are the most highly bound genes (Carr, 1999).

It is difficult to assess what fraction of transcription factors will show widespread DNA binding in vivo. The authors strongly suspect that other classes of homeoproteins in Drosophila as well as homeoproteins in other animals will bind to a very broad range of genes in vivo. It is suggested that metazoan transcription factors will show a spectrum of DNA binding, from factors that bind very selectively to those that bind as broadly as Bicoid, Paired, Eve and Ftz. The majority of transcription factor molecules in prokaryotes are predicted to be bound to DNA. Most molecules are thought to be bound in a sequence-independent manner at very low levels throughout the genome because sequence-specific DNA-binding proteins can bind any DNA sequence weakly via electrostatic interactions and because the concentration of DNA in cells is very high. It is suggested that there are several key differences between these predictions and the widespread DNA binding of homeoproteins in Drosophila. (1) In contrast to the poor discrimination between most genes shown by homeoproteins, prokaryotic regulators are predicted to bind to their target genes at levels at least 100-1000 times higher than they bind to any other region of the genome. (2) Many prokaryotic transcription factors bind with high affinity to 14-20 bp specific sequences that occur rarely in the genome, whereas homeoproteins bind to degenerate 6 bp sequences that are found in most Drosophila genes at a density of 5-10 sites per kb of DNA. (3) The low levels of prokaryotic regulators bound to most genes do not affect transcription, whereas the widespread binding of homeoproteins may play a direct role in regulating the expression of a large proportion of genes. Understanding how homeoproteins control development will require a detailed analysis of how this widespread DNA binding affects transcription (Carr, 1999).

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterized Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity (Janody, 2000b).

The results indicate that early anterior expression of a labral determinant, cnc, and of two foregut determinants, wg and hh, is repressed when zygotic expression of hb is allowed to persist at the anterior pole of the Drosophila blastoderm embryo. Expression of cnc, wg and hh is under the positive regulation of bcd and torso but no zygotic gene has yet been implicated in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and that torso-induced anterior repression of hb is necessary for their positive control by torso. To determine whether the positive control of cnc, wg and hh by torso could be the result of a double negative control involving hb, expression of these genes was analysed in hb zygotic mutant embryos derived from torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb at the pole, expression of these genes should be recovered in hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is not recovered in hb minus embryos derived from torso females whereas it is normal in hb minus embryos. This indicates that, although necessary, the anterior repression of hb is not sufficient to mediate Torso positive control on cnc, wg and hh early anterior expression (Janody, 2000b).

Probing the limits to positional information; Precision for the Bicoid morphogen in the Drosophila embryo

The reproducibility and precision of biological patterning is limited by the accuracy with which concentration profiles of morphogen molecules can be established and read out by their targets. This study considered four measures of precision for the Bicoid morphogen in the Drosophila embryo: the concentration differences that distinguish neighboring cells, the limits set by the random arrival of Bicoid molecules at their targets (which depends on absolute concentration), the noise in readout of Bicoid by the activation of Hunchback, and the reproducibility of Bicoid concentration at corresponding positions in multiple embryos. Through a combination of different experiments, it was shown that all of these quantities are 10%. This agreement among different measures of accuracy indicates that the embryo is not faced with noisy input signals and readout mechanisms; rather, the system exerts precise control over absolute concentrations and responds reliably to small concentration differences, approaching the limits set by basic physical principles (Gregor, 2007b).

The development of multicellular organisms such as Drosophila is both precise and reproducible. Understanding the origin of precise and reproducible behavior, in development and in other biological processes, is fundamentally a quantitative question. Two broad classes of ideas can be distinguished. In one view, each step in the process is noisy and variable, and this biological variability is suppressed only through averaging over many elements or through some collective property of the whole network of elements. In the other view, each step has been tuned to enhance its reliability, perhaps down to some fundamental physical limits. These very different views lead to different questions and to different languages for discussing the results of experiments (Gregor, 2007b).

The goal of this study was to locate the initial stages of Drosophila development on the continuum between the 'precisionist' view and the 'noisy input, robust output' view. To this end the absolute concentration of Bcd proteins was measured and these measurements were used to estimate the physical limits to precision that arise from random arrival of these molecules at their targets. The input/output relation between Bcd and Hb was measured, and it was found that Hb expression provides a readout of the Bcd concentration with better than 10% accuracy, very close to the physical limit. The mean input/output relation is reproducible from embryo to embryo, and direct measurements of the Bcd concentration profiles demonstrate that these too are reproducible from embryo to embryo at the ~10% level. Thus, the primary morphogen gradient is established with high precision, and it is transduced with high precision (Gregor, 2007b).

Analysis of the Bcd/Hb input/output relations is similar in spirit to measurements of noise in gene expression that have been done in unicellular organisms. The morphogen gradients in early embryos provide a naturally occurring range of transcription factor concentrations to which cells respond, and the embryo itself provides an experimental 'chamber' in which many factors that would be considered extrinsic to the regulatory process in unicellular organisms are controlled. Perhaps analogous to the distinction between intrinsic and extrinsic noise in single cells, this study has distinguished between noise in the responses of individual nuclei to morphogens within a single embryo and the reproducibility of these input signals across embryos. Although there are many reasons why antibody staining might not provide a quantitative indicator of protein concentration, the results show that coupling classical antibody staining methods with quantitative image analysis allows a quantitative characterization of noise in the potentially more complex metazoan context. This approach should be more widely applicable (Gregor, 2007b).

A central result of this work is the matching of the different measures of precision and reproducibility. Near its point of half-maximal activation, the expression level of hb provides a readout of Bcd concentration with better than 10% accuracy. At the same time, the reproducibility of the Bcd profile from embryo to embryo and from one cycle of nuclear division to the next within one embryo, is also at the ~10% level. Importantly, these different measures of precision and reproducibility must be determined by very different mechanisms. For the readout, there is a clear physical limit which may set the scale for all steps. This limiting noise level is sufficient to provide reliable discrimination between neighboring nuclei, thus providing sufficient positional information for the system to specify each 'pixel' of the final pattern (Gregor, 2007b).

Previous work has shown that the Bcd profile scales to compensate for the large changes in embryo length across related species of flies, but evidence for scaling across individuals within a species has been elusive, perhaps because the relevant differences are small. This study found that the Bcd profile is sufficiently reproducible that it can specify position along the anterior-posterior axis within 1%-2% when position is expressed in units relative to the length of the embryo. But embryos have a standard deviation of lengths. Even if the Bcd profile were perfectly reproducible as concentration versus position in microns, this would mean that knowledge of relative position would be uncertain by 4%, which is more than what was see. This suggests that the Bcd profile exhibits some degree of scaling to compensate for length differences. New experiments will be required to test this more directly (Gregor, 2007b).

The results suggest that communication among nearby nuclei, perhaps through a diffusable messenger, plays a role in the suppression of noise. The messenger could be Hb itself since in the blastoderm stages the protein is free to diffuse between nuclei, and hence the Hb protein concentration in one nucleus could reflect the Bcd-dependent mRNA translation levels of many neighboring nuclei. This model predicts that precision will depend on the local density of nuclei and hence will be degraded in earlier nuclear cycles unless there are compensating changes in integration time. Such averaging mechanisms might be expected to smooth the spatial patterns of gene expression, which seems opposite to the goal of morphogenesis; the fact that Hb can activate its own expression may provide a compensating sharpening of the output profile. There is a theoretically interesting tradeoff between suppressing noise and blurring of the pattern, with self-activation shifting the balance. Note that the idea of spatial averaging, although employed in this study in a syncitial embryo, can be extended to nonsyncitial systems (e.g., via autocrine signaling or via small molecules that can freely pass through cell membranes or gap junctions) (Gregor, 2007b).

The reproducibility of absolute Bcd concentration profiles from embryo to embryo literally means that the number of copies of the protein is reproducible at the ~10% level. Understanding how the embryo achieves reproducibility in Bcd copy number is a significant challenge. Feedback mechanisms, explored for other morphogens, could compensate for variations in mRNA levels, but the linear response of the Bcd profile to halving the dosage of the Bcd-eGFP transgene argues against such compensation. The simplest view consistent with all these data is that mRNA levels themselves are reproducible at the ~10% level, and this should be tested directly (Gregor, 2007b).

At a conceptual level the results on Drosophila development have much in common with a stream of results on the precision of signaling and processing in other biological systems. There is a direct analogy between the approach to the physical limits in the Bcd/Hb readout and the sensitivity of bacterial chemotaxis or the ability of the visual system to count single photons. In each case the reliability of the whole process is such that the randomness of essential molecular events dominates the reliability of the macroscopic output. There are several examples in which the reliability of neural processing reaches such limits, and it is attractive to think that developmental decision making operates with a comparable degree of reliability. The approach to physical limits places important constraints on the dynamics of the decision making circuits. Finally, it is noted that the precision and reproducibility which observed in the embryo are disturbingly close to the resolution afforded by the measuring instruments (Gregor, 2007b).

Anterior-posterior positional information in the absence of a strong Bicoid gradient

The Bicoid (Bcd) transcription factor is distributed as a long-range concentration gradient along the anterior posterior (AP) axis of the Drosophila embryo. Bcd is required for the activation of a series of target genes, which are expressed at specific positions within the gradient. This study directly tested whether different concentration thresholds within the Bcd gradient establish the relative positions of its target genes by flattening the gradient and systematically varying expression levels. Genome-wide expression profiles were used to estimate the total number of Bcd target genes, and a general correlation was found between the Bcd concentration required for activation and the positions where target genes are expressed in wild-type embryos. However, concentrations required for target gene activation in embryos with flattened Bcd were consistently lower than those present at each target gene's position in the wild-type gradient, suggesting that Bcd is in excess at every position along the AP axis. Also, several Bcd target genes were positioned in correctly ordered stripes in embryos with flattened Bcd, and it is suggested that these stripes are normally regulated by interactions between Bcd and the terminal patterning system. These findings argue strongly against the strict interpretation of the Bcd morphogen hypothesis, and support the idea that target gene positioning involves combinatorial interactions that are mediated by the binding site architecture of each gene's cis-regulatory elements (Ochoa-Espinosa, 2009).

This study used genetic and transgenic manipulations to create pure populations of embryos with flattened Bcd gradients. These manipulations expanded specific subregions of the body plan, which reduced the complexity of cell fates in the embryo compared with wild type, and increased signal-to-noise ratios in the microarray experiments. The three levels of Bcd generated in these experiments, ≈4%, 11%, and ≈40%, cover the lower half of the full range of the Bcd gradient, and these experiments identified 13 of the 18 known Bcd target genes (Ochoa-Espinosa, 2009).

The 13 known Bcd target genes are included in a set of 242 genes that are differentially activated by increasing levels of Bcd. Ninety-seven of these genes have been tested for expression in the early embryo, and 48 are expressed differentially along the AP axis. Of these, 30 are likely to be direct targets based on known or predicted Bcd-dependent CRMs. If a linear extrapolation of this number is used to take into account the full set of 242 genes, the genome-wide estimate is ≈74 genes, and if the fact that these experiments did not identify five previously known Bcd target genes (27%), the estimate increases to ≈103 genes (Ochoa-Espinosa, 2009).

Six other genes were identified as Bcd targets based on the microarray experiments and the presence of nearby clusters of Bcd sites, but these genes are either expressed ubiquitously or in dorsal-ventral patterns, with no apparent modulation along the AP axis. It is possible that Bcd-dependent activation may partially contribute to these patterns by activating expression in anterior regions, which is consistent with recent studies that showed ChIP-chip binding of DV transcription factors to AP-expressed genes and vice versa. If these are real target genes, they would slightly increase the estimate of the total number of Bcd target genes (Ochoa-Espinosa, 2009).

Bicoid has been considered as one of the best examples of a gradient morphogen. Several lines of evidence suggest that Bcd does indeed function as a morphogen, including the coordinated shifts of morphological features and target gene expression patterns in embryos with different copy numbers of the bcd gene, and the ability of bcd mRNA to establish anterior cell fates when microinjected into ectopic positions. Furthermore, manipulations of the Bcd-binding sites in the hb P2 promoter and synthetic constructs with defined Bcd sites showed that cis-regulatory elements can be designed to be more or less sensitive to Bcd-mediated transcription. These studies led to the hypothesis that differential sensitivity to Bcd binding may control the relative positioning of different target genes (Ochoa-Espinosa, 2009).

The current findings suggest that differential sensitivity to Bcd binding is not the primary mechanism that controls the relative positioning of its target genes. Though some target genes respond in an all-or-none fashion to different levels of flattened Bcd, the levels required for activation are much lower than those present in the wild-type gradient in the regions where those genes are activated. These findings suggest that Bcd concentrations are in excess of those required for activation at every position along the length of the wild-type gradient (Ochoa-Espinosa, 2009).

It was also shown that the head gap genes otd, ems, and btd are expressed in correctly ordered stripes in embryos containing flattened Bcd gradients. This is most dramatically demonstrated by the mirror-image duplication of otd, ems, and btd stripes in the posterior region of 6B (6 copies) vas exu embryos, where the Bcd gradient slopes in the opposite direction to the order of striped expression. It is proposed that these genes are patterned by the terminal system in the absence of a Bcd gradient, and though Bcd function is required for their activation, the Bcd gradient does not play a major role in establishing their relative positions along the AP axis (Ochoa-Espinosa, 2009).

Bcd seems capable of bypassing the terminal system if expressed at high levels. For example, the anterior defects in terminal-system mutants can be partially rescued by increasing bcd copy number. Also, in 6B (6 copies) vas exu embryos, higher levels of Bcd are present throughout the embryo, with a relatively weak gradient along the AP axis. This causes expansions of the anterior otd, ems, and btd expression patterns into central regions of the embryo. The posterior boundaries of these patterns are positioned correctly, suggesting that the Bcd protein gradient is sufficient to position these target genes in regions where the terminal system does not reach. This is consistent with the observation that microinjected bcd mRNA can autonomously specify anterior structures (Ochoa-Espinosa, 2009).

These data are consistent with previous studies that failed to find a strong correlation between the relative positioning of target genes and the Bcd-binding 'strength' of their associated cis-regulatory elements. They further support a model in which Bcd functions as only one component of an integrated patterning system that establishes gene expression patterns along the AP axis. A second major component is maternal Hb, which is expressed in an AP protein gradient. Hb synergizes with Bcd in the activation of several specific target genes. In vas exu embryos, the loss of vas causes ectopic translation of maternal hb in posterior regions, so Hb protein is ubiquitously expressed and available for combinatorial activation with Bcd. This combination is likely sufficient to lead to the near ubiquitous expression of zygotic hb and Kr in 1B vas exu embryos, and gt in 2B vas exu embryos (Ochoa-Espinosa, 2009).

A third major component is the terminal system, which seems to affect the expression patterns of Bcd target genes in two ways. First, it causes a repression of all known Bcd target genes at the anterior pole by a mechanism that is not clearly understood. Second, the data suggest that the terminal system functions with Bcd for the establishment of the posterior boundaries of the head gap genes. This interaction appears to be important for regulating at least two other target genes, gt and slp1, which are expressed in anterior domains that shift toward the anterior pole in terminal system mutants. Both gt and slp1 are also activated in anterior and posterior stripes in embryonic regions containing low levels of flattened Bcd. These findings suggest that interactions with the terminal system may be required for positioning most Bcd target genes. The only known target genes that may not be directly influenced by the terminal system are zygotic hb and Kr, which are expressed in middle embryonic regions, far from the source of the terminal system activity (Ochoa-Espinosa, 2009).

How synergy between Bcd and the terminal system is achieved for each target gene is not clear. One possibility is that the Torso phosphorylation cascade directly modifies the Bcd protein, increasing its potency as a transcriptional activator. Mutations in Bcd's MAP-kinase phosphorylation sites partially reduce the ability of Bcd to activate otd, consistent with this hypothesis. Alternatively, the terminal system has been shown to repress the activities of ubiquitously expressed repressor proteins. Perhaps repression by the terminal system creates posterior to anterior gradients of these proteins, which then compete with Bcd-dependent activation mechanisms to establish posterior boundaries of target gene expression (Ochoa-Espinosa, 2009).

Interactions between Bcd, maternal Hb, and the terminal system may be critical for the initial positioning of target gene expression patterns, but it is clear that other layers of regulation are required for creating the correct order of gene expression boundaries in the anterior part of the early embryo. Almost all known Bcd target genes are transcription factors, and there is evidence that they regulate each other by feed-forward activation and repression mechanisms. Each target gene contains one or more CRMs, each of which is composed of a specific combination and arrangement (code) of transcription factor binding sites. Unraveling the mechanisms that differentially position Bcd target will require the detailed dissections of CRMs that direct spatially distinct expression patterns (Ochoa-Espinosa, 2009).

Live imaging of bicoid-dependent transcription in Drosophila embryos

The early Drosophila embryo is an ideal model to understand the transcriptional regulation of well-defined patterns of gene expression in a developing organism. In this system, snapshots of transcription measurements obtained by RNA FISH on fixed samples cannot provide the temporal resolution needed to distinguish spatial heterogeneity from inherent noise. This study used the MS2-MCP reporter transgene system to visualize in living embryos nascent transcripts expressed from the canonical hunchback (hb) promoter under the control of Bicoid (Bcd). The hb-MS2 reporter is expressed as synchronously as endogenous hb in the anterior half of the embryo, but unlike hb it is also active in the posterior, though more heterogeneously and more transiently than in the anterior. The length and intensity of active transcription periods in the anterior are strongly reduced in absence of Bcd, whereas posterior ones are mostly Bcd independent. This posterior noisy signal decreases progressively through nuclear divisions, so that the MS2 reporter expression mimics the known anterior hb pattern at cellularization. It is proposed that the establishment of the hb pattern relies on Bcd-dependent lengthening of transcriptional activity periods in the anterior and may require two distinct repression mechanisms in the posterior (Lucas, 2013).

The data indicate that the establishment of the precise border of endogenous hb gene expression results from at least three distinct processes. First, Bcd is responsible for a strong and persistent expression in nuclei localized in the anterior half of the embryo. This Bcd-dependent expression does not seem to control the instantaneous activity of the gene in a spatially graded fashion but sets a rough boundary of maximal activation. Interestingly, the transcription initiation time is constant at each interphase and along the AP axis. This observation suggests that the postmitotic delay of transcription reactivation is not limited by the Bcd physical parameters, but more probably by the assembly of the transcription machinery after decondensation of mitotic chromosomes and the delay of transcribing sufficient numbers of MS2 stem loops for signal detection. Mechanistically, as shown by the Bcd-dependent lengthening of activity events in the anterior, as a transcription activator, Bcd may be critical to maintain the flux of polymerases initiating transcription. Second, transcriptional repression in the posterior initiates mildly during interphase 11 and progresses over cycles 12 and 13. In the posterior, as the number of active loci decreases from one cycle to the next, initiation times of activity events become more variable, suggesting a posterior repressor becoming stronger. This putative repressor does not necessarily require Bcd as the overall repression exhibits the same feature in an embryo lacking Bcd. Third, as early as interphase 10, a 'silencing' mechanism prevents the erratic posterior expression of the canonical hb promoter observed upon insertion as a reporter transgene in the genome. As endogenous hb is not expressed in the posterior, this third regulation mechanism must be encoded in the genomic DNA outside of the canonical promoter and could involve the newly identified distal shadow or stripe enhancers of hb. This last silencing mechanism together with the Bcd induced activation of transcription are likely responsible for the sharp border observed for endogenous hb as early as cycle 11. In absence of this early silencing in the posterior, as exemplified by the hb-MS2 reporter, a second unidentified mechanism of repression (discussed in the second point) can rescue the formation of the sharp boundary by cycle 13 (Lucas, 2013).

The ability to observe the early transcription of developmental genes in live embryos opens news perspectives for the understanding of the patterning processes. Despite not recapitulating all the features of the endogenous regulation, access to new quantitative measurements sheds light on this critical biological process. At the mechanistic level, this approach indicates how the Bcd transcription factor could activate transcription: it is not absolutely required for transcription initiation at the promoter and it does not allow faster initiation at the promoter after mitosis, but it is essential for the maintenance of the activity event once the latter has been initiated (Lucas, 2013).

Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning

Spatiotemporal patterns of gene expression are fundamental to every developmental program. The resulting macroscopic domains have been mainly characterized by their levels of gene products. However, the establishment of such patterns results from differences in the dynamics of microscopic events in individual cells such as transcription. It is unclear how these microscopic decisions lead to macroscopic patterns, as measurements in fixed tissue cannot access the underlying transcriptional dynamics. In vivo transcriptional dynamics have long been approached in single-celled organisms, but never in a multicellular developmental context. This study directly addressed how boundaries of gene expression emerge in the Drosophila embryo by measuring the absolute number of actively transcribing polymerases in real time in individual nuclei, using a Bicoid driven hb enhancer-P2 promoter-reporter transgene. Specifically, this study showed that the formation of a boundary cannot be quantitatively explained by the rate of mRNA production in each cell, but instead requires amplification of the dynamic range of the expression boundary. This amplification is accomplished by nuclei randomly adopting active or inactive states of transcription, leading to a collective effect where the fraction of active nuclei is modulated in space. Thus, developmental patterns are not just the consequence of reproducible transcriptional dynamics in individual nuclei, but are the result of averaging expression over space and time (Garcia, 2013).

Regulation of gap genes by Bicoid

Continued: Bicoid Targets of Activity part 2/2


bicoid: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Miscellaneous Interactions | Developmental Biology | Effects of Mutation | References

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