bicoid
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).
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).
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).
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).
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).
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).
The homeodomain (HD) protein Bicoid (Bcd) is thought to function as a gradient morphogen that positions boundaries of target genes via threshold-dependent activation mechanisms. This study analyzed 66 Bcd-dependent regulatory elements, and their boundaries were shown to be positioned primarily by repressive gradients that antagonize Bcd-mediated activation. A major repressor is the pair-rule protein Runt (Run), which is expressed in an opposing gradient and is necessary and sufficient for limiting Bcd-dependent activation. Evidence is presented that Run functions with the maternal repressor Capicua and the gap protein Kruppel as the principal components of a repression system that correctly orders boundaries throughout the anterior half of the embryo. These results put conceptual limits on the Bcd morphogen hypothesis and demonstrate how the Bcd gradient functions within the gene network that patterns the embryo (Chen, 2012).
This study identified 32 enhancers that respond to Bcd-dependent activation and form expression boundaries at different positions along the AP axis of fly embryos. Adding these elements to the 34 previously known enhancers constitutes the largest data set of in vivo-tested and -confirmed enhancers regulated by a specific transcription factor in all of biology (Chen, 2012).
The 32 confirmed enhancers were identified among 77 tested genomic fragments, which were selected because they showed in vivo-binding activity, or they conformed to a stringent homotypic-clustering model for predicted Bcd-binding sites, or both. All seven previously unknown fragments showing in vivo binding and a predicted site cluster directed Bcd-dependent transcription in the early embryo. Other fragments from the top 50 ChIP-Chip signals (which do not conform to the clustering model) were also very likely (21 of 26) to test positive in the in vivo test, but this likelihood drops significantly (9 of 25) in a set of fragments from lower on the list of ChIP-Chip fragments. Interestingly, of 19 tested fragments that contain clusters of predicted sites, but no in vivo binding activity, not a single one tested positive in vivo. These results suggest that in ;vivo binding assays are much better predictors of regulatory function than simple site-clustering algorithms alone (Chen, 2012).
One explanation for the failure of these predicted site clusters to bind Bcd in vivo is that they lie in heterochromatic regions of the genome that prevent site access. However, because they fail to function when taken out of their normal context (in reporter genes), whatever is preventing activation must be a property of the fragment itself and not its location in the genome. Interestingly, a number of Bcd site cluster-containing fragments drive expression later in development. It is proposed that these fragments fail to bind Bcd because they lack sites for cofactors that facilitate Bcd binding. In preliminary experiments it was observed that Bcd-activated fragments contain on average more binding sites for the ubiquitous activator protein Zelda (Zld) than those that fail to activate. Zld has been shown to be critical for timing the zygotic expression of hundreds of genes in the maternal to zygotic transition (Chen, 2012).
These results suggest strongly that a gradient of Run protein plays a major role in limiting Bcd-dependent activation. Run seems to work as part of a repression system that also includes Cic and possibly Kr. Expression boundaries in the region anterior to the presumptive cephalic furrow shift toward the posterior in run and cic mutants, and the double mutant causes boundaries that are normally well separated to collapse into a single position (Chen, 2012).
The use of multiple repressors permits flexibility in binding site architecture within enhancers that establish boundaries at similar positions. For example type I enhancers show overrepresentations of both Run and Cic sites, but 27% lack strong matches to the Cic PWM, and 12% lack strong matches to the Run PWM. Importantly, however, all type I enhancers lacking Cic sites contain Run sites, and those lacking Run sites contain Cic sites. Multiple Kr sites were observed in a large number of Bcd-dependent enhancers, which suggests that Kr is also a major component of the repression system that orders Bcd-dependent expression boundaries. Taken together, these data suggest that antagonistic repression of Bcd-mediated activation is a key design principle of the system that organizes the AP body plan. The repressors identified so far (Run, Cic, and Kr) are expressed in overlapping domains with gradients at different positions, consistent with the formation and ordering of a relatively large number of boundaries throughout the anterior half of the embryo (Chen, 2012).
The close linkage between repressor sites and Bcd sites within discrete enhancers suggests that repression occurs via short-range interactions that interfere directly with Bcd binding or activation. Interestingly, Cic also shows repressive effects that seem to be binding site independent. For example some type I enhancers do not contain recognizable Cic sites, but their expression boundaries expand posteriorly in cic mutants. This could be caused by the reduced expression of run and Kr in cic mutants. However, genetically removing both Kr and run causes a less dramatic expansion than that seen in the absence of cic. This suggests that Cic binds these enhancers via suboptimal sites or that it is required for the correct patterning of another unknown repressor. Another possibility is that these expansions are caused indirectly by changing the balance of MAPK phosphorylation events that control terminal patterning (Chen, 2012).
These results do not strictly falsify the Bcd morphogen hypothesis, but they support the idea that the Bcd gradient can establish only a 'rough framework that is elaborated by the interaction of the zygotic segmentation genes'. What is the nature of this framework, and what role does it play in the network that precisely positions target gene boundaries (Chen, 2012)?
One component of the system, the Cic repression gradient, is maternally produced and formed by downregulation at the poles via the terminal patterning system. This gradient is formed independently of Bcd but is critical for establishing boundaries of Bcd-dependent target genes. In contrast, Bcd is involved in activating the expression patterns of run and Kr and in repressing them in anterior regions. Both run and Kr expand anteriorly in bcd mutants. There is no evidence that Bcd functions directly as a transcriptional repressor, so these repressive activities are probably indirect. Previous work showed that the Bcd target gene gt is involved in setting the anterior Kr boundary, and it is hypothesized that another Bcd target gene, slp1, encodes a forkhead domain (FKH) protein that sets the anterior boundary of the early run pattern. slp1 is expressed in a pattern reciprocal to the run pattern and was previously shown to position the anterior boundaries of several pair-rule gene stripes including run stripe 1 (Chen, 2012).
These results suggest that a major function of the Bcd gradient is the differential positioning of two repressors, Slp1 and Gt, which set the positions of the Run and Kr repression gradients, which then feedback to repress Bcd-dependent target genes. How are slp1 and gt differentially positioned? One possibility is that slp1 and gt enhancers respond to specific concentrations within the Bcd gradient, consistent with the original model for morphogen activity. However, the fact that the slp1 and gt expression domains form boundaries at the same positions in embryos lacking the Cic and Run repressors argues against this model for these genes (Chen, 2012).
It was also shown that Bcd target genes normally expressed in cephalic regions form and correctly position posterior boundaries in embryos containing flattened Bcd gradients. Run is still expressed in these embryos, specifically in a domain that consistently abuts the boundaries of the anterior Bcd target genes, regardless of copy number. This suggests that a mutually repressive interaction between Slp1 and Run is maintained in these embryos but does not explain how these boundaries are consistently oriented perpendicularly to the AP axis. The answer might lie in the fact that the flattened Bcd gradients in these embryos are not completely flat but are present as shallow gradients with slightly higher levels in anterior regions. In these embryos the slight changes in concentration along the AP axis might cause a bias that enables the orientation of the mutual repression interaction. In wild-type embryos, Bcd is much more steeply graded, which makes this bias stronger and the boundary between these mutual repressors more robust (Chen, 2012).
These results suggest that antagonistic repression precisely orders Bcd-dependent expression boundaries. However, repression may not be required for the activity of all morphogens. For example the extracellular signal activin has been shown to activate target genes in a threshold-dependent manner in isolated animal caps from frog embryos. Also, a gradient of the transcription factor Dorsal (Dl) is critical for setting boundaries between different tissue types along the dorsal-ventral (DV) axis of the fly embryo. It is thought that the major mechanism in Dl-specific patterning is threshold-dependent activation, which is quite different from the system described in this paper. One major difference between Bcd and Dl is the number of boundaries specified: three for Dl and more than ten for Bcd. It is proposed that the robust ordering of more boundaries simply requires a more complex system (Chen, 2012).
In general, though, it seems that antagonistic mechanisms are involved in controlling the establishment or interpretation of most morphogen activities. For example in the Drosophila wing disc, the TGF-N2 signal Dpp forms an activity gradient that is refined by interactions with multiple extracellular factors. Also, in vertebrates the signaling activity of the extracellular morphogen Sonic hedgehog (Shh) is affected by positive and negative interactions with specific molecules on the surfaces of receiving cells (Chen, 2012).
There is some evidence that transcriptional repression is also used for refining the patterning activities of extracellular molecules. Dpp acts as a long-range morphogen that activates two major target genes (optomotor blind [omb] and spalt [sal]) in nested patterns with boundaries at different positions with respect to the source of Dpp. Although these boundaries could in theory be formed by differential responses to the morphogen, it is clear that the transcriptional repressor Brinker (Brk), which is expressed in an oppositely oriented gradient, also plays an important role. The Brk gradient is itself positioned by Dpp activity in a manner analogous to positioning of the Run and Kr repressor gradients by Bcd. Also, a similar transcriptional network functions in Shh-mediated patterning of the vertebrate neural tube, where a series of spatially oriented repressors feeds back to limit the expression boundaries of Shh-mediated cell fate decisions (Chen, 2012).
Conceptually, these more complex systems are reminiscent of the reaction-diffusion model proposed by Turing, in which a localized activator would activate a repressor, which would diffuse more rapidly than the activator, and feed back on its activity. These systems strongly suggest that the patterning activity of a single monotonic gradient is insufficiently robust for establishing precise orders of closely positioned expression boundaries. By integrating gradients with repressive mechanisms that refine gradient shape or influence outputs, systems are generated that ensure consistency in body plan establishment while still maintaining the flexibility required for complex systems to evolve (Chen, 2012).
Continued: Bicoid Targets of Activity part 2/2
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