Targets of Activity (part 2/2)

Regulation of gap genes by Bicoid

Together with the terminal system, bicoid is required to establish the head (acron). Both bcd and the terminal system are required to activate the anterior-dorsal stripe of tailless that is correlated with the formation of the acron (Pignoni, 1992).

The activity of bicoid is required for the development of larval head and thoracic structures, and the activity of the maternal gene torso (tor) for the development of the unsegmented region of the head (acron). orthodenticle expression responds to the activity of the maternal tor gene and to Bicoid at the anterior pole of the embryo (Finkelstein, 1990).

Anterior patterning of the Drosophila embryo is specified by the localized expression of the gap genes, which are controlled by the bicoid gradient . hunchback can substitute for bcd in the thorax and abdomen. hb is required for bcd to execute all of its functions, including activation of head gap genes. Removal of both maternal and zygotic hb produces embryos with disrupted polarity that fail to express all known bcd target genes correctly. Proper expression of hb and the head gap genes requires synergistic activation by hb and bcd. It is the combined activity of bcd and hb, not just bcd alone, that forms the morphogenetic gradient that specifies polarity along the embryonic axis and patterns the embryo. bcd may be an evolutionarily newly acquired Drosophila gene, which is gradually replacing some of the functions performed by maternal hb in other species (Simpson-Brose, 1994).

Anterior repression of orthodenticle is carried out by Huckebein which in turn receives input for the torso system, from Dorsal and from Bicoid. Dorsal functions in the anterior repression of otd expression. The repression function of Dorsal is mediated, at least in part, through Huckebein, since anterior hkb expression is lost in dorsal mutants. Contrary to early models of embryonic pattern formation, high levels of Bicoid are not required for otd activation or for the establishment of anterior head structures (Gao, 1996).

Cis-acting elements for the expression of buttonhead head stripe expression are contained in a 1 kb DNA fragment, located about 3 kb upstream of the promoter, The four maternal coordinate systems are necessary for correct btd head stripe expression, most likely by acting through the 1 kb cis-acting control region. Expression of the btd head stripe depends on bicoid. bcd-dependent activation also involves the activity of the morphogens of the posterior and dorsoventral systems, hunchback and dorsal, respectively, which act together to control the spatial limits of the expression domain. Finally, tailless, a torso dependent repressor of btd, takes part in the regulation of btd head stripe expression by enhancing activation at low levels of activity and repression at high levels of activity (Wimmer, 1995).

The anterior, the dorsoventral and terminal systems, are required for the activation of crocodile expression and for the spatial control of the anterior cap domain, while the posterior system is not required for the regulation of the croc expression pattern. In the absence of bcd activity, croc fails to be expressed in the anterior cap domain. Conversely, an increase in bcd activity in embryos leads to an expansion of croc expression toward the posterior. However, this expansion is one-sided with respect to the dorsoventral axis of the embryo. Thus, although Bcd acts in a concentration-dependent manner, croc expression can only be expanded ventrally in the absence of dorsal activity. In fact, the lack of dorsal activity causes a strong reduction of the croc expression domain to a single spot, corresponding in position to the peak of bicoid activity at the anterior pole. Conversely, Dorsal activity along the entire dorsoventral axis, as in embryos laid by Toll mutant females, causes an expansion of the croc expression domain towards the dorsal-most position. In embryos lacking tor activity, croc expression is abolished in the dorsal region. However, if tor is activated ectopically due to a dominant tor mutation, the croc expression domains are expanded significantly on the ventral side. Thus Dorsal postively regulates croc and Bcd requires Dl to set the spatial limit of the croc anterior expression domain (Häcker, 1995).

The initial expression of the gap gene Krüppel (Kr) occurs in a precisely bounded central region of the Drosophila blastoderm embryo. According to genetic analysis, the spatial limits of the Kr expression domain are controlled by the morphogenetic activities of the anterior organizer gene bicoid and the anterior gap gene hunchback . A 730 bp KR control element drives gene expression in place of the endogenous KR central domain. This cis-acting element, Kr730, is composed of BCD and HB responsive sequences. This fragment represents a target for the redundant activator/repressor system provided by the anterior morphogens BCD and HB (Hoch, 1991).

Three different maternal morphogen gradients regulate expression of tailless, a gap gene required to establish the acron and telson of the Drosophila embryo. Activation of tailless in the anterior is through the maternal protein Bicoid (Liaw, 1992 and Pignoni, 1992). Regions mediating both activation and repression by Bicoid, and repression by Dorsal have been identified. Binding sites of BCD protein in a 0.5 kb region, revealed by DNaseI footprinting, could be crucial for the BCD-dependent activation of tll expression in the anterior stripe (Liaw, 1993).

The early expression of the empty spiracles gene is controlled by Bicoid. Using gene fusion, a cis-acting element that is a target for the bcd gene product has been identified (Walldorf, 1992).

sloppy paired, in addition to its roles as a segment polarity gene and as a pair rule gene, acts like a gap gene in the head. All three maternal systems are active in the cephalic region and are required for proper slp expression. High levels of the terminal system (torso) inhibit slp through bicoid, by an unknown mechanism, not involving huckebein or tailless . This may occur through the phosphorylation of Bicoid (Ronchi, 1993). Low levels of terminal system activity seem to potentiate bcd as an activator of slp in more posterior positions. Dorsal, the morphogen of the dorsoventral system, and the head-specific gap gene empty spiracles, act respectively as repressor and corepressor in the regulation of slp (Grossnicklaus, 1994) .

The Bicoid morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. According to an early model, the affinity of Bcd-binding sites in the promoters of target genes determines the posterior extent of their expression. This hypothesis, referred to as the Gene X model, predicts that genes expressed specifically in the head primordium will contain low affinity Bcd sites, so that high levels of Bcd protein are required for their activation. Higher affinity Bcd sites would permit gene expression extending into the thoracic primordium. Other parameters, such as the spacing between Bcd sites and cooperative binding have also been proposed to affect bcd target gene regulation. However, the importance of all these factors in the regulation of actual bcd target genes has not been determined. A small regulatory region upstream of the cephalic gap gene orthodenticle is shown to be sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. This element is sufficient to generate early otd-like expression. This indicates that this second fragment must contain binding sites for a different activator of early head expression. However, since lacZ expression driven by this 173 bp fragment is eliminated in embryos lacking bcd, this activator must, at least in Drosophila, be bcd-dependent. The only clue regarding the functional specificity of this activator is the reiterated sequence motif required for the activity of this regulatory element. This study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation (Gao, 1998).

To localize the control elements required for embryonic head expression, a series of lacZ reporter fusions were constructed spanning the otd genomic region. A 7.6 kb fragment extending upstream of the otd transcriptional start site is sufficient to recapitulate the endogenous pattern of otd head expression. The pattern of endogenous otd expression was compared to that driven by the 7.6 kb regulatory fragment. otd is expressed initially at relatively low levels in an anterior cap-like region of the syncytial blastoderm embryo. The posterior boundary of this early expression domain is not sharp, but is graded in intensity. Expression quickly fades from the anterior terminus, leaving a stripe extending from 75%- 92% egg length (EL) in the cellular blastoderm embryo. During this period, ventral expression also disappears. By this stage, the anterior and posterior boundaries of otd expression are sharply defined. During germ band extension, otd expression becomes more complex, appearing at the ventral midline and in other regions of the embryo. In the germ band-retracted embryo, expression can be seen in the anterior brain and in midline CNS cells. This expression persists through embryogenesis. In the blastoderm embryo, lacZ expression driven by the 7.6 kb fragment is indistinguishable from endogenous otd expression. Later in embryogenesis, lacZ expression in the anterior head and in midline cells is similar, but not identical to otd expression at equivalent developmental stage. Expression of the transgene is less localized within the head primordium, and significantly weaker in midline cells. This suggests that additional regulatory elements are required for correct late expression. The results described above indicate that the 7.6 kb fragment contains the regulatory elements that control otd expression in the blastoderm head primordium. Further dissection of the 7.6 kb fragment reveals that a 900 bp sub-fragment is the smallest contiguous regulatory region capable of driving strong head expression, and this is referred to as the Early Head Enhancer (EHE). The EHE responds to maternal cues in a similar fashion to otd expression (Gao, 1998).

To understand how the EHE functions, it was mapped at higher resolution. Progressive 5' deletions show that a 186 bp element at its 5' end is critical for maintaining the intensity of early head expression. This region contains the three putative Bcd sites in the EHE. Deletion of this element significantly decreases the intensity of lacZ expression, without significantly affecting its spatial extent. Further 5' deletions, which removed a putative Hb site, have no obvious effect on the level or position of lacZ expression. 3' deletions reveal a second important control element at the opposite end of the EHE. Removal of 173 bp from the 3' end of the 900 bp fragment also reduced the intensity of lacZ expression. Again, the spatial extent of early head expression is not significantly altered. These experiments revealed that the activity of the EHE resides primarily within two small regulatory elements, each sufficient to drive otd-like expression in the head primordium. The 186 bp element contains three candidate Bcd binding sites. Each of these sites contains 6 of the 9 nucleotides defined as a high affinity Bcd site in the hb promoter. In particular, each site contains the TAATC core critical for the recognition of purified Bcd protein in vitro. The presence of these sequences suggested that Bcd binds directly to the 186 bp fragment. As described, the removal of the single putative Hb site does not obviously affect the function of the EHE. This is consistent with previous observations that hb plays a relatively minor role in otd activation. In contrast, the loss of a putative dorsal site that lies between the 186 bp and 173 bp fragments prevents the ventral retraction of lacZ expression. This is consistent with previous findings that dorsal is required for this retraction. The EHE also contains possible binding sites for the product of the terminal gap gene huckebein, which is involved in repressing otd expression at the anterior terminus of the embryo (Gao, 1998).

One of the goals of this study was to determine whether Bcd directly activates otd in the head primordium. Purified Bcd protein indeed binds to the three Bcd consensus sites in the 186 bp fragment and these sites are required for its activity in vivo. In its original form, the Gene X model predicted that a gene expressed specifically in the head primordium would have lower affinity Bcd sites than genes expressed more posteriorly. In addition to the affinity of isolated Bcd sites, subsequent studies show that intersite spacing, the number of sites, and cooperative binding effects all contribute to the affinity of regulatory regions for Bcd. The overall affinity of the 186 bp element for Bcd was compared to that of a 250 bp enhancer from the hb promoter. The hb enhancer drives lacZ expression across both the head and thoracic primordia and binds Bcd with high affinity. Significantly higher Bcd levels are found to be required in gel retardation assays to shift the labeled 186 bp fragment than the labeled hb regulatory element. Consistent with the Gene X model, the overall affinity of the otd regulatory element for Bcd is lower than that of the hb enhancer (Gao, 1998).

The 1.8 kb regulatory fragment contains two candidate Bcd and one Hb site that lie upstream of the EHE. Deletion of the region containing these sites causes a slight decrease in the intensity of expression. To determine whether this region is sufficient to drive early head expression, more fusion constructs were generated and their functions tested in vivo. Unexpectedly, it was found that a 526 bp EcoRV- HincII fragment containing these sites drive both anterior and posterior lacZ expression. This expression resembles that of the terminal gap gene tll, suggesting that this fragment contains terminal system response elements. Consistent with this idea, the anterior and posterior expression domains specified by this fragment both expand in embryos derived from torD females, bearing a constitutively active Egf receptor, triggering excess tll activation. Since otd is not expressed at the posterior pole, it was hypothesized that additional regulatory elements exist that prevent posterior expression. To test this idea, expression driven by a larger regulatory fragment extending to the 5' end of the EHE was examined. This fragment drives expression only in the head primordium, indicating that it contains a negative regulatory element that represses posterior expression. These result strongly suggests that Bcd participates directly in the regulation of otd (Gao, 1998).

Unlike gap genes in the trunk region of Drosophila embryos, gap genes in the head were presumed not to regulate each other's transcription. However, in tailless loss-of-function mutants the empty spiracles expression domain in the head expands, whereas it retracts in tll gain-of-function embryos. A 304bp element in the ems-enhancer is sufficient to drive expression in the head and brain; it contains two Tll and two Bcd binding sites. Transgenic reporter gene lines containing mutations of the Tll binding sites demonstrate that tll directly inhibits the expression of ems in the early embryonic head and the protocerebral brain anlage. These results are the first demonstration of direct transcriptional regulation between gap genes in the head (Hartmann, 2002).

The protein product of the anterior maternal system gene, bcd, is a morphogen and differentially directs the expression pattern of the first zygotic genes in the anterior region of the embryo. This is thought to be achieved by differences in the affinity of the Bcd binding sites within the promotors of these zygotic genes. Thus, the broadly expressed gap gene hb contains strong Bcd binding sites and requires only a low level of Bcd for its activation. In contrast, the cephalic gap genes ems, orthodenticle (otd), buttonhead (btd) and sloppy paired (slp), whose expression patterns are restricted to anterior regions of the embryo, are presumed to contain low affinity Bcd binding sites requiring high levels of Bcd for their activation. So far, Bcd binding sites have only been mapped for otd, and it is assumed that these binding sites have a low affinity for Bcd. A 304 bp fragment of the ems enhancer that is sufficient to generate an ems like expression pattern in the head primordium contains two Bcd consensus sites that bind Bcd in vitro. These sites in the ems enhancer element are medium affinity binding sites. This might reflect the fact that ems is expressed posterior to otd and thus requires a lower threshold level of Bcd for its activation compared to otd. Mutations of these Bcd binding sites show that they are also essential for the in vivo function of this enhancer element during early head patterning. This suggests that Bcd, or a protein with similar binding specificity, directly activates ems expression in the head primordium. The only known protein with a similar binding specificity as Bcd is Otd. Since ems activation is independent of otd, it is posited that Bcd directly regulates early ems expression (Hartmann, 2002).

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

Dynamical analysis of regulatory interactions in the gap gene system of Drosophila

Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. A detailed analysis of regulatory interactions involved in gap gene regulation is presented based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. The models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. These models predict activation of Kr by Cad and clarify several other regulatory interactions. This analysis suggests a central role for repressive feedback loops between complementary gap genes. Repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, these models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system (Jaeger, 2004).

Although activating contributions from Bcd and Cad show some degree of localization, positioning of gap gene boundaries during cycle 14A is largely under the control of repressive gap-gap cross-regulatory interactions. Thereby, activation is a prerequisite for repressive boundary control, which counteracts broad activation of gap genes in a spatially specific manner. In addition, gap genes show a tendency toward autoactivation, which increasingly potentiates activation by Bcd and Cad during cycle 14A. Autoactivation is involved in maintenance of gap gene expression within given domains and sharpening of gap domain boundaries during cycle 14A (Jaeger, 2004).

Regulatory loops of mutual repression create positive regulatory feedback between complementary gap genes, providing a straightforward mechanism for their mutually exclusive expression patterns. Such a mechanism of 'alternating cushions' of gap domains has been proposed previously. The results suggest that this mechanism is complemented by repression among overlapping gap genes. Overlap in expression patterns of two repressors imposes a limit on the strength of repressive interactions between them. Accordingly, repression between neighboring gap genes is generally weaker than that between complementary ones. Moreover, repression among overlapping gap genes is asymmetric, centered on the Kr domain. Posterior to this domain, only posterior neighbors contribute functional repressive inputs to gap gene expression, while anterior neighbors do not. This asymmetry is responsible for anterior shifts of posterior gap gene domains during cycle 14A (Jaeger, 2004).

Repression by Tll mediates regulatory input to gap gene expression by the terminal maternal system. Tll provides the main repressive input to early regulation of the posterior boundary of posterior gt, and activation by Tll is required for posterior hb expression. Note that these two features form only during cycle 13 and early cycle 14A, while other gap domain boundaries are already present at the transcript level during cycles 10-12 and largely depend on the anterior and posterior maternal systems for their initial establishment. The delayed formation of posterior patterning features and their distinct mode of regulation are reminiscent of segment determination in primitive dipterans and intermediate germ-band insects, supporting a conserved dynamical mechanism across different insect taxa (Jaeger, 2004).

The set of regulatory interactions presented here provides a consistent and sufficient dynamical mechanism for gap gene expression. In summary, this set of interactions consists of the following five basic regulatory mechanisms: (1) broad activation by Bcd and/or Cad, (2) autoactivation, (3) strong repressive feedback between mutually exclusive gap genes, (4) asymmetric repression between overlapping gap genes, and (5) feed-forward repression of posterior domain boundaries by the terminal gap gene tll. In the following subsections, evidence is discussed concerning specific regulatory interactions involved in each of these basic mechanisms in some detail (Jaeger, 2004).

Activation by Bcd and Cad: Activation of gap gene expression by Bcd and Cad is supported by the following. Bcd binds to the regulatory regions of hb, Kr, and kni. The kni regulatory region also contains binding sites for Cad. The anterior domains of gt and hb are absent in embryos from bcd mothers. The posterior domain of gt is missing in embryos mutant for both maternal and zygotic cad, while the posterior domain of kni is absent in embryos mutant for maternal bcd plus maternal and zygotic cad. These results suggest partial redundancy of activation of kni by Bcd, consistent with evidence from zygotic cad embryos from bcd mothers, where maternally provided Cad is sufficient to activate kni (Jaeger, 2004).

Kr expression expands anteriorly in embryos from bcd mothers, which is due to the absence of the anterior gt and hb domains. Bcd has been shown to activate expression of Kr reporter constructs. The fact that Kr is still expressed in embryos from bcd mutant mothers has been attributed to activation by general transcription factors or low levels of Hb. In contrast, the models predict that this activation is provided by Cad. Although Kr expression is normal in embryos overexpressing cad, repressive control of Kr boundaries could account for the lack of expansion of the Kr domain in such embryos (Jaeger, 2004).

The activating effect of Cad on hb found in gap gene circuits is likely to be spurious. The anterior hb domain is absent in embryos from bcd mutant mothers, which show uniformly high levels of Cad. Moreover, the complete absence of the posterior hb domain in tll mutants suggests activation of posterior hb by Tll rather than by Cad. It is believed that this spurious activation of hb by Cad is due to the absence of hkb in gap gene circuits. The posterior hb domain fails to retract from the posterior pole in hkb mutants, suggesting a repressive role of Hkb in regulation of the posterior hb border. Consistent with this, the posterior boundary of the posterior hb domain never fully forms in any of the circuits. Moreover, Tll is constrained to a very small or no interaction with hb due to the absence of the posterior repressor Hkb, since activation of hb by Tll would lead to increasing hb expression extending to the posterior pole (Jaeger, 2004).

Autoactivation:: A role for autoactivation in the late phase of hb regulation is supported by the fact that the posterior border of anterior hb is shifted anteriorly in a concentration-dependent manner in embryos with decreasing doses of zygotic Hb. Weakened and narrowed expression of Kr in mutants encoding a functionally defective Kr protein suggests Kr autoactivation. Similarly, a delay in the expression of gt in mutants encoding a defective Gt protein indicates gt autoactivation. However, the results suggest that gt autoactivation is not essential. It is generally weaker than autoactivation of other gap genes, and circuits lacking gt autoactivation show no specific defects in gt expression. Finally, in the case of kni, there is no experimental evidence for autoactivation, while some authors have even suggested kni autorepression. No such autorepression has been detected in any gap gene circuit (Jaeger, 2004).

Repression between complementary gap genes: Mutual repression of gt and Kr is supported by the following. gt expression expands into the region of the central Kr domain in Kr embryos. In contrast, Kr expression is not altered in gt mutants before germ-band extension. However, Gt binds to the Kr regulatory region, and the central domain of Kr is absent in embryos overexpressing gt. Moreover, Kr expression extends further anterior in hb gt double mutants than in hb mutants alone. The above is consistent with this analysis, which shows no significant derepression of Kr in the absence of Gt even though repression of Kr by Gt is quite strong (Jaeger, 2004).

Hb binds to the kni regulatory region, and the posterior kni domain expands anteriorly in hb mutants. Embryos overexpressing hb show no kni expression at all, and embryos misexpressing hb show spatially specific repression of kni expression.There is no clear posterior expansion of kni in hb mutants. This could be due to the relatively weak and late repressive contribution of Hb on the posterior kni boundary or due to partial redundancy with repression by Gt and Tll. The posterior hb domain expands anteriorly in kni mutants, but anterior hb expression is not altered in these embryos. Nevertheless, a role of Kni in positioning the anterior hb domain is suggested by the fact that misexpression of kni leads to spatially specific repression of both anterior and posterior hb domains. Moreover, only slight posterior expansion of anterior hb is observed in Kr mutants, while hb is completely derepressed between its anterior and posterior domains in Kr kni double mutants (Jaeger, 2004).

Repression between overlapping gap genes: gt, kni, and Kr show repression by their immediate posterior neighbors hb, gt, and kni, respectively. Retraction of posterior Gt from the posterior pole during midcycle 14A fails to occur in hb mutants, and no gt expression is observed in embryos overexpressing hb. The posterior kni boundary is shifted posteriorly in gt mutant embryos, and kni expression is reduced in embryos overexpressing gt. Note that these effects are very subtle and were not reported in similar studies by different authors. A weak but functional interaction of Gt with kni is consistent with these results. This interaction was found to be essential even in a circuit where it was deemed below significance level. Finally, Kni has been shown to bind to the Kr regulatory region, and the central Kr domain expands posteriorly in kni mutants (Jaeger, 2004).

In contrast, no effect of Kr on hb was detected. However, hb expression expands posteriorly in Kr mutants. This effect is likely to involve repression of hb by Kni. Kni levels are reduced in Kr embryos. hb is completely derepressed between its anterior and posterior domains in Kr kni double mutants, whereas anterior hb does not expand at all in kni mutants alone. Taken together these results suggests that there is direct repression of hb by Kr in the embryo, but it is at least partially redundant with repression of hb by Kni (Jaeger, 2004).

Unlike repression by posterior neighbors, no or only weak repression was found of posterior kni, gt, and hb by their anterior neighbors Kr, kni, and gt, respectively. Most gap gene circuits show weak activation of hb by Gt. Graphical analysis failed to reveal any functional role for such activation. Moreover, no functional interaction was found between gt and Kni. Although relatively weak repression of kni by Kr was found in 6 out of 10 circuits, no specific patterning defects could be detected in the other 4. Consistent with the above, expression of posterior hb is normal in gt mutants, and both the anterior boundaries of posterior gt and kni are positioned correctly in kni and Kr mutant embryos, respectively (Jaeger, 2004).

Note that activation of kni by Kr, which has been proposed to explain decreased expression levels of kni in Kr mutants, was never found. The results strongly support the view that this interaction is indirect through Gt, which is further corroborated by the fact that kni expression is completely restored in Kr gt double mutants compared to that in Kr mutants alone (Jaeger, 2004).

A significant repressive effect of Hb on Kr was found. Consistent with this, Hb has been shown to bind to the Kr regulatory region, and the central Kr domain expands anteriorly in hb mutants. However, partial redundancy of this interaction is suggested by correct positioning and shape of the anterior Kr domain in a circuit that does not show repression of Kr by Hb (Jaeger, 2004).

It has been proposed that Hb plays a dual role as both activator and repressor of Kr. In the framework of the gene circuit model, concentration-dependent switching of regulative action could be implemented by allowing genetic interconnection parameters to switch sign at certain regulator concentration thresholds. The current model explicitly does not include such a possibility. Nevertheless, circuits have been obtained that reproduce Kr expression faithfully, suggesting that a dual role of Hb is not required for proper Kr expression. Moreover, activation of Kr by Hb was ever observed in any of the circuits. Therefore, the results support a mechanism in which the activation of Kr by Hb is indirect through derepression of kni (Jaeger, 2004).

Repression by Tll: Only a few earlier theoretical approaches have considered terminal gap genes. Gap gene circuits accurately reproduce tll expression. However, in gene circuits, tll is subject to regulation by other gap genes, which is inconsistent with experimental evidence. In contrast, the correct expression pattern of tll in gap gene circuits allows its effect on other gap genes to be studied in great detail. Strong repressive effects of Tll on Kr, kni, and gt have been found. Tll binding sites have been found in the regulatory regions of Kr and kni. In tll mutants, Kr expression is normal, whereas expression of kni expands posteriorly, and the posterior gt domain fails to retract from the posterior pole. No expression of Kr, kni, or gt can be detected in embryos overexpressing tll under a heat-shock promoter (Jaeger, 2004).

Reverse engineering the gap gene network of Drosophila

A fundamental problem in functional genomics is to determine the structure and dynamics of genetic networks based on expression data. A new strategy is described for solving this problem and it is applied to recently published data on early Drosophila development. The method is orders of magnitude faster than current fitting methods and allows fitting of different types of rules for expressing regulatory relationships. Specifically, this approach is sused to fit models using a smooth nonlinear formalism for modeling gene regulation (gene circuits) as well as models using logical rules based on activation and repression thresholds for transcription factors. The technique also allows inference of regulatory relationships de novo or testing network structures suggested by the literature. A series of models is fitted to test several outstanding questions about gap gene regulation, including regulation of and by hunchback and the role of autoactivation. Based on the modeling results and validation against the experimental literature, a revised network structure is proposed for the gap gene system. Interestingly, some relationships in standard textbook models of gap gene regulation appear to be unnecessary for, or even inconsistent with, the details of gap gene expression during wild-type development (Perkins, 2006).

The regulatory structure of the Combined model is itself sufficient to reproduce all six gap gene domains using either the gene circuit or logical formalisms for production rate functions. Support is cited for the Combined model, and then consider the results of the individual models in light of several outstanding questions about gap gene regulation are discussed (Perkins, 2006).

The maternal proteins Bcd and Cad are largely responsible for activating the trunk gap genes, with Bcd being more important for the anterior domains and Cad more important for the posterior domains. Bcd is a primary activator of the anterior hb domain, the anterior gt domain, and the Kr domain. Cad activates posterior gt. The kni domain is present in bcd mutants and in cad mutants, but not in bcd;cad double mutants. This suggests redundant activation by the two maternal factors. Such redundant activation of kni is present in the Unc-GC model. For the other models, the optimization selected one or the other as activators, but not both. Tll is crucial for activating the posterior hb domain, while it represses Kr, kni, and gt, preventing their expression in the extreme posterior. All the regulatory relationships between the gap genes in the Combined model are repressive. The complementary gap gene pairs, hb-kni and Kr-gt are known to be strongly mutually repressive, as was found in nearly all the models. [Repression of hb by Kni is not part of the Rivera-Pomar and Jäckle (RPJ) regulatory relationships (Rivera-Pomar, 1996a), but the unconstrained gene circuit (Unc-GC) model and Unc-Logic model (that employs the regulatory structure discovered by the unconstrained gene circuit fit, except that Gt activation of hb and Kni activation of gt were removed) included the link.] The models also suggest that mutual repression between hb and Kr helps to set the boundary between those two domains. A chain of repressive relationships, hb-gt-kni-Kr, causes the shifts in the Kr, kni, and posterior gt domains. Autoactivation by hb is well-established, and there is also some evidence for autoactivation by Kr and gt (Perkins, 2006).

Does Hb have a dual regulatory effect on Kr? There is a long-running debate about whether or not low levels of Hb activate Kr. In hb mutants, the Kr domain expands anteriorly, suggesting that Hb represses Kr. However, Kr expression in these mutants is lower than in wild-type and expands posteriorly in embryos overexpressing Hb. Further, in embryos lacking Bcd and Hb, the Kr domain is absent, but can be restored in a dosage-dependent manner by reintroducing Hb. These observations suggest that Hb activates Kr. It has been suggested, therefore, that low levels of Hb activate Kr while high levels repress it. An alternative explanation, however, is that the apparently activating effects of Hb are indirect, via Hb's repression of kni and Kni's repression of Kr. Optimization of the Unc-GC model, which could have resulted in activation or repression of Kr by Hb, but not both, resulted in repression. The RPJ models allow for a dual effect, but activation by Hb was eliminated during optimization of the RPJ-Logic model. The RPJ-GC model retained functional activation and repression of Kr by Hb. However, Kr expression in this model is defective. Kr is not properly repressed in the anterior. Further, Kr is ectopically expressed in a small domain in the posterior of the embryo. Thus, the current models provide no support for activation of Kr by Hb. The only support found, which is crucial in all models except Unc-Logic and also consistent with the mutant and overexpression studies, is for repression of Kr by Hb (Perkins, 2006).

What represses hb between the anterior and posterior domains? Another point of disagreement in the literature is what prevents the expression of hb between its two domains. In the model of Rivera-Pomar and Jäckle (1996a), repression by Kr is the explanation. The RPJ models confirm that this mechanism is sufficient. Specifically, in these models Kr repression prevents hb expression just to the posterior of the anterior hb domain. Between the Kr and posterior hb domains, there is no explicit repression of hb. Rather, Hb is not produced simply because of a lack of activating factors. In contrast, the models of Jaeger (2004a and b) detected no effect of Kr and attributed repression solely to Kni. The Unc-GC and Unc-Logic models found repression by Kni, but in addition to repression by Kr, not instead of it. Kr is more responsible for repression near the anterior hb domain and Kni is more responsible for repression near the posterior hb domain. This is consistent with observations of expression in mutant embryos. Embryos mutant for Kr show slight expansion of the anterior hb domain, while kni embryos show expansion of the posterior hb domain. In Kr;kni double mutants, hb is completely derepressed between its two usual domains. This suggests, as seen in the Unc-GC and Unc-Logic models, that Kr and Kni are both repressors of hb, that their activity is redundant in the center of the trunk, and that Kr and Kni are the dominant repressors for setting the boundaries of the anterior and posterior domains, respectively. This interpretation was also favored by Jaeger (2004a and b), on the basis of the mutant data, even though Jaeger's models did not find repression by Kr (Perkins, 2006).

The posterior hb domain. In all of the current models, the posterior hb domain is activated by Tll and sustained by Tll and hb autoactivation. Rivera-Pomar (1996a) did not consider the posterior hb domain, and did not include activation by Tll in his model. That link was added to the RPJ network structure because otherwise it was not possible to capture the posterior hb domain. The model of Jaeger (2004a and b) captured the domain without Tll activation by substituting activation from cad. However, there is no confirming evidence for such an interaction. The absence of posterior hb in tll mutants and the inability of the models to explain posterior hb by other means, leads to the straightforward hypothesis that Tll activates posterior hb. Posterior hb is unique in that the domain begins to form later than the other five domains modeled. In the RPJ models, this happens simply because high levels of Tll are needed to activate hb -- levels that are reached only at about t = 30 min. The Unc-GC and Unc-Logic models also employ repression by Cad to slightly delay Hb production in the posterior. However, there is no confirming evidence for such repression, and it is omitted from the Combined model (Perkins, 2006).

Shifting of the Kr, kni, and posterior gt domains. Domain shifting was first observed by Jaeger (2004a and b) and attributed to a chain of repressive regulatory relationships, hb-gt-kni-Kr. The current models largely support the importance of this regulatory chain, particularly the final two links. Repression of Kr by Kni was significant in all of the current models. Repression of kni by Gt was present in all models except RPJ-Logic, where it would be of little impact anyway, since RPJ-Logic has a defective posterior gt domain. Consistent with these findings, Kni binds to the regulatory region of Kr, and the Kr domain expands towards the posterior in kni mutants. Similarly, the kni domain expands posteriorly in gt mutants, while embryos overexpressing gt show reduced kni expression (Perkins, 2006).

Repression of gt by Hb is not as well supported by the current models. The Unc-GC model included the link, though the regulatory weight was the smallest of all those in the model. The link was eliminated from Unc-Logic and, of course, not present in the RPJ network structure. Instead, the models utilized decreasing activation by Cad (Unc-GC, Unc-Logic) and repression by Tll (Unc-GC, RPJ-GC) to shift the posterior gt domain. Even with these links, however, shifting of the domain is not well-captured. RPJ-GC appears to capture the posterior gt shift best (Figure 3E). However, it relies on its small ectopic Kr domain to repress gt, a completely incorrect mechanism. Interestingly, a gene circuit fit using the network structure of Sanchez and Thieffry (2001), captured the shift of posterior gt better than any of the other current models, and it did so using repression of gt by Hb, providing additional modeling support for the relationship. There also is strong mutant evidence in favor of the relationship. In hb mutants, the posterior gt domain does not retract from the posterior pole. Further, Gt is absent in embryos that have ubiquitous Hb, such as maternal oskar or nanos mutants or embryos expressing Hb ubiquitously under a heat-shock promoter. Thus, sufficient evidence was found to include a repressive link from hb to gt in the Combined model (Perkins, 2006).

Activating or repressing links that oppose the direction of the repressive chain were eliminated by optimization of the Unc-Logic, RPJ-GC, and RPJ-Logic models. In agreement with this result, the boundaries of the kni and posterior gt domains are correctly positioned in Kr and kni mutants, respectively. Thus, the simplest picture supported by the current models and consistent with the mutant studies is that there is no regulation from Kr, kni, or posterior gt to any of their immediate posterior neighbors, and that the repressive chain highlighted by Jaeger (2004a and b) is indeed responsible for domain shifting (Perkins, 2006).

Do gap genes autoregulate? All four of the current models include autoactivation by hb. This is supported by the observation that late anterior hb expression is absent in embryos lacking maternal and early zygotic Hb 47. The models suggest hb autoactivation also plays a crucial role in sustaining the posterior domain, once it has been initiated by Tll, a role not previously emphasized. Autoactivation for the other genes was found by the Unc-GC model, but is not part of the RPJ network structure. It included autoactivation only for Kr and gt in the Combined model, on the basis of a weakened and narrowed Kr domain in embryos producing defective Kr protein and a delay in gt expression in embryos producing defective gt protein. Interestingly, the gene circuit models of Jaeger (2004a and b) also found autoactivation for all four gap genes, but they considered autoactivation by gt to be the weakest and least certain. In contrast, the Unc-Logic model retained gt autoactivation while eliminating autoactivation for Kr and kni. The RPJ-Logic model was unable to reproduce the posterior gt domain. However, it was found that by adding gt autoactivation to the model, it was able to create and sustain posterior gt correctly, bringing the error of the model down to 15.34. This suggests that, after hb, gt is the most likely candidate for autoactivation. However, even this is not strictly necessary. The RPJ-GC model is able to reproduce and sustain the posterior gt domain without autoactivation by relying on cooperative activation from Bcd and Cad (Perkins, 2006).

Comparison of regulatory architectures. The regulatory relationships proposed by Rivera-Pomar and Jäckle (1996a) are not fully consistent with the data and require amending. Repression of gt by Kni, which contradicts the mechanism of domain shifts described by Jaeger (2004a and b), was eliminated by the optimization in both of the current models based on the RPJ regulators. Activation of kni by Kr was never observed. No support was found for a dual regulatory effect of Hb on Kr. Activation of Kr at low levels of Hb was eliminated in the RPJ-Logic model. It was retained in the RPJ-GC model, but resulted in serious patterning defects. Inclusion of Tll as an activator of hb was sufficient to produce the posterior hb domain. Based on the current fits and the primary experimental literature, there are likely other regulatory links missing from the model of Rivera-Pomar and Jäckle, though they are not strictly required to reproduce the wild-type gap gene patterns. Foremost is repression of hb by Kni, which appears important for eliminating hb expression anterior of the posterior domain. Fits based on the Sanchez and Thieffry (2001) regulatory relationships also support these conclusions (Perkins, 2006).

In contrast, the regulatory relationships in the Combined model and both the Unc-GC and Unc-Logic models are able to capture the wild-type gap patterns without gross defects. The relationships in the Unc-GC model are very similar to those obtained by Jaeger (2004a and b). For example, the regulation of Kr and kni is qualitatively equivalent in both models, and there is a single minor difference in the regulation of gt. The optimizations correctly identified activation of hb by Tll, which was missed by Jaeger (2004a and b), though the current models did less well at capturing shifting of the posterior gt domain. These regulatory relationships are also similar to those found by Gursky (2004), though that study was based on gap gene expression data with much lower accuracy and temporal resolution than the data used in this study. These similarities show that differences in the mathematical formulations of these models-as ordinary versus partial differential equations, how diffusion and nuclei doubling are modeled, and choice of boundary conditions and other simulation parameters-are not important for the reproduction of the gap gene patterns nor for the inference of regulatory relationships from the data (Perkins, 2006).

The role of binding site cluster strength in Bicoid-dependent patterning in Drosophila

The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient that is critical for patterning the anterior-posterior (AP) body plan in Drosophila. Previous work identified several target genes that respond directly to Bcd-dependent activation. Positioning of these targets along the AP axis is thought to be controlled by cis-regulatory modules (CRMs) that contain clusters of Bcd-binding sites of different 'strengths.' A combination of Bcd-site cluster analysis and evolutionary conservation has been used to predict Bcd-dependent CRMs. Tested were 14 predicted CRMs by in vivo reporter gene assays; 11 showed Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the most anterior part of the embryo, whereas others extend into middle and posterior regions. However, no strong correlation is detected between AP position of target gene expression and the strength of Bcd site clusters alone. Rather, binding sites for other activators, including Hunchback and Caudal correlate with CRM expression in middle and posterior body regions. Also, many Bcd-dependent CRMs contain clusters of sites for the gap protein Krüppel, which may limit the posterior extent of activation by the Bcd gradient. It is proposed that the key design principle in AP patterning is the differential integration of positive and negative transcriptional information at the level of individual CRMs for each target gene (Ochoa-Espinosa, 2005).

Previous studies of segmentation gene regulation led to the identification of several CRMs that are directly activated by Bcd. All Bcd-dependent CRMs identified so far contain clusters of Bcd sites. Simultaneous scans trained on the general structures of the known CRMs were used to search for more Bcd target elements. This method correctly identified many previously known Bcd target elements and previously uncharacterized Bcd site clusters located throughout the genome. Thirteen previously uncharacterized clusters were found within 15 kb of genes known to be expressed in the early embryo, and all of these were tested for regulatory activity in vivo. One strong cluster located within the sixth intron of the bl gene was tested. The sizes of tested fragments were determined in part by cross-species comparisons (Ochoa-Espinosa, 2005).

In reporter gene assays, 11 of the 14 tested fragments directed expression patterns in wild-type embryos that recapitulate all or part of the endogenous patterns of the associated genes. These experiments identified several elements that control segmentation genes, including three new gap gene CRMs. Two CRMs were found in the genomic region that lies 5' of the gap gene gt. One CRM (gt23) is initially expressed in a broad anterior domain and then refines into two stripes. A second CRM (gt1) is expressed later in a small dorsal domain very near the anterior tip. Double stain experiments indicated that the timing and spatial regulation of both patterns are indistinguishable from the anterior expression domains of the endogenous gt gene. A CRM 3' of the gap gene tll was identified that drives expression similar to the anterior tll domain (Ochoa-Espinosa, 2005).

Four novel CRMs were identified near known pair rule genes. One CRM was detected in the 3' region of hairy and drives expression of a small anterior dorsal domain similar to the hairy 0 stripe of the endogenous gene. Another CRM is located 3' of the paired gene and directs expression of an early broad domain that coincides with the later position of the native paired stripes 1 and 2. Two more CRMs (slpA and slpB) were identified in the slp locus, which contains the two related genes, slp1 and slp2. Both slpA and slpB faithfully reproduce parts of the early slp1 and slp2 expression patterns (Ochoa-Espinosa, 2005).

Four other CRMs were identified near the genes bowl, CG9571, D/fsh, and bl/Mir7. In three cases (bowl, CG9571, and D/fsh), the newly identified CRMs direct patterns similar to their associated endogenous genes. The final CRM (bl/Mir7) is located in the sixth intron of the bl gene and directs a strong anterior domain of expression. However, the endogenous bl gene is expressed nearly ubiquitously , which makes it an unlikely target of regulation by this CRM. One potential target of this element is the microRNA gene (Mir7), which is located 7 kb downstream in the eighth intron of bl. Four of the CRMs reported here (gt1, gt23, slpA, and D/fsh) were also identified in a recent genome-wide search for new patterning elements based on clusters of combinations of different binding sites including Bcd. The fragments used in that study were significantly larger in size but show very similar patterns to those in this study (Ochoa-Espinosa, 2005).

Three fragments that were predicted to be CRMs did not show any embryonic expression in reporter gene assays. One fragment contains a third Bcd site cluster in the slp locus; the other two fragments are located near the goosecoid gene, which is expressed in a Bcd-dependent anterior domain. It is not clear why these elements failed to direct expression in the embryo, but the chosen fragments may lack critical sequences for activation (Ochoa-Espinosa, 2005).

To confirm that the 11 CRMs are bona fide Bcd target elements, males carrying each reporter gene were crossed to bcdE1 mutant females. In all cases, the lacZ reporter expression patterns were abolished in embryos from these crosses. These results suggest that Bcd activity is required for activation of each identified CRM (Ochoa-Espinosa, 2005).

The discovery of these elements brings the total number of experimentally validated Bcd-dependent CRMs to 21. This set of CRMs permitted an examination of the mechanisms involved in AP patterning by the Bcd morphogen. Specifically, it was of interest to correlate the PBPs of the patterns driven by each CRM with the relative binding 'strength' of each cluster. First, attempts were made to correlate specific characteristics of the CRMs with their PBPs. Three features were tested: site number, average PWM score above a cutoff of 5.0, and the single highest PWM score. There was no correlation between any of these features and the PBPs of reporter gene expression. However, individual CRMs showed very different rankings among themselves when different features were tested. This inconsistency suggests that no single characteristic tested so far accurately reflects the in vivo binding strength of the CRM (Ochoa-Espinosa, 2005).

CLUSTERVIEW software was used to generate a graphical depiction that simultaneously accounts for several binding characteristics. This program was first tested on 4-kb genomic regions surrounding the previously characterized Bcd-dependent CRMs, otd early and hb P2. The otd region contains more sites than the hb P2 region, and most sites have intermediate or low PWM scores, whereas two sites in the hb P2 region have very high PWM scores. Also, the sites in the otd region are dispersed over a relatively large genomic region compared with the hb P2 region. These differences are accurately reflected in the associated color graphs. Furthermore, in silico mutations of single sites in the hb P2 sequence cause dramatic changes in the graphical appearance of the cluster (Ochoa-Espinosa, 2005).

CLUSTERVIEW was next used to analyze all known Bcd-dependent CRMs and attempts were made to correlate the resulting graphs with the Posterior border positions (PBPs) of reporter gene expression. In general, no correlation was detected. For example, there are seven CRMs whose PBPs lie between 79% and 70% EL. Within this group, three Bcd clusters (CG9571, bl/Mir7, and slpA) appear very strong, one (otd early) appears very weak, and three (slpB, bowl, and tll) lie somewhere in between. The other four groups contain fewer CRMs but also show significant differences in the depictions of individual clusters. Together, these results strongly suggest that differential binding alone does not play a major role in determining the limits of expression of most Bcd target genes (Ochoa-Espinosa, 2005).

An alternative to the simple gradient mechanism is a combinatorial mechanism, which is supported by several previous studies of Bcd-dependent CRM. To determine whether regulatory inputs by Hb and/or Kr are general characteristics of Bcd-dependent CRMs, all known elements were scanned for clusters of Hb and/or Kr sites and they were grouped into distinct classes according to their binding site composition. Interestingly, only five CRMs (Class 1) seem to be regulated primarily by Bcd alone. The expression patterns driven by all five are restricted to the most anterior 30% of the embryo (average PBP, 75.4% EL; PBP range, 85% to 72% EL). Four CRMs (Class 2) also contain strong Hb clusters but very few, if any, Kr sites. Expression patterns driven by this class extend more posteriorly (average PBP, 52.5% EL; PBP range, 88% to 30% EL), consistent with the hypothesis that synergistic interactions between Bcd and Hb increases CRM sensitivity to the Bcd gradient. Six Bcd-dependent CRMs (Class 3) also contain relatively strong clusters of Kr sites but very little, if any, Hb binding. Compared with class 1 CRMs (regulated by Bcd alone), these elements direct transcription in more posterior regions (average PBP, 62.6% EL; PBP range, 70% to 56% EL). This finding is quite interesting in light of the fact that Class 3 elements do not contain significant Hb-binding clusters. Finally, six CRMs (Class 4) showed strong binding clusters for Bcd, Hb, and Kr. PBPs of the patterns driven by these elements varied considerably, ranging from 70% to 25% EL (Ochoa-Espinosa, 2005).

In summary, most Bcd-dependent CRMs appear to contain significant contributions from Hb and/or Kr. Several CRMs (including kni and hairy 7) also contain clusters of binding sites for Caudal (Cad), which is expressed in a posterior gradient. Previous work suggests that Cad functions with Bcd to activate expression of these CRMs (Ochoa-Espinosa, 2005).

Probing intrinsic properties of a robust morphogen gradient in Drosophila

A remarkable feature of development is its reproducibility, the ability to correct embryo-to-embryo variations and instruct precise patterning. In Drosophila, embryonic patterning along the anterior-posterior axis is controlled by the morphogen gradient Bicoid (Bcd). This article describes quantitative studies of the native Bcd gradient and its target Hunchback (Hb). The native Bcd gradient is highly reproducible and is itself scaled with embryo length. While a precise Bcd gradient is necessary for precise Hb expression, it still has positional errors greater than Hb expression. Analyses are described further probing mechanisms for Bcd gradient scaling and correction of its residual positional errors. The results suggest a simple model of a robust Bcd gradient sufficient to achieve scaled and precise activation of its targets. The robustness of this gradient is conferred by its intrinsic properties of 'self-correcting' the inevitable input variations to achieve a precise and reproducible output (He, 2008).

In a developing embryo, cells need to make unambiguous decisions in choosing their own fates by expressing distinct sets of genes. Such decisions must be reproducible from embryo to embryo, despite individual and environmental differences. In Drosophila, cells adopting the anterior fate express Hb, a direct target of the Bcd morphogen gradient. Despite embryo size variations, Hb expression boundary is precise and scaled with embryo length. How Hb precision is achieved directly affects understanding of developmental scaling and reproducibility. Although live-imaging study has provided unprecedented new insights into both the dynamics and precision of the Bcd gradient, it had to rely on a GFP-Bcd hybrid protein. This article describes quantitative studies to analyze the behaviors of the native Bcd gradient and its target Hb. The results show that: (1) the native Bcd gradient is precise and scaled with embryo length; (2) a precise Bcd gradient is necessary for Hb precision; and (3) a precise Bcd gradient still has positional errors that are greater than Hb boundary variations. The results uncover correlated 'self-correcting' input variations as the underpinnings of a robust gradient system sufficient for scaled and precise target gene activation (He, 2008).

A major finding of the current studies is that native Bcd profiles are not only reproducible, but also scaled with embryo length. Unlike previous embryo staining studies, this study: (1) used raw Bcd intensity data captured within a linear range; (2) specifically measured background intensities under identical experimental conditions; and (3) avoided any normalization or adjustment of Bcd intensity data (except background subtraction when necessary). These and other improvements have enabled accurate measurement of Bcd profiles in stained embryos. The studies reveal Bcd properties expected of scaling. In particular, Bcd intensities are more precise when measured as a function of normalized A-P position than without such normalization. Moreover, Bcd intensity in the anterior (B0) is correlated with L. This correlation drops rapidly as a function of normalized A-P position (x/L), effectively preventing its propagation toward normalized xHb and beyond. A B0-Lcorrelation is sufficient to account for the observed scaling properties of Bcd gradient in WT embryos. Currently it is not known exactly the source(s) of the observed B0-L correlation. If the amount of bcd mRNA deposited into an egg during oogenesis is proportional to the egg volume, it could represent a source for the observed B0-L correlation. This simple model of Bcd gradient scaling contrasts with an alternative model, in which Bcd gradient precision is maintained throughout the A-P length by 'counting' the nuclear number, rather than measuring distance. A fundamental difference between these two models reflects how Bcd intensity variations near the anterior are interpreted: while the model suggests that such variability is biologically meaningful and responsible for size scaling through the observed B0-L correlation, the alternative model interprets it as a mere consequence of the difference in the locations (but not L-correlated amounts/rates) of Bcd protein synthesis (He, 2008).

Analysis of stau embryos demonstrates that a precise Bcd gradient is necessary for precise Hb expression. Bcd profiles in stau embryos are more variable than in WT embryos, most likely resulting from the increased variations in bcd mRNA localization and/or amount. Concurrently, Hb expression is more variable in stau embryos and exhibits properties indicative of a loss of scaling. More importantly, normalized xHb position in stau embryos is positively correlated with Bcd level at the mean normalized xHb, a correlation that is further improved for embryos at a more uniform developmental stage. These results suggest that increased Hb variability in stau embryos is a direct consequence of increased Bcd gradient variations. The observed Bcd gradient behaviors in stau embryos are different from those described previously, and these and other differences are attributed to methods in detecting and analyzing Bcd intensities. On a technical note, it is suggested -- based on the following two observations -- that stained embryos are suitable for studying developmental precision when data are captured and analyzed properly. Bcd intensities detected in stained WT embryos have variations comparable to live-imaging data. In addition, Bcd intensity variability for a group of stained WT embryos is comparable to that for neighboring nuclei of single embryos (He, 2008).

As demonstrated by these studies, positional information of a precise Bcd gradient is still more variable than the observed Hb precision. At the Hb boundary position, the Bcd gradient has already become very shallow and, thus, any Bcd intensity variations, even for a very precise gradient, would correspond to significant positional errors that reflect its intrinsic properties. In this study, the two parameters that directly describe the relationship between Bcd and Hb both exhibit variations. These variations could either reflect the true nature of the Bcd-Hb system, or may result merely from measurement uncertainties. The former possibility is favored, although it is not currently known exactly the source(s) of this observed correlation. However, there have been examples of coupling between an activator's stability and its ability to activate transcription. In addition or alternatively, it is sensible to imagine that stochastic, embryo-to-embryo variations in chromatin structure may affect both Bcd diffusion and its effective DNA binding affinity. Regardless of the details that remain to be uncovered, both possibilities support a link between Bcd gradient formation and activation in embryos, a notion consistent with the idea that nuclei are important for both degradation and diffusion properties of Bcd (He, 2008).

The studies described here suggest that the sources of Hb scaling and precision can be directly traced to the behaviors of the native Bcd gradient. This study identifies two intrinsic properties of Bcd relevant to developmental precision: (1) formation of a precise and scaled Bcd gradient resulting from a correlation between B0 and L; and (2) correction of its own positional errors through a link between gradient formation and activation (i.e., BxHb-λ correlation). Simulation studies show that a Bcd gradient with these two observed properties is sufficient to achieve a precise and scaled Hb boundary without theoretically provoking the involvement of any additional factors. Consistent with experimental observations, the Bcd gradient model based on these properties is robust: it is insensitive to embryo length variations, and its precise action is applicable to targets with distinct boundary positions. The robustness of this Bcd gradient model stems from mechanisms that self-correct the system's inevitable input variations arising from embryo-to-embryo differences. In particular, while egg size (L) variations are corrected by Bcd amount (B0) to achieve scaling, variations in gradient formation (λ) are corrected by target recognition/activation (BxHb) to enhance precision. According to this simple model, other factors, such as gap gene products, may affect the mean position of the Hb boundary, but they are not required for Hb precision and scaling, a notion fully consistent with experimental data. Furthermore, since the two observed properties (correlations) are sufficient for the Bcd gradient to achieve a precise and scaled output, as shown by simulation studies, foreign activators (such as the yeast activator Gal4) expressed as A-P gradients in Drosophila embryos are expected to activate their targets in a precise and scaled manner if they possess these same properties. It is relevant to note that the yeast activator Gal4 does possess a property that couples its degradation to its activation function, and, furthermore, its effective affinity for target DNA sites in vivo is regulated by its activation potency. Finally, it has been shown that the nuclear concentration of Bcd has already become stable prior to nuclear cycle 14, and, therefore, the robust properties of the Bcd gradient should be applicable throughout the entire relevant period of development (He, 2008).

The observations of a highly reproducible Bcd gradient have recently led to the suggestion that the system may be so precise that it approaches the limits set by basic physical principles. The current results show that, while the Bcd gradient is highly reproducible, the system still faces input variations arising from embryo-to-embryo differences. A hallmark feature of biological systems is, to their advantage, the interconnections among the operating components and processes. These studies suggest a robust Bcd gradient system that can self-correct its own inevitable input noise to achieve a precise and reproducible output. This work thus underscores the importance of input variations, because their self-correcting properties are actually responsible for conferring the robustness to the system. This simple model provides a new framework for developmental scaling and precision, and understanding its molecular and dynamic details represents future challenges (He, 2008).

A multiscale investigation of bicoid-dependent transcriptional events in Drosophila embryos

Morphogen molecules form concentration gradients to provide spatial information to cells in a developing embryo. Precisely how cells decode such information to form patterns with sharp boundaries remains an open question. For example, it remains controversial whether the Drosophila morphogenetic protein Bicoid (Bcd) plays a transient or sustained role in activating its target genes to establish sharp expression boundaries during development. This study describes a method to simultaneously detect Bcd and the nascent transcripts of its target genes, including hunchback, in developing embryos. This method allows investigation of the relationship between Bcd and the transcriptional status of individual copies of its target genes on distinct scales. On three scales analyzed concurrently -- embryonic, nuclear and local, the actively-transcribing gene copies are associated with high Bcd concentrations. These results underscore the importance of Bcd as a sustained input for transcriptional decisions of individual copies of its target genes during development. It was also shown that the Bcd-dependent transcriptional decisions have a significantly higher noise than Bcd-dependent gene products, suggesting that, consistent with theoretical studies, time and/or space averaging reduces the noise of Bcd-activated transcriptional output. Finally, this analysis of an X-linked Bcd target gene reveals that Bcd-dependent transcription bursts at twice the frequency in males as in females, providing a mechanism for dosage compensation in early Drosophila embryos. This study represents a first experimental uncovering of the actions of Bcd in controlling the actual transcriptional events while its positional information is decoded during development. It establishes a sustained role of Bcd in transcriptional decisions of individual copies of its target genes to generate sharp expression boundaries. It also provides an experimental evaluation of the effect of time and/or space averaging on Bcd-dependent transcriptional output, and establishes a dosage compensation mechanism in early Drosophila embryos (He, 2011; full text of article).

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

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

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

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

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

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

Estimating binding properties of transcription factors from genome-wide binding profiles

The binding of transcription factors (TFs) is essential for gene expression. One important characteristic is the actual occupancy of a putative binding site in the genome. In this study, an analytical model is proposed to predict genomic occupancy that incorporates the preferred target sequence of a TF in the form of a position weight matrix (PWM), DNA accessibility data (in the case of eukaryotes), the number of TF molecules expected to be bound specifically to the DNA and a parameter that modulates the specificity of the TF. Given actual occupancy data in the form of ChIP-seq profiles, copy number and specificity are backwards inferred for five Drosophila TFs during early embryonic development: Bicoid, Caudal, Giant, Hunchback and Kruppel. The results suggest that these TFs display thousands of molecules that are specifically bound to the DNA and that whilst Bicoid and Caudal display a higher specificity, the other three TFs (Giant, Hunchback and Kruppel) display lower specificity in their binding (despite having PWMs with higher information content). This study gives further weight to earlier investigations into TF copy numbers that suggest a significant proportion of molecules are not bound specifically to the DNA (Zabet, 2014: 25432957).

Temporal and spatial dynamics of scaling-specific features of a gene regulatory network in Drosophila

A widely appreciated aspect of developmental robustness is pattern formation in proportion to size. But how such scaling features emerge dynamically remains poorly understood. This study generated a data set of the expression profiles of six gap genes in Drosophila melanogaster embryos that differ significantly in size. Expression patterns exhibit size-dependent dynamics both spatially and temporally. A dynamic emergence of under-scaling in the posterior was uncovered, accompanied by reduced expression levels of gap genes near the middle of large embryos. Simulation results show that a size-dependent Bicoid gradient input can lead to reduced Kruppel expression that can have long-range and dynamic effects on gap gene expression in the posterior. Thus, for emergence of scaled patterns, the entire embryo may be viewed as a single unified dynamic system where maternally derived size-dependent information interpreted locally can be propagated in space and time as governed by the dynamics of a gene regulatory network (Wu, 2015).

back to Bicoid Targets of Activity part 1/2

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

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