hunchback


REGULATION

cis-Regulatory Sequences and Functions

There are two promoter regions. The posterior regulatory region is several kb upstream of the P1 (maternal & zygotic) transcript. The anterior regulatory region is upstream of the P2 (zygotic) transcript within the large maternal intron.

hunchback expression in the tail initiates from two promoters. A cap, covering the terminal 15% of the embryo, is composed only of mRNA from the distal transcription initiation site (P1). A posterior stripe generated later is composed of mRNA from both the distal and proximal (P2) transcription initiation sites, upstream of the P2 site of initiation. The posterior regulation region, a 1.4 kb fragment of the hb upstream region is both necessary and sufficient for posterior expression. Sequences within this fragment mediate activation by the terminal gap gene tailless and repression by terminal gap gene huckebein, which together direct the formation of the posterior hb stripe. The TLL protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by TLL (Margolis, 1995).

In adult Drosophila females maternal transcripts of hunchback are produced only by the distal (P1) promoter. This expression is largely restricted to the ovarian nurse cells. A deletion analysis of the hb promoter using lacZ reporter constructs defines a 1.2-kb genomic DNA fragment surrounding the P1 promoter sufficient to reproduce the wild-type pattern of hb ovarian transcript accumulation (Margolis, 1994).

The maternal determinant Bicoid (Bcd) represents the paradigm of a morphogen that provides positional information for pattern formation. However, since bicoid seems to be a recently acquired gene in flies, the question has been raised as to how embryonic patterning is achieved in organisms with more ancestral modes of development. Because the phylogenetically conserved Hunchback (Hb) protein acts as a morphogen in abdominal patterning, it was asked which functions of Bcd could be performed by Hb. By reestablishing a proposed ancient regulatory circuitry in which maternal Hb controls zygotic hunchback expression, it has been shown that Hb is able to form thoracic segments in the absence of Bcd (Wimmer, 2000).

A functional hb transgene has been generated that is missing all P2 promoter sequences and relies solely on the P1 promoter (hbP1only). hbP1only constructs do not respond to bcd and do not mediate gene expression in the anterior cap domain. Therefore, hbP1only uncouples the direct link between the Bcd and Hb morphogen systems. Zygotic hb mutants derived from heterozygous parents do not develop labial or thoracic structures, and they also show a fusion of abdominal segments A7 and A8. When one copy of the hbP1only transgene is provided zygotically (by the father) to a hb mutant embryo, it rescues the posterior phenotype, and A7 and A8 developed normally. The labial/thoracic phenotype is not rescued. However, when hbP1only is provided as one copy by the mother to a hb mutant embryo, the posterior and part of the anterior phenotype are rescued. These embryos exhibit normal labial and prothoracic (T1) segments, and only lack meso- and meta-thoracic segments (T2 and T3). The anterior rescue is due to the maternal contribution of hbP1only because sibling embryos that do not inherit the hbP1only construct zygotically also exhibit the partial anterior (but not the posterior) rescue. This indicates that restoring high levels of maternal hb expression (i.e., two copies: one wild type plus one copy of hbP1only) is sufficient to rescue the labial and prothoracic segments in the zygotic hb mutant progeny. Therefore, the lack of zygotic hb leads only to the loss of T2 and T3 and to the fusion of A7 and A8, whereas the previously reported zygotic hb phenotype represents a combination of a haploinsufficient maternal plus a zygotic phenotype (Wimmer, 2000).

The loss of zygotic hb activity affects regions of the embryo that correspond to the two late stripes of zygotic hb expression: The A7-A8 fusion corresponds to the posterior stripe, whereas the loss of T2 and T3 corresponds to the PS4 stripe, which starts as a fairly wide domain covering the anlagen of T2 and T3. This correlation between the zygotic hb phenotype and the late stripe expression pattern led to a reconsideration of the importance of the early bcd-dependent anterior cap domain. Under some conditions, hbP1only (maternal hb contribution plus stripe expression) might suffice for normal segmentation of head and thorax, making superfluous the bcd-dependent anterior cap domain. Hence, the hb PS4 stripe is activated without bcd-dependent hb expression. This stripe is repressed by the knirps abdominal gap-gene product and is activated by high levels of Hb itself, either directly or indirectly (through repression of kni). Embryos that lack the bcd-dependent hb cap domain have been generated that contain an increased maternal hb contribution (to four copies) and kni reduced to one copy. These embryos display a range of partially rescued hb phenotypes, including some embryos with a full set of head and thoracic segments. Thus, bcd-dependent hb expression can in principle be dispensable for embryonic segmentation, and the only critical anterior domain of zygotic hb expression appears to be the PS4 stripe, with the bcd-dependent cap domain serving to activate this stripe. This role is likely achieved by the maternal hb contribution in species where zygotic hb is not under the control of bcd or where a bcd homolog might not exist (Wimmer, 2000).

The rescue of T2 and T3 structures by bcd-independent hb expression raises the question of whether these structures could develop in a completely bcd-independent manner. Embryos derived from bcd mutant mothers develop ectopic tail structures that replace head and thorax and exhibit a disruption of some abdominal segments. Although previous work has shown that, in the absence of bcd, high levels of maternal hb can rescue a normal abdomen and some thoracic structures, no complete thoracic segments can be induced. A bcd-independent source for high levels of zygotic hb expression was introduced into a bcd mutant background. By establishing this artificial zygotic Hb gradient, two notable results were obtained, with variable expressivity: (1) about 20% of the embryos exhibit rescued T2 and T3 segments. The maintenance of high Hb levels that lead to the rescue of thoracic segments is likely due to the activation of the hb stripe element because the hbP1AB reporter is activated as a stripe where T2 and T3 form. (2) Most of the ectopic tail structures that are anteriorly duplicated in bcd mutants are suppressed, suggesting further redundancy between Hb and Bcd. However, Hb and Bcd must act at different levels in suppressing these tail structures, which depend on the activity of the caudal (cad) gene: Bcd acts by repressing cad mRNA translation, whereas Hb does not but might instead interfere with Cad protein function. This bcd-independent suppression of cad function might be important in organisms where the Cad gradient only forms late and represents another variation as to how cad activity is suppressed at the anterior of the embryo (Wimmer, 2000).

Different species use various strategies for repression of Cad function: In Drosophila, translational repression of CAD mRNA involves the Bcd homeoprotein, whereas in Caenorhabditis elegans repression involves the KH-domain protein MEX-3. In vertebrates, a mutually antagonistic relation between otd-like and cad-like genes has been proposed to reflect an ancestral system to pattern the anteroposterior axis of the embryo. In arthropods, ancestral head determinants are probably encoded by otd-like genes as well. Thus, in the beetle Tribolium, where no bcd homologs but Bcd-like activities have been found, these activities are probably also covered by Otd or KH-domain proteins. This is consistent with the Otd-like DNA binding specificity of Bcd, which is atypical for a factor encoded by a gene duplication in the Hox cluster. This change in specificity was probably crucial for Bcd to acquire its key role in anterior patterning, because it allowed Bcd to function both as an RNA binding protein and as an Otd-like transcription factor. In this respect, it is not surprising that the zinc-finger protein Hb cannot completely replace Bcd in the head region. Even the highest levels of Hb obtained in these experiments were not able to induce head formation in the absence of Bcd. However, Hb is required for the posterior head region (maxillary and labial segment) and supports anterior head development by synergizing with Bcd. It will be interesting to see whether such a synergism can also take place between Hb and other more ancestral head determinants (Wimmer, 2000).

These results indicate that the two morphogenetic systems, Bcd and Hb, do not need to be directly linked. Hence, the direct regulation of hb by Bcd might represent a recent evolutionary addition to the insect body plan. In Drosophila, the abundance of bcd-dependent hb expression eventually renders superfluous the maternal hb contribution, which is widespread within arthropods. Consistent with the idea that the bcd-dependent hb expression represents a recent evolutionary acquisition, the P2 promoter contains only activator sites that allow the direct response to a specific threshold level of a morphogen. This might be a unique situation, given that most other developmentally regulated promoters contain, in addition to activator sites, repressor elements for setting the exact borders of gene expression. By tinkering with the rather plastic mechanisms of early development, the ontogeny of Drosophila could be changed toward an inferred ancestral state where maternal Hb controls zygotic hb. This change could be brought about by altering patterns and levels of gene expression; this presents the most likely variation on which evolutionary processes are based (Wimmer, 2000).

Interplay between positive and negative activities that influence the role of Bicoid in hunchback transcription

The Drosophila mophogenetic protein Bicoid (Bcd) can activate transcription in a concentration-dependent manner in embryos. It contains a self-inhibitory domain that can interact with the co-repressor Sin3A. A Bcd mutant, BcdA57-61, that has a strengthened self-inhibitory function and is unable to activate the hb-CAT reporter in Drosophila cells, has been used to analyze the role of co-factors in regulating Bcd function. Increased concentrations of the co-activator dCBP in cells can switch this protein from its inactive state to an active state on the hb-CAT reporter. The C-terminal portion of BcdA57-61 is required to mediate such activity-rescuing function of dCBP. BcdA57-61 has a normal ability to bind to a single TAATCC site when analyzed in vitro. Although capable of binding to DNA in vitro, BcdA57-61 is unable to access the hb enhancer element in cells, suggesting that its DNA binding defect is only manifested in a cellular context. Increased concentrations of dCBP restore not only the ability of BcdA57-61 to access the hb enhancer element in cells but also the occupancy of the general transcription factors TBP and TFIIB at the reporter promoter. These and other results suggest that an activator can undergo switches between its active and inactive states through sensing the opposing actions of positive and negative co-factors (Fu, 2005).

As a molecular morphogen, Bcd can undergo switches, in a concentration-dependent manner, between its active and inactive states in activating transcription of its target genes. The experiments described in this report suggest another mechanism that can facilitate on-off switches of Bcd activity in a Bcd concentration-independent manner. In particular, the mutant BcdA57-61 is incapable of activating the hb-CAT reporter gene in S2 cells at all concentrations tested. The inability of this mutant Bcd to activate the hb-CAT reporter reflects a distinct functional state of this protein rather than its defects in protein stability. In fact, this same mutant protein is only modestly weaker than the wt protein on another reporter gene, kni-CAT, which contains the Bcd-responsive kni enhancer element. These and other results suggested that the A57-61 mutation may cause its functionally inactive state on hb-CAT by more efficiently interacting with a co-repressor protein(s), such as Sin3A and its associated complex(es). The experiments described in this report show that increased concentrations of dCBP can restore activity to BcdA57-61 on the hb-CAT reporter in cells. These results suggest that the opposing actions of positive and negative co-factors can facilitate Bcd to switch between its active and inactive states in a manner that is Bcd concentration-independent (Fu, 2005).

Although BcdA57-61 can bind to both a single site and natural enhancer elements in vitro, it is unable to access the hb enhancer element in cells. These results suggest that the DNA binding defect of this mutant protein is only manifested in a cellular context. This notion is consistent with the finding that the PAH domains of Sin3A do not exhibit any increased ability to reduce DNA binding by BcdA57-61 in vitro when compared with wt Bcd. It is proposed that other co-repressors or those that are associated with Sin3A, such as the HDACs, can reduce the ability of Bcd to access a natural enhancer in cells. It is possible that the enzymatic HDAC activity that is more stably associated with BcdA57-61 makes it unable to negotiate with histones for accessing DNA. It is also possible that a more stable Bcd-co-repressor complex may sterically hinder the interaction between BcdA57-61 molecules and prevent cooperative binding to the enhancer element in cells (Fu, 2005).

The most striking finding of this report is that high levels of dCBP can switch BcdA57-61 from its inactive state to an active one on the hb-CAT reporter in cells. ChIP data further show that dCBP increases both the ability of BcdA57-61 to access the hb enhancer element in cells and the occupancy of GTFs at the reporter promoter. How does dCBP switch the activity states of BcdA57-61 on hb-CAT in cells? Since Bcd and dCBP can physically interact with each other through multiple domains, it is possible that dCBP may increase the DNA binding ability of Bcd in cells by stabilizing the interaction between Bcd molecules and thus enhancing its cooperativity. It is also possible that dCBP may physically compete with co-repressor complexes in interacting with Bcd. Co-IP results suggest that dCBP may negatively affect the interaction between Bcd and Sin3A in cells. dCBP could also play a role in facilitating the interaction between Bcd and the transcription machinery. For all these actions, dCBP may play a structural (rather than enzymatic) role. Finally, the fact that the HAT-defective mutant of dCBP does have a reduced ability to restore activity to BcdA57-61 indicates that its enzymatic activity has a positive role, possibly through modifications of histones. It is likely that dCBP can affect the BcdA57-61 activity through multiple mechanisms that may be weak individually but, when combined, can lead to a dramatic switch from its inactive state to an active one on the hb-CAT reporter in cells (Fu, 2005).

Currently, it is poorly understood how precisely Bcd activates transcription. Previous studies suggest that much of its activation function is conferred by the C-terminal portion of Bcd. This portion of the protein contains several domains, including the acidic, glutamine-rich and alanine-rich domains, that are characteristic of activation domains capable of interacting with components of the transcription machinery. Interestingly, the alanine-rich domain previously thought to play an activation role was shown recently to exhibit an inhibitory function instead. The C-terminal domain of Bcd can also interact with dCBP, and the results show that this domain is responsible for mediating the activity-switching function of dCBP. Although much of the activation function of Bcd is provided by its C-terminal domain, the N-terminal portion of the protein also contains some activation function. Studies have shown that Bcd(1-246), a derivative lacking the entire C-terminal portion of Bcd, can rescue the bcd- phenotype when expressed at high levels. These results suggest that Bcd can achieve its activation function through multiple domains presumably by interacting with different proteins, including co-activators and components of the transcription machinery. The results described in this report further support the importance of dCBP in facilitating activation by Bcd (Fu, 2005).

Bcd is a morphogenetic protein whose behavior can be regulated not only by its own concentration but also by the enhancer architecture. On the kni and hb enhancer elements, the N-terminal domain of Bcd is preferentially used for either cooperative DNA binding or self-inhibition, respectively. It is proposed that the interaction between Bcd molecules bound to the kni enhancer element, through its N-terminal domain, can interfere with its interaction with co-repressors, such as Sin3A. Co-activators such as dCBP and co-repressors such as Sin3A can also functionally antagonize each other, possibly by competing for Bcd interaction as part of the mechanisms. Bcd is more sensitive to the self-inhibitory function on the hb enhancer element than on the kni enhancer element: consistent with dCBP's antagonistic role, dCBP increases the activity of Bcd more robustly on the hb enhancer element than on the kni enhancer element. However, the interplay between positive and negative activities that regulate Bcd functions is probably far more complex than the simple physical competition: as already discussed above, dCBP can affect Bcd activity through multiple mechanisms in both HAT-dependent and independent manners. Moreover, in the presence of exogenous dCBP, high levels of BcdA57-61 cause a reduction in its activity on the hb-CAT reporter in cells, a reduction that is not observed with wt Bcd, suggesting that the optimal concentration ratio between Bcd and dCBP may vary depending on the strengths of the self-inhibitory function and interaction with co-repressors. In addition, high concentrations of dCBP can rescue the inactive derivative BcdA57-61, but not another inactive derivative lacking the C-terminal portion, Bcd(1-246; A57-61), suggesting that the Bcd-dCBP interaction strength can also influence the balance between positive and negative activities that regulate Bcd function (Fu, 2005).

The experiments described in this report suggest that an activator's function is subject to intricate controls by both positive and negative activities in cells. A fine balance between these activities is critical for normal cellular and developmental processes. Transgenic experiments show that both BcdA57-61, which has a strengthened self-inhibitory function, and BcdA52-56, which has a weakened self-inhibitory function, cause embryonic defects. In addition, embryos with reduced dCBP activity exhibit defects in early expression patterns of a Bcd target gene, even-skipped. Finally, mutations affecting SAP18, a component of the Sin3A-HDAC complex, can alter Bcd function and anterior patterning in embryos. In addition to the co-factors discussed in this study (Sin3A, dCBP and SAP18), Bcd likely has the ability to interact with many other proteins, including not only regulatory proteins but also components of the transcription machinery. Precisely how all these different proteins harmoniously regulate and facilitate the execution of Bcd functions during development remains to be determined. Recent studies have shown that the Bcd gradient in embryos possesses a strikingly sophisticated ability to activate its target genes in a precise manner. These findings further underscore the need of intricate control mechanisms that facilitate Bcd to switch between its active and inactive states in target gene activation. These studies suggest that on-off switches of Bcd activity can be achieved not only in a Bcd concentration-dependent manner but also in a Bcd concentration-independent manner. It remains to be investigated whether and how Bcd interacting proteins, including those yet to be identified, participate in the precision control of target gene activation during development (Fu, 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Precision of hunchback expression in the Drosophila embryo

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

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

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

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

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

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

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

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

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

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

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

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

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

Live imaging of bicoid-dependent transcription in Drosophila embryos

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

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

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

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

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

Identification of hunchback cis-regulatory DNA conferring temporal expression in neuroblasts and neurons

The specification of temporal identity within single progenitor lineages is essential to generate functional neuronal diversity in Drosophila and mammals. In Drosophila, four transcription factors are sequentially expressed in neuroblasts and each regulates the temporal identity of the progeny produced during its expression window. The first temporal identity is established by the Ikaros-family zinc finger transcription factor Hunchback (Hb). Hb is detected in young (newly-formed) neuroblasts for about an hour and is maintained in the early-born neurons produced during this interval. Hb is necessary and sufficient to specify early-born neuronal or glial identity in multiple neuroblast lineages. The timing of hb expression in neuroblasts is regulated at the transcriptional level. This study identified cis-regulatory elements that confer proper hb expression in 'young' neuroblasts and early-born neurons. The neuroblast element contains clusters of predicted binding sites for the Seven-up transcription factor, which is known to limit hb neuroblast expression. Highly conserved sequences were identified in the neuronal element that are good candidates for maintaining Hb transcription in neurons. These results provide the necessary foundation for identifying trans-acting factors that establish the Hb early temporal expression domain (Hirono, 2012).

Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo

Metazoan genes are embedded in a rich milieu of regulatory information that often includes multiple enhancers possessing overlapping activities. This study employed quantitative live imaging methods to assess the function of pairs of primary and shadow enhancers in the regulation of key patterning genes - knirps, hunchback, and snail-in developing Drosophila embryos. The knirps enhancers exhibit additive, sometimes even super-additive activities, consistent with classical gene fusion studies. In contrast, the hunchback enhancers function sub-additively in anterior regions containing saturating levels of the Bicoid activator, but function additively in regions where there are diminishing levels of the Bicoid gradient. Strikingly sub-additive behavior is also observed for snail, whereby removal of the proximal enhancer causes a significant increase in gene expression. Quantitative modeling of enhancer-promoter interactions suggests that weakly active enhancers function additively while strong enhancers behave sub-additively due to competition with the target promoter (Bothma, 2015).

This quantitative analysis of hb and kni expression provides seemingly opposing results. For kni, additive, sometimes even super-additive, action of the two enhancers was observed within the presumptive abdomen. In contrast, the two hb enhancers do not function in an additive fashion in anterior regions but are additive only in central regions where expression abruptly switches from 'on' to 'off'. It is proposed that 'weak' enhancers function additively or even super-additively, whereas 'strong' enhancers can impede one another (Bothma, 2015).

Additional support for this view is provided by the analysis of sna. The removal of the proximal enhancer significantly augments expression, consistent with the occurrence of enhancer interference within the native locus. It is also conceivable that a single strong enhancer (e.g., hb proximal or sna distal) already mediates maximum binding and release of Pol II at the promoter, and additional enhancers are therefore unable to increase the levels of expression. However, the increase in the levels of sna expression upon removal of the primary enhancer is inconsistent with this explanation. Perhaps, the proximity of the proximal enhancer to the sna promoter gives it a 'topological advantage' in blocking access of the distal enhancer. The proximal enhancer might mediate less efficient transcription than the distal enhancer and thereby reduce the overall levels of expression. It is not believed that this proposed difference is due to differential rates of Pol II elongation since published and preliminary studies suggest that different enhancers and promoters lead to similar elongation rates (~2 kb/min; T Fuyaka and M Levine, unpublished results). A nonexclusive alternative possibility is that deletion of the proximal enhancer removes associated sna repression elements, thereby augmenting the efficiency of the distal enhancer (Bothma, 2015).

A minimal model of enhancer–promoter associations provides insights into potential mechanisms. In the parameter regime where such interactions are infrequent the two enhancers display additive behavior. However, in the regime of frequent interactions, enhancers compete for access to the promoter resulting in sub-additive behavior. Enhancer–promoter interaction parameters are likely to vary not only between different enhancers but also as the input patterns are modulated in time and space during development (Bothma, 2015).

This simple model explains the switch from sub-additive to additive enhancer activities for hb and sna. However, in order to explain the super-additive behavior of the kni enhancers, it would be necessary to incorporate an additional state in the model, whereby both enhancers form an active complex with the same target promoter. Such a complex would have a more potent ability to initiate transcription than individual enhancer–promoter interactions (Bothma, 2015).

In summary, it is proposed that enhancers operating at reduced activities ('weak enhancers') can function in an additive manner due to relatively infrequent interactions with their target promoters. In contrast, 'strong' enhancers might function sub-additively due to competition for the promoter. For hb, this switch between competitive and additive behavior occurs as the levels of Bicoid activator diminish in central regions where the posterior border of the anterior Hb domain is formed. Similarly, stress might reduce the performance of the sna enhancers to foster additive behavior under unfavorable conditions such as increases in temperature. This study highlights the complexity of multiple enhancers in the regulation of gene expression. They need not function in a simple additive manner, and consequently, their value may be revealed only when their activities are compromised (Bothma, 2015).

Transcriptional Regulation

Bicoid activates hunchback's anterior to posterior zygotic gradient (Tautz, 1988 and Struhl, 1989).

Bcd contains three putative activation domains: a glutamine-rich region, which interacts in vitro with TAFII110; an alanine-rich domain, which targets TAFII60, and a C-terminal acidic region, which has an unknown role. Transcriptional activation of a bcd target, the hb promoter, is synergistically enhanced in vitro by Bcd and Hb. However, this effect is observed only when both TAFII60 and TAFII110 are present. It has been suggested that the synergy observed in vivo is because of the corecruitment of TAFII110 and TAFII60 by Bcd and Hb, respectively. Flies were generated carrying bcd transgenes lacking one or several of these domains to test their function in vivo. Surprisingly, a bcd transgene that lacks all three putative activation domains is able to rescue the bcdE1 null phenotype to viability. Moreover, the development of these embryos is not affected by the presence of dominant negative mutations in TAFII110 or TAFII60. This means that the interactions observed in vitro between Bcd and TAFII60 or TAFII110 aid transcriptional activation but are dispensable for normal development (Schaeffer, 1999).

Bicoid (Bcd), the anterior determinant of Drosophila, controls embryonic gene expression by transcriptional activation and translational repression. Both functions require the homeodomain (HD), which recognizes DNA motifs at target gene enhancers and a specific sequence interval in the 3' untranslated region of Caudal (CAD) mRNA. The Bcd HD has been shown to be a nucleic acid-binding unit. Its helix III contains an arginine-rich motif (ARM), similar to the RNA-binding domain of the HIV-1 protein REV, needed for both RNA and DNA recognition. Replacement of arginine 54, within this motif, alters the RNA but not the DNA binding properties of the HD. Corresponding BCD mutants fail to repress CAD mRNA translation, whereas the transcriptional target genes are still activated (Niessing, 2000).

In order to characterize portions and individual amino acid residues of the Bcd HD that are specifically required for one or both Bcd regulatory functions, transgenes expressing wild-type or mutant bcd cDNAs were placed into the genome of homozygous bcd mutant females and their ability to rescue wild-type zygotic hb activation and cad mRNA translation in their embryos was assayed. Such embryos, referred to as 'bcd embryos,' fail to exert Bcd-dependent transcriptional activation of the zygotic target gene hb in their anterior half. Instead, the embryos show a duplication of the posterior Bcd-independent stripe of hb expression in the anterior region (Niessing, 2000).

Expressed Bcd mutant proteins that lack the helices I and II of the HD (BcdDeltaH1-2) or the amino acid interval between positions 42 and 51 in helix III (BcdT42-N51) fail to restore Bcd-dependent hb transcriptional activation and translational repression of CAD mRNA in the anterior region of bcd embryos. This indicates that the integrity of the Bcd HD is necessary for the control of transcription and translation. Transgene-dependent expression of BcdhIIIAntp, in which the C-terminal half of the Bcd HD is exchanged for the corresponding sequence of the Antennapedia (Antp) HD, rescues Bcd-dependent hb expression in the anterior region of bcd embryos, but no Cad gradient is formed. Bcd mutations in which two adjacent arginines at positions 53-54 and 54-55 of the HD, respectively, were replaced, fail to control Bcd-dependent transcription and translation. Thus, helix III of the Bcd HD is necessary for both transcriptional activation and translational repression, and amino acids within helix III are essential for specifying not only DNA binding but also RNA recognition by the HD. This proposal is consistent with the observation that part of the helix III of the Bcd HD has characteristics of an arginine-rich motif (ARM) (Niessing, 2000).

To test whether the conserved amino acids of Bcd's ARM are indeed required for RNA target recognition and whether single amino acid replacements may allow the DNA and RNA binding properties to separate, alanine replacement mutants of the Bcd HD were generated and their in vitro binding properties assayed. The Bcd HD (HDwt) binds both DNA and RNA, whereas HDK50A, HDN51A, HDR53A, and HDR55A failed to bind to both targets. Bcd HDR54A, which contains alanine in place of arginine in position 54 of the HD, bound DNA properly, but its RNA binding was reduced by more than one order of magnitude. The binding properties of HDK57A were indistinguishable from HDwt. In summary, arginine at position 54 of the HD is critical for specifying RNA versus DNA binding, and its replacement shifts the binding property of the HD to prefer DNA over RNA recognition (Niessing, 2000).

In order to test the in vivo relevance of these binding studies, the corresponding Bcd HD mutants were examined by transgene-dependent expression in bcd embryos. The Bcd mutants were generated in the context of an 8.7 kb genomic DNA fragment spanning the entire bcd locus, which fully rescues bcd embryos after P element-mediated transformation. The transgene-expressed BcdK57A protein, which contains an HD with normal DNA and RNA binding properties, causes Bcd-dependent hb expression and Cad gradient formation, and the embryos developed into normal-looking larvae and fertile adults. BcdN51A, BcdR53A, and BcdR55A, which contain HD mutations that cause the loss of DNA and RNA binding properties in vitro, fail to activate Bcd-dependent hb transcription and to repress translation of CAD mRNA; such embryos develop a bcd mutant phenotype. The BcdR54A mutant, which contains an HD with DNA, but no RNA, binding properties, was able to activate the transcription of hb but not to repress the translation of CAD mRNA. This observation is consistent with the result obtained using the transgene bearing the BcdR54S mutation, which contains a serine residue in place of arginine at position 54. Thus, both Bcd mutants that contain a replacement of arginine at position 54 of the HD fail to control CAD mRNA translation but do activate transcription of hb (Niessing, 2000).

Nanos functions as a localized determinant of posterior pattern. Nanos RNA is localized to the posterior pole of the maturing egg cell. It encodes a protein that emanates from this localized source. Nanos acts as a translational inactivator of hunchback and thereby establishes the early anterior to posterior gradient of Hunchback (Curtis, 1995).

Terminal gap genes tailless and huckebein direct the formation of the posterior Hunchback stripe. The TLL protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by TLL. The anterior border of the posterior HB stripe is determined by TLL concentration in a manner analogous to the activation of anterior hb expression by Bicoid. In the posterior expression pattern of hb, the transcription factor serves as a (so-called) secondary gap gene regulated by "primary" gap genes tailless and huckebein, regulated in turn by torso (Margolis, 1995).

The Krüppel binds to the sequence AAGGGGTTAA. Binding sites are present for KR upstream of the two hb promoters. These could mediate the repression of hb by KR and perhaps allow hb to influence its own expression. A 10 Kb genomic DNA fragment contains the hb coding sequence and both promoters. The proximal promoter directs early zygotic expression of hb in the anterior part of the embryo The distal hb promoter is transcribed maternally and also directs later zygotic expression . This latter fragment contains the KR binding sites. 300 bp upstream of the transcription start of the 2.9 kb transcript are sufficient for normal regulation of the expression of this transcript. The two KR binding sites are located at -676 and -359 bp from the proximal hb promoter (Treisman, 1989).

The asymmetric distribution of the gap gene knirps (kni) in discrete expression domains is critical for striped patterns of pair-rule gene expression in the Drosophila embryo. To test whether these domains function as sources of morphogenetic activity, the stripe 2 enhancer of the pair-rule gene even-skipped was used to express kni in an ectopic position. Manipulating the stripe 2-kni expression constructs and examining transgenic lines with different insertion sites led to the establishment of a series of independent lines that display consistently different levels and developmental profiles of expression. Individual lines show specific disruptions in pair-rule patterning that are correlated with the level and timing of ectopic expression. No effect on the expression patterns of giant or Krüppel could be observed at any level of kni misexpression. However, the ectopic kni did significantly alter the hunchback pattern. Stripe 2-kni, centered on PS3, completely prevents the expression of the PS4 hb stripe. This expression occurs even in embryos that contain the lowest levels of ectopic kni (Kosman, 1997).

High local concentrations of NOS protein in the posterior of the embryo are necessary to inhibit translation of the transcription factor Hunchback in this region, and thus permit expression of genes required for abdomen formation (Gavis, 1994).

Nanos prevents the repressor hunchback from acting in the posterior half of the embryo. This allows Caudal to activate the gap genes giant and knirps (Rivera-Pomar, 1995).

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

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

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

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, 2000).

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, 2000).

During embryonic development, orderly patterns of gene expression eventually assign each cell in the embryo its particular fate. For the anteroposterior axis of the Drosophila embryo, the first step in this process depends on a spatial gradient of the maternal morphogen Bicoid (Bcd). Positional information of this gradient is transmitted to downstream gap genes, each occupying a well defined spatial domain. The precision of the initial process has been determined by comparing expression domains in different embryos. The Bcd gradient displays a high embryo-to-embryo variability, but this noise in the positional information is strongly decreased ('filtered') at the level of hunchback (hb) gene expression. In contrast to the Bcd gradient, the hb expression pattern already includes the information about the scale of the embryo. Genes known to interact directly with Hb are not responsible for its spatial precision, but the maternal gene staufen may be crucial (Houchmandzadeh, 2002).

Among all the mutations studied, the only ones that affect Hb boundary precision are certain alleles of staufen. In embryos from mothers homozygous for either stauHL or staur9, the Hb boundary position shows a variability of 6%, comparable to the observed Bcd variability. Surprisingly, this variability is largely reduced (to 2%) in another strong allele of stau, D3. Mutations in stau disrupt bcd and osk mRNAs and decrease Bcd protein level about twofold. Whether the effect of stau on Hb is simply an indirect effect of its variable effect on bicoid was tested. From the pool of embryos in stauHL background that were double stained for Bcd and Hb, two populations were selected: one that displayed an anterior Hb boundary shift, and one that displayed a posterior shift. The corresponding average Bcd profiles for these two populations are very similar, both in the Bcd level and in its spatial distribution. Thus, the observed variability in the Hb boundary position may reflect an activity of staufen independent of bcd. The disruption of Hb precision in stauHL is transmitted to downstream genes, and is not corrected before gastrulation. For instance, double staining for Hb and Kr shows that the variability of the Kr boundary in the stauHL background is similar to that of the Hb boundary. Moreover, the positions of these two boundaries remain tightly correlated, as in the wild type (Houchmandzadeh, 2002).

By quantitatively analyzing the protein profiles of maternal morphogens and zygotic gap genes in numerous wild-type and mutant embryos, two phenomena that take place in the early Drosophila development have been demonstrated: (1) at a very early stage, noise associated with the maternal gradient of Bcd is filtered out, and (2) at the same time, the genetic network, which includes the Hb gap gene, establishes spatial proportions (scaling) in the embryo. It is potentially significant that staufen, the one gene affecting the process, makes a product that localizes to both poles of the egg. More work is needed to establish the mechanisms that control the spatial scaling and precision. It would then be interesting to investigate whether similar phenomena are present in other developmental processes in Drosophila and other organisms (Houchmandzadeh, 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).

Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline: Silencing of hunchback

In Drosophila, the germline precursor cells, i.e. pole cells, are formed at the posterior of the embryo. As observed for newly formed germ cells in many other eukaryotes, the pole cells are distinguished from the soma by their transcriptional quiescence. To learn more about the mechanisms involved in establishing quiescence, a potent transcriptional activator, Bicoid (Bcd), was ectopically expressed in pole cells. Bcd overrides the machinery that downregulates transcription, and activates not only its target gene hunchback but also the normally female specific Sex-lethal promoter, Sxl-Pe, in the pole cells of both sexes. Unexpectedly, the terminal pathway gene torso-like is required for Bcd-dependent transcription. However, terminal signaling is known to be attenuated in pole cells, and this raises the question of how this is accomplished. Evidence is presented indicating that polar granule component (pgc) is required to downregulate terminal signaling in early pole cells. Consistently, pole cells compromised for pgc function exhibit elevated levels of activated MAP kinase and premature transcription of the target gene tailless (tll). Furthermore, pgc is required to establish a repressive chromatin architecture in pole cells (Deshpande, 2004).

A number of maternally derived gene products are likely to contribute to transcriptional quiescence in the pole cells of Drosophila. One of these is Germ cell less (Gcl), a component of the germ plasm that is necessary for the formation of pole cells. gcl appears to be involved in the establishment of transcriptional quiescence and in embryos lacking gcl activity, newly formed pole buds are unable to silence the transcription of genes such as sisterless-a and scute. Conversely, when Gcl protein is ectopically expressed in the anterior of the embryo it can downregulate the transcription of terminal group genes such as tailless (tll) and huckebein (Leatherman, 2002). A second maternally derived gene product involved in transcriptional quiescence is Nanos. In the soma, Nanos, together with Pumilio, plays a key role in posterior determination by blocking the translation of maternally derived hunchback (hb) mRNA. Nanos (Nos) also plays a role in down-regulating transcription in pole cells, and in embryos produced by nos mutant mothers: genes that are normally active only in somatic nuclei are inappropriately transcribed in pole cells. These include the pair-rule genes fushi tarazu and even skipped, and the somatic sex determination gene Sex-lethal (Deshpande, 2004 and references therein).

Ectopic expression of Bcd in pole cells can induce the transcription of the bcd target gene hb. In addition to activating hb transcription, Bcd protein perturbs the migration of the pole cells to the primitive somatic gonad and causes abnormalities in cell cycle control. These effects on germ cell development resemble those observed in embryos from nos mutant females. Moreover, as in the case of nos- pole cells, the Sxl promoter Sxl-Pe is also turned on in pole cells by Bcd in a sex-nonspecific manner. Surprisingly, transcriptional activation in pole cells by Bcd requires the activity of the terminal signaling system. This observation is unexpected, since previous studies have established that the transcription of a downstream target gene of the terminal pathway, tailless (tll) is shut down completely in pole cells. Moreover, the doubly phosphorylated active isoform of MAP kinase ERK, which serves as a sensitive readout of the terminal pathway, is nearly absent in pole cells. Taken together, these findings argue that the activity of terminal signaling pathway in pole cells of wild-type embryos must be substantially attenuated, but not shut off completely. What mechanisms are responsible for downregulating terminal signaling in the presumptive germline? Evidence indicates that polar granule component (pgc) functions to attenuate the terminal pathway in newly formed pole cells. pgc encodes a non-translated RNA that is localized in specialized germ cell-specific structures called polar granules (Nakamura, 1996). Loss of pgc function in newly formed pole cells results in the ectopic phosphorylation of ERK and the activation of the ERK dependent target gene tll. pgc is required to block the establishment of an active chromatin architecture in pole cells (Deshpande, 2004).

Thus Bcd protein expressed from a bcd-nos3'UTR transgene (the 3' UTR of nos serves to localize the bcd message to pole cells) can activate the transcription of its target gene hb in pole cells, overcoming whatever mechanisms are responsible for transcriptional quiescence. In addition to activating transcription of hb, Bcd has other phenotypic effects. It prevents the pole cells from properly arresting their cell cycle and disrupts their migration to the somatic gonad. Because similar defects in pole cell development can be induced by the inappropriate expression of Sxl protein in these cells, one plausible hypothesis is that Bcd not only activates the hb promoter, but also turns on the Sxl establishment promoter, Sxl-Pe. Consistent with this idea, the Sxl-Pe:lacZ reporter is turned on in the pole cells of male and female bcd-nos 3' UTR embryos and Sxl protein accumulates in these cells. Although previous studies indicate that Sxl-Pe is responsive to Bcd, it is somewhat surprising that Sxl-Pe is not only inappropriately turned on in pole cells by Bcd, but that it is activated in both sexes. This suggests that Bcd activation of Sxl-Pe in pole cells must proceed by a mechanism that bypasses the X/A chromosome counting system which controls Sxl-Pe activity in the soma. It is interesting to note that the activation of Sxl-Pe in pole cells in the absence of nos function also seems to depend upon a mechanism(s) that circumvents the X/A chromosome counting system (Deshpande, 2004).

That Bcd protein depends upon other ancillary factors to turn on transcription in pole cells is demonstrated by the requirement for tsl function in the activation of both the hb and Sxl-Pe promoters. tsl is a component of the maternal terminal signaling pathway that activates the zygotic genes, tll and huckebein (hkb), at the poles of the embryo. In addition, the terminal pathway has opposing effects on the expression of bcd-dependent gap genes. At the anterior pole, where terminal signaling activity is highest, Bcd targets such as hb and orthodenticle (otd) are repressed. At a distance from the anterior pole, where both the concentration of Bcd protein and the strength of the terminal signaling cascade is much lower, the terminal pathway has an opposite, positive effect on hb and otd expression. Two mechanisms are thought to account for the positive effects of the terminal pathway on bcd target genes: (1) Bcd is a direct target for phosphorylation by the terminal signaling cascade; (2) regulatory regions of bcd target genes have sites for other transcription factors whose activity can be directly modulated by the terminal system (Deshpande, 2004).

Susceptibility of hunchback to RNAi

Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindleE, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte (Kennerdell, 2002).

To analyze the effects of dsRNA on mRNA stability in Drosophila oocytes, dsRNAs corresponding to the maternally expressed genes bicoid and hunchback were used. These genes were chosen because their mRNAs are synthesized, processed, and localized to the cytoplasm of oocytes during mid- to late oogenesis. To test the sensitivity of bicoid and hunchback to RNAi, fertilized eggs were initially injected with dsRNA. bicoid dsRNA reduces the expression of Bicoid protein and induces a bicoid loss-of-function phenotype in which embryos have partial transformation of anterior structures to posterior identities. The effect is robust enough that dsRNA-coated gold particles randomly introduced into fertilized eggs by a gene gun generate mutant phenotypes. hunchback dsRNA induces phenotypes in which embryos are missing thoracic and head segments. These phenotypes resemble mutant embryos generated when maternal and zygotic hunchback gene activity is reduced. To determine if dsRNA injection causes mRNA degradation, endogenous mRNA levels were measured using a semiquantitative RT-PCR assay. The level of bicoid mRNA was reduced about fourfold 40 min after injection of bicoid dsRNA. Likewise, injection of hunchback dsRNA resulted in a reduction of hunchback mRNA levels. Coinjection of a pan-specific ribonuclease inhibitor, vanadyl-ribonucleoside, with bicoid dsRNA results in no reduction of bicoid mRNA, indicating the effect requires a ribonuclease activity (Kennerdell, 2002).

Whether and when transcripts become sensitive to dsRNAs during oogenesis was determined. dsRNA was injected into staged oocytes and their consequent levels of bicoid and hunchback mRNAs were examined. Although oocytes earlier than stage 14 could not be injected, stage 14 oocytes could be examined for RNAi activity. Levels of bicoid and hunchback mRNAs were unchanged in stage 14 oocytes after injection of dsRNA, indicating that oocytes at this stage are unable to carry out RNAi (Kennerdell, 2002).

Oocytes of most animals arrest at species-specific stages of meiosis while differentiation of the oocytes occurs. Drosophila oocytes arrest transiently in prophase I while the oocytes are loaded with RNAs and proteins. Some of these molecules are differentially localized within the oocyte, imparting positional information to be used for embryonic axis formation. When Drosophila oocytes reach stage 14, they undergo meiotic arrest once more, this time at metaphase I. These arrested oocytes remain translationally quiescent in the ovary, potentially for weeks. Arrest is relieved as in most animal eggs by the process of maturation or activation that precedes fertilization. In the case of Drosophila, it appears that ovulation triggers activation of the oocyte to resume meiosis. When oocytes are activated, meiosis is completed and translation of maternal RNAs is dramatically elevated. Shortly thereafter, the oocyte is fertilized as it passes into the uterus (Kennerdell, 2002).

RNAi-like effects are not detected in arrested stage 14 oocytes injected with dsRNA. Was this a general feature of the female germ line? To explore this issue, dsRNA was injected into mature activated oocytes. Injection of dsRNA causes reduction in bicoid and hunchback mRNA levels comparable to those seen in embryos. To confirm that mRNA sensitivity to dsRNA is strictly coincident with oocyte maturation, arrested stage 14 oocytes were isolated from dissected ovaries and the oocytes were activated in vitro. This maturation procedure reactivates meiosis, mRNA translation, and vitelline membrane cross-linking. After maturation, oocytes were injected with bicoid dsRNA and assayed for bicoid mRNA levels. These oocytes showed a decrease in bicoid mRNA. Thus, immature Drosophila oocytes that are coordinately blocked for meiosis and translation are resistant to RNAi, and the block to these processes can be released by maturation or activation of oocytes (Kennerdell, 2002).

There are several possible ways in which RNAi might be blocked in arrested oocytes. One possibility is that an essential component of the RNAi machinery might be missing at this stage. Oocyte maturation would then involve synthesis of the component. To address if synthesis of a missing component is responsible, oocytes were activated in the presence of the protein synthesis inhibitor cycloheximide. Arrested stage 14 oocytes were preincubated with cycloheximide and then activated in vitro in the presence of cycloheximide. This treatment inhibits >95% of the protein synthesis that occurs during maturation. These oocytes were injected with bicoid dsRNA and, strikingly, they showed a decrease in bicoid mRNA levels that was comparable to that of normal mature oocytes. RNAi is established during oocyte maturation even when protein synthesis is blocked. Thus, RNAi establishment during oocyte maturation does not likely occur by synthesis of an essential protein component of the RNAi machinery (Kennerdell, 2002).

The stage 14 oocyte is coordinately blocked in both translation and RNAi. The two processes are released near simultaneously from this block, suggesting perhaps that a shared mechanism links their regulation. To test this possibility, the effectiveness of dsRNA was examined against a transcript that is present but not translated after oocyte maturation. The alphaTubulin67C gene encodes one of three alpha-tubulin proteins synthesized during oogenesis and embryogenesis. Transcript accumulates and is actively translated in early immature oocytes. However, after oocyte maturation, no translation of alphaTubulin67C mRNA occurs, even though transcripts at this stage are associated with ribosomes and are competent to drive translation in vitro. The stable pool of alphaTubulin67C mRNA is comparable to levels of bicoid and hunchback mRNA in mature oocytes. When two nonoverlapping dsRNAs against alphaTubulin67C transcript were independently injected into mature activated oocytes, no destruction of mRNA was detected. This suggests that the ability of dsRNAs to destroy transcripts during oogenesis is coupled to the translation activity of the transcript. Successful translation of transcripts is perhaps necessary to link a transcript to dsRNA-triggered degradation (Kennerdell, 2002).

Several Drosophila genes have been identified that affect translation of maternal mRNAs during oogenesis. One of these genes, aubergine (aub), encodes a protein with a PIWI and PAZ domain. To determine whether Aub has any role for RNAi in oocytes, the effect of aub mutations on RNAi activity was examined. bicoid and hunchback dsRNAs were injected into aub mutant oocytes that were activated in vitro. Degradation of bicoid and hunchback mRNAs was not observed in aub mutants, indicating that Aub is necessary for germ-line RNAi. Two independent aub alleles in heteroallelic combination produced the same result, indicating that the effect was not due to the influence of linked modifiers (Kennerdell, 2002).

The aub gene is a member of a family of genes implicated in RNAi and PTGS. Indeed, aub has been implicated in PTGS regulation of the Stellate repeats and Su(Ste) genes on X and Y chromosomes. Another member of the family, piwi, has been implicated in PTGS within somatic cells. A third family member, Ago2, is a subunit of the mRNA-cleaving complex that mediates RNAi in Drosophila embryonic cells. Thus, several members of this gene family in Drosophila have been implicated in RNAi and PTGS at various steps (Kennerdell, 2002).

It was of interest to determine if other translational regulatory genes are involved in RNAi. To test this possibility, two genes that possibly act through interactions with RNA were examined. vasa and spindle-E (spn-E) encode DexH-box RNA helicases. When activated spn-E mutant oocytes were injected with bicoid or hunchback dsRNAs, no reduction in cognate mRNA levels occurred. In contrast, activated vasa mutant oocytes injected with bicoid dsRNA were found to show transcript degradation comparable to wild type. It is concluded that activation of RNAi in oocytes is dependent on the activity of Spn-E but not Vasa (Kennerdell, 2002).

Arrested Drosophila oocytes are unable to generate RNAi silencing of endogenous maternal mRNAs, but selectively establish this capability upon egg maturation. How is RNAi activated by egg maturation? It is argued that RNAi is linked in some way to translation of maternal mRNAs, which is also specifically activated by egg maturation. Establishment of RNAi is probably not caused by translation of a missing RNAi component. Rather, the complete RNAi apparatus may be present and poised for action but is unable to target homologous substrate mRNAs until egg maturation. Translational masking of mRNAs, a mechanism that operates on maternal Drosophila gene expression, may conceivably be one way in which mRNA is blocked from RNAi attack. Alternatively, targeting of mRNA might require transcripts be assembled onto active polysomes. This may be the case, because siRNA-containing RISC complexes physically fractionate with polysomes, and siRNAs associate with polysomes in Trypanosoma brucei. There is no evidence to indicate that dsRNA-targeting requires ribosome translocation on transcripts, because it is found that cycloheximide inhibition of ribosome translocation does not block RNAi activity in activated mature oocytes (Kennerdell, 2002).

Coupling RNAi to translated mRNA might facilitate base-pairing interactions between siRNAs and an unfolded mRNA target, or it might simply be a means to mark RNAs to be scanned for destruction. The key evidence suggesting that transcript translation is linked to transcript degradation by RNAi comes from experiments in which dsRNA against the alphaTubulin67C message was tested. dsRNA is ineffective against the untranslated alphaTubulin67C transcript in mature activated oocytes, which are nevertheless competent to carry out RNAi against translated bicoid and hunchback transcripts. Thus, there is a correlation between the ability of a transcript to be translated and its ability to be destroyed by dsRNA (Kennerdell, 2002).

Thus Aub and Spn-E are required for RNAi in Drosophila oocytes. Aub and Spn-E might play a specific role in gene silencing mechanisms, including RNAi, that nevertheless have a widespread impact on many features of development. Alternatively, Aub and Spn-E could be required for RNAi because they activate translation of germ-line transcripts including those for bicoid and hunchback. Although there is no evidence for translational control of bicoid mRNA in aub mutants, these mutants may perturb steps in the translation of transcripts that are essential for triggering RNAi. Future experiments should define the specific roles for Aub and Spn-E in dsRNA-mediated destruction and its relationship to translation control (Kennerdell, 2002).

Probing the limits to positional information; the noise in readout of Bicoid by the activation of Hunchback

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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007).

seven-up controls switching of transcription factors that specify temporal identities of Drosophila neuroblasts

Drosophila neuronal stem cell neuroblasts (NB) constantly change character upon division, to produce a different type of progeny at the next division. Transcription factors Hunchback (Hb), Kruppel (Kr), Pdm, and Castor are expressed sequentially in each NB and act as determinants of birth-order identity. How any NB switches its expression profile from one transcription factor to the next is poorly understood. The Hb-to-Kr switch is directed by the nuclear receptor Seven-up (Svp). Svp expression is confined to a temporally restricted subsection within the NB's lineage. Loss of Svp function causes an increase in the number of Hb-positive cells within several NB lineages, whereas misexpression of svp leads to the loss of these early-born neurons. Lineage analysis provides evidence that svp is required to switch off HB at the proper time. Thus, svp modifies the self-renewal stem cell program to allow chronological change of cell fates, thereby generating neuronal diversity (Kanai, 2005).

The expression profile of Svp in the CNS is extremely dynamic. For example, at stage 11, Svp is expressed in NB2-4 but not in NB7-3 just after NB2-4 formation. After NB7-3 has divided, Svp is expressed in NB7-3 and in the GMC that it has generated but Svp is no longer detectable in NB2-4. Thus, the expression of Svp is confined to temporally restricted subsections of the NB lineage. While Svp is expressed in many NB and GMCs, only a small number of neurons are Svp positive. This indicates that, unlike Hb and Kr, the expression profile of Svp in the NBs is not maintained in their neuronal progeny (Kanai, 2005).

In the svp mutant, NB7-3 does not switch its expression pattern from the Hb, Kr double-positive state to the Kr single-positive state until one division after the normal transition period. This prolonged expression of Hb results in overproduction of Hb-positive neurons exhibiting characteristics of early-born neurons. The timing of the expression of Svp protein in NB7-3 coincides with the transition in the expression of Hb to Kr, and precocious expression of Svp causes the loss of Hb expression within the lineage. These results indicate that Svp has an instructive role in determining the period of Hb expression in the NB and the proper generation of neuronal diversity. While this work places Svp upstream of Hb, how the expression of Svp itself is regulated is not well understood. In hb mutant embryos, Svp is still expressed transiently at the time that NB7-3 produces its first GMC. Thus, it is unlikely that the temporal delay of Svp expression with respect to Hb is due to a negative feedback loop in which Hb induces its own repressor (Kanai, 2005).

Svp is a well-conserved nuclear receptor whose human homolog, COUP, has been shown to act as a transcriptional repressor. Because a reporter gene that contains only an enhancer element of the hb gene also responded to Svp, Svp can affect hb expression at the level of its transcription. It is thus possible that Svp directly represses hb transcription by binding to its cis-element. Interestingly, misexpression of Svp in postmitotic neurons does not affect their Hb expression, consistent with the observation that the regulatory mechanism of Hb expression differs between the NBs and their progeny. The repressor activity of Svp on hb expression likely requires other factors that are present in precursor cells of neurons (Kanai, 2005).

In svp mutant embryo, augumented expression of Hb was seen in many NBs, resulting in overproduction of early-born neurons in at least three NB lineages. This suggests that Svp may have a common function in many NB lineages regulating hb expression. However, of 30 NBs within each hemisegment, four do not express svp. Indeed, in an svp-negative NB1-1 lineage, the number of the early-born neurons aCC and pCC in svp mutant embryo is normal. How do these NBs generate birth-order-dependent progeny without svp expression? Since some NBs are known to start their lineage without expressing Hb, they may not need Svp to regulate Hb expression. Indeed, svp-negative NB6-1, which expresses Cas at the time of formation never expresses Hb. It is also possible that there are other factors or mechanisms to regulate hb expression. In the nematode C. elegans, hb homolog lin57/hbl-1 (which controls developmental timing as a heterochronic gene) is regulated by a micro RNA that binds its 3'UTR. Since Drosophila hb 3' UTR contains putative micro RNA binding sites, transcription factor switching in Drosophila NBs might also be regulated posttranscriptionally by micro RNAs (Kanai, 2005).

While the overproduction of Hb-positive neurons is consistent with the idea that prolonged expression of Hb in svp mutant NBs causes production of supernumerary GMC-1s, examination of postmitotic neurons reveals that the number of neurons with particular identity does not always correspond to duplicated GMC-1s. In the NB7-3 lineage, GMC-1 divides to produce two neurons, EW1 and GW, whereas GMC-2 gives rise to EW2 neuron and its sibling which undergoes programmed cell death. In svp mutant, two EW1 neurons are present consistent with duplicated GMC-1, but only one GW-like neuron is observed. Likewise, when Hb is misexpressed in the NB7-3 lineage, not all GMCs that were transformed toward GMC-1 produced GW neurons. These data suggest that the fate of postmitotic progeny from GMCs is dependent not only on the birth-order identity of GMCs determined by transcription factors such as Hb and Kr, but is also influenced by signals that come from outside of the NB lineage. Since the decision for the sibling of the EW2 neuron to undergo cell death depends on the activation of Notch signaling, it is possible that signals for Notch activation originate outside the NB7-3 lineage, and are not affected by genetic manipulations altering the birth-order identity of the GMCs (Kanai, 2005).

In addition to the increase in the number of early born neurons, svp mutant embryos display another phenotype, the reduction of late-born neurons that express Zfh-2 (NB7-3), Runt, and Cas (NB7-1). This phenotype is dramatically enhanced when Kr is inactivated, freezing the lineage such that only Hb-positive cells are produced. One interpretation of this phenotype is that Svp somehow cooperates with Kr to generate the late part of the lineage. In fact, this seems to be the only known genetic situation in which loss of gene function eliminates the late born neurons. However, because this phenotype is completely suppressed by removing Hb, the idea is favored that Svp does not have a direct role in activating the transcription factors that specify the late-born identity, but rather acts through repressing Hb, which can repress PDM expression. Thus the apparent requirement of Svp in the generation of the late-born neurons deduced from the svp loss-of-function phenotype is due to its primary function in mediating the Hb-to-Kr switch, whose failure secondarily blocks the initiation of the late lineage program. The results also show that the late lineage can be produced in the absence of Hb and Kr (and Svp), suggesting that it may be the 'default' state. It is possible that primitive lineage consisted only of the late lineage program, to which Svp was recruited to add the early program involving Hb and Kr, thereby generating the birth-order-dependent neuronal diversity (Kanai, 2005).

Timing of identity: spatiotemporal regulation of hunchback in neuroblast lineages of Drosophila by Seven-up and Prospero

Neural stem cells often generate different cell types in a fixed birth order as a result of temporal specification of the progenitors. In Drosophila, the first temporal identity of most neural stem cells (neuroblasts) in the embryonic ventral nerve cord is specified by the transient expression of the transcription factor Hunchback. When reaching the next temporal identity, this expression is switched off in the neuroblasts by seven up (svp) in a mitosis-dependent manner, but is maintained in their progeny (ganglion mother cells). svp mRNA is already expressed in the neuroblasts before this division. After mitosis, Svp protein accumulates in both cells, but the downregulation of hunchback (hb) occurs only in the neuroblast. In the ganglion mother cell, svp is repressed by Prospero, a transcription factor asymmetrically localised to this cell during mitosis. Thus, the differential regulation of hb between the neuroblasts and the ganglion mother cells is achieved by a mechanism that integrates information created by the asymmetric distribution of a cell-fate determinant upon mitosis (Prospero) and a transcriptional repressor present in both cells (Seven-up). Strikingly, although the complete downregulation of hb is mitosis dependent, the lineage-specific timing of svp upregulation is not (Mettler, 2006).

The up- and down-regulation of Hb in the NBs is regulated on the transcriptional level, and it is switched off by the activity of Svp, a member of the orphan receptor family of zinc finger transcription factors. However, svp mRNA expression has already started before the NB divides, after which hb expression is terminated. As a result, both progeny, the NB and the GMC, inherit svp mRNA and produce Svp protein, although the GMC continues to express hb. This suggests that one or more GMC-specific factors are able to suppress the Svp-mediated repression of hb within the GMC. Good candidates for such factors are the asymmetrically segregating cell-fate determinants Numb and Prospero (Pros). Both of these proteins form a basal crescent within the NB prior to division, and both are inherited only by the newly formed GMC. Therefore, hb expression was analyzed in loss-of-function alleles of these genes. Although no obvious difference was found in the number of Hb-positive (Hb+) cells in the absence of Numb function, there was a strong reduction in the number of these cells in pros mutant embryos (Mettler, 2006).

pros codes for a homeodomain transcription factor that enters the nucleus of the GMC after mitosis, subsequently regulating GMC-specific gene expression. To confirm that the observed reduction of Hb+ cells is indeed due to a lack of hb maintenance within the GMCs and their progeny, the timing of hb expression was compared within different lineages between wild type and the pros loss-of-function alleles prosC7 and pros17. The lineages of the thoracic NB2-4T and NB6-4T, as well as the abdominal NB7-3, were analyzed. As in wild type, NB7-3 in both pros alleles is initially Hb+ and generates a Hb+ GMC (GMCa) after its first division. At early stage 12, the NB is Hb-negative (Hb-) and generates a second GMC (GMCb) that is Hb- too. At this stage, GMCa maintains hb expression in wild type, whereas this is reduced or already undetectable in pros mutants. In stage 14 pros mutant embryos, 100% of the NB7-3 derived cell clusters do not show any Hb+ cells, whereas there are two cells in wild type, the EW1 and GW neurons. To rule out that the lack of Hb+ cells in NB7-3 is due to a loss of these cells by programmed cell death, prosC7 was recombined with the deficiency H99 to prevent apoptosis. Again, only Hb- progeny of NB7-3 were found in later stages, confirming that the phenotype is indeed due to lack of hb maintenance. Consistent with its role as a repressor of hb, the opposite phenotype is seen in svp mutants: here the NB stays Hb+ after its first division and produces at least one additional Hb+ GMC before becoming Hb- (Mettler, 2006).

A similar result was obtained for the thoracic NB6-4T lineage. This NB is special because its first division produces a glial precursor instead of a GMC that gives rise to three glial cells. Again, the glial precursor and its progeny normally maintain hb expression, whereas it is switched off in the parental NB. As is expected in pros mutants, the hb expression in the glial cells is not maintained, whereas there is a considerable delay in switching off hb in svp mutants. This lack of svp function leads to one additional Hb+ glial cell in 56% of the hemineuromeres. Concomitantly, a reduction is observed of the number of NB6-4T-derived neurons in almost all hemineuromeres in svp mutants (Mettler, 2006).

Because NB7-3 and NB6-4T terminate hb expression after their first division, it was next asked whether pros is also necessary for hb maintenance in NBs that produce two Hb+ GMCs. NB7-1 generates such a lineage and it has already been shown that it also produces additional Hb+ progeny in svp mutant embryos. Unfortunately this lineage could not be analyzed in pros mutants because the expression of even skipped (eve), which is needed as a marker for the detection of the first NB7-1 progeny, is itself pros dependent. Therefore, NB2-4T, which was also found to be a neuroblast generating two Hb+ GMCs leading to four Hb+ neurons, was analyzed. In this lineage too, hb expression stays switched on longer in svp-mutant embryos, and as a result there are about five to eight Hb+ cells in 86% of the analysed thoracic hemineuromeres. In pros mutants, the hb expression within the NB2-4T lineage seems initially to be normal, but at stage 14, in about 53%, there are only two Hb+ neurons detectable. Thus, in all lineages analysed Pros seems to counteract the hb-downregulating activity of Svp (Mettler, 2006).

To test whether Pros is not only necessary but also sufficient for hb maintenance, use was made of the GAL4/UAS-system to express pros ectopically within the NBs. engrailed-GAL4 (en-GAL4) was used to drive pros expression within NB7-3 and its progeny. Ectopic Pros caused a precocious stop in cell divisions within this lineage in all hemineuromeres analysed. This was expected, since Pros has been shown to activate dacapo, which subsequently inhibits further mitotic divisions. Nevertheless, in some hemineuromeres three NB7-3-derived cells could be identified. In most of these cases, all three cells were Hb+. This shows that Pros activity is indeed sufficient for maintaining hb expression, because one of these cells must be the NB that has divided at least once (Mettler, 2006).

The opposite phenotypes of svp and pros mutants suggest that hb maintenance in the GMC is due to Pros activity, which inhibits the repressive function of svp. If this is the case, a concomitant loss of Pros and Svp function should show a svp-like phenotype. To test this, a svpe22, prosC7 double mutant was generated and the embryonic CNS was stained for hb expression. Generally more Hb+ cells were found, which is similar to the phenotype in svp single mutant embryos. This was also confirmed on the lineage level: in the NB7-3 derived cluster of stage 14 svp-mutant embryos, there were three or four Hb+ cells in 75% of the hemineuromeres. This is similar to the double mutants, which showed this in 67% of the hemineuromeres, thus supporting the hypothesis that Pros antagonises Svp activity in the GMC. But on which level does this occur? One possibility is that svp transcription, which is initiated before mitosis, is suppressed by Pros in the GMC after division. Alternatively, Pros could suppress the activity of the Svp protein. To distinguish between these two possibilities, the dynamics of svp mRNA expression was analysed in the NB7-3 lineage in wild-type and pros mutant embryos. In both genotypes, svp expression in the NB starts before its first division and svp mRNA is still present in the NB after mitosis. However, when svp mRNA expression was examined in GMCa before the NB divides again, a difference was found between the wild-type and pros mutant embryos. In wild type, 70% of these GMCs were negative for svp mRNA. In contrast to that, all GMCs examined in pros mutants expressed svp mRNA, although on a lower level than the NBs did. After the birth of GMCb, there is detectable svp mRNA in only eight out of 21 cases in GMCa in wild type, whereas in pros mutants 13 out of 20 are still positive for this transcript. This suggests that Pros might participate in the GMC-specific transcriptional downregulation of svp. However, overexpression of pros could not eliminate svp expression within the NBs (Mettler, 2006).

Interestingly, the observed difference in svp mRNA expression between wild-type and pros mutant NB7-3 lineages was not seen in the Svp protein distribution; there was no or only a weak level of Svp protein found in NB7-3 before division in both genotypes. Likewise, after division both cells are always Svp+. After the second neuroblast division, GMCa remains positive for Svp protein in nearly all cases in wild type, as well as in pros mutant embryos. At this stage, Hb protein normally has completely vanished from the NB but is maintained in GMCa despite the presence of Svp protein. Taken together, this suggests that Pros acts on both a transcriptional and a post-transcriptional level to downregulate Svp activity in the GMC (Mettler, 2006).

It has been shown that hb downregulation in the NB is mitosis dependent, because Hb is maintained in string (stg) mutant NBs, where mitosis is blocked at the G2/M transition. However, in NB7-3, svp mRNA begins to be expressed prior to the division that leads to hb downregulation. This timing of svp expression seems to be a general feature, since this is also seen in other lineages. NB6-4T, which generates only one hb-positive progeny, switches on svp expression before its first division, whereas NB2-4T and NB7-1, which both generate two Hb+ GMCs, start svp expression before the Hb+ GMCb is born. This suggests that either there is no svp mRNA expression in stg mutant NBs, or that the svp-mediated hb-repressing activity is post-transcriptionally upregulated after division (Mettler, 2006).

To distinguish between these two possibilities, svp mRNA expression was analyzed in stg mutant embryos in Eg-positive NBs at different developmental time points. Z normal onset of svp expression was found within NB2-4T and NB7-3, showing that lack of hb-downregulation in stg mutants in these NBs is not due to a lack of svp transcription. To test whether the regulation could be on the level of protein translation, stg mutant embryos were examined for the presence of Svp protein in the NBs. Indeed, only a low or undetectable amount of this protein was found in these cells up to early stage 12, suggesting that the translation of the svp mRNA is very low. The reason for this might be the unusual localisation of the svp mRNA: when comparing the distribution of hb and svp mRNAs, it was realised that almost all of the visible svp mRNA is localised in the nucleus, whereas the hb mRNA is enriched in the cytoplasm. This nuclear localisation of the svp mRNA is also evident in the in situ hybridisation for svp mRNA combined with the antibody staining for Hb protein in stg mutant embryos. It is assumed that this localisation might prevent efficient translation of the Svp protein, which takes place in the cytoplasm. However, some of the svp mRNA molecules seem to escape from the nucleus, since a low level of Svp protein was detected in NBs from around stage 12 onwards. This seems to lead to a reduction of hb expression because the amount of hb mRNA and protein in the svp-expressing NBs is lower than in the other cells (Mettler, 2006).

The observation that, in stg mutants, not only NB6-4T and NB7-3 but also NB2-4T expressed svp mRNA was unexpected because, in this NB, svp mRNA is normally only detectable after the birth of the first GMC. The generation of this cell is obviously not necessary for svp upregulation because otherwise NB2-4T would remain svp mRNA negative in stg mutant embryos. The same was observed for the En+ NB7-1; although normally becoming svp positive after the birth of its first GMC at the beginning of stage 10, svp expression started at exactly the same time in stg mutants, despite of lack of cell division. Thus, in contrast to hb downregulation, the timing of svp upregulation is mitosis independent in the analysed lineages (Mettler, 2006).

Common to all genes of the temporal specification cascade is the fact that after division they are downregulated within the NB but remain expressed in the newly generated GMCs and their progeny. For hb, this downregulation is dependent on Svp, whose mRNA is already expressed within the neuroblast before the generation of the Hb+ GMC and is symmetrically distributed to both cells after NB division. Why then does svp downregulate hb only within the NB and not in the GMC? This is due to the activity of Pros, a homeodomain transcription factor that is asymmetrically distributed only to the GMC. Earlier work by other groups has suggested that Pros is involved in the regulation of GMC-specific gene activity. In principle this is also true for the function of pros in the context of hb regulation, because it inhibits the NB-specific svp-mediated downregulation of hb. How is this antagonistic activity of Svp and Pros achieved at the molecular level? In one case, the data suggest that Pros downregulates svp transcription, because the svp mRNA in the first GMC of NB7-3 is present longer in pros mutants than in wild type. In the other, it seems likely that Pros also inhibits Svp activity, because the Hb+ GMC often possesses Svp protein even after the parental NB is Hb-. An attractive model for this would be that Pros neutralises Svp repressor function by binding to the same regulatory region of hb. In fact, an evolutionarily conserved enrichment of putative Svp-binding sites was found in the vicinity of a potential Pros-binding site, within a regulatory region that is necessary for neural hb regulation (J. Margolis, PhD thesis, University of California at San Diego, 1992, cited in Mettler, 2006). Whether these sequences are indeed functional in the proposed context is currently being studied (Mettler, 2006).

Because blocking the transition between the G2 and M phase prevents hb from being downregulated, the repressing activity of svp must somehow depend on mitosis. This regulation cannot be at the level of the transcriptional activation of svp because its mRNA is already present before the NBs enter the decisive M-phase. Moreover, in stg mutant embryos, where the G2/M transition is blocked, co-expression of svp mRNA and Hb is found for several hours, although at later stages the average amount of Hb molecules seems to be generally lower than in cells without svp expression. At the protein level the situation is somewhat different: in wild-type embryos hardly any Svp protein is seen before the NB divides, suggesting that the svp mRNA cannot be efficiently translated before mitosis occurred. This might be due to a low translation rate, because in stg mutants only a slowly increasing Svp protein level was found in the NBs despite a permanently strong svp mRNA expression. One reason for this might be the nuclear localisation of the svp mRNA, which was found in NBs of stg mutant embryos as well as in wild type. This localisation might be able to largely prevent svp mRNA from becoming translated before the cell divides. Clearly, further work is needed to test this interesting hypothesis (Mettler, 2006).

The fact that Svp protein is found in NBs in stg mutants that reduces but does not switch off hb expression might offer an explanation as to how the different fates of two Hb+ GMCs might be determined. A well-studied case is NB7-1, where the first GMC gives rise to the Zfh2-negative U1 neuron, whereas the second generates a Zfh2-positive U2 neuron. Earlier work provided evidence that U2 is specified by a reduction of hb activity within the NB or GMC. Thus, it is possible that a low level of Svp protein present in the NB before the second GMC is born might be responsible for this. Indeed, when svp is expressed in NB7-1 prematurely before the birth of the first GMC, there is no U1 neuron and the chain of U neurons often starts with only one Hb+ neuron, which has a U2 identity. According to the hypothesis, this would be due to a reduced Hb activity caused by the premature svp expression. Likewise, in the absence of svp function the NB first produces many additional Hb+ U1 neurons before it eventually generates the other U neurons starting with U2. In this case, hb expression level might initially remain high resulting in the production of several U1 neurons before it drops down leading to the specification of a U2 neuron (Mettler, 2006).

It has been shown that the lineage-specific timing of the switching on of svp expression defines the end of the Hb+ time window, and thereby the number of the progeny generated during this phase. How is this timing regulated? In one group of NBs, the expression of svp already starts before its first division (e.g., NB7-3 and NB6-4T). This could be directly dependent on the activity of proneural genes. Indeed, the early expression of svp within the developing Malphigian tubules has been shown to be regulated by these genes. In this context, it is interesting to note that, in Drosophila, svp expression in certain NB lineages has already begun in their proneural clusters within the neuroectoderm. A second group of NBs show svp upregulation after the generation of their first GMCs (e.g. NB2-4T and NB7-1), suggesting that the mitotic division is the trigger for this event. Surprisingly, this is not the case: in stg mutant embryos, NB7-1 upregulates svp at the same time as in wild type, although no division has occurred. The same was found for NB2-4T. Thus, lineage-specific timing of svp expression is independent of the number of cell divisions. However, currently it cannot be ruled out that earlier stages of the cell cycle, like the S-Phase, could be the trigger instead. Interestingly, the sequential transitions of the temporal specification genes acting after hb expression have recently been shown to occur independently of the cell cycle. According to the current results, this might be also true for the timing of svp expression (Mettler, 2006).

The regulatory interactions between hb, svp and pros are the first example where mitosis-dependent gene activity acts together with an asymmetric cell fate determinant to regulate differential gene expression in space and time. It is currently not known whether such a regulation also exists in other organisms. Interestingly, Svp shows a high homology with COUP-TF orphan receptors from vertebrates, which are also necessary for CNS development. Prox1, the vertebrate homologue of Pros is not asymmetrically distributed during division but is expressed and needed during neurogenesis. During retinal development, Prox1 is involved in the specification of the fate of the early born horizontal neurons. Future investigations will show whether during vertebrate CNS development these homologous factors play a role comparable to Svp and Pros in Drosophila (Mettler, 2006).

Regulation of neuroblast competence: multiple temporal identity factors specify distinct neuronal fates within a single early competence window

Cellular competence is an essential but poorly understood aspect of development. Is competence a general property that affects multiple signaling pathways (e.g., chromatin state), or is competence specific for each signaling pathway (e.g., availability of cofactors)? This study has found that (1) Drosophila neuroblast 7-1 (NB7-1) has a single early window of competence to respond to four different temporal identity genes (Hunchback, Krüppel, Pdm, and Castor); (2) each of these factors specifies distinct motor neuron identities within this competence window but not outside it, and (3) progressive restriction to respond to Hunchback and Krüppel occurs within this window. This work raises the possibility that multiple competence windows may allow the same factors to generate different cell types within the same lineage (Cleary, 2006).

To determine whether NB7-1 undergoes progressive restriction in competence to respond to Kr, similar to that observed for Hb, pulses of Kr were generated at progressively later points in the NB7-1 lineage. Both hsp70-Kr and hsp70-hb were used to allow precise comparison of the effects of both genes. Progressively later pulses of Hb produce a decreasing frequency of U1/U2 neurons. Similarly, progressively later Kr pulses generate decreasing frequencies of extra U3 at each subsequent stage, with the exception of the earliest portion of the lineage, where Hb is known to be dominant to Kr. Thus, NB7-1 shows progressive restriction in competence to respond to both Hb and Kr, and competence to respond to both Hb and Kr is lost at the same point in the lineage (after five divisions) (Cleary, 2006).

An independent method was used to measure the competence window in the NB7-1 lineage. prospero-gal4 was used to induce expression of Kr within the NB7-1 lineage from the fourth division onward. When one copy of UAS-Kr was used at 22°C, which provides relatively low levels of Kr, only five to six Eve+ U neurons were observed, mostly U1, U2, and three U3 neurons (91%), but also U1, U2, and four U3 neurons (9%). Thus, NB7-1 loses competence to respond to prolonged Kr expression after five to six cell divisions, similar to results from the Kr pulse experiments described above. Prolonged expression of Hb using the same conditions (prospero-gal4, one copy of UAS-Hb, 22°C) also results in just five to six Eve+ U neurons. It is concluded that NB7-1 has a single competence window for generating U1-U3 neurons in response to Hb and Kr (Cleary, 2006).

Next to be tested was whether the later-expressed temporal identity factors Pdm and Cas share the same early competence window with Kr, or if they have distinct competence windows. Pdm specifies the U4 neuronal identity, while Pdm/Cas together specify U5 neuronal identity. scabrous-gal4 was used to prolong Kr expression for a variable length of time within the NB7-1 lineage (two copies of UAS-Kr at 29°C), which delayed but did not prevent the sequential expression of Kr, Pdm, and Cas. This experiment allowed NB7-1 competence to be assayed when presented with Kr, Pdm, or Cas at different times in its lineage (Cleary, 2006).

It was found that the scabrous-gal4 UAS-Kr embryos always had a total number of seven to eight Eve+ U neurons, although ectopic U3 neurons ranged from two to six in number. Interestingly, hemisegments with only two ectopic U3 neurons typically had U4/U5 neurons; those with three ectopic U3 neurons had only a U4 neuron, and those with four or more ectopic U3 neurons lacked both U4/U5 neuronal fates. These data are interpreted in the following way: in segments where Kr declines the fastest (fewest ectopic U3 neurons), there is time for Pdm to induce U4 fate and Pdm/Cas to induce U5 fates prior to loss of competence; however, in segments where Kr lasts the longest, both Pdm and Cas expression occur after the competence window and no U4/U5 fates are produced. Taken together, this experiment allows several conclusions to be drawn: (1) prolonged Kr expression can partially extend the neuroblast competence window (from five to six divisions to seven to eight divisions); (2) competence to respond to Kr, Pdm, and Cas is simultaneously lost at the end of this competence window, suggesting that there is a single competence window for responding to multiple temporal identity factors, and (3) each temporal identity factor specifies different U1-U5 motor neuron identities within the competence window, but not outside it. It is currently an open question as to how prolonged expression of one factor (Kr or Hb) can extend the competence window to respond to three distinct factors (Kr, Pdm, and Cas) (Cleary, 2006).

The previous experiment showed that prolonging Kr expression (scabrous-gal4 UAS-Kr) in NB7-1 lineage can only partially extend neuroblast competence. Interestingly, similar experiments prolonging Hb expression (scabrous-gal4 UAS-hb) revealed that the neuroblast maintains full competence for as long as Hb is expressed, in some cases over 15 divisions, with normal U3-U5 fates appearing after Hb levels decline. Thus, extended Hb expression (but not extended Kr expression) can maintain the neuroblast in a young, fully competent state. This raised the possibility that down-regulation of Hb is required for loss of neuroblast competence; alternatively, Hb may be more potent than Kr in maintaining neuroblast competency (Cleary, 2006).

To distinguish these models, the effect was tested of high-level Hb or Kr expression beginning at the fourth neuroblast division (prospero-gal4, 2x UAS-hb or UAS-Kr, 29°C), which would allow Hb down-regulation and permit comparison of the efficacy of Hb versus Kr in extending neuroblast competence. Performing this experiment with Hb resulted in a partial extension of neuroblast competence and the production of an average of 9.1 Eve+ U neurons: U1-U3, 6.1 extra U1, and no U4/U5. Performing the experiment with Kr resulted in an almost identical phenotype of 9.8 Eve+ U neurons: U1/U2, 7.8 U3s, and no U4/U5. Thus, Hb and Kr appear equally efficient at extending neuroblast competence; this is supported by their equivalent effect when expressed under heat shock or lower level prospero-gal4 control (competence lost after five divisions). More importantly, a comparison of the scabrous-gal4 UAS-hb and prospero-gal4 UAS-hb experiments shows that Hb down-regulation is critical for loss of neuroblast competence. When Hb is maintained from the beginning of the lineage (scabrous-gal4 UAS-hb), competence persists for the length of Hb expression, in some cases over 15 divisions; when Hb down-regulation occurs followed by permanent Hb re-expression one division later (prospero-gal4 UAS-hb), then competence is lost after approximately nine divisions. It is concluded that down-regulation of Hb, but not Kr, initiates progressive restriction in neuroblast competence that is normally complete after five divisions (Cleary, 2006).

Thus far, how neuroblast competence changes over multiple rounds of cell division was investigated. Now, how competence changes during neuronal differentiation is considered. Kr was expressed in high levels in the newborn post-mitotic U1-U5 neurons (eve-gal4 UAS-Kr). In these embryos, Kr is first detected just as the U1-U5 neurons are born. Despite high levels of Kr protein, no change in U1-U5 fate was ever detected. Conversely, transient expression of Kr in NB7-1/GMCs can occasionally generate ectopic U3 neurons that do not maintain Kr expression, despite the ability of Kr to positively autoregulate within the CNS. Thus, mitotic progenitors but not post-mitotic neurons are competent to respond to Kr. Similar results have been observed for competence to respond to Hb (Cleary, 2006).

These experiments, combined with previous studies, allow four major conclusions to be drawned.

  1. A single early competence window is used by multiple temporal identity factors. The molecular basis for the early competence window is unknown, but it must be general enough to modulate response to four distinct transcription factors rather than being factor specific. Perhaps loss of competence leads to restricted access of Hb, Kr, Pdm, and Cas to target loci involved in U1-U5 neuronal specification; other loci may remain unaffected, allowing these four transcription factors to induce different cell fates later in the neuroblast lineage. Identifying Hb and Kr target genes, and investigating how or whether they undergo chromatin modifications during the process of progressive restriction will help resolve this question, and may provide insight into the mechanism of progressive restriction in mammalian neural progenitors (Cleary, 2006).
  2. Each temporal identity factor specifies distinct motor neuron fates within the competence window, but not outside of it. Within the early competence window, each temporal identity factor specifies a unique U1-U5 neuronal identity: high Hb, U1; low Hb, U2; Kr, U3; Pdm, U4; Pdm/Cas, U5. The loss of competence to generate U1-U5 fates may allow each of these transcription factors to be 'reused' later in the NB7-1 lineage to generate different subsets of neurons. This model is supported by the fact that a second round of Kr and Cas neuroblast expression is observed later in embryonic development. These findings suggest that neuroblasts have the potential for cycling through distinct competence windows, and may provide a model for understanding how successive competency states are established (e.g., in vertebrate retinal progenitors) (Cleary, 2006).
  3. NB7-1 undergoes progressive restriction in competence to respond to both Hb and Kr. Competence to respond to both Hb and Kr is progressively restricted early in the lineage, then completely lost after five divisions of NB7-1. Progressive restriction may be regulated autonomously in the neuroblast or by changing environmental cues, such as inhibitory feedback from GMC or neuronal progeny. A lineage-intrinsic mechanism is favored because different neuroblasts lose competence to respond to Hb at different times (e.g., NB7-1 remains competent to respond to Hb for five divisions, whereas the adjacent NB1-1 is only competent to respond to Hb for two to three divisions). A feedback inhibition model would have parallels with vertebrate retinal progenitors, where differentiated amacrine cells send an inhibitory feedback signal to terminate amacrine cell production. In this case, the signal would likely depend on the number of progeny produced rather than the type of progeny, because loss of competence can occur without production of the last-born neurons in the competence window (U4/U5) (Cleary, 2006).
  4. Down-regulation of Hb but not Kr initiates progressive restriction and loss of competence. Neuroblast competence is maintained if Hb is expressed from the beginning of the lineage. However, neuroblast competence is not maintained when Kr is expressed from the beginning of the lineage (where Hb is down-regulated normally) or when Hb or Kr are expressed later in the lineage after normal Hb down-regulation. It is proposed that Hb down-regulation initiates progressive restriction in neuroblast competence, ultimately leading to a complete loss of competence (Cleary, 2006).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development

Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of Hb->Kr->Pdm->Cas->Grh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a 'switching factor'. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, a novel allele of svp was isolated. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage (Benito-Sipos, 2011).

This study has found that Svp is expressed in two pulses and plays two different roles in the NB5-6T lineage. Initially, Svp is expressed briefly in the early part of this lineage, where it acts to control the downregulation of the first temporal factor, Hb. Subsequently, Svp is expressed in the late part of this lineage, in the Ap window, in a highly dynamic fashion: initiated in all four Ap neurons, to be downregulated in the first- and last-born Ap cells. In the second expression phase, Svp acts to suppress Col and Dimm, thereby preventing the first-born Ap neuron fate, Ap1/Nplp1, from being established in the subsequently born Ap2 and Ap3 neurons. Misexpression studies further indicate that Svp also suppresses the last-born Ap neuron fate, Ap4/FMRFa, from being established in Ap2/3 (Benito-Sipos, 2011).

Previous studies of Svp demonstrated that it is expressed in a brief pulse in the majority of early embryonic neuroblasts, where it acts to suppress Hb, thereby allowing for the switch to the next stage of temporal competence. Recently, studies have identified additional factors involved in the downregulation of Hb: the pipsqueak-domain proteins Distal antenna and Distal antenna-related (herein referred to collectively as 'Dan'). Dan is expressed somewhat earlier than Svp, and is also maintained in a longer pulse. svp and dan do not regulate each other, and although they can be activated by ectopic hb expression, neither Svp nor Dan expression is lost in hb mutants. This raises the intriguing questions of how Svp and Dan are activated during early stages of lineage progression, and how they become downregulated at the appropriate stage (Benito-Sipos, 2011).

Another interesting complexity with respect to Svp expression and function pertains to the fact that the Hb window is of different size in different lineages. For example, in NB6-4T and NB7-3, Hb is downregulated in the neuroblast immediately after the first division, whereas in NB5-6T, Hb expression is evident during three divisions. In line with this, no Svp expression is observed in NB5-6T until stage 10, when the neuroblast has already gone through two rounds of division. How the on- and offset of Svp, and perhaps Dan, expression is matched to the specific lineage progression of each unique neuroblast lineage, to thereby allow for differing Hb window sizes, is an interesting topic for future studies (Benito-Sipos, 2011).

Svp is re-expressed in the NB5-6T lineage in a second pulse. In contrast to the early pulse of Svp expression, where there is no evidence for temporal genes controlling Svp, it was found that the second pulse of Svp expression is regulated by the temporal genes cas and grh. However, it was not found that svp is important for the expression of Cas or Grh. Instead, svp participates in the sub-division of the Cas/Grh temporal window, i.e. the Ap window. Based upon the idea that Svp is regulated by temporal genes, and acts to sub-divide a broader temporal window, it could be referred to as a 'sub-temporal' factor in the latter part of the NB5-6T lineage (Benito-Sipos, 2011).

The expression of Svp is dynamic also in the second pulse of expression, commencing in the neuroblast at stage 14 -- after the three first Ap neurons are born -- and being maintained in the neuroblast until it exits the cell cycle at stage 15. At late stage 14 and 15, Svp expression becomes evident in all four Ap neurons, but it is rapidly downregulated from Ap1 and Ap4 during stages 16 and 17. Svp is, however, maintained in the Ap2 and Ap3 neurons into late embryogenesis. The role of svp in the Ap window appears to be to ensure proper specification of the Ap2/3 interneurons, generated in the middle of the Ap window. This is achieved by svp suppressing the first- and last-born Ap neuron fates: the Ap1/Nplp1 and Ap4/FMRFa fates. With regard to the suppression of the Ap1 fate, one important role for svp is to suppress Col expression in Ap2/3. Importantly, the temporal delay in Svp expression when compared to Col -- commencing two stages after Col in the Ap neurons -- allows for col to play its critical early role in Ap neuron specification: activating ap and eya. The timely suppression of Col in Ap2/3 is mediated also by sqz and nab, and the loss of Nab expression in svp mutants may be a contributing factor to the failure of Col downregulation in svp. However, the potent function of svp in suppressing Ap1/Nplp1 fate when misexpressed postmitotically from apGal4 does not appear to require Nab, as Nab is not ectopically expressed in these experiments. Thus, svp may act via several routes to prevent Ap1/Nplp1 fate from being established in the Ap2/3 cells: by suppressing Col and by activating Nab (Benito-Sipos, 2011).

Regarding the second role of svp in the Ap window -- the suppression of the Ap4/FMRFa fate -- it is less clear what the target(s) may be. However, a common denominator for both the Ap1/Nplp1 and the Ap4/FMRFa neurons is the expression of Dimm. Dimm, a basic-helix-loop-helix protein, is a critical determinant of the neuropeptidergic cell fate, and also controls high-level neuropeptide expression in many neuropeptide neurons. Both svp loss and gain of function results in robust effects upon Dimm expression in the NB5-6T lineage, indicating that Dimm is an important target for svp. However, dimm mutants show only reduced levels of FMRFa expression, and thus svp is likely to regulate additional targets to prevent the Ap4/FMRFa cell fate in the Ap2/3 neurons (Benito-Sipos, 2011).

Another interesting phenotype in svp mutants, pertaining to the second pulse of Svp expression in the NB5-6T lineage, is the finding of one to two extra Ap neurons. This indicates that the NB5-6T neuroblast undergoes one to two extra rounds of division, and that the expression of Svp in the neuroblast during stage age 14-16 is important for precise cell cycle exit. Interestingly, the other temporal (cas and grh) and sub-temporal (sqz and nab) genes acting in the latter part of the NB5-6T lineage also play roles in controlling cell cycle exit. Moreover, studies of neuroblast cell cycle exit in other neuroblasts, both embryonic and postembryonic, have also shown roles for grh and svp in these decisions. Thus, a picture is emerging in which late temporal and sub-temporal genes may be broadly involved in controlling timely cell cycle exit of many neuroblasts (Benito-Sipos, 2011).

The early role of svp, in its first expression pulse, is to suppress Hb expression. Svp is expressed transiently by most if not all neuroblasts, and the regulation of Hb also appears to be a global event. Similarly, the second pulse of Svp expression has been observed in many lineages, although the role for svp in this later pulse was hitherto unknown. The findings of a role for svp as a sub-temporal gene in the latter part of the NB5-6T lineage indicates that svp may play such roles in many lineages. However, it should be noted that global changes in Col, Dimm and Eya expression in the embryonic central nervous system (CNS) are not seen. Thus, unlike the more universal role of svp in regulating Hb during the first pulse, the putative sub-temporal function of the second pulse of svp expression in other lineages must be highly context-dependent and involve other targets (Benito-Sipos, 2011).

In mammals, the svp orthologues COUP-TFI and -II are expressed dynamically in the developing CNS. Functional studies reveal a number of important roles for COUP-TFI/II during nervous system development, and mutant mice display aberrant neuro- and gliogenesis, accompanied by axon pathfinding defects. Intriguingly, recent studies have revealed that COUP-TFI/II acts in a temporal manner to control the timing of generation of sub-classes of neurons and glia in the developing mouse brain. Given that the other genes described in this study are also conserved, it is tempting to speculate that temporal and sub-temporal cascades similar to those outlined in this study are also used in the mammalian CNS during development (Benito-Sipos, 2011).

The pipsqueak-domain proteins Distal antenna and Distal antenna-related restrict Hunchback neuroblast expression and early-born neuronal identity

A fundamental question in brain development is how precursor cells generate a diverse group of neural progeny in an ordered manner. Drosophila neuroblasts sequentially express the transcription factors Hunchback (Hb), Krüppel (Kr), Pdm1/Pdm2 (Pdm) and Castor (Cas). Hb is necessary and sufficient to specify early-born temporal identity and, thus, Hb downregulation is essential for specification of later-born progeny. This study shows that distal antenna (dan) and distal antenna-related (danr), encoding pipsqueak motif DNA-binding domain protein family members, are detected in all neuroblasts during the Hb-to-Cas expression window. dan and danr were identified in a forward genetic screen of ~100 second and third chromosomal deficiency lines for mutants that had altered numbers of Even-skipped (Eve)+ early-born neurons. Dan and Danr are required for timely downregulation of Hb in neuroblasts and for limiting the number of early-born neurons. Dan and Danr function independently of Seven-up (Svp), an orphan nuclear receptor known to repress Hb expression in neuroblasts, because Dan, Danr and Svp do not regulate each other and dan danr svp triple mutants have increased early-born neurons compared with either dan danr or svp mutants. Interestingly, misexpression of Hb can induce Dan and Svp expression in neuroblasts, suggesting that Hb might activate a negative feedback loop to limit its own expression. It is concluded that Dan/Danr and Svp act in parallel pathways to limit Hb expression and allow neuroblasts to transition from making early-born neurons to late-born neurons at the proper time (Kohwi, 2011).

Dan and Danr are required to limit Hb expression in neuroblasts and restrict the number of early-born neurons generated in multiple neuroblast lineages. The orphan nuclear hormone receptor protein Svp also functions to limit Hb expression in neuroblasts, and the current data strongly suggest that Dan/Danr and Svp function in parallel pathways that are each independently required. First, the temporal expression patterns of Dan and Danr versus Svp do not suggest their coordinated activity: Dan and Danr are expressed from the time of neuroblast formation (stage 9), beyond Hb downregulation (stage 10), until the time of strong Castor expression (stage 12). By contrast, Svp protein is very transiently detected in neuroblasts only at the onset of Hb downregulation. Second, Dan/Danr and Svp are not in a linear transcriptional hierarchy: neither mutant affects expression of the other gene. Third, dan danr and svp mutants have distinct phenotypes: for example, compared with the dan danr mutant, the svp mutant has many more early-born neurons in the NB7-1 lineage, whereas it does not have any extra early-born neurons in the NB1-1 lineage. Fourth, the dan danr svp null triple mutant has the summed phenotypes of the dan danr double mutant and the svp single mutant. Fifth, misexpression of Svp, but not Dan, can repress hb transcription in neuroblasts. The fact that neither one appears to have an effect on cell fate when misexpressed in postmitotic neurons suggest that both Svp and Dan function at the level of the mitotic precursors. Taken together, it appears that Dan/Danr and Svp are each required to downregulate hb expression in neuroblasts, but do so using separate mechanisms. The data are consistent with Svp directly repressing neuroblast hb transcription (although this has not been shown) whereas Dan and Danr act more indirectly (Kohwi, 2011).

Do Dan, Danr and Svp have lineage-specific functions? Despite the widespread expression of Dan and Danr in early neuroblasts and the widespread transient expression of Svp in most neuroblasts, it is likely that each has lineage-specific functions. For example, in the NB1-1 lineage, ectopic early-born neurons are generated in dan danr mutants, but not in svp mutants. Further comparing Dan versus Danr in this lineage, it appears that Danr is more important than Dan, because the danrex35 single mutant phenocopies the dan danrex56 double mutant in the number of ectopic aCC/pCC neurons generated and the number of hemisegments affected per embryo. In contrast to the NB1-1 lineage, Dan and Danr each appear to be required for limiting the number of early-born neurons in the NB7-1 lineage, as danrex35 single mutants had a weaker phenotype than the dan danr double mutant. Additionally, there are more early-born neurons in the NB7-1 lineage in svp mutants than in dan danr mutants, highlighting their lineage-specific differences. These differences might be due to different levels or functions of each protein in distinct neuroblasts. For example, there is variability in dan and danr mRNA levels between neuroblasts, suggesting that distinct neuroblasts might have different levels of Dan and/or Danr protein (although Dan protein levels appear constant between newly formed neuroblasts), or that they express Dan and/or Danr protein for different durations. Alternatively, or in addition, the lineage-specific variation might be due to unique cofactors present in different neuroblasts. This seems likely, as Hb misexpression in all neuroblasts has varying effects within different lineages. For example, NB1-1 generates only one to three ectopic early-born neurons in response to Hb misexpression, whereas NB7-1 generates ~20 ectopic early-born neurons. Consistent with the notion that co-factors can alter the functional output of transcriptional regulators, recent evidence shows that the co-regulator CtBP forms complexes with distinct eye specification factors, including Dan and Danr, to regulate proliferation versus differentiation during eye development in Drosophila (Kohwi, 2011).

Do Dan and Danr function redundantly? This hypothesis could not be rigorously tested owing to the lack of a dan null single mutant, but the available evidence suggests that they do have redundant functions. First, they have nearly identical expression patterns. But most crucially, overexpression of Dan in the dan danr double mutant can nearly completely rescue the CNS phenotype, suggesting that high enough levels of Dan can compensate for loss of Danr. However, a danr null single mutant shows a strong phenotype in the NB1-1 lineage and a partial phenotype in the NB7-1 lineage, suggesting that endogenous levels of Dan are insufficient for normal CNS development. The most parsimonious explanation is that each protein has equivalent function, but that both genes are required to generate sufficient levels of Dan/Danr protein (Kohwi, 2011).

Hb overexpression can activate expression of both Dan and Svp. What is the significance of this activation? Previous work has shown that Hb can function both as a transcriptional activator and a repressor. Although its repressive functions are required for the neuroblast to specify early-born fates and maintain neuroblast competence, its activator functions remain elusive. One possibility is that Hb-mediated activation of Svp, and the subsequent Svp-mediated downregulation of Hb, create a negative feedback loop to ensure timely progression of the neuroblast to later temporal fates. This is not unlike what has been observed for Cas, which activates feed-forward and feed-back transcriptional cascades to regulate temporal identity in the NB5-6 lineage. By contrast, Hb activation of Dan expression might be part of the mechanism by which Hb maintains neuroblast competence, because Dan is unlikely to repress hb expression directly (Kohwi, 2011).

What might be the mechanism by which Dan and Danr function to restrict the duration of Hb expression in neuroblasts? Some clues might come from the fact that Dan and Danr are found in a subgroup of pipsqueak-domain containing nuclear proteins that have been proposed to regulate higher order chromatin structure by targeting distal DNA elements. Pipsqueak, the founding member of the family, has been shown to recruit Polycomb group complexes to specific regions of the genome to mediate gene silencing. Perhaps Dan and Danr modify chromatin structure through recruitment of chromatin remodeling complexes, which indirectly affects hb transcription by changing the accessibility of the hb locus to other transcriptional regulators. Such a function in modulating chromatin architecture might not be restricted to regulating just hb expression, but can extend to other temporal identity factors as well. Indeed, in NB7-1, the initial Hb->Cas 'competence window', during which the U1-U5 motor neurons are generated, matches nearly exactly the window of Dan and Danr expression. This raises the possibility that Dan and Danr might have a more global role in NB temporal progression by stabilizing 'transition states' between successive temporal identity factors (e.g. Hb->Kr, Kr->Pdm or Pdm->Cas). Such a function might explain the low frequency misregulation of later-born neuron numbers in several lineages (7-1, 3-1, 7-3), in addition to the extra early-born neurons phenotypes. Future experiments that address the role of Dan and Danr in later temporal identity transitions will provide a better understanding of the mechanisms that control the progression of temporal identity in neuroblasts (Kohwi, 2011).

Developmentally regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila

Stem and/or progenitor cells often generate distinct cell types in a stereotyped birth order and over time lose competence to specify earlier-born fates by unknown mechanisms. In Drosophila, the Hunchback transcription factor acts in neural progenitors (neuroblasts) to specify early-born neurons, in part by indirectly inducing the neuronal transcription of its target genes, including the hunchback gene. Using vivo immuno-DNA FISH the hunchback gene was found to move to the neuroblast nuclear periphery, a repressive subnuclear compartment, precisely when competence to specify early-born fate is lost and several hours and cell divisions after termination of its transcription. hunchback movement to the lamina correlated with downregulation of the neuroblast nuclear protein, Distal antenna (Dan). Either prolonging Dan expression or disrupting lamina interfered with hunchback repositioning and extended neuroblast competence. It is proposed that neuroblasts undergo a developmentally regulated subnuclear genome reorganization to permanently silence Hunchback target genes that results in loss of progenitor competence (Kohwi, 2012). .

The Drosophila embryo undergoes a reorganization of genome architecture that is gene, cell type, and developmental stage specific. As neuroblasts age, the hb genomic locus becomes repositioned to the nuclear periphery, which marks the end of the neuroblast competence window to specify early-born cell fates. Why can ectopic hb not induce early-born fates after the close of the competence window? It is proposed that hb is just one of many genes that move to the nuclear lamina at the end of the early competence window -- that a genome-wide reorganization shifts the neuroblasts into a state in which hb is unable to regulate the same targets it could during the competence window. In support of this model, misexpression of hb in the NB5-6 lineage was shown to have no effect on the activation of late-born cell fate factors, consistent with a new genome organization that is refractory to Hb-induced early-born neuronal identity (Kohwi, 2012). .

The data lead to a proposal that neural progenitors undergo a developmentally regulated reorganization of genome architecture as they age, potentially changing the palette of genes available to specify cell fate in aging progenitors. The following three-step model for neuroblast competence is proposed. (1) In the newly formed NB7-1, the hb gene is in the nuclear interior and is transcriptionally active; this is the time when early-born Hb+ U1/U2 neurons are generated. (2) After the second division of NB7-1, the transcriptional repressor Svp terminates hb transcription; however, the hb gene remains accessible in the nuclear interior where ectopic hb protein can indirectly induce hb neuronal transcription to generate extra U1/U2 neurons. (3) After the fifth division of NB7-1, Dan protein is downregulated, resulting in the movement of hb (and probably many other hb target genes) to the nuclear lamina; at this point, ectopic hb in the neuroblast can no longer induce transcription of hb, and the competence window is closed (Kohwi, 2012).

The results are consistent with growing evidence from multiple organisms that repositioning of a gene to the nuclear lamina can cause transcriptional repression. For example, forced tethering of reporter genes to Lamin can repress reporter expression , and Lamin depletion can derepress silent genes. An important difference in the current work, however, is that hb movement to the lamina occurs 3 hr after termination of hb transcription, when hb undergoes an additional level of repression to become permanently silenced. Does lamina targeting induce permanent hb gene silencing or vice versa? Depletion of the nuclear envelope protein Lamin displaces hb away from the lamina, decreases hb silencing, and increases neuroblast competence; this strongly suggests that lamina targeting is an early and essential step in permanent hb gene silencing and the loss of neuroblast competence. However, the possibility cannot be ruled out that hb positioning at the nuclear periphery might maintain, rather than establish, the permanently silenced state (Kohwi, 2012).

The results show that neuroblast cell fate specification and neuroblast competence are independently regulated temporal programs. Prolonged expression of Dan can extend the competence window but cannot induce neuronal identity. Conversely, hb can specify early-born neuronal identity but cannot extend the competence window. Importantly, coexpression of Dan and hb act synergistically: Dan extends the neuroblast competence window, and hb 'fills' this window with U1/U2 neurons, thereby specifying more early-born identity neurons than prolonging hb alone. The mechanism and function of Dan is poorly understood. Why might Dan be sufficient, but not necessary, to promote neuroblast competence? One common finding is that dan and dan related (danr) genes show weak double mutant phenotypes, but strong misexpression phenotypes, in all tissues examined. For example, dan danr double mutants can live to adulthood with weak antennal and eye defect and show no change in the neuroblast competence window. These findings are consistent with a redundant protein or pathway that can compensate for loss of Dan/ Danr. A second model arises from comparing the dan danr mutant and Dan overexpression phenotypes. dan danr double mutants have a slight delay hb transcription termination at stage 10 (distinct from permanent hb silencing at stage 12) (Kohwi, 2011) but no effect on hb gene movement to the nuclear periphery. Conversely, prolonged Dan expression has no effect on hb transcription but blocks hb gene movement and extends the competence window. Thus, during the competence window, Dan may promote a genome organization that allows timely access of the Svp transcriptional repressor to the hb locus, whereas after the competence window, Dan must be eliminated to allow a new genome organization to form. This model emphasizes the role of Dan in maintaining a genome organization in which hb and hb target genes can be efficiently regulated. A third, not mutually exclusive, model is that Dan binds DNA via its Pipsqueak domain to competitively inhibit other Pipsqueak-like factors from recruiting hb and other loci to the nuclear lamina. Indeed, the founding member of the Pipsqueak- motif family, Pipsqueak, is a GAGA-binding factor, and recent work has shown an enrichment for GAGA motifs in Lamin-associated DNA sequences. Consistent with this model, Dan protein is dispersed throughout the nucleoplasm, where it could associate with hb and other loci (Kohwi, 2012).

What is the normal function of a competence window? Competence windows could provide both flexibility and limitations on the production of neural diversity. They could serve as a substrate for natural selection by allowing variation in neuronal subtype numbers through fluctuations in the length over which a progenitor is exposed to a temporal identity cue. Conversely, a competence window could also prevent stochastic fluctuations in the expression of a temporal identity cue from generating a neuronal subtype at a completely inappropriate time (e.g., an early-born fate at the end of a progenitor lineage), thereby limiting potentially deleterious effects. Another potential function of competence windows is that successive competence windows may allow the same cell fate determinant to generate different cell types. Indeed, during spinal cord development, Olig2 first promotes neurogenesis and later induces oligodendrogenesis, and retinal progenitors are thought to progress through multiple competence states during which they can specify only limited cell fates. In support of this model, previous studies have shown that Dan has two waves of neuroblast expression, one at stages 9-late 12 and a second at stages 13-16 (Kohwi, 2011). Bimodal Dan expression may produce two competence windows in which the same temporal identity factors can access different genomic targets to generate additional neuronal diversity. For example, during the first Dan expression window, Hb, Kr, Pdm, Castor, and Svp specify U1-U5 motoneuron identities in the NB7-1 lineage; during the second Dan expression windows, Kr, Castor, and Svp are re-expressed and a different population of neurons is produced. Mammalian Ikaros, an hb homolog, is expressed in young progenitors in which it specifies early-born retinal ganglion cell (RGC) identity. As with Hb, re-expression of Ikaros in older progenitors in vivo cannot induce specification of the early-born RGCs, although Ikaros misexpression in late retinal progenitors cultured in vitro can activate RGC-specific genes. Perhaps in vitro cultured progenitors are lacking an extrinsic cue that closes the competence window, allowing Ikaros to generate more early-born neurons. Interestingly, Dan downregulation could also be regulated by an extrinsic cue, because Dan downregulation occurs nearly simultaneously in the entire neuroblast population, despite each neuroblast being at a different stage of its cell lineage. Mammalian retinal and cortical progenitor cells change competence over time, generating an ordered series of distinct neural cells. In the future, it would be interesting to determine whether the genes expressed in early-born cortical or retinal cell types are repositioned to the nuclear lamina in mammalian neural progenitors as competence to specify that these cells are lost over time. Determining the mechanisms underlying loss of competence and identifying the molecular players in this process would have important implications for understanding normal brain development, adult tissue homeostasis, and tissue repair (Kohwi, 2012).

Dampened regulates the activating potency of Bicoid and the embryonic patterning outcome in Drosophila

The Drosophila morphogen gradient of Bicoid (Bcd) initiates anterior-posterior (AP) patterning; however, it is poorly understood how its ability to activate a target gene may have an impact on this process. This paper reports an F-box protein, Dampened (Dmpd) as a nuclear cofactor of Bcd that can enhance its activating potency. A quantitative platform was established to specifically investigate two parameters of a Bcd target gene response, expression amplitude and boundary position. Embryos lacking Dmpd have a reduced amplitude of Bcd-activated hunchback (hb) expression at a critical time of development. This is because of a reduced Bcd-dependent transcribing probability. This defect is faithfully propagated further downstream of the AP-patterning network to alter the spatial characteristics of even-skipped (eve) stripes. Thus, unlike another Bcd-interacting F-box protein Fates-shifted (Fsd), which controls AP patterning through regulating the Bcd gradient profile, Dmpd achieves its patterning role through regulating the activating potency of Bcd (Liu, 2013).

Morphogen gradients such as those of Bcd are excellent experimental paradigms for dissecting the mechanistic operations of the regulatory networks that control patterning decisions. They provide a unique window to probing the regulatory impacts of F-box proteins both spatially and temporally at a fine resolution. Increasing evidence supports the notion that F-box proteins - there are up to 45 of them in Drosophila and 75 in humans - belong to a critical class of regulatory proteins; however, few of them have been studied in native developmental contexts. This work reports the identification of a nuclear cofactor of Bcd, Dmpd, that can enhance the potency of Bcd as an activator. Dmpd thus joins an expanding list of F-box proteins that can act as transcriptional co-activators; it remains to be determined whether the co-activator role of Dmpd for Bcd in the embryo is dependent on a functional SCF complex and the E3 ligase activity. The results show that embryos lacking Dmpd have a lower amplitude of Bcd-activated hb expression, a defect that can be passed further downstream of the network, causing an enlargement of ΔELeve3-4, the spacing between eve expression stripes that are sensitive to absolute concentrations of Hb. In embryos that lack another Bcd-interacting F-box protein Fsd, neither the hb expression amplitude nor ΔELeve3-4 is affected. This stems from the fact that, unlike Dmpd, Fsd regulates the Bcd gradient profile through a proteolytic pathway without a detectable co-activator function. The contrasting functions of these two F-box proteins thus document that a normal AP-patterning outcome is subject to regulation by two distinct mechanisms. These mechanisms control two distinct properties of Bcd that are indispensable to its morphogen action: the formation of a concentration gradient and the activation of its target genes (Liu, 2013).

A critical feature of morphogen gradients is their ability to induce downstream responses in a concentration-dependent manner. This particular feature has been subjected to extensive investigations, in part because of a significant interest in the question of what morphogen gradients do. It has been proposed recently that the concentration-dependent input-output relationship between Bcd and hb also contributes directly to the formation of AP patterns that are scaled with the length of the embryo. By contrast, the regulation of the amplitude of a response to the morphogen input has been relatively underexplored. Its importance may be better appreciated from the perspective of regulatory networks that control the patterning outcome. In the case of hb as a direct target gene of Bcd, its encoded protein Hb acts as an input, in a concentration-dependent manner, for genes (such as eve) that are further downstream of the AP-patterning network. The regulation of the expression of these downstream genes allows the regulatory network to refine and evolve towards the desired final outcome of patterning. Since these downstream genes respond to absolute concentrations of Hb, the boundary position for hb expression in response to the Bcd gradient input has become no longer directly relevant to the decision-making processes of these genes. However, as shown by this study, another feature of the hb response to the Bcd gradient input, namely its expression amplitude, remains directly relevant to the continued operation of the AP-patterning network (Liu, 2013).

Analysis of the impact of the dmpd mutation on active hb transcription reveals important mechanistic insights into the regulation of the transcription process in a native developmental context. As documented recentl, Bcd-activated hb transcription becomes detectable immediately upon entering the nc 14 interphase; however, it is shut off within a few minutes. Intron-staining results show that, at time classes t1 and t2, the hb-transcribing probability at the plateau region is largely unaffected by the dmpd mutation. They suggest that the onset of active hb transcription upon entering the nc 14 interphase is largely insensitive to dmpd mutation. The reduction in ρplat (the plateau region of the hb expression domain) at t3 and subsequent time classes is thus consistent with the possibility that dmpd embryos might have a hastened shutdown of active hb transcription at the nc 14 interphase. To test this possibility, quantitative hb mRNA FISH was performed in wt and dmpd embryos with an exclusive focus on nc 13. Embryos at this stage already have a significant accumulation of hb mRNA suitable for quantitative measurements that are necessary for effective comparisons between wt and dmpd embryos. In addition and importantly, active hb transcription is known to span the entire interphases prior to nc 14. Thus, if the defect of dmpd embryos is specific to hb shutdown at the nc 14 interphase, hb mRNA level should remain unchanged prior to this shutdown-that is, at nc 13. This prediction is supported the results. An implication of these findings is that altering the hb expression amplitude at, and only at, the last interphase (nc 14) prior to cellularization and gastrulation can still have an impact on the AP-patterning outcome, suggesting that this interphase represents a critical time period in making patterning decisions forward (Liu, 2013).

An important feature of developmental systems is that their desired spatial properties must be attained within the allotted periods of time when an entire system is progressing along the irreversible temporal axis. This interconnection between the spatial and temporal aspects of the developmental systems poses significant constraints on their operation. In Drosophila, the early embryo undergoes rapid cycles of nuclear division. This poses a constraint on the transcription process itself and the decoding of maternal gradient inputs, since mitosis is known to abort transcription. It has been documented that active hb transcription can resume almost immediately upon entering the interphase in the blastoderm embryo; however, it remains unknown precisely how this can be achieve. For developmental systems evolving rapidly along the temporal axis such as the early Drosophila embryo, patterning decisions may need to be made before true steady states could be achieved. In a recent study, it was shown that how quickly a gene can resume efficient transcription upon entering the nc 14 interphase can affect the amount (the amplitude) of the gene products at a later time when such products are needed for action. Thus, it was shown that a slowed onset of snail transcription led to gastrulation defects. In the case of dmpd mutation investigated in this report, while the onset of active hb transcription upon entering the nc 14 interphase is unaffected, a hastened shutdown reduces the amplitude of hb expression products, a defect that alters the spatial characteristics of the patterning outcome. Together, these two latest examples of dynamic regulation of transcription illustrate the importance of understanding the actual transcriptional decisions in a developmental system through the prism of time, an area of research that has only begun to be explored (Liu, 2013).

Three recent studies have reported investigations of the dynamics of transcription in early embryos. Two of the studies were based on a live-imaging technique using the MS2. For evaluating the onset of transcription upon entering an interphase, the use of this system requires an adjustment by the delay between transcription initiation and detection of fluorescent signals at the reporter locus, as dictated by the time necessary for RNA polymerase to transcribe through the MS2 stem loop repeats and for the MS2 coat protein-green fluorescent protein to bind to these RNA repeats. With this and detection limit-imposed delay adjustments, both of the live-imaging studies are consistent with a quick onset of Bcd-dependent transcription initiation upon entering an interphase as documented in the current and previous studies. A live-imaging study also supports a role of Bcd in directly lengthening the time period of active transcription during an interphase. At the nc 14 interphase, two studies support a shutdown of hb. Importantly, an observation that a reporter gene driven by the ~250-bp Bcd-responsive hb enhancer element is shut down at nc 14 in a manner that is broadly similar to the endogenous hb shutdown is consistent with the documented role of Dmpd and the activating potency of Bcd in influencing this shutdown process (Liu, 2013).

A contribution of the current work is the establishment of a quantitative platform for specifically (and simultaneously) analysing the amplitude and expression boundary of a target response to the Bcd gradient input. Under the current experimental framework, these two parameters are primarily subjected to regulation by two distinct mechanisms. The results document that Dmpd has a role in enhancing the activating potency of Bcd as an activator and in regulating the AP-patterning outcome. Interestingly, a detectable, although small, posterior shift in the hb expression boundary (xhb) in dmpd embryos, suggests that Dmpd may have regulatory roles beyond its primary role of regulating the amplitude of hb expression. This posterior shift in xhb cannot be simply explained by the Bcd gradient profile properties because a smaller B0 (Bcd concentration at the anterior) in dmpd embryos, if biologically meaningful, would have predicted a small shift in xhb towards the anterior. This small shift is detectable in embryos not yet exhibiting a significant sign of PS4 expression, suggesting that it is related to Bcd-dependent hb transcription. It remains to be determined mechanistically whether Dmpd may have a meaningful role in regulating the affinity of Bcd for the hb enhancer during development. It is anticipated that, as more experimental tools are developed and more regulatory players are identified, it will be able to further improve mechanistic knowledge about how the AP-patterning network operates in space and time at an even finer resolution and precision (Liu, 2013).

In silico evolution of the hunchback gene indicates redundancy in cis-regulatory organization and spatial gene expression

Biological development depends on the coordinated expression of genes in time and space. Developmental genes have extensive cis-regulatory regions which control their expression. These regions are organized in a modular manner, with different modules controlling expression at different times and locations. Both how modularity evolved and what function it serves are open questions. This paper presents a computational model for the cis-regulation of the hunchback (hb) gene in the fruit fly (Drosophila). Evolution was simulated (using an evolutionary computation approach from computer science) to find the optimal cis-regulatory arrangements for fitting experimental hb expression patterns. The cis-regulatory region was found to tend to readily evolve modularity. These cis-regulatory modules (CRMs) do not tend to control single spatial domains, but show a multi-CRM/multi-domain correspondence. The CRM-domain correspondence seen in Drosophila evolves with a high probability in the model, supporting the biological relevance of the approach. The partial redundancy resulting from multi-CRM control may confer some biological robustness against corruption of regulatory sequences. The technique developed on hb could readily be applied to other multi-CRM developmental genes (Zagrijchuk, 2014).

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).

Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo

The Drosophila embryo at the mid-blastula transition (MBT) experiences a concurrent receding of a first wave of zygotic transcription and surge of a massive second wave. It is not well understood how genes in the first wave become turned off transcriptionally and how their precise timing may impact embryonic development. This study perturbed the timing of the shutdown of Bicoid (Bcd)-dependent hunchback (hb) transcription in the embryo through the use of a Bcd mutant that has a heightened activating potency. A delayed shutdown increases specifically Bcd-activated hb levels that alter spatial characteristics of the patterning outcome and cause developmental defects. This study thus documents a specific participation of the maternal activator input strength in timing molecular events in precise accordance with the MBT morphological progression (Liu 2015).

A fundamental feature of animal development is the control of gene expression to achieve specific spatial and temporal patterns in a highly coordinated way. There are two aspects of the temporal dynamics of a gene's transcription in a developmental system, its onset and duration. Proper control of the temporal dynamics of transcription is particularly important for developmental systems that progress rapidly, such as the early Drosophila embryo. It is well known that alterations in transcription activation of early zygotic genes can cause morphological defects in the Drosophila embryo. The results show that the timing of hb transcription shutdown is associated with the MBT and can be perturbed specifically in embryos containing a Bcd mutant defective in sumoylation. A postponement of hb shutdown at nc14 can elongate the duration of active transcription, leading to increased levels of hb gene products and patterning defects. These results show that the precise timing in the shutdown phase of Bcd-activated hb transcription at nc14 is important for normal development. The effects of the Bcd sumoylation mutant on hb shutdown are highly specific, and they are restricted to the shutdown phase (without affecting the onset phase) only at nc14 and only on Bcd-activated hb transcription. It should be noted that the current results do not show that Bcd sumoylation is a temporally regulated event during the MBT, although it represents an attractive possibility that remains to be tested in the future. Temporally regulated Bcd sumoylation could directly account for the temporally restricted effects of the Bcd mutant, but 'constitutive' Bcd sumoylation can also exert time-dependent actions in association with the global events of the system (e.g., mitotic cycles and morphological progression of the MZT) (Liu 2015).

The results provide new insights into how Bcd-activated hb transcription becomes shut down at nc14. Evaluations of the bcd6-lacZ reporter gene demonstrate that neither the P2 promoter of hb nor any of its cis-regulatory elements is required for the shutdown of Bcd- activated transcription at nc14. Importantly, hb shutdown takes place at a time when the Bcd concentration gradient remains intact. It has been suggested that specific pathways can become activated at the MBT to cause a quick degradation of maternal proteins such as Twine. If hb shutdown were to merely reflect a decaying Bcd gradient at nc14, a shutdown process would be expected that initiates near mid-embryo (where Bcd concentration is low) and 'spread' toward the anterior (with increasing Bcd concentrations). But the results do not support this prediction. In addition, neither the length constant nor the amplitude of the Bcd gradient profile is affected by the Bcd mutation. Thus the results show that the timing of Bcd-activated transcription during nc14 does not require either a physical disappearance of this maternal activator or the accumulating activities of sequence-specific zygotic repressors. Instead it is the functional potency of the maternal activator Bcd that is a part of the mechanism in timing the molecular events in accordance with the MZT morphological progression. Importantly, the potency of Bcd activator can be either strengthened (this paper) or weakened to tune--in opposite directions--the hb shutdown timing and hb expression level. It is noted that the bcd6-lacZ reporter results do not formally exclude the possibility that the shutdown of Bcd-activated transcription at nc14 involves a zygotic repressor(s) that operates by competing with Bcd binding to its DNA sites. But this possibility is not favored because the position-independent and quick features of the shutdown event would likely require an unknown zygotic repressor(s) not only to have the same/overlapping Bcd binding specificity but also to accumulate in a spatially non-restricted (i.e., covering the entire hb expression domain) and temporally sudden way at nc14 (Liu 2015).

As part of the receding of the first wave of zygotic transcription in association with the MBT, hb is among a group of genes that exhibit a shutdown phase at nc14. These genes play key roles in different processes that are ongoing during the MBT, suggesting a possibility that a global or general mechanism may regulate the shutdown events of transcription in a coordinated manner. This study shows that the timing of the shutdown can be postponed by an elevated activating potency of Bcd, but only to a degree. For genes that are activated by combinatorial sets of maternal inputs, it remains to be determined whether it is also the activators' functions, as opposed to protein availability, that are regulated during transcription shutdown at the MBT. Genes that are transcriptionally active prior to the MBT tend to share promoter features that are distinct from those of the genes that become activated during the MBT. An intriguing possibility exists where the MBT might be associated with a systematic change in the composition of the transcription machinery. But the fact that a synthetic reporter containing a different core promoter also exhibits a shutdown phase at nc14 indicates that the hb P2 promoter is not required (Liu 2015).

A recent study reveals an interplay between zygotic transcription and DNA replication at the MBT. It has been proposed that euchromatin DNA is replicated within a few minutes into the nc14 interphase. Thus the DNA replication time coincides broadly with the time of hb shutdown, raising a question of whether DNA replication at the hb locus might trigger its transcription shutdown. A single embryo was captured in which nuclei contain more than two intron dots. The existence of nuclei with more than two dots is a positive indicator of DNA replication at the hb gene locus. The strong intron staining detectable at the anterior part of the embryo indicates that Bcd-activated hb transcription has not yet been turned off (nuclear height measurements suggest that this embryo belongs to time class t2). These results thus suggest that DNA replication at the hb locus does not directly trigger its transcription shutdown at nc14 (Liu 2015).

Sumoylation is a posttranslational modification that regulates a variety of biological processes through mechanisms that may involve protein-protein interactions, subcellular localization, and protein stability. From the perspective of developmental biology, many transcriptional activators with important developmental roles are substrates of sumoylation. It has been reported that sumoylation of Medea (Med), an intracellular transducer of Drosophila morphogen Decapentaplegic (Dpp), triggers Med nuclear export and therefore, restricts the range of the Dpp signaling. The lengthening of the duration of Bcd-activated hb transcription caused by the Bcd sumoylation mutation increases the amplitude of hb expression without extending its expression boundary. Thus, sumoylation of proteins involved in morphogen functions can alter either the action range (in space) or the output level (due to action time). In yeast, sumoylation has been suggested to play a role in terminating inducible activation events by evicting activator molecules from promoters. For example, disruption of Gcn4 sumoylation can extend its promoter association and increase the expression level of the target gene ARG1. Whether sumoylation of Bcd plays a mechanistically equivalent role in evicting Bcd molecules from the hb enhancer at nc14 remains an open question and speculative possibility (Liu 2015).

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

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


hunchback: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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