Promoter Structure

The runt promoter has a TATA box (Kania, 1990). Runt has an extended cis-regulatory region. There are multiple elements that make quantitative contributions to runt regulation during segmentation. Sequences that are more than 8.5 kb upstream of the runt promoter are necessary for normal expression during the post-blastoderm stages of embryogenesis (Butler, 1992).

Analysis of the runt promoter reveals three different modes of regulation responsible for segmental expression. Upstream elements resemble the stripe-specific enhancers of even-skipped. The interval between -14 and -9 kb contains site specific elements for stripes 1, 3 and 5. Gap genes giant, hunchback, knirps, and Krüppel function as the major input for these stripe specific enhancers. A second mode of regulation is carried out by DNA extending 5 kb upstream of the transcription initiation site. Termed the "7-stripe region", this interval reveals few distinct subelements, suggesting that regulatory sites responsible for early stripe formation are spread diffusely over the whole region. The pair-rule gene even-skipped shows the strongest input for the expression regulated by the 7-stripe region, with additional contributions of bicoid and tailless. Of the pair-rule gene regulatory elements identified so far, the one most similar to the runt 7-stripe region is the zebra element of fushi tarazu. A third mode of regulation is carried out by a late element between +3 and +6 kb (i.e., downstream of the runt transcription start site). This "six-stripe element" is responsible for the narrow late stripes appearing between the 7 early stripes, giving rise to the late segment polarity pattern of runt expression. The 6-stripe element is not an independent enhancer, as it requires promoter-proximal sequences (Klingler, 1996).

Pair-rule genes serve two important functions during Drosophila development: they first initiate periodic patterns, and subsequently interact with one another to refine these patterns to the precision required for definition of segmental compartments. A pair-rule input region of the runt gene characterizes this through the use of reporter gene constructs and by comparison with corresponding sequences from Drosophila virilis. Many but not all regulatory properties of this '7-stripe region' are functionally conserved. All primary pair-rule genes (hairy, eve and runt itself) are known to affect the expression from this element. Nevertheless, the interspecies comparision reveals that the conservation between homologous sequences is surprisingly low. Fourteen conserved blocks can be identified which together comprise 419 bp, i.e. 6.7% of the total sequence. The largest of these blocks encompasses the basal promoter region with the transcription start site and TATA box. Without the TATA box area, i.e. when just regarding the upstream region, only 346 bp appear to be conserved, which corresponds to 5.6% of the total. Hairy and Bicoid are shown to bind to conserved sequence blocks in vitro, and Tailless binding sites are also conserved. While expression in the early central domain and the early stripe pattern are largely conserved between the 7-stripe regions of the two species, this is not the case for the narrowing of the stripes and the transition to a segmental expression. This function change in the upstream region of the runt genes in the two species is reflected by the phylogenetic footprinting results, which do not identify conserved sites in the sequence that mediates this regulation in Drosophila melanogaster. When compared to similar data for gap gene input elements in eve and hairy, these data suggest that pair-rule target sequences of runt are less constrained during evolution, and that functional elements mediating pair-rule interactions can be dispersed over many kilobases (Wolff, 1999).

With respect to runt's function in segmentation, promoter constructs identify two subelements that are essential for the stripe pattern. (1) A general activating sub-element is situated close to the TATA box, between -0.7 and -0.1 kb. Constructs without the DNA lack expression in stripes almost completely, and they also do not form an early central domain. (2) Sequences between -.07 and -1.3 kb negatively regulate the stripe pattern during gastrulation. Experiments suggest that the DNA between -1.2 and -.07 kb mediates the inhibitory effects of fushi tarazu on runt expression. At later stages of development, the 7-stripe region also gives rise to neural expression, first in head spots, and also at the fully extended germ band stage in the ventral nerve cord. Both of these expression aspects can be attributed to distinct elements. The regulation in the CNS is provided by DNA between 5 and 6 kb upstream of the transcription start site, and the head spot expression by DNA between -1.3 and -1.9 kb. In both cases, expression does not require the presence of the general activating element immediately upstream of the basal promoter. These regulatory regions are functionally conserved, for the most part, in D. virilis. Nevertheless, the subelements mediating repression by ftz and activation in the head spots appear not to be conserved (Wolff, 1999).

How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation.

The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The 'primary' pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas 'secondary' pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, a comprehensive systems-level examination was conducted of the regulatory architecture underlying pair-rule stripe formation. runt (run), fushi tarazu (ftz) and odd skipped (odd) were found to establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, long-range cis-regulation was observed across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, this study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo (Schroeder, 2011).

The transition from non-periodic to periodic gene expression patterns is a key step in the establishment of the segmented body plan of the Drosophila embryo. The pair-rule genes that lie at the heart of this process have been the subject of much investigation, but important questions, in particular regarding the organization of cis-regulation, have remained unresolved. We have revisited the issue in a comprehensive fashion by combining computational and experimental cis-dissection with mutant analysis and a detailed characterization of expression dynamics, both of endogenous genes and of cis-regulatory elements. This systems-level analysis under uniform conditions reveals important insights and gives rise to a refined and in some respects substantially revised view of how periodic patterns are generated (Schroeder, 2011).

The most important finding of this study is that ftz and odd, which had been regarded as secondary pair-rule genes, closely resemble eve, h and run in nearly all respects and should thus be co-classified with them as primary pair-rule genes. Expression dynamics clearly subdivide the pair-rule genes into two distinct groups, with eve, h, run, ftz and odd all showing an early and non-synchronous appearance of most stripes, whereas the 7-stripe patterns of prd and slp1 arise late and synchronously. The early expression of stripes is associated with the existence of stripe-specific cis-elements that respond to positional cues provided by the maternal and gap genes. Unlike previously thought, maternal and gap input is used pervasively in the initial patterning not only of eve, h and run, but also of ftz and odd. Aided by computational predictions, eight new stripe-specific cis-elements were identifed for ftz, odd, and run. Although molecular epistasis experiments reveal a stronger role for eve, h and run in pair-rule gene cross-regulation, odd clearly affects the blastoderm patterning of other primary factors as well and ftz affects the patterning of odd (Schroeder, 2011).

The revised grouping brings the classification of pair-rule genes in line with their role in transmitting positional information to the subsequent tiers in the segmentation hierarchy. By the end of cellularization, the expression patterns of h, eve, run and ftz/odd are offset against each other to produce neatly tiled overlaps of four-nuclei-wide stripes. In combination, these patterns define a unique expression code for each nucleus within the two-segment unit that specifies both position and polarity in the segment and is read off by the segment-polarity genes. ftz (as an activator of odd) and odd together represent the fourth essential component of this positional code and are thus functionally equivalent to h, eve and run. Placing all components of this code under the same direct control of the preceding tier of regulators presumably increases both the speed and the robustness of the process (Schroeder, 2011).

Although the results support a subdivision between primary and secondary pair-rule genes, they also reveal surprising complexities in the regulatory architecture that are not captured by any rigid dichotomy. The five primary pair-rule genes all have different cis-regulatory repertoires: stripe formation in h appears to rely solely on stripe-specific cis-elements, whereas eve employs a handoff from stripe-specific elements to a late-acting 7-stripe element. In run, ftz and odd, by contrast, the emergence of the full 7-stripe pattern is the result of joint action by stripe-specific elements and early-acting 7-stripe elements, with ftz and odd requiring the 7-stripe element to generate a subset of stripes. This difference in the importance of 7-stripe elements in stripe formation is supported by the results of rescue experiments: whereas the 7-stripe elements of both run and ftz provide partial rescue of the blastoderm expression pattern when the stripe-specific elements are missing, this is not the case for eve. Interestingly, run appears to occupy a unique and particularly crucial position among the five genes: it has both a full complement of stripe-specific elements and an early-acting 7-stripe element, and thus serves as an early integration point of maternal/gap input and pair-rule cross-regulation. Its regulatory region is particularly large and complex, with cis-elements acting over long distances across intervening genes and with partial redundancy between elements. Moreover, the removal of run has the most severe effects on pair-rule stripe positioning among the five genes. Another complexity lies in the fact that the early anterior expression of slp1 and prd, which otherwise exhibit all the characteristics of secondary pair-rule genes, has a clear role in patterning the anteriormost stripes of the primary pair-rule genes. In fact, evidence is provided that the most severe defect observed in the primary pair-rule gene mutants, namely the loss of pair-rule gene stripes 1 or 2 under eve loss-of-function conditions, is an indirect effect attributable to the regulation of these stripes by slp1 (Schroeder, 2011).

This investigation provides important new insight regarding the relative roles of maternal/gap input and pair-rule gene cross-regulation in stripe formation. As described, nearly all stripes of the primary pair-rule genes are initially formed through stripe-specific cis-elements. Cross-regulatory interactions between the pair-rule genes then refine these patterns by ensuring the proper spacing, width and intensity of stripes, as revealed by mutant analysis. However, these two aspects or layers of regulation are not as clearly separated as has often been thought. For example, 7-stripe elements have been regarded primarily as conduits of pair-rule cross-regulation; by contrast, this study found that the 7-stripe elements of run, ftz and odd are activefrom phase 1 onwards and contain significant input from the maternal and gap genes, resulting in modulated or partial 7-stripe expression early. In the case of the KR binding sites predicted within the run 7-stripe element, mutational analysis showed that this input is indeed functional. Conversely, stripe-specific cis-elements receive significant regulatory input from pair-rule genes. This is particularly clear in the case of eve and h: expression dynamics indicate that their 7-stripe patterns become properly resolved without the involvement of 7-stripe elements, and the defects in the h and eve expression patterns that were observe in pair-rule gene mutants occur at a time when only stripe-specific elements are active. In a few cases, pair-rule input into stripe-specific elements has been demonstrated directly, but the comparative analysis of expression dynamics underscores that it is indeed a pervasive feature of pair-rule cis-regulation (Schroeder, 2011).

An important question arising from these findings is how the different types of regulatory input are integrated, both at the level of individual cis-elements and at the level of the locus as a whole. Binding sites for maternal and gap factors typically form tight clusters, supporting a modular organization of cis-regulation. Such modularity is crucial for the 'regional' expression of individual stripes, which can be achieved only if repressive gap inputs can be properly separated between cis-elements. By contrast, binding sites for the pair-rule factors appear to be more dispersed across the cis-regulatory regions and not tightly co-clustered with the maternal and gap inputs. Unlike the gap factors, which are thought to act as short-range repressors, with a range of roughly 100 bp, most pair-rule genes act as long-range repressors, with an effective range of at least 2 kb. Pair-rule factor inputs may therefore influence expression outcomes at greater distances along the DNA, which would allow a single cluster of binding sites to affect multiple cis-elements. Remarkably, the 7-stripe elements of ftz, run, eve, prd and odd all combine inputs from promoter-proximal and more distal sequence over distances of at least 5 kb, and, is seen in the case of odd, the combination can involve non-additive effects. Thus, the refinement of expression patterns through pair-rule cross-regulation might rely on interactions over greater distances, consistent with the 'global' role of pair-rule genes as regulators of the entire 7-stripe pattern. How such interactions are realized at the molecular level and how the inputs from stripe-specific and 7-stripe elements are combined to produce a defined transcriptional outcome is currently unknown (Schroeder, 2011).

Taken together with previous studies, these data support a coherent and conceptually straightforward model of how stripes are made in theDrosophila blastoderm. In the trunk region of the embryo, the maternal activators BCD and CAD form two overlapping but anti-correlated gradients; they coarsely specify the expression domains of region-specific gap genes, which become refined by cross-repressive interactions among the gap factors. The result is a tiled array of overlapping gap factor gradients, centered around the bilaterally symmetric domains of KR and KNI, which are flanked on either side by the bimodal domains of GT and HB. The same basic principles are used again in the next step: following the initial positioning of stripes by maternal and gap factor input, cross-repressive interactions among the primary pair-rule genes serve to refine the pattern and ensure the uniform spacing and width of expression domains. The significant correlations that were observe between the strength of KR/KNI input and the position of the resulting stripes relative to the respective gradients supports the notion that these factors function as repressive morphogens in defining the proximal borders of the nascent pair-rule stripes. Importantly, owing to the symmetry of the KR and KNI gradients, combined with the largely symmetric positioning of the flanking GT and HB gradients, the same regulatory input can be used to specify two distinct positions, one on either slope of the gradient; this is exploited in a systematic fashion by dual-stripe cis-elements, which account for the majority of stripe-generating elements. Thus, the key to stripe formation is the translation of transcription factor gradients into an array of narrower, partially overlapping expression domains that are stabilized by cross-repressive interactions; the iteration of this process, combined with the duplication of position through bilateral gradient symmetry, creates a periodic array of stripes that is sufficient to impart a unique identity to each nucleus in the trunk region of the embryo (Schroeder, 2011).

With the exception of the anteriormost pair-rule gene stripes, which are subject to more complex regulation (as evidenced by the crucial role of slp1), this model accounts for most of the stripe formation process. Particularly striking is the similarity between the central gap factors and the primary pair-rule factors with respect to regulatory interactions and expression domain positions. The offset arrangement of two pairs of mutually exclusive gap domains, KNI/HB and KR/GT, is mirrored at the pair-rule gene level, with the anti-correlated and mutually repressive stripes of HAIRY and RUN phase-shifted against the similarly anti-correlated expression patterns of EVE and ODD. The parallel suggests that this regulatory geometry is particularly suited to robustly specify a multiplicity of positions. However, such a circuitry of cross-repressive relationships is compatible with a range of potentially stable expression states and thus is insufficient by itself to uniquely define position along the anterior-posterior axis. Therefore, the initial priming of position by the preceding tier of the regulatory hierarchy is crucial. At the level of the pair-rule genes, the extensive repertoire of maternal/gap-driven cis-elements that initiate stripe expression for all four components of the array is thus necessary to ensure that the cross-regulatory dynamics will drive the correct overall pattern. Finally, to achieve a smooth transition between the tasks of transmitting spatial information from the preceding tier of the hierarchy and refinement/stabilization of the pattern, the two layers of regulation need to be closely integrated. This is likely to be facilitated by the fact that in each of the mutually repressive pairs, one gene generates stripes purely through stripe-specific elements (h, eve), whereas the other has an early-acting 7-stripe element that mediates pair-rule cross-repression and acts concurrently with stripe-specific elements (run, odd) (Schroeder, 2011).

Stripe positioning is not always symmetric around the central gap factor gradients. In such cases, the conflicting needs for appropriate regulatory input into the relevant cis-elements appear to have driven the separation of ancestral dual-stripe elements into more specialized elements optimized for generating a single stripe. The co-localization of the cis-elements that generate stripes 1 and 5 within all pair-rule gene regulatory regions, be it in the form of dual-stripe elements or of adjacent but separable single-stripe elements, provides strong evidence that this is indeed a key mechanism underlying the emergence of single-stripe cis-elements. Given the evolutionary plasticity of regulatory sequence, it is not difficult to envision such a separation and subfunctionalization of cis-elements. How the crucial transition from a non-periodic to periodic pattern is achieved in other insects is a fascinating question. Intriguingly, most of the molecular players and general features of their expression patterns are well conserved beyond Diptera, and it is tempting to speculate that dual-stripe cis-regulation might have arisen through co-option as the blastoderm fate map shifted to include more posterior positions. It will be interesting to see to what extent the mechanisms and principles of stripe formation that we have outlined here apply in other species (Schroeder, 2011).

Huckebein is part of a combinatorial repression code in the anterior blastoderm

The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated in this study, led to a hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. This study investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods deviations from the wild-type patterns of the anterior-most pair-rule stripes were detected in different genetic backgrounds, consistent with Hkb-mediated repression. Moreover, an image processing tool was developed that, for the most part, confirmed the assumptions. Using an hkb misexpression system, specific repression on anterior stripes was detected. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1 when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes (Andrioli, 2012).

The aim of this study was to understand the mechanisms underlying the regulation of the anterior pair-rule stripes. The model tested was first proposed for eve 2 regulation. Transcriptional activators do not give enough patterning information, and the presence of repressors is instructive for determining the precise positioning of a particular stripe. The hypothesis was that transcription repressors could be working in a combinatorial manner to determine the correct positioning of the anterior stripes and prevent, in a spatial and temporal manner, the expression of stripe CRMs in the more anterior regions of the embryo by counteracting the activity of activators. There is plenty of evidence supporting this hypothesis, which was further confirmed in this study (Andrioli, 2012).

Regarding activators, computational analysis predicted Bcd, Hb and Btd binding sites are part of significant clusters in the anterior-most stripe CRM. These predictions agree well with previous genetic data and in vivo DNA binding data from ChIP/chip experiments. Thus, Btd, and above all the widely spread maternal factors Bcd and Hb, might activate anterior stripe CRMs early in the anterior blastoderm. Alternatively, the early broad expression patterns of pair-rule genes could be under the control of dedicated CRMs, although no such elements have yet been reported. It is possible that other regulatory elements could contribute to the expression detected early in the anterior blastoderm, for instance, the CRM responsible for the expression of h head patch or the CRMs responsible for eve 3, eve 5 and h 5, which were proposed to be activated by the maternal factor DSTAT (Drosophila Signal Transducer and Activator of Transcription), which is ubiquitously expressed in the embryo (Andrioli, 2012).

The expression of several gap domains covering all of the anterior regions of the embryo ahead of the seven-striped patterns is consistent with the expected subsequent local repression of pair-rule CRMs activated in the head region. Of these gap domains, Slp1 is a common repressor for anterior pair-rule stripes, but other repressors besides Slp1 were predicted to be necessary for correctly determining the borders of the anterior-most stripes. This study investigated hkb, which, in addition to tll, is the other major gap gene target of the Torso signaling regulation in the terminal system. In the anterior region, hkb is required for the proper formation of the foregut and midgut. Its domain at the anterior tip coincides with the region where the diffused early expression patterns of pair-rule genes first fade. These observations are consistent with local repression roles of Hkb. However, it was not possible to detect derepression of pair-rule genes in the anterior pole of hkb- embryos. One possibility is that the progressive non-detection of the expression of pair-rule genes might correspond to a failure in activation. In fact, Bcd activation was shown to be down-regulated by the Torso-signaling cascade at the anterior tip. Nevertheless, other data suggest that the Torso pathway might induce a repression mechanism at the anterior tip that would be parallel and redundant with Torso-induced inhibition of Bcd. Thus, one might predict that another repressor might still able to act on Hkb targets in the absence of Hkb protein (Andrioli, 2012).

Although no pair-rule derepression was detected in the anterior pole, it was possible to detect subtle deviations in the positioning of eve 1 in hkb- embryos, which was confirmed by morphological measurements using the image processing tool. Enhanced derepression effects were also detected for all anterior-most stripes investigated in slp-;hkb- double-mutant embryos compared to the effects observed in slp- embryos; these results were statistically significant. With the hkb misexpression system, repression effects were detected for h 1, eve 1, run 1 and ftz 1. With the exception of gt repression, no other gap domain disruption was detected in these assays. These results strongly suggest direct repression by Hkb on the CRMs of these stripes. In vivo binding data confirms this possibility. Moreover, with the bioinformatics analysis it was verified that Hkb, along with putative activators, increased the already high significance values of predicted clusters for activators that match these stripe CRMs. Therefore, the combined data suggest that Hkb acts as a repressor for a specific group of anterior pair-rule stripes (Andrioli, 2012).

These data also suggest that there is another possible mechanism underlying the repression that involves the activity of repressors further away from their original sources. One example of this mechanism is expression detected for the ectopic hkb domain, demonstrating that target CRMs are sensitive to Hkb-mediated repression even in the presence of low expression levels of Hkb. The prediction is that low concentrations of Hkb that have diffused away from its endogenous domain could still repress these CRMs. For this mechanism, repressors could fulfill additive repression roles at different anterior subdomains or even contribute to the definition of the anterior borders of stripes that are distantly positioned from where gap domains are detected. Thus, the increased derepression observed in slp-;hkb- embryos would be expected if a combinatorial additive mechanism existed in which each repressor had a small contribution to the overall repression. Following the same rationale, one can predict that at least one other repressor is still responsible for setting anterior border stripes in slp-;hkb- embryos (Andrioli, 2012).

The complexity of the regulation of genes involved in early patterning was postulated to be a condition that is necessary for sensing relatively small differences in the concentrations and combinations of many regulatory factors, which is likely the environment found in the syncytial blastoderm. In agreement with that hypothesis, recent studies revealed that the protein gradients of factors such as Bcd and Dorsal alone are not sufficient to determine all of the spatial limits of target gene expression and that these gradients might combine with other factors to pattern the early embryo. In the head region, it has been suggested that Bcd and the terminal system-mediated activities interact at the level of the target CRMs to generate the proper patterning for the head region of the embryo. In contrast to these studies that focused on gap genes, the current data shed light on a mechanism that is involved in the regulation of the anterior stripe CRMs, with the putative participation of hkb (Andrioli, 2012).

The correct positioning of the anterior pair-rule stripes must be a critical issue in the early developmental patterning of the fly. Even a slightly incorrect positioning of the anterior stripes, for instance, results in the non-formation of the mandibular segment in the slp null mutant. Thus, a complex repression mechanism is necessary to shape the stripes and to avoid inappropriate expression of their CRMs. Therefore, Hkb, Slp1 and other repressors are likely involved in a combinatorial repressive activity in the CRMs of the anterior stripes. Other experiments are necessary to test this hypothesis further and to reveal the underlying molecular mechanisms involved in this regulation (Andrioli, 2012).

Transcriptional Regulation

There are several distinct phases of runt expression in the early embryo. Each phase depends on a different set of regulators. The first phase of expression is a broad-field of mRNA accumulation in the central regions of syncytial blastoderm stage embryos. This pattern is due to terminal repression by the anterior and terminal maternal systems. The effect of the terminal system, even at this early stage, is mediated by two zygotic gap genes, tailless and huckebein. A 7 stripe pattern of Runt mRNA accumulation emerges during the process of cellularization. The initial formation of this pattern depends on position-specific repression by zygotic gap genes.

In a second phase, three pair-rule genes, hairy, even-skipped, and runt itself, affect Runt's 7 stripe pattern. The autoregulatory effects of runt are stripe specific; the effects of hairy are more uniform; and the patterns obtained in even-skipped mutant embryos show a combination of both stripe specific and uniform regulatory effects.

In a third distinct phase of expression, at the onset of gastrulation, runt is expressed in 14 stripes . fushi tarazu plays a negative regulatory role in generating this pattern, whereas the pair-rule genes paired and odd-paired are required for either activating or maintaining runt expression during these stages (Klingler, 1993).

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 (Kosman, 1997).

It is likely the KNI functions as a repressor to set the posterior border of eve stripe three. To test whether the early repression of eve stripe 3 is mediated through the eve stripe three enhancer, stripe 2-kni constructs were crossed with a line carrying lacZ under the control of this enhancer. Ectopic kni specifically represses the stripe 3 enhancer in a dose-dependent manner. Stripe 2-kni causes disruption of runt stripes 2 and 3, but has no effect on stripe 1. The repression of stripe 3 increases in proportion to the level of ectopic kni, a response similar to that seen for eve stripe 3. Different levels of ectopic kni cause disruptions of fushi tarazu stripes 2 and 3, but have no effect on the expression of ftz stripe 1. It is possible that these effects are indirect and may be mediated through other segmentation genes but this possibility is made unlikely by the fact that hairy expression is virtually unaffected in stripe 2-kni embryos. These results suggest that the ectopic domain of kni acts as a source for morphogenetic activity that specifies regions in the embryo where pair-rule genes can be activated or repressed. Evidence is presented that the level and timing of expression, as well as protein diffusion, are important for determining the specific responses of target genes (Kosman, 1997).

The early bell-shaped gradient of even-skipped expression is sufficient for generating stable parasegment borders. The anterior portion of each early stripe has morphogenic activity, repressing different target genes at different concentrations. These distinct repression thresholds serve to both limit and subdivide a narrow zone of paired expression. Within this zone, single cell rows express either engrailed, where runt and sloppy-paired are repressed, or wingless, where they are not (Fujioka, 1995).

Although many of the genes that pattern the segmented body plan of the Drosophila embryo are known, there remains much to learn in terms of how these genes and their products interact with one another. Like many of these gene products, the protein encoded by the pair-rule gene odd-skipped (Odd) is a DNA-binding transcription factor. Genetic experiments have suggested several candidate target genes for Odd, all of which appear to be negatively regulated. Pulses of ectopic Odd expression have been used to test the response of these and other segmentation genes. Three different phenotypes are generated in embryos in which odd is expressed from a heat shock promoter: head defects only, a pair-rule phenotype and a pair-rule phenotype restricted to the dorsal half of the embryo. The head defects only phenotype prevails when Odd is induced between 2:10 and 2:30 hours after egg laying (AEL). The second phenotype is generated when Odd is induced between 2:30 and 2:50 AEL, while the third phenotype prevails when heat shocks are administered between 2:50 and 3:10 AEL. The results are complex, indicating that Odd is capable of repressing some genes wherever and whenever Odd is expressed, while the ability to repress others is temporally or spatially restricted (Dréan, 1998).

Two of the seven pair-rule genes tested do not show significant changes in expression at the stages examined. These include the genes odd-paired (opa) and, surprisingly, ftz. In odd minus embryos, ftz stripes do not resolve properly, remaining about 3 cells wide until well into the process of germ band extension. This suggests that Odd may be a repressor of ftz. However ectopic Odd does not repress ftz expression. Also unexpected was the fact that ectopic Odd has effects on all three of the ‘primary’ pair-rule genes. These were previously thought not to be regulated by Odd. In stage 5 embryos, stripe 1 of hairy is efficiently repressed by ectopic Odd. The first stripe of eve is also repressed at this stage. Repression of h stripe 1 continues in older embryos and is accompanied by weaker repression of stripes 2-6. These effects of Odd on h correlate with what appears to be a modest broadening of h stripes in odd-minus embryos, particularly stripe 1. Early repression of the first stripes of h and eve likely accounts for the cuticular head defects that arise from early pulses of ectopic Odd expression. Interestingly, in odd-minus embryos, the entire 7-stripe pattern of h appears to expand, both anteriorly and posteriorly. This is also true of eve and runt stripes. These data provide no explanation for this, but it may explain the fairly consistent spacing of h stripes, despite their apparent broadening (Dréan, 1998).

The gene, hopscotch (hop), the Drosophila JAK kinase homolog, is required maternally for the establishment of the normal array of embryonic segments. In hop mutant embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu. The effect of hop on the expression of these genes is stripe-specific (Binari, 1994).

Ectopic expression of the 69 kDa TTK protein significantly represses even-skipped, odd-skipped, hairy and runt. The 88 kDa form does not act to repress these genes (Brown, 1993).

Pair-rule gene expression is disrupted in Dichaete mutants. Expression of the gap genes Krüppel, knirps, and giant are normal, indicating that Dicaetae acts in parallel or downstream of these gap genes. the so-called primary pair-rule gene even-skipped, Hairy, and runt each show reductions in levels of expression in Dichaete mutants, with variable stripe specific effects on eve, fushi tarazu, hairy and runt. Since the stripes of pair rule genes generally occur in the correct anterior-posterior position in Dichaete mutants, the gene is unlikely to provide key positional information; it is more likely to be required in the maintainance or establishment of appropriate levels of pair-rule gene expression in the central region of the embryo (Russell, 1996 and Nambu, 1996).

Neuroblast expression of runt is restricted to single cells through the action of the Notch pathway (Kania, 1990).

At least some aspect of dosage compensation is not carried out by Male-specific-lethals, including MSL-2. Early runt dosage compensation is directed by the product of the early promoter of Sex lethal. Thus the early transcripts of Sex lethal have a role in addition to splicing, that is, in directing the early stages of dosage compensation. runt dosage compensation is a consequence of early Sex lethal expression in females. Since MSL-1 and MSL-2 begin to associate with the X chromosome during the cellular blastoderm, it is likely that MSL-independent compensation of genes such as runt and MSL-mediated compensation of other early-acting X-linked genes could either be separated by a very short developmental period or could occur simultaneously. The mechanism of early Sex lethal directed dosage compensation is unknown (McDowell, 1996 and Bernstein, 1994).

Control of photoreceptor axon target choice by transcriptional repression of Runt

Drosophila photoreceptor neurons (R cells) project their axons to one of two layers in the optic lobe, the lamina or the medulla. The transcription factor Runt (Run) is normally expressed in the two inner R cells (R7 and R8) that project their axons to the medulla. The relationship between Run and the ubiquitously expressed nuclear protein Brakeless (Bks), which has previously been shown to be important for axon termination in the lamina, has been examined. Bks represses Run in two of the outer R cells: R2 and R5. Expression of Run in R2 and R5 causes axonal mistargeting of all six outer R cells (R1-R6) to the inappropriate layer, without altering expression of cell-specific developmental markers (Kaminker, 2002).

As an axon navigates toward a target region during development, it alters its course based on attractive or repulsive molecular signals in its environment. There are at least two phases in the establishment of neuronal connections. First, axons project to and distinguish between regions or layers and second, once within the target layer, axons fine-tune their projections. This second step involves precise interactions between growth cones and target cells and has been well studied, particularly for the participation of cell-surface molecules and their associated signal transduction machinery. This study investigates the role of two transcription factors, Run and Bks, in the first phase of target layer selection for differentiating R cells in the Drosophila optic lobe (Kaminker, 2002).

The expression pattern of several R cell-specific differentiation markers is normal in bks mutants. In striking contrast to other markers, however, Run is ectopically expressed in two extra R cells per cluster in somatic loss-of-function clones of bks mutant tissue. In bks clones, Run expression is expanded from its normal R7/R8 pattern to also include R2 and R5. This suggests that Bks represses Run in R2 and R5 cells. Cells along the edges of bks clones were analyzed for the expression of Run. In 196 ommatidia counted along clone boundaries, R2/R5 expression of Run was never seen in a cell that is wild type for bks. It is concluded that the repression of run by Bks is cell-autonomous (Kaminker, 2002).

R1-R6 photoreceptor axons misproject to the medulla in bks loss-of-function mutants. To determine whether this axonal targeting defect is due to the relief of Run repression in R2 and R5, the GAL4/UAS system was used to express Run in these cells. When Run is expressed in R2, R5 and R8 using the MT14-GAL4 driver, all innervating R-cell axons bypass the lamina and projected through to the medulla. When Run is misexpressed in R2 and R5 alone, the defect is as severe as when Run is misexpressed in all R cells using the GMR-GAL4 driver. Run over-expression in R8 alone, where it is normally expressed, does not affect axonal projections. In addition, misexpression of Run in R1, R6 and R7 using lz-GAL4 or in R3 and R4 using sal-GAL4 does not give rise to a comparable axonal misprojection phenotype. The severe axonal mistargeting phenotype in MT14-GAL4/UAS-run flies is attributable to Run expression in R2 and R5 (Kaminker, 2002).

Unlike the ordered wild-type array, thick bundles of axons are seen entering the medulla when Run is mis-expressed in R2 and R5. The axons do not project into deeper areas of the brain, but stop within the medulla. The phenotype observed in this genetic background is very similar to that for bks. Therefore, in both bks loss-of-function and MT14-GAL4/UAS-run genetic backgrounds, Run expression in R2 and R5 results in the mistargeting of all retinal axon types to the medulla. This also suggests that the targeting of R2 and R5 axons affects axonal pathfinding of other outer R cells. For technical reasons, it was not possible to generate marked, double-lethal clones of run and bks (Kaminker, 2002).

The mistargeting of R-cell axons could, in principle, result from the conversion of all R-cell fates to R7 and R8. Markers for every R-cell type and for cone cells were therefore analyzed, both in mosaic clones of null bks mutant tissue and in the context of MT14-GAL4/UAS-run. In each of these backgrounds, the Run-expressing R2 and R5 cells did not express the R8-specific antigen, Bride of Sevenless (Boss) or the R7 marker, Prospero (Pros). Therefore, the projection phenotype of R cells to the medulla in these backgrounds does not result from the conversion of these R cells to the R7/R8 type during their development. The R1/R6 marker Bar and the R2/R5/R3/R4 marker Rough are also unaffected in these backgrounds. It is concluded that Run reprograms the projection pattern of outer R cells without affecting the expression of developmental markers of cell identity (Kaminker, 2002).

Consistent with the R cell marker expression in larval tissue, plastic sections of adult eyes show that Run misexpression in R2 and R5 during development does not perturb adult R cells or ommatidial structure. The correct complement and arrangement of R cells was found. Strikingly, these seemingly normal adult R cells misproject their axons to the inappropriate optic layer. Axon termination in the lamina region is virtually absent and all R-cell axons project to the medulla. This adult phenotype is also identical to that reported in bks mutant clones in which R cells are unchanged in the expression pattern of Rhodopsins. These data provide strong evidence that Run expression in R2 and R5 causes mistargeting of all outer R-cell axons without changing their individual R-cell fates, as determined by developmental markers and by the adult morphology of rhabdomeres. In spalt (sal) mutants, cell fate is altered without changes in axonal connectivities. Similarly, in sal-GAL4/UAS-run flies, some change in R3/R4 fate to R7 cell type is evident without a significant perturbation of outer R-cell projections. Hence, transcriptional events that control cell identity are separable from those that control axonal targeting (Kaminker, 2002).

R-cell axons provide critical anterograde signals to the lamina target region to induce the proliferation and differentiation of lamina neurons, and to induce the differentiation and migration of glial cells to their correct position adjacent to the lamina plexus. In turn, the glial cells provide positional information that directs R1-R6 axons to terminate in the lamina, and in the absence of glia these axons project into the medulla. The development of the lamina target region was assessed using neuronal and glial cell differentiation markers. Wild-type brains stained with anti-Dachshund (Dac) antibody show a large area of labeled cells corresponding to maturing lamina precursor cells (LPCs) and differentiated lamina neurons. This remains unchanged in GMR-GAL4/UAS-run brains, although the R-cell axons do not target properly. Additionally, the three rows of glial cells that delineate the lamina plexus in wild type also remain unaltered when Run is misexpressed. It is concluded that the abnormal pathfinding of R cells is due to defects intrinsic to R cells and not to a global disruption of lamina target neurons or glia (Kaminker, 2002).

Thus, this study highlights two important characteristics of neuronal pathfinding in the optic lobe. (1) A mechanism involving Bks exists in wild-type flies for repressing Run expression specifically in R2 and R5 cells. The bks loss-of-function causes relief of run repression in these two cells, which entirely abolishes the distinct targeting of R cells to the two optic ganglia. Perhaps additional genes are responsible for repressing run in the other R cells. (2) The mistargeting of R2 and R5 alone is sufficient for all outer R cells to project to the medulla. It appears that R2 and R5 cells, the first two outer R cells to be specified, provide pioneering axons whose tracts the other axons follow. In rough loss-of-function, R2 and R5 are converted to R8 cells. The resulting phenotype, however, is not as severe as that described in this study. Presumably, a residual pioneering function is maintained. The mechanism underlying the interaction between R2/R5 and R3/R4/R1/R6 axons in regulating target layer selection is unclear, although interaction between R1-R6 axons regulates a later step in axonal pathfinding: the fine-tuning of R-cell connections in the lamina (Kaminker, 2002).

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

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