Promoter Structure

paired expression in stripes is directed by a 10 kb paired 5'-flanking region, a 5kb 3' region and the intron. A 383 bp "paired zebra element" was found at the proximal promoter (including the 264 bp 5'-UTR). Individual stripe specific regulation can also be found (Gutjar, 1994).

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

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

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

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

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).

Transcriptional Regulation

Analysis of the initial paired expression in the primary pair-rule mutants even-skipped, runt and hairy, and in all gap mutants suggests that the products of the gap genes hunchback, Krüppel, knirps and giant activate paired expression in stripes. With the exception of stripe 1, which seems to be activated by even-skipped, and stripe 8, which depends upon runt, the primary pair-rule proteins are required for subsequent modulation rather than activation of the paired stripes. The factors activating paired expression in the pair-rule mode appear to interact with those activating it along the dorsoventral axis (Gutjahr, 1993).

paired is a direct target of even-skipped (Gutjahr, 1993). Two tiers of response times appear to distinguish between genes that are direct (fushi tarazu, odd-skipped, runt, paired, and wingless) and indirect (eve, hairy, and engrailed) targets of Eve. Genes that appear to be directly regulated by Eve are differentially repressed in a concentration-dependent fashion. (Manoukian, 1992).

paired is repressed by eve and hairy at the interstripes; the genes responsible for the late split pattern of 14 stripes are odd-paired, odd-skipped and perhaps runt as well (Gutjahr, 1993, 1994).

At least two pair-rule genes, paired and sloppy paired, and all segment-polarity genes analysed to date are under the control of Tenascin major, the extracellular protein related to vertebrate tenascin. Tenm initiates a signal transduction cascade acting (either via odd-paired or in concert with odd-paired) on downstream targets such as prd, slp, gooseberry, engrailed and wingless, leading to an opa-like phenotype (Baumgartner, 1994).

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).

Groucho acts as a co-repressor for several Drosophila DNA binding transcriptional repressors. Several of these proteins have been found to contain both Groucho-dependent and -independent repression domains, but the extent to which this distinction has functional consequences for the regulation of different target genes is not known. The product of the pair-rule gene even skipped contains a Groucho-independent repression activity. In the Eve, outside the Groucho-independent repression domain, a conserved C-terminal motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Hückebein, has been identified. Eve interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo. Eve with a mutated Groucho interaction motif, which abolishes binding to Groucho, shows a significantly reduced ability to rescue the eve null phenotype when driven by the complete eve regulatory region. Replacing this motif with a heterologous Groucho interaction motif restores the rescuing function of Eve in segmentation. Further functional assays demonstrate that the Eve C terminus acts as a Groucho-dependent repression domain in early Drosophila embryos. This novel repression domain is active on two target genes that are normally repressed by Eve at different concentrations: paired and sloppy paired. When the Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining. Thus, the ability of Eve to repress different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho. The accumulation of multiple domains of similar function within a single protein may be a common evolutionary mechanism that fine-tunes the level of activity for different regulatory functions (Kobayashi, 2001).

What is the significance of the two distinct Eve repression activities, only one of which is dependent on Gro? Gro could be required to repress a subset of Eve targets, whereas repression of other target genes might be Gro independent. Alternatively, the two repression activities might function cooperatively, in which case both activities might be required for repression of each target gene. Extensive molecular and genetic studies have identified several target genes that are likely to be directly repressed by Eve. The best characterized of these genes are sloppy paired (slp), paired (prd) and odd skipped (odd). The posterior boundaries of expression of slp and prd correspond to the anterior and posterior borders, respectively, of the odd-numbered en stripes. As these en stripes shift posteriorly, both when the dose of gro is reduced and when the Groucho interaction domain (GID) is mutated, the boundaries of slp and prd may be coordinately shifted. slp and prd expression were examined in embryos rescued with a GID-mutated transgene. Both slp and prd expression were expanded in the eve domains, relative to wild-type embryos. The degree of expansion of each gene correlates with the shift of en stripes. Furthermore, both the width of individual en stripes and their spacing are very similar to those in eve hypomorphs. Thus, the removal of the GID has an effect that is similar to that of a general reduction of eve activity on both targets, slp and prd. This expansion of slp and prd expression is reversed, in each case, when the Eve GID motif is replaced by that of Hairy. These results suggest that Gro is required by Eve to a similar degree for its repression activity on each of these genes (Kobayashi, 2001).

Repression of another Eve target gene (odd) is required for the establishment of the even-numbered (ftz-dependent) en stripes. Intriguingly, these are established more or less normally in embryos rescued by the GID-mutated transgene, and examination of odd expression in those embryos has showen it to be normal in the even-numbered parasegments. However, repression of odd and the establishment of even-numbered en stripes are also normal when eve function is reduced in other ways (e.g. in the hypomorph), suggesting that a lower threshold of Eve activity is required for this eve function than for proper repression of slp and prd. Therefore, this assay did not allow for a full assessment of the contribution of Gro to odd repression by Eve (Kobayashi, 2001).

Regulation of paired expression by the sex hierarchy

In virtually all animals, males and females are morphologically, physiologically and behaviorally distinct. Using cDNA microarrays representing one-third of Drosophila genes to identify genes expressed sex-differentially in somatic tissues, an expression analysis was carried out on adult males and females that: (1) were wild type; (2) lacked a germline; or (3) were mutant for sex-determination regulatory genes. Statistical analysis identified 63 genes sex-differentially expressed in the soma, 20 of which (thus far) have been confirmed by RNA blots. In situ hybridization experiments with 11 of these genes showed they were sex-differentially expressed only in internal genital organs. The nature of the products these genes encode provides insight into the molecular physiology of these reproductive tissues. Analysis of the regulation of these genes revealed that their adult expression patterns are specified by the sex hierarchy during development, and that doublesex probably functions in diverse ways to set their activities (Arbeitman, 2004).

When does sex hierarchy regulation of the 11 selected genes occur? There are two known mechanisms by which sex-differential gene expression in adults is generated: (1) the sex hierarchy actively regulates gene expression in adults, as is the case for Yolk protein 1 (Yp1); or (2) the hierarchy functions earlier in development to specify which sex-specific adult tissues will be formed but does not regulate gene expression in those tissues in the adult. Temperature-sensitive tra2 alleles were used; this allowed switching between the male and female mode of splicing dsx. In chromosomally XX tra-2ts animals, female development occurs at the permissive temperature (16°C), whereas male development occurs at the non-permissive temperature (29°C). Animals were raised at one temperature, collected 0-24 hours after eclosion and maintained at their original temperature for one more day. Then half of each group was switched to the other temperature (16°C to 29°C, or 29°C to 16°C). All animals were maintained for three more days, and RNA was then extracted. Under these conditions, expression of Yp1 (the positive control, responded to temperature shifts as expected; the Yp1 transcript was reduced when animals were switched from 16°C to 29°C and induced when animals were switched from 29°C to 16°C. By contrast, expression of the 11 other genes analyzed did not change substantially over the three days following the temperature shifts. Thus, sex-differential expression of all 11 genes is the consequence of the developmental action of the sex hierarchy and is independent of the hierarchy during adult stages (Arbeitman, 2004).

paired (prd) is expressed in the male accessory gland, and is of interest as its expression is required during both development and adulthood for accessory gland formation and physiology. The role of dsx in prd expression was examined by means of in situ hybridization of frozen sections of XX and XY dsx null individuals, and XX and XY wild-type controls. Comparable levels of prd expression were observed in the accessory glands of XY wild-type animals and both XX and XY intersexual animals, with no visible expression in XX wild-type animals. These observations suggest that male-specific expression of prd is not a consequence of positive regulation by DSXM, but rather of negative action by DSXF to prevent the formation of male accessory glands, as was observed for the other accessory gland genes (Arbeitman, 2004).

Targets of Activity

The three paired-box and homeobox genes paired, gooseberry and gooseberry neuro have distinct developmental functions in Drosophila embryogenesis. During the syncytial blastoderm stage, the pair-rule gene prd activates segment-polarity genes (such as gsb, wingless, and engrailed) in segmentally repeated stripes. Despite the functional difference and the considerably diverged coding sequence of these genes, their proteins have conserved the same function. The essential difference between these genes may reside in their cis-regulatory regions (Li, 1994).

There are several distinct phases of runt expression in the early embryo. Each phase depends on a different set of regulators. In a third distinct phase of expression, at the onset of gastrulation, runt becomes 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 activating or maintaining runt expression during these stages (Klingler, 1993).

The Drosophila Paired (Prd) transcription factor has homeodomain (HD) and paired domain (PD) DNA-binding activities required for in vivo function. Correspondingly, Prd activation of late even-skipped (eve) expression occurs through a conserved target sequence (PTE) with HD and PD half sites, both of which are required for activation. To investigate the relationship between the HD and PD, and their roles in conferring specificity to Prd function, altered versions of the Prd protein and of the PTE target site were investigated using in vivo assays in embryos. It was found that function through PTE is constrained by the targeting specifications of both the HD and PD as well as the spatial relationship between these two domains. PTE function is also constrained by the spacing between the target half sites for the PD and HD, although surprisingly, late eve activation is retained when PTE is replaced by in vitro optimized binding sites for either the PD alone or for an HD dimer. In contrast to late eve regulation, other Prd targets tolerate more changes in the Prd protein, suggesting that their target sequences may be qualitatively different from PTE (Lan, 1998).

paired determines the posterior boundary of engrailed stripes (Gutjahr, 1993), and is also involved in the activation of wingless (Ingram, 1993).

The exact positioning of neuroblasts in the neuroectodermal region giving rise to the CNS is regulated by a combination of pair-rule genes. Proneural achaete-scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Specifically, paired mutants lack achaete producing cells in the fourth row of neuroblasts in odd-numbered segments. paired mutants also exhibit fusion of second and fourth rows in even-numbered segments. (Skeath, 1992)

DPTP61F is a non-receptor protein tyrosine phosphatase that is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein which are targeted to different subcellular locations. The transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression along the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation along the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes. In hairy mutants, the complement of DPTP62Fm stripes is reduced by half, to approximately eight wide stripes. It is presumed that odd numbered stripes have been deleted. Within embryos homozygous for a strong eve allele, odd stripes are absent except for stripe 1. In odd paired mutants every even stripe is decreased. In paired mutants odd numbered domains of expression are shifted anteriorly towards the even numbered domains. wingless, hedgehog, naked and patched are involved in refining the pattern of mRNA accumulation within each parasegment (Ursuliak, 1997).

The response kinetics of known and putative target genes of Fushi tarazu has been examined in order to distinguish between direct and indirect Ftz targets. This kinetic analysis was achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (less than 10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely to be important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes have been analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy (Nasiadka, 1999).

In contrast to engrailed, the segment polarity gene wingless has been identified genetically as a negative target of Ftz. This negative interaction has also been demonstrated in HSFtz embryos. Although all wg stripes are repressed in these embryos, the predominant effect is on odd-numbered stripes, which are completely repressed following Ftz induction. Repression of even-numbered stripes is much less efficient. To assess whether this repression is direct, the kinetics of repression were examined. The differential repression of odd- versus even-numbered stripes of wg was a helpful tool and an internal control for recognizing affected embryos. This curve follows very closely that of ftz autoregulation, with the midpoint of both curves occuring at 18 minutes post-heat shock. This indicates that repression of wg by Ftz is also likely to be direct, and that Ftz can act as both an activator and repressor of transcription. Repression is not the only response exhibited by wg in HSFtz embryos. Weak activation within most of each odd-numbered parasegment is also detected. The kinetic curve of this activation response is considerably delayed relative to the kinetics of the other three responses measured thus far. This suggests that wg activation results from an indirect genetic interaction. A likely intermediary gene in this response is the paired gene. prd is genetically required for the proper initiation of all 14 wg stripes; all 14 wg stripes expand rapidly in HSPrd embryos (Nasiadka, 1999).

To test whether the prd gene acts as an intermediate in the positive response of wg to ectopic Ftz, the spatial and temporal responses of prd were determined in HSFtz embryos. If prd does function as an intermediary gene, its expression should be induced in odd-numbered parasegments where wg activation is later observed. Moreover, the induction of prd transcripts should occur with the same rapid kinetics as the ftz, en and early wg responses. The prd expression pattern was examined 20 minutes after ectopic expression of Ftz. Stripes are significantly wider than those in similarly staged wild-type embryos. Using the most posterior stripe of prd as a landmark, it was seen that each of the expanded stripes had broadened at its anterior edge. These regions of expansion comprise most of each odd-numbered parasegment, which is exactly where ectopic expression of wg occurs. The time course of prd mRNA induction was assessed as described for endogenous ftz, en and wg. Although the slopes of the prd and ftz activation curves differ, the initial responses occur at about the same time, suggesting that the interaction between Ftz and prd is also direct. The differences in the slopes of the two curves are likely due to the autoregulatory nature of the ftz response: for a short time, Ftz is expressed from both heat shock and endogenous promoters, and then maximal expression is sustained via autoregulation at the endogenous locus. In contrast, prd activation takes place in regions of the embryo where neither ftz nor prd autoregulates. Hence, prd transcripts do not accumulate as quickly as those of ftz and soon disappear due to degradation of the ectopically expressed Ftz activator (Nasiadka, 1999).

The role of prd as an intermediary factor in wg activation was tested further by examining wg expression in a HSFtz;prd munus background. In the absence of prd, wg stripes should no longer expand. Ectopic Ftz does indeed fail to activate ectopic wg in the absence of prd. The expression pattern in HSFtz;prd minus embryos is essentially identical to the pattern of wg expressed in prd- embryos: odd-numbered stripes are weak and even-numbered stripes are essentially absent. This result is consistent with the proposed role of prd as a direct activator of wg and as a genetic intermediate between Ftz and wg during ectopic stripe broadening (Nasiadka, 1999).

To verify that the prd protein (Prd) is a direct activator of wg, the nature of the temporal delay between prd and wg activation was analysed. Specifically, the temporal accumulation of Prd protein with respect to prd and wg transcripts was examined. If the interaction between prd and wg is direct, then one would expect that much of the interval between accumulation of the two transcripts would be occupied by synthesis and nuclear transport of the Prd protein. The kinetic curve for ectopic Prd induction closely resembles that of WG mRNA activation except that it is shifted by 1-2 minutes to the left (earlier). This indicates that most of the delay observed between the accumulation of prd and wg transcripts (about 8 minutes) is consumed by the synthesis and localization of Prd protein. The time required (~6-7 minutes) may be fairly typical of other segmentation proteins expressed at this stage. Indeed, the delay between detection of en transcript and protein responses is also 6-7 minutes, with curves that are virtually identical to those of prd transcripts and protein. These data do not exclude the possibility that there are genes in addition to prd that are required for ectopic activation of wg. However, if such gene products are required, their rates of synthesis or removal do not appear to supercede the temporal limitations imposed by the synthesis of Prd (Nasiadka, 1999).

The early expression of the Drosophila segment polarity gene gooseberry is under the control of the pair-rule genes. A 514-bp enhancer, -5.3 to -4.8 kb interval (called fragment IV), has been identified that reproduces the early gsb expression pattern in transgenic flies. The transcription factor Paired (Prd) is the main activator of this enhancer in all parasegments of the embryo. It binds to paired-and homeodomain-binding sites, which are segregated on the enhancer. Using site-directed mutagenesis, sites critical for Prd activity have been identified. Negative regulation of this enhancer is mediated by the Even-skipped protein (Eve) in the odd-numbered parasegments and by the combination of Fushi-tarazu (Ftz) and Odd-skipped proteins in the even-numbered parasegments. The organization of the Prd-binding sites, as well as the necessity for intact DNA binding sites for both the paired- and homeodomain-binding sites, suggests a molecular model whereby the two DNA-binding domains of the Prd protein cooperate in transcriptional activation of gsb. This positive activity appears to be in competition with Eve and Ftz on Prd homeodomain-binding sites (Bouchard, 2000).

Prd, like Gsb, contains a paired domain and a paired-type homeodomain. These domains are able to bind DNA in vitro in an independent as well as in a cooperative manner. This peculiar feature gives the members of this family of transcription factors a great DNA binding versatility. The gsb early transcriptional enhancer studied here presents an interesting case in which Prd uses both DNA-binding domains to interact with homeo- and paired domain recognition sequences segregated on the enhancer. An enhancer fragment lacking either all homeodomain-binding sites (IV.2345M) or a specific paired domain-binding site (IV.7M) loses all activity. These results suggest that the two DNA-binding domains of Paired cooperate for its proper activity on fragment IV. Whether the different sites of fragment IV are bound by the same molecule or by two different molecules is currently unclear. The model involving only one Prd molecule is supported by an experiment showing that a combination of two mutant prd transgenes (under the control of the prd promoter) that mutate in the paired domain and in the homeodomain, respectively, cannot rescue the early expression pattern of gsb in a prd mutant background, whereas a wild-type prd transgene is able to do so (Bouchard, 2000).

The onset of gsb expression first occurs in seven stripes at the cellular blastoderm stage. These stripes appear in a first row of cells at the posterior border of the even-numbered parasegments. The expression rapidly expands to a second row of cells at the anterior border of the odd-numbered parasegments. The gsb stripes coincide with the posterior border of Prd expression in the odd-numbered parasegments. It has been suggested that the early bell-shaped expression of Eve acts as a morphogenetic gradient regulating the posterior border of prd expression. Although the posterior border of gsb expression in the odd-numbered parasegments could be specified by Prd expression alone, it is likely that Eve also acts directly on the gsb control region. Indeed, the Eve-binding sites overlap with some of the Prd-binding sites, suggesting a competition at the DNA-binding level (Bouchard, 2000).

In principle, the consensus sequences identified by DNase I protection with Eve could represent binding sites of other homeodomain proteins regulating gsb expression. However, the fact that Eve and Ftz are the only known homeodomain proteins expressed in a double segment periodicity at the blastoderm stage strongly argues against this. Moreover, a direct action of Eve on gsb regulation is supported by short-pulse heat-shock experiments that favor direct regulatory effects. Using this assay, the ectopic overexpression of prd could override the repression by Eve in the odd-numbered parasegments, while a heat-shock eve could abrogate Prd activation of gsb in all parasegments. Altogether these results suggest that gsb responds to the Eve morphogenetic gradient in the odd-numbered parasegments (Bouchard, 2000).

The endogenous even-numbered gsb stripes appear at stage 6 with a slight delay compared to the odd-numbered stripes. It is now clear that Prd is essential for the activation of these stripes since neither the endogenous gsb transcription nor the fragment IV expression is observed in the trunk during germ-band extension in a prd embryo. Transcriptional activity is also lost in tissue culture assays and in vivo (transgenic lines) upon removal of Prd-binding sites. A similar conclusion concerning the activity of Prd in all parasegments has been reached using ectopic overexpression of Prd. The necessity of Prd for gsb activation does not exclude the potential requirement of another factor such as Odd-paired. Alternatively, it is possible that Opa is involved in the maintenance of gsb by activating wg in the even-numbered stripes. The activity of Opa on gsb through the Wg signal could account for the remnants of even-numbered stripes observed at late stage 11 in a prd transgenic line IV embryo. Indeed, wg is known to depend on prd in the odd-numbered stripes, whereas it depends on opa in the even-numbered stripes (Bouchard, 2000).

The establishment of the posterior border of gsb in the even-numbered parasegments requires an efficient mechanism of repression, since Prd is present throughout all all even-numbered parasegments at the time of gsb initiation. The expression of transgenic line IV-LacZ is derepressed in ftz and odd mutant embryos. Moreover, Prd activity is directly competed by Ftz and Odd in tissue cultured cells. These data identify Ftz and Odd proteins as being responsible for the establishment of gsb expression borders in the even-numbered parasegments (Bouchard, 2000).

In the genetic analysis of fragment IV, it was observed that neither ftz nor odd mutant embryos show a complete derepression in the even-numbered parasegments. An odd embryo shows an anterior widening of the odd-numbered gsb stripes, suggesting a more important role for Odd in the region of low Ftz concentration. This result also indicates that Ftz is a potent repressor in the embryo since it is still able to partially repress gsb, even though Prd levels remain high in the central part of the even-numbered parasegments in an odd embryo, as opposed to its gradual repression in this region in a wild-type background. In a ftz embryo, a posterior widening of two to three cells in the even-numbered stripes is observed. This limited expansion can be explained by the action of Odd in the posteriormost portion of the parasegment combined with the fact that Prd is fading exclusively in this region in a ftz mutant embryo. The true repressor effect of Odd on fragment IV is possibly masked in these genetic experiments by the fact that, in an odd mutant embryo, Ftz is not properly repressed in the posterior portion of the parasegment. In such an embryo, Ftz is thus compensating for the absence of Odd. At the molecular level, the mechanism of action of Odd is unclear. It is possible that Odd binds directly to fragment IV via its zinc-finger domain, but this interaction would have been missed due to insufficient binding activity in vitro. Alternatively, Odd could bind Prd via protein-protein interaction and thereby interfere with its transactivation properties (Bouchard, 2000).

Protein Interactions

Protein-protein interactions are critical in homeodomain protein function. A homeodomain-deleted Fushi tarazu polypeptide (Ftz delta HD) is incapable of binding DNA in vitro, but can regulate endogenous ftz gene expression. Ftz delta HD can directly regulate ftz-dependent segmentation, suggesting that it can control target gene expression through interactions with other proteins. A likely candidate is the pair-rule protein Paired. Ftz delta HD binds directly to Prd protein in vitro and requires Prd to repress wingless in vivo (Copeland, 1996).

The function of the paired domain is still not completely understood. One study shows that the paired domain binds DNA and is required for function. Thus, Paired has two DNA binding domains: a homeodomain and a paired domain (Wilson, 1993).

There are seven Pax genes in Drosophila and nine Pax genes known in mouse and human. Different Pax proteins use multiple combinations of the HTH motifs to recognize several types of target sites. Drosophila Paired protein can bind, in vitro exclusively through its PAI domain (the N-terminal portion of the bipartite paired domain), or through a dimer of its Homeodomain, or through cooperative interaction between PAI domain and HD. However, paired function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD (such as Pax-5) appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein Lune, may rely on the RED domain and HD. Thus Pax protein appear to recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire (Jun, 1996).

While Paired can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain (the N-terminal segment of the bipartite paired domain) and the homeodomain. This requirement is the same for three genes regulated by Paired protein, engrailed, wingless and gooseberry. Both the HD and the PAI domain are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the paired domain (the RED domain) appears to be dispensable. Although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue of phenotype in paired mutants. A Paired protein lacking the C-terminal PRD repeat is functionally impaired, but still able to rescue vitality (Bertuccioli, 1996)

Mutations in either the paired domain or homeodomain abolish the normal regulation of Engrailed target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that the in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when two gene were placed in the same embryo, one with mutated PD and the other with mutated HD, Paired function was restored, indicating that the two DNA binding activities need not be present in the same molecule. Quantitation of this effect shows that the PD mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer (Miskiewicz, 1996).

The orphan nuclear receptor alpha Ftz-F1, which is deposited in the egg during oogenesis, is an obligatory cofactor for Fushi tarazu. Mutation of Ftz-F1 causes a pair-rule phenotype even though the maternal alphaFTZ-F1 gene product is uniformly distributed through the embryo. Surprisingly, patterns of FTZ mRNA and protein expression in the alphaFTZ-F1 mutants are indistinguishable from wild type. In Ftz-F1 mutant embryos, as in ftz mutant embryos, Ftz-dependent engrailed stripes fail to be expressed, and wingless stripes expand. Thus alphaFtz-F1 is required for all Ftz activities tested except that for which it was first identified: regulation of the ftz promoter. Given this result, a test was made for direct interaction between Ftz protein and alphaFtz-F1. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. The conserved motif is independent of the Ftz homeodomain and is located in the central portion of the protein, flanked by prolines. Deletion of this motif disrupts all but one of the the Ftz activities described above, that is, it is still capable of broadening endogeneous ftz expression stripes. Thus removal of the alphaFtz-F1 interaction domain from the Ftz polypeptide results in the same loss of Ftz activities as removal of alphaFtz-F1. The Ftz-mediated repression of wingless requires both Paired and alphaFtz-F1. This interaction could involve either simultaneous or competitive interactions among the three proteins, as Prd also contacts residues 101-150 of Ftz. Paired protein may be a cofactor of Ftz or Ftz-F1 that is required for target genes that are repressed by Ftz, because prd is required for Ftz-dependent wingless repression, but not for Ftz-dependent activation of engrailed or ftz auto-regulation (Guichet, 1997).

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

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

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

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

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

Selective spatial regulation of gene expression lies at the core of pattern formation in the embryo. In Drosophila, localized transcriptional regulation accounts for much of the embryonic pattern. Properties of a newly identified gene, partner of paired (ppa), suggest that localized receptors for protein degradation are integrated into regulatory networks of transcription factors to ensure robust spatial regulation of gene expression. The Ppa protein interacts with the Pax transcription factor Paired (Prd) and contains an F-box, a motif found in receptors for ubiquitin-mediated protein degradation. In normal development, Prd functions only in cells in which ppa mRNA expression has been repressed by another segmentation protein, Even-skipped (Eve). When ppa is expressed ectopically in these cells, Prd protein, but not mRNA, levels diminish. When ppa function is removed from cells that express PRD mRNA, Prd protein levels increase. It is concluded that Ppa coordinates Prd degradation and is important for the correct localization of expressed Prd. In the presence of Ppa, Prd protein is targeted for degradation at sites where its mis-expression would disrupt development. In the absence of Ppa, Prd is longer-lived and regulates downstream target genes (Raj, 2000).

To gain further insight into the combinatorial regulation by Prd and Eve, a yeast two-hybrid screen was performed for a cDNA library derived from 0-12 hour old embryos, using as bait a 140 amino-acid fragment of Prd that included its homeodomain. From a total of 2.4 x 106 primary transformants, 22 classes of clones were identified by restriction analysis; mRNA in situ hybridization analysis of representatives from each class indicated that one of the cDNAs from the screen is expressed in a pair-rule pattern of stripes. This cDNA was named ppa. To test the specificity of the interaction of Ppa with Prd, the ppa cDNA was retransformed into yeast and tested using a mating assay against a panel of different baits, including Prd. Ppa interacts either with the original homeodomain-containing Prd fragment, or a fragment containing both the homeodomain and the Prd domain, but not with homeodomain-containing fragments of Ftz or Bicoid, or with several unrelated control baits. In addition, Ppa does not interact with Prd containing a Ftz homeodomain substitution, suggesting that the Prd homeodomain sequences are required for the protein interaction (Raj, 2000).

Sequence analysis of ppa provided insights into its possible functions. The Ppa open reading frame (ORF) contains 11 leucine-rich repeats (LRRs). These 20-29 amino-acid motifs have Leu residues at characteristic positions and have been implicated in protein-protein interactions. Indeed, the carboxy-terminal 92 amino acids of Ppa, encompassing three LRRs, is sufficient for the interaction with Prd in the two-hybrid screen. Sequence alignments indicate that the LRRs in Ppa are similar to those found in yeast glucose repression regulator 1 (Grr1), C. elegans CO2F5.7, an ORF of unknown function, and a human hypothetical ORF that has been named Ppa because it is 63% identical (78% similar) to the carboxy-terminal 392 amino acids of the Drosophila protein (Raj, 2000).

Like GRR1, C. elegans CO2F5.7, and the human ORF, Drosophila Ppa also contains an F-box motif amino-terminal to the LRRs. Previously characterized F-box proteins, including Grr1 and yeast Cdc4, have been shown to be receptors that target their substrates for ubiquitin-mediated protein degradation. These proteins interact through their F-boxes with Skp1, which associates with Cdc53/Cullin, forming an SCF complex (Skp1/Cullin/F-box). The SCF complex functions as a ubiquitin ligase enzyme (E3), which facilitates transfer of ubiquitin from a ubiquitin conjugation enzyme (E2) to the substrate. The F-box proteins provide a vital link between this machinery and specific substrates to be degraded, the substrate interaction typically being mediated through WD40 or LRR protein-interaction motifs within the F-box protein. Thus, the F-box proteins provide for specificity of substrate choice. Unlike Grr1, CO2F5.7 or other described F-box proteins, Ppa also contains a region rich in Ala, His and Pro, which is similar to Ala-rich domains observed in previously identified transcriptional repressor proteins, including Kruppel, Knirps, Eve and En. The presence of the F-box and Ala/His/Pro motifs suggests that Ppa might function as a receptor for protein degradation, or as a transcriptional co-repressor, or both (Raj, 2000).

The ppa mRNA is not detected in unfertilized embryos, suggesting that ppa is not expressed maternally. Uniform expression throughout the embryo is first detected at nuclear cycle 10, and gradually increases in intensity during cycles 11-14. Ppa expression diminishes in the pole regions during cycle 13. During cycle 14 and early gastrulation, the expression of ppa transformed into a pair-rule striped pattern with the formation of interbands within which ppa expression is lost. This is followed during germ-band elongation by splitting of the ppa stripes to generate a one-segment-repeated pattern of reiterated interbands. The ppa stripes do not have sharp borders. Expression of ppa is lost throughout the ventral region of the embryo, which contributes to the ventral furrow during gastrulation, presumably as a result of dorsoventral regulators. The ppa mRNA is localized in the basal regions of cells, in contrast to the apical localization of most pair-rule gene mRNAs (Raj, 2000).

To assess the possible functional relationships with Prd and Eve, embryo fillets were double-stained for ppa mRNA and Prd or Eve protein. During the early stages of cycle 14, when ppa expression is being restricted to stripes, there are significant levels of ppa expression overlapping the stripes of Prd protein. As cycle 14 proceeds, the posterior regions of the forming ppa stripes transiently overlap the anterior regions of the primary Prd stripes but, by early gastrulation, the Prd and ppa stripes are almost distinct. This transient but limited overlap in the expression of ppa and Prd is consistent with the model that Ppa negatively regulates Prd protein function (Raj, 2000).

Comparison of ppa mRNA with Eve protein shows almost reciprocal expression of the two genes (ppa interbands coincide with Eve stripes), raising the possibility that Eve might repress ppa expression, thereby giving rise to the ppa interbands. This interpretation is supported by examination of eve mutant embryos, which have uniform instead of striped ppa expression during cycle 14 and germ-band elongation. Moreover, adding back a transgene (P[eve.2,3,7] that expresses eve stripes 2, 3 and 7 in an otherwise eve mutant background, results in ppa interbands corresponding to these three Eve stripes (Raj, 2000).

The spatial expression and sequence of ppa suggest that Ppa might negatively regulate Prd, either by transcriptional co-repression or degradation of the Prd protein. To test these possibilities, ppa was ectopically expressed in the Prd-expressing cells to determine whether activation of en transcription or levels of Prd protein are affected. A transgene with the full ORF of ppa driven by an hsp70 promoter (hs-ppa) was introduced into embryos. Heat treatment of hs-ppa embryos during cycle 14 has pronounced effects. The odd-numbered, Prd-dependent, en stripes are weakened or completely absent, suggesting that Prd activation of these stripes is repressed. This is not observed in heat-treated wild-type embryos processed in parallel (Raj, 2000).

Because the Drosophila embryo develops very quickly, the segmentation gene products are expected to be short lived. This is indeed the case for those products examined and is also likely to be true for the Prd protein, perhaps even in the absence of ppa function. Indeed, it is difficult to assess whether Prd protein levels are reduced in hs-ppa embryos because of the fairly broad range of immuno-staining signals observed between different embryos, a problem inherent to the detection technique. To overcome this problem, ppa was ectopically expressed over only part of the embryo, so that the effects of ectopic ppa could be assessed relative to regions of the same embryo where ppa expression is normal. eve mutant embryos with a transgene P[eve.2,3,7] that expresses only eve stripes 2, 3 and 7 have well-formed ppa interbands at these locations. Thus, it is possible to compare Prd protein expression at stripe 2, which overlaps the ppa interband at eve stripe 2, with Prd expression at stripe 4, where ppa is expressed ectopically. Examination of prd mRNA signals in eve-;P[eve.2,3,7] embryos reveals strong expression of stripe 4 when compared with stripe 2, consistent with previous observations that eve represses prd transcription, thereby contributing to refinement of Prd stripes. In contrast, Prd protein signal at stripe 4 is significantly lower than at stripe 2, correlating with the ectopic ppa expression at stripe 4, and suggesting that Ppa regulates Prd protein levels. Even though there is 50% more mRNA signal at stripe 4 than stripe 2 after ppa upregulation, there is 25% less protein. Note that it is formally possible that the reduced Prd protein levels result from changes in genes other than ppa that are regulated by eve. Nevertheless, these analyses of hs-ppa and ppa mutant embryos suggest that regulation by ppa is responsible (Raj, 2000).

The equivalent analysis of wild-type embryos shows similar mRNA signals at stripes 4 and 2, whereas the Prd protein signal at stripe 4 is somewhat reduced compared with stripe 2, correlating with the residual ppa expression normally still present at gastrulation at the ppa interband corresponding to Prd stripe 4. This decrease in Prd protein is less pronounced than in eve-;P[eve.2,3,7] embryos, presumably because the difference in ppa expression at stripes 2 and 4 is smaller. To confirm the interpretation that Ppa regulates Prd protein levels, the stripe 4 to stripe 2 ratios for mRNA and protein would be expected to be similar in heat-treated hs-ppa embryos, in which ppa is expressed ectopically at both stripes 2 and 4. This was indeed observed, supporting the conclusion that ppa regulates Prd protein levels. Because Ppa has an F-box, this regulation is most likely through targeted protein degradation rather than translational repression. Consistent with these data, Western analysis of embryo extracts indicates that Prd protein levels are reduced by approximately 50% in hs-ppa embryos, as compared with the wild type (Raj, 2000).

To confirm the role of Ppa in Prd degradation, a small chromosomal deletion was generated that removes the Ppa ORF, starting 345bp upstream and ending 304bp downstream of the ORF. Consistent with the observation that high levels of ppa are normally only observed in regions where Prd function is not required, the homozygous ppa mutants survive to adulthood but with reduced viability and abnormal nuclear cycling. To analyze the mutants, advantage was taken of the normal anterior-posterior progression of ppa stripe development in wild-type embryos: at the gastrulation stage, the more anterior Prd stripes (for example, stripe 2) has little overlap with ppa expression, whereas the more posterior stripes (for example, stripes 4 and 6) still have significant overlap. When prd mRNA and protein signals at stripes 4 and 6 are measured relative to stripe 2 in the same embryos, the protein signals at stripes 4 and 6 are found to be significantly lower than the corresponding mRNA levels in wild-type embryos. In ppa mutant embryos, however, the mRNA and protein signals are similar, indicating that ppa normally reduces Prd protein expression (Raj, 2000).

Supporting the conclusion that Ppa mediates Prd degradation, Ppa was found to interact with Drosophila Skp1, the component of the protein degradation machinery that is predicted to link Ppa to the ubiquitin-mediated degradation pathway. Expressed-sequence-tag (EST) cDNAs for Drosophila Skp1 were identified from the Berkeley Drosophila Genome Project; yeast two-hybrid assays show that the Skp1 protein interacts with Ppa (amino acids 131-538, which lacks the Ala/His/Pro-rich region). As expected, the smaller fragment of Ppa (amino acids 447-538) originally identified in the two-hybrid screen does not interact with Skp1, presumably because it has no F-box. The interaction between Ppa and Skp1 was confirmed by co-immunoprecipitation analysis of yeast cell extracts. Immunoprecipitation of a hemagglutinin (HA) epitope-tagged Ppa fragment (amino acids 131-538) using anti-HA antibody also brings down LexA-tagged Skp1, which was detected with anti-LexA antibody. The Ppa-Skp1 interaction was also observed by immunoprecipitation with anti-LexA antibody, and the interaction of Ppa with Prd was also verified in these experiments (Raj, 2000).

This analysis of ppa function indicates that, when it is expressed ectopically in Prd-expressing cells, the levels of Prd protein diminish about twofold. A similar change in substrate stability (2-4-fold) is observed when GRR1, the yeast gene most similar to ppa, is mutated. It is also possible that, in addition to reducing Prd protein levels, Ppa might function as a transcriptional co-repressor, interacting with Prd to reduce its activation of en. Together, these two repression functions would ensure robust negative regulation of Prd in the Ppa-expressing cells (Raj, 2000).

Ppa is the first example of an F-box receptor localized in stripes. Loss- and gain-of-function analyses show that, while the presence of ppa expression in stripes is important for embryo development, it is the absence of ppa expression in the interbands that is crucial. Homozygous ppa minus mutants survive to adulthood, but show 50%-70% lethality, consistent with the fact that Ppa works in conjunction with the transcriptional repressors slp and run to localize Prd function. The partial lethality may be due to altered Prd expression or abnormal nuclear cycling. In contrast, in embryos with a functional ppa gene, it is absolutely essential that its expression be spatially regulated (by eve). Even basal expression of one of the hs-ppa transgenes (two copies without heat treatment) causes complete lethality, whereas the same transformant is not lethal before removal of an FRT cassette that blocks transcription. This predicts that cis-regulatory mutations in ppa causing loss of spatial regulation will have profound detrimental effects on embryogenesis, and this could also apply to the vertebrate homologs of ppa, which have such striking sequence similarity (Raj, 2000).

With the recent cloning of F-box proteins and the realization that they provide specific links between substrates and the protein degradation machinery, it has been predicted that F-box proteins would play important roles in development. Because F-box-regulated degradation normally depends on phosphorylation of substrates, localized action of signal transduction systems can, in principle, lead to localized protein degradation. This is likely to be the case for the signal-dependent localized degradation of Drosophila Cactus, a homolog of vertebrate IkappaB, whose degradation is a prerequisite for nuclear import of the Dorsal transcription factor (a homolog of NFkappaB) in the ventral portion of the embryo. Degradation of Cactus is mediated by the F-box protein Slimb (a homolog of ß-TrCP), which is also implicated in Wingless and Hedgehog pathways. In contrast to these signal transduction systems, the localized protein degradation in the Ppa system depends on spatially regulated expression of the Ppa F-box protein itself. By having its transcription regulated by a segmentation protein (Eve), and by targeting other segmentation proteins for degradation (Prd), the Ppa F-box protein forms an integrated link in the segmentation protein regulatory cascade that serves to strengthen the spatial refinement required for pattern formation. It is predicted that integration into transcriptional cascades may be a property of an important subfamily of F-box proteins, which, as suggested above, may also have recruited transcriptional repression functions to optimize their negative regulation of targeted transcription factors (Raj, 2000).

Ppa is the first example of a localized F-box receptor for protein degradation that works alongside transcription factors to ensure localized gene expression in the Drosophila segmentation cascade. These analyses suggest that Ppa targets the Prd transcription factor for degradation in cell rows in which Prd function is inappropriate, and that it is crucial that ppa expression is removed, through repression by eve, from cell rows in which Prd function is required for normal embryonic development (Raj, 2000).

paired continued: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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