Pair-rule Genes (Molecular Biology)

Pair-rule genes play critical roles in the formation of the Drosophila melanogaster body plan, which consists of fourteen contiguous segments arranged along the anterior-posterior axis. This basic plan is established by a cascade of segmentation gene activities that generate spatially repeated patterns of gene expression before the completion of cellularization. Most pair-rule genes are initially expressed in patterns of seven evenly spaced stripes about four nuclei wide that represents the first evidence of a segmental body plan. The striped patterns are established by integrating crude positional cues provided by asymmetrically localized maternal factors and proteins encoded by the gap genes. These cues are in the form of gradients of transcription factors that interact directly with pair-rule regulatory regions, resulting in position-specific activation or repression. In several cases, pair-rule genes have been shown to contain discrete enhancers that direct the expression of single stripes of pairs of stripes. Each enhancer responds in a unique way to the asymmetric cues, and the seven stripe entire pattern represents the sum of the effects of all enhancers. Once initial stripes are established, they are refined by interactions among the pair-rule genes themselves, creating a system of overlapping patterns. This system then directs the expression of segment polarity genes in patterns of fourteen stripes about one cell wide. The fourteen stripe patterns mark cells that will form borders between segmental compartments, and, thus, establish the body plan of the mature animal.

Pair-rule genes were first identified as members of a class of zygotic recessive mutations that affect the organization of the Drosophila body plan (1). Loss of function mutants in pair-rule genes contain only seven segments, and close examination of specific defects in the body plan indicates that every other segment is missing. For example, embryos with reduced even-skipped (eve) gene function contain only odd-numbered segments ("even" numbered segments are "skipped"). Other pair-rule genes lack odd-numbered segments or contiguous portions of adjacent segments that overlap every other border between two segments. These mutant phenotypes suggest that the fourteen segment body plan is established by a mechanism involving genes that specify patterns of cell fate decisions with a pair-rule periodicity.

Most pair-rule genes encode proteins with well-characterized DNA-binding motifs, and, thus, function as transcription factors in Drosophila development (Table 1). Many of these genes are expressed in patterns that appear as seven or eight transverse stripes that encircle the cellularizing blastoderm (2-4). The stripes are evenly spaced along the anterior-posterior axis in positions coincident with regions of the body plan that are disrupted in loss of function mutants. This suggests that the pair-rule genes function for the most part within their domains of expression. Interstripe regions where the genes are not expressed are also important for normal development because near reciprocal phenotypes can be generated by ubiquitous expression of pair-rule gene products (5).

Since the striped patterns of the pair-rule genes represent the first evidence of a metameric body plan, at least some of them must be generated de novo. It has been suggested that reiterated patterns such as stripes might be generated by reaction diffusion mechanisms (6-9). This model is supported by experiments in purely chemical systems that create striped patterns as the result of self-organizing processes (10). However, genetic experiments suggest that pair-rule genes are not self-organizing but are part of a hierarchy of genes that establishes polarity and segmental patterning along the anterior-posterior axis. This hierarchy is initiated by maternal effect genes that establish polarity by depositing mRNAs into the poles of the oocyte during late stages of oogenesis (11). Immediately after fertilization, the mRNAs are translated and their protein products diffuse toward the middle of the embryo creating gradients of proteins with high concentrations at the poles. These gradients direct the zygotic transcription of the gap genes, which are expressed in one or two broad domains along the anterior-posterior axis (Fig. 1). The maternal gradients and gap gene expression domains overlap, creating different combinations and concentrations of these factors according to position along the anterior-posterior axis.

Figure 1. (A) Spatial relationship between stripes of expression of the pair-rule genes even-skipped (eve) and fushi tarazu (ftz) and the positions of gap protein expression domains along the anterior posterior axis of Drosophila. Anterior is to the left. The gap genes hunchback (hb), giant (gt), Kruppel (Kr) and knirps (kni) are expressed in one or two broad domains in response to maternal gradients such as bicoid (bcd) that emanate from the poles of the embryo. (B). A double in situ hybridization experiment detects eve (red) and ftz (black) mRNAs. These genes are expressed in reciprocal patterns that establish the segmental body plan.

Striped patterns of the pair-rule genes are differentially disrupted in embryos lacking the functions of individual maternal effect, gap, and other pair-rule genes (12-16). These experiments are difficult to interpret, however, because removing the function of a single gene causes pattern shifts and disruptions in many other genes. Several experimental approaches have been used to unravel the mechanisms involved in pair-rule patterning. Regulatory interactions can be inferred from transgenic experiments that ubiquitously misexpress segmentation genes under the control of a heat shock inducible promoter (5, 17-20). When short heat pulses are used and the timing of pattern disruptions carefully monitored, it is possible to determine if a gene directly affects the expression of a given target or if intermediary genes must be expressed to induce the response (21, 22). Ectopic expression is also possible using enhancers that activate position-specific expression during early development (23-25). Finally, promoter truncation and P-element transformation assays (26) have identified two classes of cis-regulatory regions (enhancers) that are important for pair-rule patterning in the blastoderm. Enhancers of the first class specify the placement of individual stripes by responding primarily to gradients of maternal effect and gap proteins. Enhancers of the second class specify complete patterns of seven stripes but are active only after the first striped patterns are established. These enhancers respond to transcriptional cues provided by the pair-rule genes themselves and are thus important for mechanisms that maintain and refine initial patterns.

1. Regulatory Mechanisms That Control the Initiation of Individual Pair-Rule Stripes

An important breakthrough in understanding how pair-rule patterns are established arose from the analysis of hairy (h) mutations caused by translocation breakpoints in the 5′ regulatory region of the gene (27). Breakpoints located near the transcription start site cause the deletion of seven segmental units (like h null alleles), but those further away delete only a subset of segments. Thus, the regulatory elements controlling h function at different positions along the anterior posterior axis are physically separable, suggesting that individual h stripes are independently controlled by discrete enhancers. This hypothesis was confirmed by a series of P-element transformation experiment with h lacZ reporter genes, which identified discrete enhancer elements that independently control the expression of single stripes or pairs of stripes (28, 29). Similar studies led to the identification of modular enhancers in the eve regulatory region (30-32). Interestingly, enhancers that control stripes 2, 3, and 7 are located upstream of the eve coding region; those that control the other four stripes are located downstream (Fig. 2).

Figure 2. Stripe-specific enhancers in the eve locus. A map shows the positions of enhances (rectangles) identified by promoter truncation and P-element mediated transformation experiments. Simple patterns of one or two stripes of reporter gene expression are driven five discrete enhancers. The initial patterns of seven eve stripes represents the sum of the activities of these enhances. A separate enhancer directs a seven stripe pattern that appears after the initial pattern is established. This enhancer is important for maintaining and refining the pattern.

These enhancers control the positioning of individual stripes or pairs of stripes by responding to positional cues set up by the maternal effect and gap genes. The best characterized stripe enhancer controls the expression of eve stripe 2. Truncation analysis has narrowed this enhancer to a 480 base pair (bp) sequence that is sufficient to drive a stripe of lacZ reporter gene expression at the position of stripe 2(33) (Fig. 3A). Trans-acting factors involved in regulating this enhancer were identified by crossing flies containing the stripe 2-lacZ transgenes into embryos lacking maternal and gap-gene functions. These experiments suggest that the maternal effect gene bicoid (bcd) and the gap gene hunchback (hb) are required for activation, while the gap genes giant (gt) and Kruppel (kr) are required for setting the anterior and posterior borders of the stripe (13, 34). The expression patterns of the proteins encoded by these genes are consistent with their genetically identified functions (Fig. 1A). bcd and hb completely overlap the position of stripe 2, and gt and Kr about the anterior and posterior borders, respectively. Repressive interactions between gt and Kr are important for maintaining the spacing where stripe 2 is expressed (20, 25). All four proteins contain DNA-binding domains and have been shown to bind in vitro to multiple sites in the stripe 2 enhancer (34, 35) (Fig. 3B). Most binding sites for activator proteins are located within 50 bp of repressor sites. Mutating binding sites for bcd or hb causes a reduction in expression levels, and deleting gt sites causes a dramatic expansion of the stripe response into anterior regions of the embryo (33, 36, 37). These experiments suggest that the stripe 2 enhancer functions as a binary switch that controls region-specific activation by directly responding to combinations of asymmetrically localized regulatory factors.

Figure 3. (A) lac Z reporter gene expression directed by the 480 bp eve stripe 2 enhancer. Genetic experiments suggest that this enhancer is activated by bicoid (bcd) and hunchback (hb). The anterior and posterior borders of this stripe are set by repression involving the gap proteins giant (gt) and Kruppel (Kr), respectively (see Fig. 1). (B) Positions of binding sites for proteins that regulate this enhancer. Activator sites are shown below the line, with repressor sites above. These sites enable the enhancer to make on/off decisions based on the combinations and concentrations of these proteins in a given nucleus.

Several other stripe-specific enhancers have been characterized in detail (38-40). These analyses suggest a general model for the initial establishment of individual stripes (41). In this model, factors involved in activating individual stripes are broadly distributed, and stripe borders are set by repressive interactions mediated by gradients of gap proteins. Activations and repression events involve direct binding to closely linked sites within each enhancer. The close linkage of repressor to activator sites suggests that the repressors function over short distances to interfere with the binding or activity of activator proteins. Consistent with this hypothesis, increasing the spacing between activator and repressor sites to an interval greater than ~100 bp can prevent gap protein-mediated repression from occuring (42, 43).

Short-range repression is also important for ensuring that individual enhancers function independently in the context of complex promoters. This has been tested by transgenic experiments using reporter genes that contain the eve stripe 2 enhancer and another 500 bp enhancer that regulates stripes 3 and 7 (40). In the wild-type gene, these two enhancers are separated by a ~1,700 bp sequence. When this sequence is deleted so that the enhancers are juxtaposed, there is a severe disruption of the pattern driven by the reporter gene (44). Inserting shorter spacer sequences (160 bp and 300 bp) between the enhancers restores the correct expression pattern. These results suggest that transacting factors bound to one enhancer interfere with the activity of other enhancers and that spacing between enhancers prevents such interface in the wild-type gene.

2. Regulatory Mechanisms That Maintain and Refine Striped Patterns

Because different mechanisms control the establishment of individual eve and h stripes, they are expressed in a specific temporal order while the embryo is going through the process of cellularization. Each enhancer contributes to a final pattern of seven stripes that are each approximately four to five nucleus diameters wide (Figure 4A). By the end of cellularization, these initial patterns are refined until individual stripes are two or three cells wide. During this process, some seven stripe patterns are replaced by fourteen stripes, either by splitting the initial stripes or by activating new stripes in interstripe regions. Precise overlaps between specific genes are established so that each row of nuclei within a segmental unit contains a unique combination of pair-rule proteins. Genetic experiments suggest that positive interactions among the pair-rule genes maintain the stripes during this process, while negative interactions control spatial refinement. For example, there is a premature loss of eve stripes in eve and paired (prd) mutants, suggesting roles for these genes in maintaining the striped pattern (45, 46). In contrast, there is a significant posterior expansion of eve stripes in run mutants, suggesting that repression by run is important for refining the stripes (13, 31). Ubiquitous expression of run via heat shock causes a rapid repression of eve, consistent with this hypothesis (22). In contrast, ftz stripes are prematurely lost in ftz and run mutants, and expanded in h mutants, suggesting that these genes maintain and refine the ftz pattern (12, 47, 48).

Figure 4. (A) Interactions among pair-rule genes that maintain and refine initial striped patterns. Four parasegmental units are shown. Arrows represent positive interactions, blunted lines negative. The process of maintenance and refinement narrows stripes from four to five nuclei wide to two to three wide. (B) Spatial relationship between refined pair-rule patterns and the expression pattern of the segment polarity gene. en. en is expressed in cells that contain specific combinations of pair-rule proteins. B is colinear with A.

The molecular mechanisms involved in these interactions are only beginning to be understood. Promoter truncation and P-element transformation experiments have identified enhancers in eve and ftz that direct reporter gene expression in patterns of seven stripes (31, 32, 49). These stripes appear late in the process of cellularization, persisting through the stages of gastrulation and germ band elongation. This timing is consistent with a role for these enhancers in maintaining and refining initial striped patterns. An example is a 300 bp eve enhancer that directs a late pattern of seven stripes coincident with the endogenous pattern (46, 50). This enhancer is inactive in embryos that lack eve or prd function, suggesting that a combination of autoregulation and cross regulation by prd activates transcription of this enhancer. The enhancer contains binding sites for both proteins; mutating these sites causes a loss of expression, suggesting that direct DNA-binding is required for activation (46, 50).

Another autoregulatory element involved in pair-rule refinement is 430 bp ftz enhancer (AE) that contains multiple ftz binding sites; when these sites are mutated, the enhancer is inactivated (51). Further experiments strongly indicate that the in vivo activation of this enhancer involves direct binding by ftz. These experiments take advantage of the fact that the ftz protein, which contains a glutamine residue at position 50 of the homeodomain, binds in vitro with high affinity to a specific class of binding sites (CCATTA) (52, 53). Other homeodomain proteins, such as bed, contain lysine at this position and prefer a different sequence (GGATTA). Changing a single CCATTA binding site in the ftz AE to GGATTA causes a significant loss of AE activity (51). However, activity is restored in trans by a ftz rescue construct in which the glutamine at position 50 is replaced with a lysine residue. This experiment confirms the importance of this residue in specific DNA recognition by homeodomain proteins and demonstrates conclusively that direct DNA-binding by ftz is required for the activity of this enhancer.

It is worth noting, however, that the 430 bp AE represents only a small part of a complex 2,600 bp upstream element (UE) that is important for ftz stripe maintenance (54-57). A separate enhancer, the zebra element (ZE), also direct reporter gene expression in late cellularizing embryos (49). Together, these enhancers integrate positive inputs provided by ftz, the ftz-cofactor Ftz-F1, and run, as well as negative inputs mediated by h that refine the stripes from the posterior (48, 58, 59). Binding sites for gap proteins and other ubiquitously distributed factors such as tramtrack (ttk), have also been identified in these regions, but their exact roles in fiz pattering are still unclear (56, 60).

3. Functions of the Striped Patterns of the Pair-Rules Genes

Precise overlapping patterns of the pair-rule genes are established by the end of cellularization. These patterns comprise a coordinated system that controls the expression of the segment polarity class of segmentation genes. An example is the segment polarity gene engrailed (en), which is expressed in fourteen stripes one cell wide (61, 62). Cells that express en will remain segregated from neighboring cells lacking en throughout development. This segregation establishes a border between the anterior and posterior compartments within each parasegmental unit. Genetic and misexpression experiments suggest that odd and even-numbered stripes within the en pattern are regulated by different mechanisms involving pair-rule genes (21-23, 63-66) (Fig 4). Oddnumbered stripes are activated in cells that contain eve and prd protein, and the anterior and posterior borders of these stripes are set by sloppy-paired (slp) and run respectively. Even numbered stripes are activated in cells than contain ftz and prd protein, and the anterior and posterior borders of these stripes are probably set by slp and odd-skipped (odd), respectively. Very little is known, however, about the cis components that control the expression of either set of en stripes or any other segment polarity gene.

In summary, the pair-rule genes make up an integrated system that transduces asymmetrically localized positional information into fourteen stripe patters that establish the segmental body plan of Drosphila. Significant progress has been made in unraveling certain aspects of the mechanisms involved, but the complexity of the system suggests that a full understanding will not possible using only a reductionist approach. Recent attempts have been made to use computer modeling to integrate individual components of the pair-rule system into a gene circuit method (67). This method has successfully generated a complete pattern of seven stripes at the eve position using quantitative data representing the expression patterns of the maternal and gap proteins. In the future, this type of analysis will be important for synthesizing molecular data and predicting mechanisms that can then be tested experimentally.