The pair-rule class of segmentation genes was identified in a Nobel award winning genetic screen, performed in the late

1970s by Christiane NussleinVolhard, Eric Wieschaus and co-workers. Pair-rule mutants affect homologous parts of the cuticle pattern in alternate segments, resulting in the so-called "double-segment" periodicity of defects. As noted by Nusslein-Volhard and Wieschaus (1980) in their original report, the pair-rule class of segmentation genes was unexpected; the presence of this class suggests that during development, the embryo initially has a repeat unit corresponding to two segments, which are later subdivided into individual segments. Since their discovery, all eight 'classical' pair-rule genes (even-skipped, hairy, runt, fushitarazu, odd-skipped, paired, odd-paired, and sloppy-paired) have been studied at the genetic and molecular levels. Each gene encodes a transcription factor (Pankratz, 1993) that is expressed in regions corresponding to each gene's domains of function, as deduced from the gene's mutant phenotype. A hallmark of the pair-rule genes is their striped pattern of expression at the end of the cellular blastoderm stage. Interestingly, analysis of pair-rule gene expression during early cellularization indicates that, as a class, the pair-rule genes are initially expressed in domains larger than their 'final' pair-rule pattern. In fact, the products of most pair-rule genes initially accumulate throughout the entire metameric region. During cellularization, pair-rule stripes are formed by a combination of events: accumulation of products within the stripes and loss of pair-rule products in the intervening interstripes (Carroll, 1990). Formation of the interstripes must reflect a combination of transcriptional repression coupled with the turnover of gene products, whereas, establishment of stripes must reflect continued (and possibly increased) expression. Since the equivalent sequence of stripe patterns is seen for pair-rule mRNA and protein, neither translational regulation nor differential rates of protein turnover (relative to mRNA) appear likely to have a s ignificant influence on pair-rule pattern. This suggests that as a class, the pair-rule genes share a common mechanism for the rapid turnover of their gene products. Since the pair-rule genes are posited to function in discrete domains of the embryo, determining how the pair-rule gene products are specifically synthesized and degraded (or turned-over) will be important for a thorough understanding of how this class functions during development. Interestingly, although the pair-rule genes were identified genetically by their pattern defects in alternate segments, this class of segmentation genes is expressed in a wide variety of tissues during embryogenesis. One of the most fascinating examples of unexpected expression subsequent to cellularization is the 'segment-polarity' pattern of expression observed with gene products from

the even-skipped, runt, odd-skipped, pairedand odd-paired loci. In addition, select pair-rule genes are expressed in the mesoderm, gut and most notably the central nervous system. During segmentation, the pair-rule genes function as intermediates between the nonperiodic expression of gap genes and the repeated expression patterns of the segment-polarity genes. Understanding how each pair-rule gene functions within the segmentation hierarchy involves addressing at least three questions: 1. What genes are required for the correct expression of a particular pairrule gene? 2. For a given pair-rule gene, what are its downstream targets? 3. What other pair-rule genes, if any, interact with an individual pair-rule gene in regulating these targets? Initially, it was proposed that the gap genes were the sole regulators of pair-rule expression, and in turn, the segment-polarity genes were thought to be the sole targets of pair-rule function. However, studies have shown that some pair-rule genes are regulated by other members of the pair-rule class, suggesting that the pair-rule class contains both regulators and targets of pair-rule genes (Pankratz, 1993). According to this view, only a subset of pair-rule genes (even-skipped, hairy, and runt) respond directly to gap information. These so-called "primary" pair-rule genes regulate the expression of the remaining so-called "secondary" pair-rule genes (fushi-tarazu, odd-skipped, paired, odd-paired, and sloppypaired), which in turn play a more direct role in the establishment of segmentpolarity gene expression. While this probably over-simplifies the nature of these interactions, it is clear that understanding the role of a particular pair-rule gene in the segmentation hierarchy requires not only an analysis of the individual gene's role as a mediator between the gap and segment-polarity classes, but also requires identifying its function within the pair-rule class. A well-characterized target of pair-rule function is the segmentpolarity gene engrailed (en), which is expressed in 14 stripes during gastrulation and at all subsequent stages of embryogenesis. Each engrailed stripe marks the posterior margin of a segment, or the anterior margin of a parasegment. Correct establishment of the 14 engrailed stripes requires the activities of all the pair-rule genes (DiNardo, 1987). However, in general, mutations in individual pair-rule genes affect either odd- or evennumbered engrailed stripes. Thus, the correct establishment of alternate engrailed stripes requires the combined activities of a subset of pairrule genes.

How do these combinations of pair-rule activities regulate engrailed? At the time when en is initially expressed, the various pair-rule genes are present in distinct stripes that frequently overlap. It is widely held that the resulting overlapping stripes can be translated into vertical rows of cells containing unique combinations of pair-rule gene products capable of interacting with one another to establish the correct pattern of en and other segmentation genes (DiNardo, 1987). According to this model, overlapping stripes of activators (e.g. Fushi-tarazu) and repressors (such as Odd-skipped) define the narrow engrailed stripes. While this 'combinatorial model' provides a basis for understanding how narrow engrailed stripes are specified by broad pair-rule stripes, it does not define the precise interactions between the individual pair-rule genes. For example, Odd-skipped (Odd), a repressor of en, and Fushi-tarazu (Ftz), an activator of en, are proposed to interact with one another to specify the evennumbered en stripes. Two hypotheses have been proposed to account for the correct positioning of a narrow en stripe within a broad Ftz stripe. In the Hierarchy model, en responds to a critical threshold level of Ftz activity within the Ftz stripe (Lawrence, 1989). In this model, Odd and Ftz are proposed to interact in a hierarchy, with Odd protein functioning to repress ftz, presumably at the transcriptional level (Mullen, 1995). A key feature of this model is that Odd represses enindirectly by reducing Ftz levels. In the alternate view, the Combinatorial model, the presence of a negative regulator of en prevents en expression within portions of the Ftz stripe. According to this model, Odd is proposed to interact in a combinatorial (parallel) manner with Ftz. The distinguishing feature of this model is that Odd does not affect en by altering Ftz levels; rather, Odd acts to prevent en activation within the Ftz stripe. Recent experimental data (see Odd) strongly supports the Combinatorial model of interactions between Odd and Ftz (Ward, 1997 and Ward and Coulter, manuscript in prep.). Thus, in this case, two "secondary" pair-rule genes appear to act independently of one another in order to define precisely the evennumbered en stripes. Interestingly, the primary pair-rule gene even-skipped (eve) appears to directly regulate ftz and odd to define the anterior margins of these two secondary pair-rule genes, thereby allowing en to be activated in a narrow stripe (Ward, 1997, Ward and Coulter, manuscript in prep., Fujioka, 1995 and Manoukian, 1992).