The Interactive Fly

Zygotically transcribed genes

Gap Genes and Pair Rule Genes

  • Pair rule genes: the origin of the segment-polarity pattern
  • Dynamic maternal gradients control timing and shift-rates for Drosophila gap gene expression
  • An enhancer's length and composition are shaped by its regulatory task
  • Dynamic patterning by the Drosophila pair-rule network reconciles long-germ and short-germ segmentation
  • Conservation and variation in pair-rule gene expression and function in the intermediate-germ beetle, Dermestes maculatus
  • Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors
    Gap genes

    Pair rule genes

    Unclassified genes involved in regulation of gap and pair rule genes

    Pair rule genes: the origin of the segment-polarity pattern
    The pair-rule class of segmentation genes was identified in a Nobel award winning genetic screen, performed in the late 1970s by Christiane Nusslein-Volhard, 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, fushi-tarazu, 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, paired and 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 pair-rule 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 sloppy-paired), which in turn play a more direct role in the establishment of segment-polarity 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 segment-polarity 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 even-numbered engrailed stripes. Thus, the correct establishment of alternate engrailed stripes requires the combined activities of a subset of pair-rule 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 even-numbered 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 en indirectly 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 even-numbered 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).

    This essay courtesy of and copyright © 1997, Ellen J. Ward

    Dynamic maternal gradients control timing and shift-rates for Drosophila gap gene expression

    This study simulated dynamic morphogen interpretation by the gap gene network in Drosophila. Gap genes are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. This study used a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, the system was simulate in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. A newly developed method was used for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, this study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development (Verd, 2017).

    An enhancer's length and composition are shaped by its regulatory task

    Enhancers drive the gene expression patterns required for virtually every process in metazoans. It is proposed that enhancer length and transcription factor (TF) binding site composition-the number and identity of TF binding sites-reflect the complexity of the enhancer's regulatory task. In development, regulatory task complexity is defined as the number of fates specified in a set of cells at once. It is hypothesized that enhancers with more complex regulatory tasks will be longer, with more, but less specific, TF binding sites. Larger numbers of binding sites can be arranged in more ways, allowing enhancers to drive many distinct expression patterns, and therefore cell fates, using a finite number of TF inputs. This study compared ~100 enhancers patterning the more complex anterior-posterior (AP) axis and the simpler dorsal-ventral (DV) axis in Drosophila and found that the AP enhancers are longer with more, but less specific binding sites than the (DV) enhancers. Using a set of ~3,500 enhancers, enhancer length and TF binding site number were found to increase with increasing regulatory task complexity. Therefore, to be broadly applicable, computational tools to study enhancers must account for differences in regulatory task (Li, 2017).

    Dynamic patterning by the Drosophila pair-rule network reconciles long-germ and short-germ segmentation

    Drosophila segmentation is a well-established paradigm for developmental pattern formation. However, the later stages of segment patterning, regulated by the "pair-rule" genes, are still not well understood at the system level. Building on established genetic interactions, a logical model of the Drosophila pair-rule system was constructed that takes into account the demonstrated stage-specific architecture of the pair-rule gene network. Simulation of this model can accurately recapitulate the observed spatiotemporal expression of the pair-rule genes, but only when the system is provided with dynamic "gap" inputs. This result suggests that dynamic shifts of pair-rule stripes are essential for segment patterning in the trunk and provides a functional role for observed posterior-to-anterior gap domain shifts that occur during cellularisation. The model also suggests revised patterning mechanisms for the parasegment boundaries and explains the aetiology of the even-skipped null mutant phenotype. Strikingly, a slightly modified version of the model is able to pattern segments in either simultaneous or sequential modes, depending only on initial conditions. This suggests that fundamentally similar mechanisms may underlie segmentation in short-germ and long-germ arthropods (Clark, 2017).

    Conservation and variation in pair-rule gene expression and function in the intermediate-germ beetle, Dermestes maculatus

    A set of pair-rule segmentation genes (PRGs) promote the formation of alternate body segments in Drosophila melanogaster While Drosophila embryos are long-germ, with segments specified more-or-less simultaneously, most insects add segments sequentially as the germband elongates. The hide beetle, Dermestes maculatus, represents an intermediate between short- and long-germ development, ideal for comparative study of PRGs. This study shows that eight of nine Drosophila PRG-orthologs are expressed in stripes in Dermestes. Functional results parse these genes into three groups: Dmac-eve, -odd, and -run play roles in both germband elongation and PR-patterning. Dmac-slp and -prd function exclusively as complementary, classic PRGs, supporting functional decoupling of elongation and segment formation. Orthologs of ftz, ftz-f1, h, and opa show more variable function in Dermestes and other species. While extensive cell death generally prefigured Dermestes PRG RNAi cuticle defects, an organized region with high mitotic activity near the margin of the segment addition zone likely contributes to truncation of eve(RNAi) embryos. These results suggest general conservation of clock-like regulation of PR-stripe addition in sequentially-segmenting species while highlighting regulatory re-wiring involving a subset of PRG-orthologs (Xiang, 2017).

    Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors

    Long-germ insects, such as the fruit fly Drosophila melanogaster, pattern their segments simultaneously, whereas short-germ insects, such as the beetle Tribolium castaneum, pattern their segments sequentially, from anterior to posterior. While the two modes of segmentation at first appear quite distinct, much of this difference might simply reflect developmental heterochrony. This study now shows that, in both Drosophila and Tribolium, segment patterning occurs within a common framework of sequential Caudal, Dichaete, and Odd-paired expression (see Comparison of long-germ and short-germ segmentation). In Drosophila these transcription factors are expressed like simple timers within the blastoderm, while in Tribolium they form wavefronts that sweep from anterior to posterior across the germband. In Drosophila, all three are known to regulate pair-rule gene expression and influence the temporal progression of segmentation. It is proposed that these regulatory roles are conserved in short-germ embryos, and that therefore the changing expression profiles of these genes across insects provide a mechanistic explanation for observed differences in the timing of segmentation. In support of this hypothesis it was demonstrated that Odd-paired is essential for segmentation in Tribolium, contrary to previous reports (Clark, 2018).

    This study has found that segment patterning in both Drosophila and Tribolium occurs within a conserved framework of sequential Caudal, Dichaete and Odd-paired expression. In the case of Opa, there is also evidence for conserved function. However, although the sequence itself is conserved between the two insects, its spatiotemporal deployment across the embryo is divergent. In Drosophila, the factors are expressed ubiquitously within the main trunk, and each turns on or off almost simultaneously, correlating with the temporal progression of a near simultaneous segmentation process. In Tribolium, their expression domains are staggered in space, with developmentally more advanced anterior regions always subjected to a 'later' regulatory signature than more-posterior tissue. These expression domains retract over the course of germband extension, correlating with the temporal progression of a sequential segmentation process built around a segmentation clock (Clark, 2018).

    Pair-rule patterning involves several distinct phases of gene expression, each requiring specific regulatory logic. It is proposed that, in both long-germ and short-germ species, the whole process is orchestrated by a series of key regulators, expressed sequentially over time, three of which are the focus of this paper. By rewiring the regulatory connections between other genes, factors such as Dichaete and Opa allow a small set of pair-rule factors to carry out multiple different roles, each specific to a particular spatiotemporal regulatory context. This kind of control logic makes for a flexible, modular regulatory network, and may therefore turn out to be a hallmark of other complex patterning systems (Clark, 2018).

    Having highlighted the significance of these 'timing factors' in this paper, the next steps will be to investigate their precise regulatory roles and modes of action. It will be interesting to dissect how genetic interactions with pair-rule factors are implemented at the molecular level. Dichaete is known to act both as a repressive co-factor and as a transcriptional activator; therefore, a number of different mechanisms are plausible. The Odd-paired protein is also likely to possess both these kinds of regulatory activities (Clark, 2018).

    Given the phylogenetic distance between beetles and flies (separated by at least 300 million years), it is expected that the similarities seen between Drosophila and Tribolium segmentation are likely to hold true for other insects, and perhaps for many non-insect arthropods as well. It is proposed that these similarities, which argue for the homology of long-germ and short-germ segmentation processes, result from conserved roles of Cad, Dichaete and Opa in the temporal regulation of pair-rule and segment-polarity gene expression during segment patterning. This hypothesis can be tested by detailed comparative studies in various arthropod model organisms (Clark, 2018).

    This study provides evidence that a segmentation role for Opa is conserved between Drosophila and Tribolium; clear segmentation phenotypes have also been found for Cad in Nasonia, and for Dichaete in Bombyx. However, as the Tc-opa experiments reveal, functional manipulations in short-germ insects will need to be designed carefully in order to bypass the early roles of these pleiotropic genes. For example, cad knockdowns cause severe axis truncations in many arthropods, whereas Dichaete knockdown in Tribolium yields mainly empty eggs (Clark, 2018).

    It was previously thought that Tc-opa was not required for segmentation, and that the segmentation role of Opa may have been recently acquired, in the lineage leading to Drosophila. However, the current analysis reveals that Tc-opa is indeed a segmentation gene, and also has other important roles, including head patterning and blastoderm formation. Given that a similar developmental profile of opa expression is seen in the millipede Glomeris, and even in the onychophoran Euperipatoides, the segmentation role of Opa may actually be ancient (Clark, 2018).

    Head phenotypes following Tc-opa RNAi were unexpected, but both the blastoderm expression pattern and cuticle phenotypes that were observed are strikingly similar to those reported for Tc-otd and Tc-ems (Tribolium orthologues of the Drosophila head 'gap' genes orthodenticle and empty spiracles), suggesting that the three genes function together in a gene network that controls early head patterning. This function of Tc-opa might be homologous to the head patterning role for Opa discovered in the spider Parasteatoda, where it interacts with both Otd and Hedgehog (Hh) expression. Opa/Zic is known to modulate Hh signalling, and a role for Hh in head patterning appears to be conserved across arthropods, including Tribolium (Clark, 2018).

    Finally, Opa/Zic is also known to modulate Wnt signalling. In chordates, Zic expression tends to overlap with sites of Hh and/or Wnt signalling, suggesting that one of its key roles in development is to ensure cells respond appropriately to these signals. The expression domains of Tc-opa that were observed in Tribolium (e.g. in the head, in the SAZ and between parasegment boundaries) accord well with this idea (Clark, 2018).

    Similar embryonic expression patterns of Cad, Dichaete and Opa orthologues are observed in other bilaterian clades, including vertebrates. Cdx genes are expressed in the posterior of vertebrate embryos, where they play crucial roles in axial extension and Hox gene regulation. Sox2 (a Dichaete orthologue) has conserved expression in the nervous system, but is also expressed in a posterior domain, where it is a key determinant of neuromesodermal progenitor (posterior stem cell) fate. Finally, Zic2 and Zic3 (Opa orthologues) are expressed in presomitic mesoderm and nascent somites, and have been functionally implicated in somitogenesis and convergent extension. All three factors thus have important functions in posterior elongation, roles that may well be conserved across Bilateria (Clark, 2018).

    In Tribolium, all three factors may be integrated into an ancient gene regulatory network downstream of posterior Wnt signalling, which generates their sequential expression and helps regulate posterior proliferation and/or differentiation. The mutually exclusive patterns of Tc-wg and Tc-Dichaete in the posterior germband are particularly suggestive: Wnt signalling and Sox gene expression are known to interact in many developmental contexts and these interactions may form parts of temporal cascades) (Clark, 2018).

    The following outline is suggested as a plausible scenario for the evolution of arthropod segmentation; In non-segmented bilaterian ancestors of the arthropods, Cad, Dichaete and Opa were expressed broadly similarly to how they are expressed in Tribolium today, mediating conserved roles in posterior elongation, while gap and pair-rule genes may have had functions in the nervous system. At some point, segmentation genes came under the regulatory control of these factors, which provided a pre-existing source of spatiotemporal information in the developing embryo. Pair-rule genes began oscillating in the posterior, perhaps under the control of Cad and/or Dichaete, while the posteriorly retracting expression boundaries of the timing factors provided smooth wavefronts that effectively translated these oscillations into periodic patterning of the AP axis, analogous to the roles of the opposing retinoic acid and FGF gradients in vertebrate somitogenesis. Much later, in certain lineages of holometabolous insects, the transition to long-germ segmentation occurred. This would have involved two main modifications of the short-germ segmentation process: (1) changes to the expression of the timing factors, away from the situation seen in Tribolium, and towards the situation seen in Drosophila, causing a heterochronic shift in the deployment of the segmentation machinery from SAZ to blastoderm; and (2) recruitment of gap genes to pattern pair-rule stripes, via the ad hoc evolution of stripe-specific elements (Clark, 2018).

    The appeal of this model is that the co-option of existing developmental features at each stage reduces the number of regulatory changes required to evolve de novo, facilitating the evolutionary process. In this scenario, arthropod segmentation would not be homologous to segmentation in other phyla, but would probably have been fashioned from common parts (Clark, 2018).


    Carroll, S. B. (1990). Zebra patterns in fly embryos: activation of stripes or repression of interstripes? Cell 60: 9-16. PubMed Citation: 2403844

    Clark, E. (2017). Dynamic patterning by the Drosophila pair-rule network reconciles long-germ and short-germ segmentation. PLoS Biol 15(9): e2002439. PubMed ID: 28953896

    Clark, E. and Peel, A. D. (2018). Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors. Development. PubMed ID: 29724758

    DiNardo, S. and O'Farrell, P. (1987). Establishment and refinement of segmental pattern in the Drosophila embryo: spatial control of engrailed expression by pair-rule genes. Genes & Development 1: 1212-25. PubMed Citation: 3123316

    Fujioka, M., et al. (1995). Early even-skipped stripes act as morphogenetic gradients at the single cell level to establish engrailed expression. Development 121 (12): 4371-82. PubMed Citation: 8575337

    Lawrence, P. A. and Johnston, P. (1989). Pattern formation in the Drosophila embryo: allocation of cells to parasegments by even-skipped and fushi tarazu. Development. 105: 761-7. PubMed Citation: 2598812

    Li, L. and Wunderlich, Z. (2017). An enhancer's length and composition are shaped by its regulatory task. Front Genet 8: 63. PubMed ID: 28588608

    Manoukian, A. and Krause, H. (1992). Concentration-dependent activities of the even-skipped protein in Drosophila embryos. Genes & Development. 6: 1740-51. PubMed Citation: 1355458

    Mullen, J. R. and DiNardo, S. (1995). Establishing parasegments in Drosophila embryos: roles of odd-skipped and naked genes. Dev. Biol. 169: 295-308. PubMed Citation: 7750646

    Nusslein-Volhard, C. and Wieschaus, E. (1980). Mutations affecting segment number and polarity in Drosophila. Nature 287: 795-801. PubMed Citation: 6776413

    Pankratz, M. J. and Jackle, J. (1993). Blastoderm segmentation. In The Development of Drosophila melanogaster. M. Bate and A. M. Arias, eds. Cold Spring Harbor Lab Press, Vol. 1. pp. 467-516

    Verd, B., Crombach, A. and Jaeger, J. (2017). Dynamic maternal gradients control timing and shift-rates for Drosophila gap gene expression. PLoS Comput Biol 13(2): e1005285. PubMed ID: 28158178

    Ward, E.J. Dissertation. Characterization of Odd-skipped Protein Pattern of Accumulation During Embryogenesis in D. Melanogaster. 1997. St. Louis University, St. Louis, Missouri.

    Ward, E.J. and Coulter, D.E. Interactions among odd-skipped, fushi-tarazu and even-skipped define the even-numbered engrailed stripes in the Drosophila embryo (Personal communication to The Interactive Fly)

    Xiang, J., Reding, K., Heffer, A. and Pick, L. (2017). Conservation and variation in pair-rule gene expression and function in the intermediate-germ beetle, Dermestes maculatus. Development 144(24):4625-4636. PubMed ID: 29084804

    Zygotically transcribed genes

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