Primary Pair-rule Genes Research Articles

Temporal control of RNAi reveals both robust and labile feedback loops in the segmentation clock of the red flour beetle

Animals from all major clades have evolved a segmented trunk, reflected in the human spine or the insect segments. These units emerge during embryogenesis from a posterior segment addition zone (SAZ), where repetitive gene activity is regulated by a mechanism described by the clock and wavefront/speed gradient model. In the red flour beetle Tribolium castaneum, RNA interference (RNAi) has been used to continuously knock down the function of primary pair-rule genes (pPRGs), caudal or Wnt pathway components, which has led to the complete breakdown of segmentation. However, it has remained untested, if this breakdown was reversible by bringing the missing gene function back to the system. To fill this gap, we established a transgenic system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi via heat shock. We show that the T. castaneum segmentation machinery was able to reestablish after RNAi targeting the pPRGs Tc-eve, Tc-odd, and Tc-runt was blocked. However, we observed no rescue after blocking RNAi targeting Wnt pathway components. We conclude that the insect segmentation system contains both robust feedback loops that can reestablish and labile feedback loops that break down irreversibly. This combination may reconcile conflicting needs of the system: Labile systems controlling initiation and maintenance of the SAZ ensure that only one SAZ is formed. Robust feedback loops confer developmental robustness toward external disturbances.

Studying primary pair-rule gene eve in the early development of Drosophila by using Zelda as approach.

This paper mainly studies the primary pair-rule gene Even-skipped to reveal its position in the early transcription network established in the Drosophila embryo and gain a deeper view of the network. We used the genome activator Zelda as an approach. We found Zelda’s role in regulating Eve by committing CHIP- seq towards RNA polymerase and Zelda protein in wt and Zld- embryo. With the PWM matrix, we know transcription factors that might bind to eve. By further researching its TFs, we find the relationship between timing and pattern of expression of these genes involved in the early transcriptional network, while Zld is still our approach. Also, by looking into the specific gene, we find the possible timing mechanism on Eve based on the accumulation of earlier transcribed transcription factors. The role of maternally loaded transcription factors in early development is once again shown by affecting the timing and patterning of primary patterning transcription factors.

Growth zone segmentation in the milkweed bug Oncopeltus fasciatus sheds light on the evolution of insect segmentation

BackgroundOne of the best studied developmental processes is the Drosophila segmentation cascade. However, this cascade is generally considered to be highly derived and unusual, with segments being patterned simultaneously, rather than the ancestral sequential segmentation mode. We present a detailed analysis of the segmentation cascade of the milkweed bug Oncopletus fasciatus, an insect with a more primitive segmentation mode, as a comparison to Drosophila, with the aim of reconstructing the evolution of insect segmentation modes.ResultsWe document the expression of 12 genes, representing different phases in the segmentation process. Using double staining we reconstruct the spatio-temporal relationships among these genes. We then show knock-down phenotypes of representative genes in order to uncover their roles and position in the cascade.ConclusionsWe conclude that sequential segmentation in the Oncopeltus germband includes three slightly overlapping phases: Primary pair-rule genes generate the first segmental gene expression in the anterior growth zone. This pattern is carried anteriorly by a series of secondary pair-rule genes, expressed in the transition between the growth zone and the segmented germband. Segment polarity genes are expressed in the segmented germband with conserved relationships. Unlike most holometabolous insects, this process generates a single-segment periodicity, and does not have a double-segment pattern at any stage. We suggest that the evolutionary transition to double-segment patterning lies in mutually exclusive expression patterns of secondary pair-rule genes. The fact that many aspects of the putative Oncopeltus segmentation network are similar to those of Drosophila, is consistent with a simple transition between sequential and simultaneous segmentation.

Differential expression of primary pair-rule genes during bidirectional regeneration in Perionyx excavatus.

The earthworm Perionyx excavatus is a species highly capable of bidirectional regeneration. Pair-rule genes are thought to have an ancestral function in arthropod segmentation. However, orthologs in annelids (i.e. Capitella teleta) do not exhibit segmental expression in the ectoderm or mesoderm. Their role in regeneration is currently unclear. Here, we report the expression profile of primary pair-rule genes (Pex-EvxA, Pex-EvxB, Pex-RuntA, Pex-RuntB, Pex-Hes1A, Pex-Hes1B, Pex-Hes4A, and Pex-Hey) found in P. excavatus using semi-quantitative reverse transcription-polymerase chain reaction. Our results indicated these genes showed variable expression during bidirectional regeneration. Six of these genes might play diverse and potentially critical roles in head and/or tail regeneration.

Evolution of the pair rule gene network: Insights from a centipede

Comparative studies have examined the expression and function of homologues of the Drosophila melanogaster pair rule and segment polarity genes in a range of arthropods. The segment polarity gene homologues have a conserved role in the specification of the parasegment boundary, but the degree of conservation of the upstream patterning genes has proved more variable. Using genomic resources we identify a complete set of pair rule gene homologues from the centipede Strigamia maritima, and document a detailed time series of expression during trunk segmentation. We find supportive evidence for a conserved hierarchical organisation of the pair rule genes, with a division into early- and late-activated genes which parallels the functional division into primary and secondary pair rule genes described in insects. We confirm that the relative expression of sloppy-paired and paired with respect to wingless and engrailed at the parasegment boundary is conserved between myriapods and insects; suggesting that functional interactions between these genes might be an ancient feature of arthropod segment patterning. However, we find that the relative expression of a number of the primary pair rule genes is divergent between myriapods and insects. This corroborates suggestions that the evolution of upper tiers in the segmentation gene network is more flexible. Finally, we find that the expression of the Strigamia pair rule genes in periodic patterns is restricted to the ectoderm. This suggests that any direct role of these genes in segmentation is restricted to this germ layer, and that mesoderm segmentation is either dependent on the ectoderm, or occurs through an independent mechanism.

A segmentation clock operating in blastoderm and germband stages ofTriboliumdevelopment

In Drosophila, all segments form in the blastoderm where morphogen gradients spanning the entire anterior-posterior axis of the embryo provide positional information. However, in the beetle Tribolium castaneum and most other arthropods, a number of anterior segments form in the blastoderm, and the remaining segments form sequentially from a posterior growth zone during germband elongation. Recently, the cyclic nature of the pair-rule gene Tc-odd-skipped was demonstrated in the growth zone of Tribolium, indicating that a vertebrate-like segmentation clock is employed in the germband stage of its development. This suggests that two mechanisms might function in the same organism: a Drosophila-like mechanism in the blastoderm, and a vertebrate-like mechanism in the germband. Here, we show that segmentation at both blastoderm and germband stages of Tribolium is based on a segmentation clock. Specifically, we show that the Tribolium primary pair-rule gene, Tc-even-skipped (Tc-eve), is expressed in waves propagating from the posterior pole and progressively slowing until they freeze into stripes; such dynamics are a hallmark of clock-based segmentation. Phase shifts between Tc-eve transcripts and protein confirm that these waves are due to expression dynamics. Moreover, by tracking cells in live embryos and by analyzing mitotic profiles, we found that neither cell movement nor oriented cell division could explain the observed wave dynamics of Tc-eve. These results pose intriguing evolutionary questions, as Drosophila and Tribolium segment their blastoderms using the same genes but different mechanisms.

How to make stripes: deciphering the transition from non-periodic to periodic patterns inDrosophilasegmentation

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, we have conducted a comprehensive systems-level examination of the regulatory architecture underlying pair-rule stripe formation. We find that runt (run), fushi tarazu (ftz) and odd skipped (odd) 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, we observe long-range cis-regulation 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, our study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo.

Loss of Tc-arrow and canonical Wnt signaling alters posterior morphology and pair-rule gene expression in the short-germ insect, Tribolium castaneum

Wnt signaling has been implicated in posterior patterning in short-germ insects, including the red flour beetle Tribolium castaneum (Bolognesi et al. Curr Biol 18:1624-1629, 2008b; Angelini and Kaufman Dev Biol 283:409-423, 2005; Miyawaki et al. Mech Dev 121:119-130, 2004). Specifically, depletion of Wnt ligands Tc-Wnt1 and Tc-WntD/8 produces Tribolium embryos lacking abdominal segments. Similar phenotypes are produced by depletion of Tc-porcupine (Tc-porc) or Tc-pangolin (Tc-pan), indicating that the signal is transmitted through the canonical Wnt pathway (Bolognesi et al. Curr Biol 18:1624-1629, 2008b). Here we show that RNAi for the receptor Tc-arrow produced similar truncated phenotypes, providing additional evidence supporting canonical signal transduction. Furthermore, since in Tribolium segments are defined sequentially by a pair-rule gene circuit that, when interrupted, produces truncated phenotypes (Choe et al. Proc Natl Acad Sci U S A 103:6560-6564, 2006), we investigated the relationship between loss of Wnt signaling and this pair-rule gene circuit. After depletion of the receptor Tc-arrow, expression of Tc-Wnt1 was noticeably absent from the growth zone, while Tc-WntD/8 was restricted to a single spot of expression in what remained of the posterior growth zone. The primary pair-rule genes Tc-runt (Tc-run) and Tc-even-skipped (Tc-eve) were expressed normally in the anterior segments, but were reduced to a single spot in the remnants of the posterior growth zone. Thus, expression of pair-rule genes and Tc-WntD/8 are similarly affected by depletion of Wnt signal and disruption of the posterior growth zone.

Genetic regulation of engrailed and wingless in Tribolium segmentation and the evolution of pair-rule segmentation

In Drosophila, primary pair-rule genes establish the parasegmental boundaries and indirectly control the periodic expression of the segment polarity genes engrailed ( en) and wingless ( wg) via regulation of secondary pair-rule genes. Although orthologs of some Drosophila pair-rule genes are not required for proper segmentation in Tribolium, segmental expression of Tc-en and Tc-wg is conserved. To understand how these segment polarity genes are regulated, we examined the results of expressing one or two pair-rule genes in the absence of the other known pair-rule genes. Expression of one or both of the secondary pair-rule genes, Tc-sloppy-paired ( Tc-slp) and Tc-paired ( Tc-prd), activated Tc-wg in the absence of the primary pair-rule genes, Tc-even-skipped ( Tc-eve), Tc-runt ( Tc-run) and Tc-odd-skipped ( Tc-odd). Tc-eve alone failed to activate Tc-wg or Tc-en, but in combination with Tc-run or Tc-prd activated Tc-en. These results, interpreted within the pair-rule gene expression patterns, suggest separate models for the genetic regulation of the juxtaposed expression of Tc-wg and Tc-en at odd- and even-numbered parasegmental boundaries, respectively. Conserved interactions between eve and prd at the anterior boundary of odd-numbered parasegments may reflect an ancestral segmentation mechanism that functioned in every segment prior to the evolution of pair-rule segmentation.

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.

A pair-rule gene circuit defines segments sequentially in the short-germ insect Tribolium castaneum

In Drosophila, a hierarchy of maternal, gap, pair-rule, and segment polarity gene interactions regulates virtually simultaneous blastoderm segmentation. For the last decade, studies have focused on revealing the extent to which Drosophila segmentation mechanisms are conserved in other arthropods where segments are added sequentially from anterior to posterior in a cellular environment. Despite our increased knowledge of individual segmentation genes, details of their interactions in non-Drosophilid insects are not well understood. We analyzed the Tribolium orthologs of Drosophila pair-rule genes, which display pair-rule expression patterns. Tribolium castaneum paired (Tc-prd) and sloppy-paired (Tc-slp) genes produced pair-rule phenotypes when their transcripts were severely reduced by RNA interference. In contrast, similar analysis of T. castaneum even-skipped (Tc-eve), runt (Tc-run), or odd-skipped (Tc-odd) genes produced severely truncated, almost completely asegmental phenotypes. Analysis of interactions between pair-rule components revealed that Tc-eve, Tc-run, and Tc-odd form a three-gene circuit to regulate one another as well as their downstream targets, Tc-prd and Tc-slp. The complement of primary pair-rule genes in Tribolium differs from Drosophila in that it includes Tc-odd but not Tc-hairy. This gene circuit defines segments sequentially in double segment periodicity. Furthermore, this single mechanism functions in the early blastoderm stage and subsequently during germ-band elongation. The periodicity of the Tribolium pair-rule gene interactions reveals components of the genetic hierarchy that are regulated in a repetitive circuit or clock-like mechanism. This pair-rule gene circuit provides insight into short-germ segmentation in Tribolium that may be more generally applicable to segmentation in other arthropods.

Pair rule gene orthologs in spider segmentation

The activation of pair rule genes is the first indication of the metameric organization of the Drosophila embryo and thus forms a key step in the segmentation process. There are two classes of pair rule genes in Drosophila: the primary pair rule genes that are directly activated by the maternal and gap genes and the secondary pair rule genes that rely on input from the primary pair rule genes. Here we analyze orthologs of Drosophila primary and secondary pair rule orthologs in the spider Cupiennius salei. The expression patterns of the spider pair rule gene orthologs can be subdivided in three groups: even-skipped and runt-1 expression is in stripes that start at the posterior end of the growth zone and their expression ends before the stripes reach the anterior end of the growth zone, while hairy and pairberry-3 stripes also start at the posterior end, but do not cease in the anterior growth zone. Stripes of odd-paired, odd-skipped-related-1, and sloppy paired are only found in the anterior portion of the growth zone. The various genes thus seem to be active during different phases of segment specification. It is notable that the spider orthologs of the Drosophila primary pair rule genes are active more posterior in the growth zone and thus during earlier phases of segment specification than most orthologs of Drosophila secondary pair rule genes, indicating that parts of the hierarchy might be conserved between flies and spiders. The spider ortholog of the Drosophila pair rule gene fushi tarazu is not expressed in the growth zone, but is expressed in a Hox-like fashion. The segmentation function of fushi tarazu thus appears to be a newly acquired role of the gene in the lineage of the mandibulate arthropods.

The evolution of arthropod segmentation mechanisms.

The fruit fly, Drosophila melanogaster, patterns its segments rapidly and simultaneously, via a mechanism that relies on the ability of transcription factors to diffuse between blastoderm nuclei. Ancestral arthropods patterned posterior segments sequentially in a cellular environment, where free diffusion was likely to have been inhibited by the presence of cell membranes. Understanding how the Drosophila paradigm evolved is a problem that has interested evolutionary developmental biologists for some time. In this article, I review what is known about arthropod segmentation mechanisms, and present a model for the evolution of the Drosophila paradigm. The model predicts that the primary pair-rule genes of Drosophila ancestrally functioned within and/or downstream of a Notch-dependent segmentation clock, their striped expression gradually coming under the control of gap genes as the number of segments patterned simultaneously in the anterior increased and the number patterned sequentially via a segmentation clock mechanism in the posterior correspondingly decreased.

Transcriptional control in the segmentation gene network of Drosophila.

The segmentation gene network of Drosophila consists of maternal and zygotic factors that generate, by transcriptional (cross-) regulation, expression patterns of increasing complexity along the anterior-posterior axis of the embryo. Using known binding site information for maternal and zygotic gap transcription factors, the computer algorithm Ahab recovers known segmentation control elements (modules) with excellent success and predicts many novel modules within the network and genome-wide. We show that novel module predictions are highly enriched in the network and typically clustered proximal to the promoter, not only upstream, but also in intronic space and downstream. When placed upstream of a reporter gene, they consistently drive patterned blastoderm expression, in most cases faithfully producing one or more pattern elements of the endogenous gene. Moreover, we demonstrate for the entire set of known and newly validated modules that Ahab's prediction of binding sites correlates well with the expression patterns produced by the modules, revealing basic rules governing their composition. Specifically, we show that maternal factors consistently act as activators and that gap factors act as repressors, except for the bimodal factor Hunchback. Our data suggest a simple context-dependent rule for its switch from repressive to activating function. Overall, the composition of modules appears well fitted to the spatiotemporal distribution of their positive and negative input factors. Finally, by comparing Ahab predictions with different categories of transcription factor input, we confirm the global regulatory structure of the segmentation gene network, but find odd skipped behaving like a primary pair-rule gene. The study expands our knowledge of the segmentation gene network by increasing the number of experimentally tested modules by 50%. For the first time, the entire set of validated modules is analyzed for binding site composition under a uniform set of criteria, permitting the definition of basic composition rules. The study demonstrates that computational methods are a powerful complement to experimental approaches in the analysis of transcription networks.

Drosophila Topors Is a RING Finger-containing Protein That Functions as a Ubiquitin-protein Isopeptide Ligase for the Hairy Basic Helix-Loop-Helix Repressor Protein

Transcriptional repression plays an essential role in many aspects of metazoan development. Drosophila hairy is a primary pair-rule gene encoding a basic helix-loop-helix class transcriptional repressor that is required for proper segmentation. Previous characterization of Hairy-binding proteins has implicated two different classes of histone deacetylase as mediators of Hairy repression. Here, we present the characterization of a novel Hairy-interacting protein (dTopors) that binds specifically to the basic region of Hairy, but does not affect the ability of Hairy to bind DNA. By reducing the gene dose of dtopors, we demonstrate that it acts genetically as an antagonist of Hairy-mediated transcriptional repression. Consistent with this genetic interaction, we show that that recombinant dTopors protein possesses ubiquitin-protein isopeptide ligase activity in vitro and that dTopors mediates Hairy polyubiquitination and can lead to Hairy degradation. This work provides the first evidence that regulated proteolysis of Hairy is required for correct segmentation.

Expression patterns of hairy, even-skipped, and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a basal arthropod.

There is an ongoing discussion on whether segmentation in different phyla has a common origin sharing a common genetic program. However, before comparing segmentation between phyla, it is necessary to identify the ancestral condition within each phylum. Even within the arthropods it is not clear which parts of the genetic network leading to segmentation are conserved in all groups. In this paper, we analyze the expression of three segmentation genes of the pair-rule class in the spider Cupiennius salei. Spiders are representatives of the Chelicerata, a monophyletic basic arthropod group. We find that in spider embryos, the orthologues for the Drosophila primary pair-rule genes hairy, even-skipped, and runt are expressed in stripes in the growth zone, where the segments are forming, suggesting a role for these genes in chelicerate segmentation. These data imply that the involvement of hairy, even-skipped, and runt in arthropod segmentation is an ancestral character for arthropods and is not restricted to a particular group of insects.

Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression.

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

Integration of the head and trunk segmentation systems controls cephalic furrow formation in Drosophila

Genetic and molecular analyses of patterning of the Drosophila embryo have shown that the process of segmentation of the head is fundamentally different from the process of segmentation of the trunk. The cephalic furrow (CF), one of the first morphological manifestations of the patterning process, forms at the juxtaposition of these two patterning systems. We report here that the initial step in CF formation is a change in shape and apical positioning of a single row of cells. The anteroposterior position of these initiator cells may be defined by the overlapping expression of the head gap gene buttonhead (btd) and the primary pair-rule gene even-skipped (eve). Re-examination of the btd and eve phenotypes in live embryos indicated that both genes are required for CF formation. Further, Eve expression in initiator cells was found to be dependent upon btd activity. The control of eve expression by btd in these cells is the first indication of a new level of integrated regulation that interfaces the head and trunk segmentation systems. In conjunction with previous data on the btd and eve embryonic phenotypes, our results suggest that interaction between these two genes both controls initiation of a specific morphogenetic movement that separates two morphogenetic fields and contributes to patterning the hinge region that demarcates the procephalon from the segmented germ band.

Molecular characterization and embryonic expression of the even-skipped ortholog of Tribolium castaneum

In short germ insects, the procephalon and presumptive anterior segments comprise most of the embryonic rudiment which lengthens as posterior segments are added during development (Sander, K. (1976) Adv. Insect Physiol. 12, 125–238). The expression pattern of a grasshopper ortholog of the primary pair-rule gene even-skipped (eve) suggests that it is not relevant to segmentation in this short germ insect (Patel, N.H., Ball, E.E. and Goodman, C.S. (1992) Nature 357, 339–342). However in Drosophila, a long germ insect that forms all segments simultaneously, eve plays a vital role in segment formation (Nüsslein-Volhard, C., Wieschaus, E. and Klüding, H. (1984) Roux's Arch. Dev. Biol. 193, 267–282). We have characterized the eve ortholog of the beetle Tribolium castaneum. The homeodomain sequence is highly conserved between beetle, fly, and grasshopper eve orthologs. Tc eve is expressed in stripes during segmentation, but in a pattern differing in some details from that of the fly gene. This pattern is coincident with that detected with a cross-reacting antibody (Patel, N.H., Condron, B.G. and Zinn, K. (1994) Nature 367, 429–434). Thus, an ancestral even-skipped gene appears to have evolved a role in segmentation in a common ancestor of flies and beetles. Unlike vertebrate orthologs but similar to eve, Tc eve is not linked to the homeotic complex.

The Dichaete gene of Drosophila melanogaster encodes a SOX-domain protein required for embryonic segmentation.

We have cloned and characterised a member of the High Mobility Group superfamily of genes from Drosophila, Sox70D, which is closely related to the mammalian testis determining gene SRY. Sox70D corresponds to the dominant wing mutation Dichaete. Homozygous deletions of the Sox70D gene and recessive lethal Dichaete alleles have a variable embryonic segmentation phenotype. Dichaete is expressed in early embryos in a dynamic pattern reminiscent of gap and pair-rule genes and is required for the appropriate expression of the primary pair-rule genes even skipped, hairy and runt. The molecular nature of Dichaete and its expression pattern during early embryogenesis suggest that the gene plays a key role in early development; the variability in both the segmentation phenotype and the effects on pair-rule gene expression suggests that this role is to support the transcriptional regulation of key developmental genes rather than directly regulate any one of them.