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

doi:10.1016/j.ydbio.2006.09.037

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Dev Biol.Author manuscript; available in PMC 2007 February 16.

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Dev Biol. 2007 February 1; 302(1): 281–294.

Published online 2006 September 26. doi: 10.1016/j.ydbio.2006.09.037.

PMCID: PMC1800430

NIHMSID: NIHMS17182

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

Chong Pyo Choe and Susan J Brown^*

Division of Biology, Kansas State University, Manhattan, KS USA

*Corresponding author: Susan J Brown, Division of Biology, Kansas State University, Manhattan, KS 66506, USA, Email: sjbrown/at/ksu.edu, Phone: (785) 532-3935, Fax: (785) 532-6653

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Abstract

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.

Keywords: paired, sloppy-paired, segmentation, pair-rule gene, Tribolium castaneum

Introduction

Genetic studies of the segmented body plan in Drosophila and vertebrates have detailed two different segmentation mechanisms; the spatial regulation of segmentation genes by a genetic hierarchy that produces segments simultaneously in Drosophila (Ingham, 1988) and the temporal regulation of segmentation components by a segmentation clock that produces somites sequentially in vertebrates (Pourquie, 2003). While long-germ embryogenesis in Drosophila is considered to be a derived mode, most other insects display short-germ embryogenesis in which most segments are added sequentially. Because of the morphological similarity of sequential segmentation to vertebrate somitogenesis, temporal as well as spatial regulation of the segmentation process in short-germ insects and other basal arthropods has been the focus of many recent studies. Although evidence for a segmentation clock has been described for basal arthropods (Chipman et al., 2004; Stollewerk et al., 2003), there is as yet no such evidence for insects. In contrast, comparative studies on homologs of Drosophila segmentation genes in other insects have revealed that a fairly conserved hierarchical cascade of genes spatially regulates segmentation. For example, segmental expression patterns of segment polarity genes are conserved in all arthropods examined thus far (Damen et al., 1998; Nulsen and Nagy, 1999). However, despite the importance of pair-rule genes as translators of nonperiodic information from maternal and gap genes to the periodic expression of segment polarity genes in Drosophila (Niessing et al., 1997), homologs of the pair-rule genes show the most diverse expression patterns, from typical pair-rule expression to expression in every segment or even nonsegmental expression in other short-germ insects (Davis and Patel, 2002; Dawes et al., 1994; Liu and Kaufman, 2005; Patel et al., 1992). Furthermore, the systematic RNAi analysis of Tribolium homologs of Drosophila pair-rule genes that are expressed in a pair-rule manner, revealed various segmental phenotypes, from asegmental to typical pair-rule (Choe et al., 2006). Others failed to affect segmentation, confirming previous observations that expression patterns are not always consistent with function (Brown et al., 1994; Stuart et al., 1991). We observed typical pair-rule phenotypes when analyzing the homologs of two Drosophila secondary pair-rule genes (paired and sloppy-paired), leading us to hypothesize that these might be the best candidate genes to test the extent to which pair-rule mechanisms are conserved in arthropod segmentation.

In Drosophila blastoderm stage embryos, pair-rule genes initiate and maintain expression of the segment polarity genes engrailed (en) and wingless (wg) at the parasegmental boundaries to molecularly define segments (Jaynes and Fujioka, 2004; Nasiadka et al., 2001). Immediately after gastrulation, the expression of en and wg are mutually dependent upon one another to maintain parasegmental boundaries and to ultimately form segmental grooves (Martinez Arial et al., 1988).

Drosophila paired (prd), one of the earliest pair-rule genes identified, has been analyzed in detail (Frigerio et al., 1986; Kilchherr et al., 1986; Morrissey et al., 1991). It functions at the end of the pair-rule gene network as a direct activator of the segment polarity genes en and wg (Baumgartner and Noll, 1990), and a null allele produces an obvious pair-rule phenotype in which all odd-numbered trunk segments are missing (Coulter and Wieschaus, 1988). Due to these features of prd, homologs of Drosophila prd or Pax group III genes have been analyzed in various insects and some basal arthropods to understand pair-rule patterning (Davis et al., 2001; Dearden et al., 2002; Osborne and Dearden, 2005; Schoppmeier and Damen, 2005). Indeed, all known homologs of prd or Pax group III genes displayed pair-rule expression patterns in insects suggesting that prd is an ancient pair-rule gene. However, this hypothesis has yet to be functionally tested.

Drosophila has two sloppy-paired (slp) genes, slp 1 and 2, which display almost identical expression patterns and are functionally redundant (Cadigan et al., 1994a; Grossniklaus et al., 1992). In contrast to the clear pair-rule phenotype of prd null mutants, embryos lacking both slp 1 and 2 display various segmental phenotypes ranging from pair-rule to the lawn of denticles produced by wg-class segment polarity genes as well as gap-like phenotypes in the head (Grossniklaus et al., 1994; Grossniklaus et al., 1992). slp 1 and 2 are required to activate wg and repress en. Similar to prd, slp mutants that display pair-rule phenotypes are defective primarily in odd-numbered segments (Grossniklaus et al., 1992). Because of these phenotypic variations and its functional similarity to prd, homologs of Drosophila slp have not been the focus of evolutionary studies for understanding pair-rule patterning in other insects and arthropods. Only one study, on the segmental expression of the slp homolog in a spider, has been reported (Damen et al., 2005). Therefore, the role of slp homologs in pair-rule pattering in short-germ insects and other arthropods has yet to be determined.

As functional analysis via RNAi becomes available in nondrosophilid insects (Brown et al., 1999b), many noncanonical functions of segmentation genes are being reported at the level of gap and pair-rule genes, suggesting that pair-rule patterning, if functional, is quite different in other insects from Drosophila (Bucher and Klingler, 2004; Cerny et al., 2005; Liu and Kaufman, 2005; Mito et al., 2005; Patel et al., 2001). However, ethylmethane sulphonate (EMS) mutagenesis in Tribolium identified two phenotypically complementary pair-rule mutants, scratchy (scy) and itchy (icy), providing evidence that a pair-rule mechanism plays a role in Tribolium segmentation (Maderspacher et al., 1998). Their phenotypes did not suggest obvious Drosophila homologs, and a lack of molecular characterization of these mutants has restricted our understanding of pair-rule pattering in this short-germ insect. Recently, in our RNAi analysis of the Tribolium homologs of Drosophila pair-rule genes, we found that Tc-prd and Tc-slp RNAi phenocopy the mutant effects of scy and icy, respectively (Choe et al., 2006). Here we report the roles of Tc-prd and Tc-slp in Tribolium segmentation. Using RNAi to analyze the function of Tc-prd and Tc-slp revealed that Tc-prd is required for odd-numbered segment formation, while Tc-slp is required for formation of both odd- and even-numbered segments. Tc-prd activates Tc-en stripes in odd-numbered parasegments and adjacent Tc-wg stripes in even-numbered parasegments. Complementary to Tc-prd, the pair-rule function of Tc-slp activates Tc-wg stripes in odd-numbered parasegments. In addition, it is required as a segment polarity gene to maintain Tc-wg stripes. Thus, prd functions in the same parasegmental register in Drosophila and Tribolium whereas the parasegmental register of slp function is opposite in one relative to the other. We discuss the implications of these results for the evolution of secondary pair-rule gene functions and the possible use of prd and slp to study pair-rule patterning in other short-germ arthropods.

Materials and Methods

Identification and RT-PCR cloning of Tc-prd and Tc-slp

The previously cloned homeodomain fragment of Tc-prd and the forkhead domain fragment of Tc-slp (Choe et al., 2006) were used to computationally identify candidate loci in the Tribolium genome (http://www.hgsc.bcm.tmc.edu/projects/tribolium/). Initially, each full-length CDS for Tc-prd and Tc-slp was predicted manually by comparison with protein sequences from Drosophila Prd and Slp respectively. The manually predicted full-length CDS sequences were almost identical to the genes computationally predicted (Tribolium genome project, HGSC, Baylor college of medicine). A set of primers was designed from the putative 5′ and 3′-UTRs of the predicted Tribolium sequences and used to amplify fragments containing full-length Tc-prd or Tc-slp coding sequences. Total RNA was isolated from 0 – 48 hour embryos using Trizol (Invitrogen) and cDNA was synthesized from total RNA template using SuperScript™ III Reverse Transcriptase (Invitrogen). PCR was performed with Takara Ex Taq™ DNA Polymerase (Takara) and the amplicons were cloned into Promega's pGEM®-T Easy Vector (Promega). Sequences were determined on an ABI 3730 DNA Analyzer using BigDye Terminators (Kansas State University DNA Sequencing and Genotyping Facility (http://www.oznet.ksu.edu/pr_dnas/)). The cDNA sequences have been deposited in Genbank under the accession number of DQ979360 for the Tc-prd CDS and DQ979361 for the Tc-slp CDS.

Parental RNAi and embryo collection

Parental RNAi was performed as described (Bucher et al., 2002) using 500 ng/μl of Tc-prd and Tc-slp dsRNA to produce severe RNAi effects. 1 X injection buffer or 1μg/μl of Tc-ftz dsRNA was injected as a control and, as previously observed (Choe et al., 2006), did not generate any mutant phenotypes. To analyze the hypomorphic series of RNAi phenotypes, embryos were collected every 48 hours for six weeks, during which time the observed phenotypes became less and less severe until only wild type larva were produced. Embryos were incubated at 30°C for 4 days to complete embryogenesis and then placed in 90% lactic acid to assess cuticular effects. For whole-mount in situ hybridization and immunochemistry, 0–24 hour embryos were collected and fixed by standard protocols.

Whole-mount in situ hybridization and immunochemistry

Whole-mount in situ hybridization was performed as previously described (Brown et al., 1994) with some modifications. To devitellinize eggs and dissect germbands from the yolk, fixed embryos were incubated in 50% xylene and vortexed at high speed for 30 seconds every 10 minutes for 1 hour. The devitellinized and dissected embryos were immediately used for whole-mount in situ hybridization. Immunochemistry was carried out as described with a 1:5 dilution of mAbs 4D9 (anti-En) or a1:20 dilution of 2B8 (anti-Eve) from the Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa.

Molecular analysis of itchy and scratchy

Homozygous mutant icy and scy individuals were identified by visual inspection of the progeny in heterozygous male lines. Genomic DNA was isolated by grinding one larva in 50ul of squish buffer (Gloor et al., 1993) and incubating it with proteinase K for 1 hour at 25°C. 2ul of lysate from a squished larva was used as template for PCR. To survey for sequence changes in the exon of the candidate loci of the mutants, each exon was amplified from the mutants, cloned and sequenced, as described above. The sequences were aligned with wild-type exon sequences using CLUSTAL W with default parameters (Thompson et al., 1994).

Results

Tribolium paired and sloppy-paired homologues

Homologues of prd and slp were predicted by BLAST analysis of the Tribolium genome. We generated PCR clones containing full-length coding sequences for these genes from wild type cDNA. Comparison with genomic DNA confirmed the computational prediction and indicated that the Tc-prd locus is about 29 kb with 5 exons. The deduced 387 aa protein sequence contains a paired domain and a homeodomain similar to those found in Drosophila Prd (Fig. 1A). Tc-Prd does not contain the octapeptide that distinguishes Drosophila gooseberry and gooseberry-neuro, and the Schistocerca pairberry (Davis et al., 2001). There is 84.5% identity within the paired domain and 91.5% within the homeodomain between Drosophila and Tribolium.

Fig. 1

Molecular characterization of Tc-prd and Tc-slp, and identification of the mutations in scy and icy. (A) Tc-prd contains two highly conserved domains, a paired domain and a homeodomain. The amino acid substituted in scy is marked with an asterisk. (B) (more ...)

A single Tc-slp gene was found by BLAST analysis of the Tribolium genome. Similar to Drosophila, the Tc-slp locus is approximately 1.3 kb and contains a single exon encoding 312 aa. The forkhead domain and two short domains (domain II and III) are highly conserved; the forkhead domain of Tc-slp is 83.2% identical to the forkhead domain of Drosophila slp1, but 95.3% identical to that of Drosophila slp2 (Fig. 1C). Additional sequence similarity between Tc-slp and Dm-slp2 is apparent throughout the proteins, including the last 12 residues at the carboxy-terminus.

Expression patterns of Tc-prd

Previously, the expression patterns of Pax group III genes were analyzed in Tribolium with a polyclonal antibody that crossreacts with Drosophila Prd, Gooseberry and Gooseberry-neuro (Davis et al., 2001). Because the expression domains of these genes are expected to overlap in Tribolium segmentation as in Drosophila, we used whole-mount in situ hybridization to follow the expression of just Tc-prd. Anti-En antibody was used as a marker to determine the register of the Tc-prd expression domain. Transcripts of Tc-prd first appear in a narrow stripe at about 60% egg length (measured from the posterior pole) during the blastoderm stage (Fig. 2A). This stripe forms in the presumptive mandibular segment, as evidenced by the fact that it overlaps the first Tc-En stripe and extends anteriorly from it (Figs. 2A, B). Similar to the mandibular stripe of Drosophila prd, this Tribolium prd stripe does not resolve into two secondary stripes (Kilchherr et al., 1986). Immediately following condensation of the germ rudiment, the second Tc-prd stripe appears posterior to the first, and the gradient of expression within this broad stripe is strongest at the posterior boundary (Fig. 2C). This primary stripe covers an entire even-numbered parasegment and the Tc-En stripe in the next odd-numbered parasegment. It resolves into two secondary stripes by fading in the center, from posterior to anterior (Fig. 2D). Consequently, two secondary stripes of Tc-prd form; the weaker anterior stripe (Tc-prd b) corresponds to a Tc-En stripe in an even-numbered parasegment and the stronger posterior stripe (Tc-prd a) corresponds to a Tc-wg stripe and the adjacent Tc-En stripe in even- and odd-numbered parasegments respectively (Fig. 2E and summarized in Fig. 7A). These secondary stripes fade completely as the embryo develops. Similar to Drosophila, Tc-En stripes appear after the secondary Tc-prd stripes suggesting a similar role for Tc-prd as a regulator of Tc-en (Fig. 2E). During subsequent germband growth, additional Tc-prd stripes appear in the middle of the growth zone and resolve into two secondary stripes that eventually fade (Figs. 2E–I). This is similar to the dynamics of Tc-eve and Drosophila prd expression (Brown et al., 1997; Kilchherr et al., 1986; Patel et al., 1994). Therefore, we conclude that Tc-prd is expressed in a pair-rule manner. Interestingly, as the germband fully extends, a narrow Tc-prd stripe is detected in the posterior region of the germband immediately after the fifteenth Tc-En stripe (arrow in Fig. 2I). Similar to the first stripe observed in the presumptive head region at the blastoderm stage, this final stripe is not pair-rule like. It seems likely that these two Tc-prd stripes are regulated differently from the other stripes that are expressed in double segment periodicity during segmentation.

Fig. 2

Expression of Tc-prd in Tribolium embryos undergoing segmentation. (A–C and E–I) are stained with Tc-prd riboprobe (purple) and Anti-En antibody (punctuate, brown spots). (A) In the blastoderm, a narrow stripe of Tc-prd appears coincident (more ...)

Fig. 7

Summary of secondary pair-rule gene expression relative to other segmentation genes in Tribolium and the effects of secondary pair-rule gene mutations or RNAi on the expression of en and wg in Drosophila and Tribolium. (A) Pair-rule (upper) and segment (more ...)

Tc-prd is required for odd-numbered segment formation

To gain further insight into the role of Tc-prd, we extended our previous analysis of Tc-prd^RNAi embryos (Choe et al., 2006). Across a gradient of Tc-prd^RNAi effects, gnathal and thoracic segments always displayed clear pair-rule phenotypes (Figs. 3B, C). However, the series of Tc-prd^RNAi embryos showed variation in the number of abdominal segments affected (Figs. 3B, C, compare to 3A). Most Tc-prd^RNAi embryos (90.2%) were strongly affected and displayed complete pair-rule phenotypes containing only 4 or 5 abdominal segments (Fig. 3B) while weak Tc-prd^RNAi embryos (8.7%), showed deletion of 3 or fewer abdominal segments (Fig. 3C), which is similar to the common phenotypes described in the scy mutant (Maderspacher et al., 1998).

Fig. 3

Cuticle preparations and germband defects in Tc-prd^RNAi or scy. (A–C) Cuticle preparations. (D–H) Germbands undergoing segmentation. (A) Lateral view of wild-type first instar larval cuticle with head, three thoracic segments (T1–T3), (more ...)

To determine the register of segmental deletions, we followed the expression of the segment polarity genes Tc-en and Tc-wg in Tc-prd^RNAi embryos. In contrast to scy in which every other Tc-en and its adjacent Tc-wg stripes were weakly initiated with normal initiation of the alternate Tc-en and Tc-wg stripes (Maderspacher et al., 1998), every other Tc-en and its adjacent Tc-wg stripe were not activated at all in the Tc-prd^RNAi embryos (Figs. 3F, H, compare to 3E). Furthermore, double staining Tc-prd^RNAi embryos for Tc-Eve and Tc-En showed that Tc-En stripes normally expressed in the odd-numbered parasegments are missing (Fig. 3G). Thus, Tc-prd is required for formation of all odd-numbered segments through activation of Tc-en stripes in odd-numbered parasegments and the adjacent Tc-wg stripes in even-numbered parasegments (summarized in Fig. 7B). This function of Tc-prd is consistent with the alternating intensity of the secondary segmental stripes of Tc-prd in which the strong secondary stripes (Tc-prd a) overlap the Tc-En stripe in odd-numbered parasegments and the adjacent Tc-wg stripe in even-numbered parasegments while the weak stripes (Tc-prd b) overlap the Tc-En stripes in even-numbered parasegments (Figs. 7A, B). Similarly in Drosophila, prd functions as an activator of en stripes in odd-numbered parasegments and their adjacent wg stripes in even-numbered parasegments (Fig. 7B), and null alleles of prd cause a complete pair-rule phenotype where every odd-numbered segment is deleted (Ingham et al., 1988). The conserved expression and function of prd in Drosophila and Tribolium suggests that their common ancestor contained a prd gene with a similar pair-rule function in segmentation.

Expression patterns of Tc-slp

In contrast to the extensive studies of Pax group III gene expression patterns in various insects and basal arthropods, the expression pattern of slp has been reported only for Drosophila and the spider Cupiennius salei (Damen et al., 2005; Grossniklaus et al., 1992). In Drosophila, slp1 is initiated in the presumptive head region in a broad, gap-like pattern where it is required for segment formation. Soon thereafter, primary slp1 stripes appear in every even-numbered parasegment. Then secondary slp1 stripes intercalate between the primary stripes, resulting in segmental expression of slp1. slp2 is expressed in the same trunk domain as slp1 with a temporal delay, and it is not expressed in the presumptive head. In the spider, slp is expressed with a single segment periodicity instead of double segment periodicity.

To understand possible segmental functions of Tc-slp, we analyzed its expression pattern. During the blastoderm stage, a broad stripe of Tc-slp transcripts appears at about 70% egg length from the posterior pole (Fig. 4A). Soon thereafter this stripe is limited ventrally in the presumptive head lobes of the future germ rudiment (Fig. 4B), in the regions that give rise to the antennae (Fig. 4J). Before the germ rudiment condenses, a new Tc-slp stripe appears in the blastoderm (arrow head in Fig. 4C). Double staining with anti-En antibody indicates that this second stripe is expressed in the presumptive mandibular segment (Fig. 4E). Just after the germband forms, a narrow Tc-slp stripe appears in the presumptive maxillary segment (arrow head in Fig. 4D). Then a strong stripe (arrow heads in Figs. 4E, F) in the first thoracic segment appears prior to a weak narrower stripe in the labial segment (arrow Fig. 4F). During germband elongation, pairs of Tc-slp stripes appear in the anterior region of the growth zone (Figs. 4G–K). The anterior stripe (arrows in Figs. 4G–K) is narrower and weaker than the posterior stripe (arrow heads in Figs. 4G–K). As they develop, each Tc-slp stripe overlaps the anterior row of cells in a Tc-En stripe (Figs. 4G–J). To differentiate these stripes, we defined the stronger posterior stripe as Tc-slp a, most of which is in an odd-numbered parasegment, and the anterior stripe as Tc-slp b, most of which is in an even-numbered parasegment. The dynamics of the Tc-slp expression pattern is summarized in Fig. 7A. Typical of a pair-rule gene, Tc-slp stripes a and b define two segments at once during germband elongation. The difference in intensity between these two stripes suggests they may have different functions in segmentation. All Tribolium pair-rule genes reported to date show transient expression patterns; their expression initiates in the growth zone and fades away in the elongating germband (Brown et al., 1994; Brown et al., 1997; Patel et al., 1994; Sommer and Tautz, 1993). However, Tc-slp expression is not transient, but is maintained in a segmental pattern until the germband is fully elongated, which is similar to the expression of segment polarity genes. This is not unexpected, since slp genes continue to be expressed as the Drosophila germband develops (Grossniklaus et al., 1992). In summary, Tc-slp expression is similar to that of Drosophila slp 1 and 2 in that the expression pattern initiates in a pair-rule pattern and then remains during germband elongation similar to a segment polarity gene. Tc-slp expression is different in that a pair of stripes initiates simultaneously and the register of strong and weak stripes is the opposite of slp stripes in Drosophila.

Fig. 4

Expression of Tc-slp in Tribolium embryos undergoing segmentation. (A–D, F) stained with Tc-slp riboprobe (purple). (E, G–K) stained with Tc-slp riboprobe (purple) and Anti-En antibody (punctuate, brown spots). (G–K) Primary Tc-slp (more ...)

Tc-slp is required for gnathal segmentation, formation of even-numbered segments and maintenance of the odd-numbered segments in the trunk

We analyzed a graded series of Tc-slp^RNAi embryos to better understand the function of Tc-slp during segmentation. First, all the gnathal segments (mandibular, maxillary, and labial), are defective across the entire gradient of Tc-slp^RNAi embryos (Figs. 5B, C, compare to 5A) suggesting that Tc-slp performs a gap-like function in the gnathum. In Drosophila, slp1 functions as a head gap gene; a null mutant of slp1 causes defects in mandibular and pregnathal segments (Grossniklaus et al., 1994). However, Tc-slp did not show any evidence of a gap gene-like expression pattern. Instead, it is initiated as narrow stripes at the blastoderm and early germband stages (Figs. 4B–F). Thus, individual stripes in each segment, rather than gap-gene like expression, of Tc-slp appear to be required for gnathal segmentation. In addition, Tc-slp^RNAi displayed a range of phenotypes in the abdominal segments (Figs. 5B, C, compare to 5A).

Fig. 5

Cuticle preparations and germband defects in Tc-slp^RNAi or icy. (A–C) Cuticle preparations. (D–H) Germbands undergoing segmentation. (A) Ventral view of wild-type first instar larval cuticle with head, three thoracic segments (T1–T3), (more ...)

The most severe Tc-slp^RNAi embryos (8.3%) displayed a compact segmental phenotype with 4 asymmetrically incomplete segments (Fig. 5B; see 4 segments (white dots) on one side and 2 broad segments (white arrow heads) on the other side). However, most of the Tc-slp^RNAi embryos (91.7%) displayed a classical pair-rule phenotype in which T1, T3 and only 4 or 5 abdominal segments were missing (Fig. 5C).

To molecularly identify the defective segments, we followed the expression of the segment polarity genes Tc-en and Tc-wg in Tc-slp^RNAi embryos. In wild-type embryos, Tc-en and the adjacent Tc-wg stripes are initiated by pair-rule genes and then maintained by the Tc-en, Tc-hedgehog, and Tc-wg circuit during germband elongation (Farzana and Brown, unpublished data). In most Tc-slp^RNAi embryos at the elongated germband stage, all the gnathal stripes as well as every other stripe of Tc-En and Tc-wg were missing, supporting the combined head gap and pair-rule phenotypes observed in Tc-slp^RNAi cuticles. However, analysis of younger embryos revealed that Tc-slp^RNAi completely abolished the initiation of a Tc-wg stripe but not the adjacent Tc-En stripe (Fig. 5G, compare to 5E). And although it is initiated, Tc-En expression in these defective segments was not maintained, probably due to the absence of neighboring Tc-wg expression. Double staining with anti-Eve and anti-En antibodies to determine the register of the remaining Tc-En stripes demonstrated that the defective Tc-En and Tc-wg stripes are in even-numbered and adjacent odd-numbered parasegments respectively (Fig. 5H). Thus, in the trunk the missing Tc-En and Tc-wg stripes correspond to T1, T3 and the even-numbered abdominal segments (summarized in Fig. 7B). Taken together, these results indicate that Tc-slp a, which is expressed in odd-numbered parasegments, is required in there for the activation of Tc-wg stripes as well as for the maintenance of the adjacent Tc-En stripes (in even-numbered parasegments) leading to the formation of even-numbered segments (Figs. 7A, B). In Drosophila, slp functions as a pair-rule gene in combination with prd, to activate wg stripes in even-numbered parasegments (Fig. 7B), which eventually leads to the formation of odd-numbered segments (Cadigan et al., 1994b; Coulter and Wieschaus, 1988; Ingham et al., 1988). Thus, the primary requirement for slp has evolved differently in Drosophila and Tribolium.

Interestingly, in addition to the loss of Tc-wg stripes in odd-numbered parasegments and the neighboring Tc-En stripes in even-numbered parasegments, as described above, some more severely affected Tc-slp^RNAi embryos showed additional loss of the Tc-wg stripes that had formed normally in even-numbered parasegments. Although initiated, they were not properly maintained and began fading before the germband fully extended (compare the T2 Tc-wg stripes in Fig. 5G and 5E) implying that Tc-slp b, which is expressed in even-numbered parasegments, is required to maintain Tc-wg stripes in these parasegments. Furthermore, these decay dynamics provide support for the most severe Tc-slp^RNAi phenotypes in that the Tc-En stripes, which are initiated normally in odd-numbered parasegments, were not maintained sufficiently (due to the loss of Tc-wg stripes in adjacent even-numbered parasegments) to form segmental grooves (Fig. 5F, compare to 5B). Thus, the most severe Tc-slp^RNAi phenotypes appear to be caused by the combination of failing to initiate even-numbered segments and failing to maintain odd-numbered segments. In summary, we conclude that the Tc-slp a stripes are required for the formation of even-numbered segments through the activation of Tc-wg stripes in odd-numbered parasegments. Later, Tc-slp functions as a segment polarity gene to maintain Tc-wg stripes in even-numbered parasegments (Tc-slp b) and most likely all parasegments (Tc-slp a and b)(Figs. 7A, B). In Drosophila, segmentally expressed secondary (segment polarity) slp stripes are required to maintain wg stripes, and slp null individuals display a pair-rule phenotype in the thorax (T1-T2 and T3-A1 fusions) and a wg-class segment polarity phenotype in the abdomen (lawn of denticles) (Cadigan et al., 1994b). Thus, although flies require slp function in a segmental register opposite that in beetles for pair-rule patterning, the overall requirement is similar, in that it is required early for the initiation of every other segment and later for the maintenance of the remaining segments, if not all segments.

Segmental identity is not altered by the loss of Tc-prd or Tc-slp

Homeotic transformation has been reported for Tribolium gap gene mutants or in gap gene RNAi embryos (Bucher and Klingler, 2004; Cerny et al., 2005). Because it has been speculated that the homeotic defects are mediated by pair-rule genes (Cerny et al., 2005), we asked whether Tc-prd and Tc-slp are involved in determining segmental identity as well as segment formation. Cuticular phenotypes of Tc-prd^RNAi or Tc-slp^RNAi embryos did not show any homeotic defects implying that these pair-rule genes are not involved in the regulation of homeotic genes (Figs. 3B, C, 5B, C). In Tribolium, Deformed (Dfd) is expressed in the mandibular and maxillary segments (Brown et al., 1999a), Sex combs reduced in the posterior maxillary and labial segments (Curtis et al., 2001) and Ultrabithorax from T2 through the abdominal segments (Bennett et al., 1999). We performed in situ hybridization with these three homeotic genes, as markers of segmental identity in the Tc-prd^RNAi or Tc-slp^RNAi embryos. Consistent with the cuticular phenotypes, these homeotic genes were expressed normally in the Tc-prd^RNAi or Tc-slp^RNAi embryos (data not shown) except for Dfd in Tc-slp^RNAi embryos where its expression was limited to a narrow region near the head lobes (Fig. 6C, compare to 6A, B). In Drosophila, not all pair-rule genes are involved in determining segmental identity (Ingham and Martinez-Arias, 1986); ftz is required for the regulation of homeotic genes but prd is not. Even though we cannot completely exclude the possibility that other pair-rule genes are involved in the determination of segmental identity, it appears that neither Tc-prd nor Tc-slp functions to determine segmental identity.

Fig. 6

Tc-Dfd expression in Tribolium germband embryos. (A) Tc-Dfd mRNA (purple) is expressed in the mandibular and maxillary segments in this wild-type germband. (B–C) Expression of Tc-Dfd mRNA (purple) and Tc-En protein (punctuate, brown spots) in (more ...)

Scratchy and itchy are potential Tc-prd and Tc-slp mutants, respectively

Tc-prd^RNAi cuticles have maxillary palps, two pairs of legs and 4 abdominal segments; they are missing odd-numbered segments. Tc-slp^RNAi cuticles typically contain a single pair of legs and 4 abdominal segments; they lack all gnathal segments and even-numbered segments in the trunk. Interestingly, these RNAi effects phenocopy the mutant phenotypes of two complementary, EMS induced mutations in Tribolium, scy and icy (Maderspacher et al., 1998). In the scy mutant, we found a point mutation in exon 4 of Tc-prd, which causes a valine to methionine change after the homeodomain (Fig. 1B). Alignment of the protein sequences indicated that this region is not highly conserved between Drosophila and Tribolium (asterisk in Fig. 1A), making it difficult to imagine how this missense mutation may cause the scy phenotype. However, two Drosophila prd alleles, prdX3 and prdIIN indicate that this region, immediately after the homeodomain, is important for the in vivo function of Prd (Bertuccioli et al., 1996). Tc-prd transcripts are expressed in scy mutant embryos, indicating that the mutant phenotype is more likely to be due to the production of a non-functional protein than a regulatory defect (Fig. 3D). Finally, the highly variable phenotype described for scy (Maderspacher et al., 1998) is indicative of a hypomorphic mutant. Intriguingly, Tc-prd^RNAi produces the same range of phenotypes. Thus, the scy mutant might be a hypomorphic mutant of Tc-prd that is caused by the amino acid substitution in the exon 4 of Tc-prd locus.

In comparing the sequence of the Tc-slp locus in the icy mutant with that of wild type (GA-1), we detected a single nucleotide deletion in the region encoding the forkhead domain (Fig. 1D). This deletion alters the reading frame and causes truncation about half-way through the forkhead domain (53/107 aa). Considering the importance of this domain to Slp as a transcription factor, it is highly likely that this truncation within the forkhead domain causes the mutant phenotype. Furthermore, we also found that transcripts of Tc-slp are expressed in normal segmental pattern with decreased intensity in the trunk whereas the expression is irregular and almost abolished in the gnathal region in the presumptive icy embryos (Fig. 5D) indicative of nonsense mediated-degradation of the Tc-slp transcripts. Therefore, we suggest that the icy mutant might be an allele of Tc-slp that is caused by the truncation of the forkhead domain in the Tc-slp. EMS usually causes deletion of several nucleotides (Anderson, 1995) rather than deletion of a single nucleotide. However, we observed the same nucleotide deletion in six icy individuals. Truncation within an essential domain of a transcription factor is expected to produce a null phenotype. However, the icy produces a range of phenotypes, none of which are as severe as the most severe class of Tc-slp^RNAi embryos. Even though the truncation of the forkhead domain of Tc-slp and the decreased amounts of Tc-slp transcripts in the icy mutant, suggest that icy might be a Tc-slp mutant, we cannot conclude that icy is a Tc-slp mutant with certainty. Additional evidence such as positional map data or other alleles for complementation tests are required to confirm the identity of scy and icy mutants as alleles of Tc-prd and Tc-slp, respectively.

Discussion

We analyzed the expression and function of the secondary pair-rule genes prd and slp in Tribolium. Our RNAi analysis of Tc-prd and Tc-slp revealed conserved and divergent aspects of these secondary pair-rule genes relative to the function of their Drosophila homologs. The function of prd is mainly conserved between the two insects while slp displays some divergent as well as conserved functions in Drosophila and Tribolium segmentation. In addition, we discuss the possible evolution of their roles in the lineages of Drosophila and Tribolium.

The first stripe of Tc-prd expression is observed in the presumptive mandible at the blastoderm stage and seven successive stripes are formed near the middle of the growth zone as the germband elongates. Expression in the mandibular stripe is uniform while expression in the successive stripes appears in a gradient that is strongest posteriorly. Each of these stripes splits into two segmental stripes overlapping Tc-En expression and they eventually fade. In Tc-prd^RNAi embryos odd-numbered Tc-En stripes fail to initiate and the resulting cuticles displayed a typical pair-rule mutant phenotype in which odd-numbered segments are missing.

The first stripe of Tc-slp expression appears near the anterior end of the egg and is quickly restricted to the antennal region of the head lobes. The second and third stripes appear in the presumptive mandibular and maxillary segments of the blastoderm. A weak stripe appears in the labial segment after a stronger stripe has formed in T1. As the germband elongates, additional stripes of slp are added in pairs, in which the anterior stripe is weaker than the posterior one. These develop into broad segmental stripes of expression that are maintained during germband elongation. In Tc-slp^RNAi embryos the even-numbered Tc-En stripes are initiated but not maintained. In addition, in the most severe Tc-slp^RNAi embryos, odd-numbered Tc-En stripes fade later, during germband retraction. Interestingly, Tc-slp^RNAi cuticles displayed a range of phenotypes from typical pair-rule to severe segment polarity phenotypes, reminiscent of the mixed pair-rule and segment polarity phenotypes described for Drosophila slp null mutants.

Functions of prd and slp in segmentation that are conserved between Drosophila and Tribolium

In Drosophila, pair-rule genes identified by mutation were named to reflect their phenotypes (Nusslein-Volhard and Wieschaus, 1980). Subsequent molecular characterization of pair-rule genes uncovered expression patterns consistent with the mutant phenotypes, except for odd-paired (opa), which is expressed ubiquitously but correlated with a pair-rule mutant phenotype (Benedyk et al., 1994). When homologs of Drosophila pair-rule genes were shown to have pair-rule expression patterns in certain other insects and basal arthropods, but functional analysis was not available, it was reasonable to speculate that these homologs would have similar functions and thus produce similar loss of function pair-rule phenotypes. However, the systematic functional analysis of Tribolium homologs of Drosophila pair-rule genes by RNAi revealed that most of them generated phenotypes dramatically different from the pair-rule phenotypes described in Drosophila, or no segmental phenotypes, which are not easily explained by their pair-rule expression patterns (Choe et al., 2006). Our analysis indicates that Tc-prd and Tc-slp RNAi generate a range of phenotypes that include classic pair-rule phenotypes. Furthermore, they are similar to typical Drosophila pair-rule genes in that their expression patterns correlate with their mutant phenotypes. For example, the primary stripes of prd are expressed between the posterior end of odd-numbered parasegments to the anterior end of next odd-numbered parasegments in both Drosophila and Tribolium. Interestingly, in Tribolium, expression in these primary stripes is stronger toward the posterior edge of each stripe (Fig. 7A), but no such gradient of expression is described for Drosophila (Kilchherr et al., 1986). In both insects, the primary stripes split into two secondary stripes. In Tribolium the posterior stripe is stronger, but in Drosophila they appear to be of equal intensity. In both insects, the secondary stripes co-expressed with En in odd-numbered parasegments are required for segment boundary formation (Ingham et al., 1988). Considering that many homologs of Drosophila pair-rule genes show diverse expression patterns or functions in other short-germ insects, it is noteworthy that the expression pattern and function of prd are conserved between Drosophila and Tribolium and suggests that the same expression pattern and function of prd was most likely shared by their common ancestor.

Complementary to Tc-prd, Tc-slp is required as a pair-rule gene for the formation of even-numbered segments and as a segment polarity gene for the maintenance of odd-numbered segments (if not all segments). The segmental stripes of Tc-slp are expressed in the posterior region of each parasegment and slightly overlap the Tc-En stripe in the adjacent parasegment (Fig. 7A). Tc-slp is similar to Drosophila slp (Grossniklaus et al., 1992) in that both are required as pair-rule genes for the activation of alternate wg stripes and as segment polarity genes for the maintenance of the remaining wg stripes. The more intensely staining Tc-slp a stripes, are required for the activation of all gnathal Tc-wg stripes and alternate Tc-wg stripes in trunk, while the weaker Tc-slp b stripes, are required for the maintenance of the remaining Tc-wg stripes. Thus, it appears that the function of slp, to activate or maintain wg expression is conserved between Drosophila and Tribolium. However, in contrast to prd which is required in the same parasegmental register between Drosophila and Tribolium, slp is required in opposite parasegmental registers at the level of pair-rule patterning in Drosophila and Tribolium. Pair-rule function of Dm-slp is required in addition to Dm-prd for the activation of wg stripes in even-numbered parasegments, while in odd-numbered parasegments, it is required as a segment polarity gene for the maintenance of wg stripes that were activated by Dm-opa (Benedyk et al., 1994; Cadigan et al. 1994b; Ingham et al., 1988). In contrast, Tc-slp functions early as a pair-rule gene to activate Tc-wg stripes in odd-numbered parasegments, and later as a segment polarity gene in the maintenance of Tc-wg stripes that were initiated normally in even-numbered parasegments. Taken together, our data suggest that the function of slp as a pair-rule gene to activate wg or as a segment polarity gene to maintain wg has been conserved between Drosophila and Tribolium but that the parasegmental register of slp as a pair-rule gene has evolved differently in these two lineages.

Evolution of the role of slp in the network of pair-rule genes in Drosophila and Tribolium

The fact that prd is required in the same parasegmental register, while slp as a pair-rule gene is required in opposite parasegmental registers in Drosophila and Tribolium reveals an unprecedented flexibility in the pair-rule mechanism and suggests that the roles of prd and slp in the pair-rule gene network evolved differently in these insects. Since the parasegmental register for prd is conserved in Drosophila and Tribolium it is likely to be an ancestral feature. In contrast, the different parasegmental register for slp suggests the function of slp in either Drosophila, Tribolium, or both is derived. Although it is impossible to determine with certainty the ancestral state of slp function when comparing only two species, there are several lines of evidence discussed below that suggest Tribolium might more closely resemble the ancestral state.

Considering the highly derived nature of Drosophila development, it has often been implied that insects like Tribolium, which display more general modes of development, represent ancestral modes of molecular mechanisms as well. In contrast to Drosophila, all other nondrosophilid insects and basally branching arthropods examined so far have only one slp, whose sequence is more similar to Dm-slp2 than to Dm-slp1 (Damen et al., 2005). Thus, it appears that slp was duplicated in the lineage leading to Drosophila and the sequence of Dm-slp1 has diverged considerably from the other slp genes. However, despite their identical expression patterns, Dm-slp1, not Dm-slp2, functions as a pair-rule gene in Drosophila segmentation (Cadigan et al., 1994a). Later, Dm-slp2 functions redundantly as a segment polarity gene. We suggest that duplication and subsequent divergence of the slp genes are correlated with the differential function of slp genes in Drosophila and likely contributed to the evolution of the role of slp in the Drosophila pair-rule network. For example, as diagramed in Fig. 8, we can imagine that after duplication of the ancestral slp gene, one copy continued to function as a segment polarity gene, but lost its pair-rule function, and didn’t diverge much at the sequence level (Dm-slp2). The other copy, while continuing to function as a pair-rule gene required for the activation of wg, is now required in even numbered parasegments in Drosophila. In addition it has diverged at the sequence level (Dm-slp1). Furthermore, opa functions to activate wg in the odd-numbered parasegments in Drosophila while ftz is required to activate en in even numbered parasegments (Benedyk et al., 1994; Ingham et al., 1988). Neither opa nor ftz has a pair-rule function in Tribolium (Choe et al., 2006), and in Schistocerca ftz is not even expressed segmentally (Dawes et al., 1994). Thus ftz and opa may have been co-opted as secondary pair-rule genes in the lineage leading to Drosophila. Alternatively, considering the fact that Tc-ftz is expressed in a pair-rule pattern in Tribolium, the possibility exists that its function in pair-rule patterning was lost in the beetle lineage. However, if the segment polarity function of slp, which is conserved in both insects, is considered to be the ancestral function, then it is possible that the pair-rule functions of slp in Drosophila and Tribolium are both derived. The two secondary pair-rule genes, prd and slp display conserved and divergent aspects in their regulation of segment polarity genes. The expression as well as the function of prd homologs in the formation of odd-numbered segments is conserved between Drosophila and Tribolium. In contrast, differences in the functional register of slp and the acquisition or loss of ftz and opa pair-rule functions are significant to the evolution of secondary pair-rule gene interactions. Functional analysis of homologs of prd, slp, ftz, and opa in other insects and basally branching arthropods are needed to test these models for the evolution of roles of secondary pair-rule genes in segmentation.

Fig. 8

Comparison of secondary pair-rule gene functions in Drosophila and Tribolium in an evolutionary context. Across the top of the diagram, the known present pair-rule functions of secondary pair-rule genes in the formation of odd- and even-numbered segments. (more ...)

Acknowledgments

We thank M. Klingler for the scy and icy mutants. mAbs 4D9 and 2B8 developed by N.H. Patel were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. We thank members of the lab for helpful comments and discussions. This work was supported by a grant from the National Institutes of Health (R01-HD29594).

Footnotes

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References

Anderson, P. Mutagenesis. Methods Cell Biol. 1995;48:31–58. [PubMed]
Baumgartner, S; Noll, M. Network of interactions among pair-rule genes regulating paired expression during primordial segmentation of Drosophila. Mech Dev. 1990;33:1–18. [PubMed]
Benedyk, MJ; Mullen, JR; DiNardo, S. odd-paired: a zinc finger pair-rule protein required for the timely activation of engrailed and wingless in Drosophila embryos. Genes Dev. 1994;8:105–117. [PubMed]
Bennett, RL; Brown, SJ; Denell, RE. Molecular and genetic analysis of the Tribolium Ultrabithorax ortholog, Ultrathorax. Dev Genes Evol. 1999;209:608–619. [PubMed]
Bertuccioli, C; Fasano, L; Jun, S; Wang, S; Sheng, G; Desplan, C. In vivo requirement for the paired domain and homeodomain of the paired segmentation gene product. Development. 1996;122:2673–2685. [PubMed]
Brown, S; Holtzman, S; Kaufman, T; Denell, R. Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant. Dev Genes Evol. 1999a;209:389–398. [PubMed]
Brown, SJ; Hilgenfeld, RB; Denell, RE. The beetle Tribolium castaneum has a fushi tarazu homolog expressed in stripes during segmentation. Proc Natl Acad Sci USA. 1994;91:12922–12926. [PubMed]
Brown, SJ; Mahaffey, JP; Lorenzen, MD; Denell, RE; Mahaffey, JW. Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects. Evol Dev. 1999b;1:11–15. [PubMed]
Brown, SJ; Parrish, JK; Beeman, RW; Denell, RE. Molecular characterization and embryonic expression of the even-skipped ortholog of Tribolium castaneum. Mech Dev. 1997;61:165–173. [PubMed]
Bucher, G; Klingler, M. Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function. Development. 2004;131:1729–1740. [PubMed]
Bucher, G; Scholten, J; Klingler, M. Parental RNAi in Tribolium (Coleoptera). Curr Biol. 2002;12:R85–R86. [PubMed]
Cadigan, KM; Grossniklaus, U; Gehring, WJ. Functional redundancy: the respective roles of the two sloppy paired genes in Drosophila segmentation. Proc Natl Acad Sci USA. 1994a;91:6324–6328. [PubMed]
Cadigan, KM; Grossniklaus, U; Gehring, WJ. Localized expression of sloppy paired protein maintains the polarity of Drosophila parasegments. Genes Dev. 1994b;8:899–913. [PubMed]
Cerny, AC; Bucher, G; Schroder, R; Klingler, M. Breakdown of abdominal patterning in the Tribolium Kruppel mutant jaws. Development. 2005;132:5353–5363. [PubMed]
Chipman, AD; Arthur, W; Akam, M. A double segment periodicity underlies segment generation in centipede development. Curr Biol. 2004;14:1250–1255. [PubMed]
Choe, CP; Miller, SC; Brown, SJ. A pair-rule gene circuit defines segments sequentially in the short-germ insect Tribolium castaneum. Proc Natl Acad Sci U S A. 2006;103:6560–4. [PubMed]
Coulter, DE; Wieschaus, E. Gene activities and segmental patterning in Drosophila: analysis of odd-skipped and pair-rule double mutants. Genes Dev. 1988;2:1812–1823. [PubMed]
Curtis, CD; Brisson, JA; DeCamillis, MA; Shippy, TD; Brown, SJ; Denell, RE. Molecular characterization of Cephalothorax, the Tribolium ortholog of Sex combs reduced. Genesis. 2001;30:12–20. [PubMed]
Damen, WG; Hausdorf, M; Seyfarth, EA; Tautz, D. A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider. Proc Natl Acad Sci USA. 1998;95:10665–10670. [PubMed]
Damen, WG; Janssen, R; Prpic, NM. Pair rule gene orthologs in spider segmentation. Evol Dev. 2005;7:618–628. [PubMed]
Davis, GK; Jaramillo, CA; Patel, NH. Pax group III genes and the evolution of insect pair-rule patterning. Development. 2001;128:3445–3458. [PubMed]
Davis, GK; Patel, NH. Short, long, and beyond: molecular and embryological approaches to insect segmentation. Annu Rev Entomol. 2002;47:669–699. [PubMed]
Dawes, R; Dawson, I; Falciani, F; Tear, G; Akam, M. Dax, a locust Hox gene related to fushi-tarazu but showing no pair-rule expression. Development. 1994;120:1561–1572. [PubMed]
Dearden, PK; Donly, C; Grbic, M. Expression of pair-rule gene homologues in a chelicerate: early patterning of the two-spotted spider mite Tetranychus urticae. Development. 2002;129:5461–5472. [PubMed]
Frigerio, G; Burri, M; Bopp, D; Baumgartner, S; Noll, M. Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell. 1986;47:735–746. [PubMed]
Gloor, GB; Preston, CR; Johnson-Schlitz, DM; Nassif, NA; Phillis, RW; Benz, WK; Robertson, HM; Engels, WR. Type I repressors of P element mobility. Genetics. 1993;135:81–95. [PubMed]
Grossniklaus, U; Cadigan, KM; Gehring, WJ. Three maternal coordinate systems cooperate in the patterning of the Drosophila head. Development. 1994;120:3155–3171. [PubMed]
Grossniklaus, U; Pearson, RK; Gehring, WJ. The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. Genes Dev. 1992;6:1030–1051. [PubMed]
Ingham, PW. The molecular genetics of embryonic pattern formation in Drosophila. Nature. 1988;335:25–34. [PubMed]
Ingham, PW; Baker, NE; Martinez-Arias, A. Regulation of segment polarity genes in the Drosophila blastoderm by fushi tarazu and even skipped. Nature. 1988;331:73–75. [PubMed]
Ingham, PW; Martinez-Arias, A. The correct activation of Antennapedia and bithorax complex genes requires the fushi tarazu gene. Nature. 1986;324:592–597. [PubMed]
Jaynes, JB; Fujioka, M. Drawing lines in the sand: even-skipped et al. and parasegment boundaries. Dev Biol. 2004;269:609–622. [PubMed]
Kilchherr, F; Baumgartner, S; Bopp, D; Frei, E; Noll, M. Isolation of the paired gene of Drosophila and its spatial expression during early embryogenesis. Nature. 1986;321:493–499.
Liu, PZ; Kaufman, TC. even-skipped is not a pair-rule gene but has segmental and gap-like functions in Oncopeltus fasciatus, an intermediate germband insect. Development. 2005;132:2081–2092. [PubMed]
Maderspacher, F; Bucher, G; Klingler, M. Pair-rule and gap gene mutants in the flour beetle Tribolium castaneum. Dev Genes Evol. 1998;208:558–568. [PubMed]
Martinez Arias, A; Baker, NE; Ingham, PW. Role of segment polarity genes in the definition and maintenance of cell states in the Drosophila embryo. Development. 1988;103:157–170. [PubMed]
Mito, T; Sarashina, I; Zhang, H; Iwahashi, A; Okamoto, H; Miyawaki, K; Shinmyo, Y; Ohuchi, H; Noji, S. Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus. Development. 2005;132:2069–2079. [PubMed]
Morrissey, D; Askew, D; Raj, L; Weir, M. Functional dissection of the paired segmentation gene in Drosophila embryos. Genes Dev. 1991;5:1684–1696. [PubMed]
Nasiadka, A; Dietrich, BH; Krause, HM. Anterior-posterior patterning in the Drosophila embryo. Adv Dev Biol Biochem. 2002;12:155–204.
Niessing, D; Rivera-Pomar, R; La Rosee, A; Hader, T; Schock, F; Purnell, BA; Jackle, H. A cascade of transcriptional control leading to axis determination in Drosophila. J Cell Physiol. 1997;173:162–167. [PubMed]
Nulsen, C; Nagy, LM. The role of wingless in the development of multibranched crustacean limbs. Dev Genes Evol. 1999;209:340–348. [PubMed]
Nusslein-Volhard, C; Wieschaus, E. Mutations affecting segment number and polarity in Drosophila. Nature. 1980;287:795–801. [PubMed]
Osborne, PW; Dearden, PK. Expression of Pax group III genes in the honeybee (Apis mellifera). Dev Genes Evol. 2005;215:499–508. [PubMed]
Patel, NH; Ball, EE; Goodman, CS. Changing role of even-skipped during the evolution of insect pattern formation. Nature. 1992;357:339–342. [PubMed]
Patel, NH; Condron, BG; Zinn, K. Pair-rule expression patterns of even-skipped are found in both short- and long-germ beetles. Nature. 1994;367:429–434. [PubMed]
Patel, NH; Hayward, DC; Lall, S; Pirkl, NR; DiPietro, D; Ball, EE. Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation. Development. 2001;128:3459–3472. [PubMed]
Pourquie, O. The segmentation clock: converting embryonic time into spatial pattern. Science. 2003;301:328–330. [PubMed]
Schoppmeier, M; Damen, WG. Expression of Pax group III genes suggests a single-segmental periodicity for opisthosomal segment patterning in the spider Cupiennius salei. Evol Dev. 2005;7:160–169. [PubMed]
Sommer, RJ; Tautz, D. Involvement of an orthologue of the Drosophila pair-rule gene hairy in segment formation of the short germ-band embryo of Tribolium (Coleoptera). Nature. 1993;361:448–450. [PubMed]
Stollewerk, A; Schoppmeier, M; Damen, WG. Involvement of Notch and Delta genes in spider segmentation. Nature. 2003;423:863–865. [PubMed]
Stuart, JJ; Brown, SJ; Beeman, RW; Denell, RE. A deficiency of the homeotic complex of the beetle Tribolium. Nature. 1991;350:72–4. [PubMed]
Thompson, JD; Higgins, DG; Gibson, TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. [PubMed]