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

Chong Pyo Choe, Sherry C. Miller, and Susan J. Brown*

Division of Biology, Kansas State University, Manhattan, KS 66506

Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved March 7, 2006 (received for review December 4, 2005) In Drosophila, a hierarchy of maternal, gap, pair-rule, and segment (11). However, pair-rule expression of prd homologs is con- polarity gene interactions regulates virtually simultaneous blasto- served in Drosophila, Tribolium, and Schistocerca (12). These derm . For the last decade, studies have focused on results suggest that if insect segments are prepatterned in units revealing the extent to which Drosophila segmentation mecha- of double segment periodicity, then the genetic regulatory nisms are conserved in other arthropods where segments are interactions of pair-rule mechanisms differ in each species. added sequentially from anterior to posterior in a cellular envi- Interactions among pair-rule in insects other than ronment. Despite our increased knowledge of individual segmen- Drosophila have not been investigated to date. We have used tation genes, details of their interactions in non-Drosophilid insects parental RNA interference (RNAi) (13) to functionally analyze are not well understood. We analyzed the Tribolium orthologs of pair-rule gene orthologs and their interactions in the short-germ Drosophila pair-rule genes, which display pair-rule expression beetle T. castaneum. Here, we describe the genetic interactions patterns. Tribolium castaneum paired (Tc-prd) and sloppy-paired of pair-rule patterning in the short-germ insect T. castaneum and (Tc-slp) genes produced pair-rule phenotypes when their tran- discuss implications for insect segmentation. scripts were severely reduced by RNA interference. In contrast, similar analysis of T. castaneum even-skipped (Tc-eve), runt (Tc- Results run), or odd-skipped (Tc-odd) genes produced severely truncated, Two Classes of Pair-Rule Genes in Tribolium. Classic pair-rule mu- almost completely asegmental phenotypes. Analysis of interac- tant phenotypes in Drosophila include loss of alternating seg- tions between pair-rule components revealed that Tc-eve, Tc-run, ments or defects displaying double segment periodicity, which and Tc-odd form a three-gene circuit to regulate one another as are consistent with the normal expression pattern of the corre- well as their downstream targets, Tc-prd and Tc-slp. The comple- sponding gene. Because Tribolium orthologs of these genes are ment of primary pair-rule genes in Tribolium differs from Drosoph- expressed in pair-rule patterns (see Fig. 5 and Supporting Results, ila in that it includes Tc-odd but not Tc-hairy. This gene circuit which are published as supporting information on the PNAS web defines segments sequentially in double segment periodicity. Fur- site, for expression of Tc-odd, Tc-prd, and Tc-slp), we expected thermore, this single mechanism functions in the early blastoderm RNAi to produce similar phenotypes. Surprisingly, however, stage and subsequently during germ-band elongation. The peri- strong knock-down of Tc-eve, Tc-run,orTc-odd transcripts odicity of the Tribolium pair-rule gene interactions reveals com- produced truncated, almost completely asegmental ponents of the genetic hierarchy that are regulated in a repetitive instead of pair-rule phenotypes. Tc-eveRNAi embryonic cuticles circuit or clock-like mechanism. This pair-rule gene circuit provides contain labrum, antennae, and telson (Fig. 1b) but no gnathal or insight into short-germ segmentation in Tribolium that may be trunk segments. In addition to labrum and antennae, Tc-runRNAi more generally applicable to segmentation in other arthropods. cuticles contain mandibles (Fig. 1c), whereas Tc-oddRNAi cuticles contain mandibles and maxilla (Fig. 1d). Consistent with these insect segmentation ͉ even-skipped ͉ runt ͉ odd-skipped phenotypes, there are no gnathal or trunk Tc-Engrailed (Tc-En) stripes in Tc-eveRNAi germ-band embryos, and only one (man- n Drosophila, a hierarchy of maternal, gap, pair-rule, and dibular) or two gnathal (mandibular and maxillary) stripes in Isegment polarity genes regulates segmentation (1). Pair-rule Tc-run and Tc-odd RNAi embryos (Fig. 2 g, m, and s), respec- genes transform regional gradients of maternal and gap gene tively. The homeotic gene Tc-Dfd, which serves as a molecular information into cellular domains that define parasegmental marker for mandibular and maxillary segments, is expressed boundaries (2), ultimately producing segments by means of normally in Tc-odd RNAi embryos (Fig. 2s). Knock-down of any regulation of segment polarity genes. Genetic and molecular of these three genes blocked segmentation and elongation. In analyses reveal a complex pair-rule gene network, which Drosophila, eve null mutants produce asegmental cuticles, operates in units of double segment periodicity. even-skipped whereas null mutants of run or odd cause typical pair-rule (eve), hairy (h), and runt (run) are essential in setting para- phenotypes (14). The similar truncated, asegmental phenotypes segmental boundaries. These primary pair-rule genes are of Tc-eve RNAi, Tc-runRNAi, and Tc-oddRNAi embryos suggest that regulated by the maternal and gap genes; in turn, they regulate these genes function at the same level in the segmentation other, secondary pair-rule genes such as fushi-tarazu ( ftz), hierarchy. paired (prd), sloppy-paired (slp), and odd-skipped (odd) (3, 4). In contrast, Tc-prdRNAi and Tc-slpRNAi generated typical pair- In general, loss of primary pair-rule gene function affects the rule phenotypes (Fig. 1 e and f) that phenocopy previously expression of secondary pair-rule genes, whereas the expres- described mutants (15). Similar to Drosophila prd mutants (14), sion of primary pair-rule genes is not altered in secondary Tc-prdRNAi embryonic cuticles lacked odd-numbered segments, pair-rule gene mutants. Comparative studies of pair-rule gene homologs in other insects reveal a wide variety of expression patterns. In the Conflict of interest statement: No conflicts declared. grasshopper Schistocerca, homologs of eve and ftz are not This paper was submitted directly (Track II) to the PNAS office. expressed in pair-rule stripes (5, 6). In the milkweed bug Abbreviations: En, Engrailed; RNAi, RNA interference. Oncopeltus fasciatus, the eve homolog is expressed in segmental, Data deposition: The sequences reported in this paper have been deposited in the GenBank not pair-rule, stripes (7). In the beetle Tribolium castaneum, database [accession nos. DQ414246 (Tc-odd), DQ414247 (Tc-prd), and DQ414248 (Tc-slp)]. where eve, ftz, h, and run orthologs are expressed in pair-rule *To whom correspondence should be addressed. E-mail: [email protected]. stripes (8–10), loss of ftz does not produce a pair-rule phenotype © 2006 by The National Academy of Sciences of the USA

6560–6564 ͉ PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510440103 Downloaded by guest on October 2, 2021 Fig. 1. Cuticle preparations of severely affected T. castaneum pair-rule gene RNAi embryos. (a) This WT first-instar larval cuticle contains a head, three thoracic segments (T1–T3), eight abdominal segments (A1–A8), and terminal structures. Lr, labrum; Ant, antennae; Md, mandibles; Mx, maxillae; Lb, la- bium. (b) This spherical, asegmental Tc-eveRNAi cuticle contains labrum and antennae but no trunk segments. (c) In this severely affected Tc-runRNAi cuticle, the preoral and mandibular segments developed normally, but all other segments are missing, resulting in a spherical body similar to that of the Tc-eveRNAi in b.(d) Preoral, mandibular, and maxillary segments developed normally in this severely affected Tc-oddRNAi cuticle, but the ab- sence of posterior segments produced a spherical body shape similar to the Tc-eveRNAi and Tc-runRNAi embryos. (e) This severely affected Tc-prdRNAi cuticle contains maxillary, T1, T3, and four abdominal segments. (f) In this severely affected Tc-slpRNAi cuticle, T2 and four abdominal segments formed, whereas all gnathal and even-numbered trunk segments are missing.

including mandibular, labial, thoracic (T2), and four abdominal Fig. 2. Expression of Tribolium pair-rule genes in primary pair-rule gene RNAi embryos. Expression of Tc-En and pair-rule genes in WT (a–f), Tc-eveRNAi segments (Fig. 1e). Corresponding germ-band embryos lacked (g–l), Tc-runRNAi (m–r), and Tc-oddRNAi (s-x) embryos is shown. (g) Antennal and odd-numbered Tc-En stripes (Fig. 3 c and d), suggesting that intercalary Tc-En stripes formed in this severely affected Tc-eveRNAi embryo. Tc-prd is essential for the expression of Tc-En in odd-numbered (h–l) In severely affected Tc-eveRNAi embryos, expression of Tc-eve (h), Tc-run parasegments. Complementary to Tc-prdRNAi, Tc-slpRNAi cuti- (i), and Tc-odd (j) were severely reduced or abolished, and Tc-prd (k) and Tc-slp cles lacked even-numbered segments (Fig. 1f). Corresponding (l) failed to resolve into stripes. (m) In this severely affected Tc-runRNAi embryo, germ-band embryos lacked even-numbered Tc-En stripes (Fig. only antenna and mandibular Tc-En stripes formed. (n–r) In severely affected Tc-runRNAi embryos, Tc-eve (n), Tc-prd (q), and Tc-slp (r) were ectopically 3 e and f), indicating that Tc-slp is required for the expression of expressed, but Tc-run (o) and Tc-odd (p) expression was strongly reduced. (s) Tc-En in even-numbered parasegments. Interestingly, hypomor- Tc-Deformed (purple) and Tc-En are expressed normally in the mandibular and phic slp mutants in Drosophila affect odd-numbered segments maxillary segments of this severely affected Tc-oddRNAi embryo. (t–x)Inse- BIOLOGY RNAi RNAi (16), whereas Tc-slp affects even-numbered segments, im- verely affected Tc-odd embryos, Tc-eve (t) and Tc-run (u) were expressed DEVELOPMENTAL plying that the requirement for slp function is different in flies in broad continuous domains, but Tc-odd (v), Tc-prd (w), and Tc-slp (x) expres- and beetles. sion was abolished. The two classes of cuticular phenotypes seen in RNAi embryos suggest that in Tribolium, pair-rule genes may operate at two Epistasis Analysis of Tc-eve, Tc-run, and Tc-odd. To understand how functional levels, as in Drosophila. In addition, nascent stripes of genes expressed in pair-rule stripes produce truncated and Tc-run and Tc-odd appear in the posterior growth zone, whereas asegmental RNAi embryonic cuticles, we examined the RNAi stripes of Tc-prd and Tc-slp appear later in the anterior growth effects of each gene on the expression of the others. In severely zone (see Fig. 4a and Supporting Results). Taken together, these affected Tc-eveRNAi embryos, expression of Tc-run and Tc-odd data suggest that Tc-eve, Tc-run, and Tc-odd may function as was lost or greatly reduced, indicating that Tc-eve is required for primary pair-rule genes, whereas Tc-prd and Tc-slp function as the activation of Tc-run and Tc-odd (Fig. 2 h–j). The expression secondary pair-rule genes. patterns of Tc-eve and Tc-odd are almost completely comple- We also analyzed the functions of the remaining candidate mentary and show only slight overlap (Fig. 5b). Therefore, Tc-eve pair-rule genes, Tribolium h, ftz, odd-paired (opa), and Tenascin probably indirectly activates Tc-odd. In severely affected Tc- major (Ten-m). However, no segmentation defects were ob- oddRNAi embryos, the broad initial expression domains of Tc-eve served (data not shown), with the exception of Tc-hRNAi, which and Tc-run failed to resolve into pair-rule stripes (Fig. 2 t–v). produced anterior defects (Fig. 6 and Supporting Results, which Thus, Tc-odd is required for repression of Tc-eve and Tc-run to are published as supporting information on the PNAS web site). produce pair-rule stripes. However, it is unlikely that Tc-odd The truncated, asegmental phenotypes shown by Tc-eveRNAi, directly represses Tc-run, because their expression patterns Tc-runRNAi, and Tc-oddRNAi embryos; the modified pair-rule overlap (Fig. 4a and Supporting Results). Instead, Tc-odd might function of Tc-slp; and the fact that not all pair-rule gene repress Tc-run through repression of Tc-eve.InDrosophila, the orthologs participate in segmentation in Tribolium strongly initial expression of the primary pair-rule genes eve and run is not suggest that segments are prepatterned by different pair-rule altered by mutations in odd (17), a secondary pair-rule gene. The gene interactions in Tribolium and Drosophila. ectopic expression of Tc-eve and Tc-run in Tc-oddRNAi indicates

Choe et al. PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 ͉ 6561 Downloaded by guest on October 2, 2021 l, q, and r), probably because of the absence of interstripe repression. In contrast, Tc-prd and Tc-slp expression was abol- ished in the growth zone of Tc-oddRNAi embryos (Fig. 2 w and x), suggesting that Tc-prd and Tc-slp provide pair-rule functions that are secondary to those of Tc-eve, Tc-run, and Tc-odd.In addition, Tc-prd was expressed normally in Tc-slpRNAi embryos, and Tc-slp was expressed normally in Tc-prdRNAi embryos (data not shown), indicating that they do not interact with each other and are in parallel positions in the pathway. Although Tc-prd and Tc-slp were misregulated by the knock-down of the three primary pair-rule genes, it seems likely that Tc-eve and Tc-odd regulate Tc-prd and Tc-slp indirectly through Tc-run; Tc-prd and Tc-slp Fig. 3. Tc-En staining reveals pair-rule defects in severely affected secondary were misregulated by knockdown of each of the three primary T. castaneum pair-rule gene RNAi embryos. (a) Sixteen Tc-En stripes are visible pair rule genes. However, only the expression of Tc-run was in this fully elongated WT germ band. (b) Tc-run is transiently expressed in consistently correlated with the misregulation of Tc-prd and even-numbered parasegments in this elongating WT germ band. (c) There are Tc-slp in the three RNAi backgrounds (compare Fig. 2 i, o, and only seven Tc-En stripes in this fully elongated Tc-prdRNAi germ band. (d) The u with Fig. 2 l, r, and x, respectively). These results place them Tc-En stripes overlap Tc-run stripes, indicating that the odd-numbered Tc-En downstream of Tc-run. stripes are missing. (e and f) In this Tc-slpRNAi embryo, all gnathal Tc-En stripes and every even-numbered Tc-En stripe in the trunk are missing. Tribolium Pair-Rule Genes Do Not Act Upstream of Gap Genes. Depletion of eve mRNA in the milkweed bug Oncopeltus fas- that different genetic interactions between these genes evolved ciatus results in misregulation of gap genes, producing a severely in the lineages, leading to beetles and flies. Strongly affected affected head-only phenotype (7). To determine whether mis- Tc-runRNAi caused broad expression of Tc-eve as well as severe regulation of gap genes contributed to the asegmental pheno- types observed in Tc-eveRNAi, Tc-runRNAi, and Tc-oddRNAi em- reduction of Tc-odd expression in the growth zone, implying that bryos, we examined their expression in RNAi germ-band Tc-run is required for activation of Tc-odd and repression of embryos. Expression of the Tribolium orthologs of hunchback, Tc-eve (Fig. 2 n–p). However, the overlap between Tc-eve and Kru¨ppel, giant, and knirps are largely normal in the RNAi Tc-run expression (Fig. 7, which is published as supporting embryos (data not shown), suggesting that the asegmental information on the PNAS web site) suggests that the repression phenotypes generated by RNAi for Tribolium pair-rule genes are of Tc-eve by Tc-run is an indirect effect mediated by Tc-odd. not due to the misregulation of Tribolium gap genes. These interactions indicate that these three genes provide pri- mary pair-rule functions in Tribolium. Discussion We analyzed the functions and interactions of the Tribolium Tc-prd and Tc-slp Are Secondary Pair-Rule Genes. To understand homologs of Drosophila pair-rule genes by using RNAi. We whether Tc-prd and Tc-slp function as primary or secondary discovered that the Tribolium homologs of eve, run, and odd pair-rule genes, we analyzed the effect of Tc-prd or Tc-slp RNAi function as primary pair-rule genes and that prd and slp function on the expression of the others. The expression of Tc-eve, Tc-run, as secondary pair-rule genes but that h, ftz, opa, and Ten-m do or Tc-odd was not altered in Tc-prdRNAi or Tc-slpRNAi embryos not function as pair-rule genes. Severe knock-down of Tribolium (data not shown). However, the stripes of Tc-prd and Tc-slp primary pair-rule genes led to truncated, asegmental pheno- failed to resolve in Tc-eveRNAi and Tc-runRNAi embryos (Fig. 2 k, types, whereas depletion of secondary pair-rule genes produced

Fig. 4. Pair-rule patterning in Tribolium.(a) The dynamic expression of the primary and secondary pair-rule genes and their regulatory interactions are summarized. The bar at the top indicates that anterior is to the left. Newer segments forming in the growth zone are to the right. In this model of pair-rule patterning in Tribolium, two-segment units are prepatterned in the posterior region of the growth zone through one cycle of the regulatory circuit (Tc-eve, Tc-run, and Tc-odd). As the expression of Tc-run retracts anteriorly in even-numbered parasegments, the expression of Tc-prd is derepressed. Primary Tc-prd stripes resolve into two secondary stripes, showing alternatively weak and strong segmental expression. The strong secondary stripes in odd-numbered parasegments regulate Tc-En expression. Tc-run also retracts posteriorly in odd-numbered parasegments, resulting in derepression of the primary Tc-slp stripes. As Tc-run expression fades, expression of the primary Tc-slp stripe extends to the posterior border of the odd-numbered parasegment, which is required for the activation of Tc-En. GZ, growth zone; PS, parasegment; PT, posterior tip. (b) The more complex pair-rule network in Drosophila (19).

6562 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510440103 Choe et al. Downloaded by guest on October 2, 2021 classic pair-rule phenotypes. Based on these discoveries, we likely affect the regulatory interactions between pair-rule and propose a model of pair-rule patterning in Tribolium that might segment polarity genes. For example, loss of slp affects odd- explain the RNAi phenotypes and discuss major differences in numbered parasegments, whereas loss of Tc-slp affects even- the interactions of pair-rule genes in Drosophila and Tribolium. numbered parasegments. Finally, we discuss the implications of these findings on segmen- Our model provides a core mechanism for pair-rule patterning tation in short-germ insects and other arthropods. in Tribolium segmentation. However, additional components remain to be discovered. Tc-eve, Tc-run, and Tc-odd have A Model of Pair-Rule Gene Interaction in Tribolium. We describe a different anterior boundaries of expression that correspond to pair-rule gene circuit in Fig. 4a in which Tc-eve expression is the number of gnathal segments remaining in RNAi embryos. required to activate Tc-run, which, in turn, is required to activate These boundaries are likely regulated by gap genes, as in Tc-odd. Tc-odd expression in even-numbered parasegments is Drosophila. required to repress Tc-eve there, separating a primary Tc-eve By using the candidate gene approach, we determined that stripe from the broad expression domain. As Tc-eve expression orthologs of genes previously identified as pair-rule genes in is repressed in even-numbered parasegments, the posterior Drosophila function in Tribolium segmentation. However, the edges of Tc-run and then Tc-odd expression fade. Tc-eve expres- gene(s) responsible for resolution of primary pair-rule Tc-eve sion is also repressed in odd-numbered parasegments (regulated stripes into secondary segmental stripes as well as genes that by an as yet unknown gene) to produce segmental Tc-eve limit the expression of Tc-run within the Tc-eve domain and secondary stripes that are coincident with Tc-En stripes (8, 18). Tc-odd within the Tc-run domain have yet to be determined. Loss of Tc-eve expression in odd-numbered parasegments causes Furthermore, we do not yet know which genes function to Tc-run stripes to fade from their anterior edge, resulting in activate Tc-prd and Tc-slp. It still must be determined how the narrow Tc-run stripes that are coincident with every even- pair-rule gene circuit is initiated in blastoderm embryos and numbered Tc-En stripe. For reasons yet unknown, all three stopped after elongation. If this pair-rule gene circuit is regu- genes remain coexpressed with Tc-En in even-numbered para- lated by genes involved in anterior–posterior patterning, Tc- segments. Consequently, a two-segment unit is prepatterned caudal is a likely candidate. It is strongly expressed in the growth through one cycle of this primary pair-rule gene circuit. Restric- zone throughout germ-band elongation (21, 22) and produces a tion of Tc-run expression leads to the derepression of Tc-prd and severely affected RNAi phenotype (23) that is identical to that Tc-slp, which are responsible for the activation of Tc-En in odd- described for Tc-eve. Gap genes such as Tc-hunchback, which is and even-numbered parasegments, respectively. expressed in the posteriormost regions of the elongating germ The asegmental phenotypes produced by RNAi analysis of band (24), may be involved in regulating the pair-rule gene Tc-eve, Tc-run, and Tc-odd are readily explained by this model. circuit there. However, because pair-rule patterning occurs in a The knock-down of Tc-eve abolishes Tc-run expression, which cellular environment in Tribolium, it is possible that intercellular induces ectopic expression of both Tc-prd and Tc-slp. Tc-En signaling pathways are involved in regulating the pair-rule is not properly regulated to define the paraseg- circuit as components or targets of a segmentation clock. Indeed, mental borders, which results in an asegmental phenotype. the sequential function of the pair-rule gene circuit during Similarly for Tc-runRNAi, in the absence of Tc-run, Tc-prd and Tribolium segmentation provides evidence for regulation by Tc-slp are expressed ectopically, Tc-En is not activated, and some type of periodic mechanism in insects. In vertebrates, segmental grooves are not formed. However, the mechanism somitogenesis is regulated by a segmentation clock (25). Ho- that generates the asegmental phenotype in Tc-oddRNAi em- mologs of vertebrate segmentation clock components, such as bryos is different from that in Tc-eveRNAi or Tc-runRNAi Notch and Delta, are required for proper segmentation in basally embryos; the knock-down of Tc-odd leads to ectopic expres- branching arthropods such as the spider Cupiennius and have led sion of Tc-eve, which induces ectopic expression of Tc-run.As to the speculation that this mode of segmentation might be very a result, Tc-prd and Tc-slp are fully repressed, which leads to ancient (26). Although a Notch homolog has not been implicated misregulation of Tc-En expression and produces the asegmen- in insect segmentation (27), other signaling molecules may BIOLOGY

tal Tc-oddRNAi phenotype. Thus, either loss or ectopic expres- provide the regulatory link between pair-rule genes and a DEVELOPMENTAL sion of Tc-prd or Tc-slp leads to misregulation of Tc-En, segmentation clock. ultimately resulting in asegmental phenotypes. Primary Pair-Rule Genes in Germ-Band Elongation. In Tribolium,a Major Differences of Pair-Rule Interactions Between Drosophila and short, wide germ rudiment elongates into a long, narrow germ Tribolium. Our model of pair-rule interactions in Tribolium is band during segmentation (28). In the absence of concerted cell not predicted by simple application of the Drosophila pair-rule division, this morphological change may be due to cell movement gene paradigm (19) (Fig. 4b). In Drosophila, the three primary and intercalation, similar to convergent extension in Drosophila pair-rule genes (h, eve, and run) are key players in initiating (29). Germ-band elongation is not disrupted in Tc-prd and Tc-slp pair-rule patterning. However, Tc-h seems not to function as RNAi embryos; the classic pair-rule phenotypes result from loss a pair-rule gene at all. Although odd is a secondary pair-rule of patterning in alternating segments. In contrast, defective gene in Drosophila that is repressed by eve, Tc-odd functions as elongation in Tc-eve, Tc-run, and Tc-odd RNAi embryos pro- a primary pair-rule gene in Tribolium that represses Tc-eve. duces short, amorphous germ bands in which posterior segments Repression of slp and odd by eve is critical to activate prd- are not initiated. These results, taken together with their WT dependent odd-numbered and ftz-dependent even-numbered expression patterns, implicate primary (but not secondary) en stripes, respectively, in Drosophila (19, 20) (Fig. 4b). In pair-rule genes in elongation as well as segmentation. Interest- contrast, Tc-eve is required for the activation of Tc-odd, which ingly, eve and run have been implicated in convergent extension in turn represses Tc-eve to prepattern a two-segment unit. of the Drosophila germ band (29). Furthermore, Tc-run, which is induced by Tc-eve, is important for the formation of Tc-prd-dependent odd-numbered and One Segmentation Mechanism Functions in the Blastoderm and During Tc-slp-dependent even-numbered Tc-en stripes. Drosophila ftz Elongation. In Tribolium, up to three pair-rule stripes form in the is a secondary pair-rule gene that activates even-numbered en cellular blastoderm, prepatterning the three gnathal and three stripes, but Tc-ftz does not function in segmentation (11). thorax segments; abdominal segments are subsequently added Differences in the primary pair-rule genes result in different from the growth zone during germ-band elongation. Gap gene genetic interactions between primary and secondary genes and RNAi and mutant phenotypes display specific homeotic pheno-

Choe et al. PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 ͉ 6563 Downloaded by guest on October 2, 2021 types in the gnathum and thorax with severely disrupted seg- TBLASTN analysis of Drosophila protein sequences. PCR ampli- mentation in the abdomen (30, 31). These results have led to the cons from total embryonic RNA were cloned to use as templates hypothesis that segmentation mechanisms differ between the for in situ probes or dsRNA. blastoderm and elongation phases of short-germ development. The pair-rule gene circuit that we describe prepatterns segments Parental RNAi. Parental RNAi was performed as described in ref. in double segment periodicity from the gnathum through the 13. Injection of 900 ng͞␮l(Tc-eve), 500 ng͞␮l(Tc-run, Tc-prd, abdomen, providing continuity between the blastoderm and and Tc-slp), or 350 ng͞␮l(Tc-odd) into pupae produced strong germ-band elongation phases. Thus, it appears that the biggest RNAi effects. Injection buffer (1ϫ)or1␮g͞␮l Tc-ftz dsRNA was difference between these phases occurs at the level of the gap injected and produced no mutant effects. genes. Whole-Mount in Situ Hybridization and Immunocytochemistry. Several Insights into Segmentation in Other Short-Germ Arthropods. Whole-mount in situ hybridization was carried out as in refs. 8 Our results provide several insights into segmentation in Tribo- and 9 with digoxigenin-labeled RNA probes. The anti- lium that may apply to other short-germ arthropods in general. digoxigenin antibody conjugated with alkaline phosphatase ͞ First, a smaller complement of genes may comprise the core (Roche Diagnostics) was preadsorbed and used at a 1 2,000 pair-rule mechanism. Second, primary and secondary genes may dilution. Immunocytochemistry was performed as described in ͞ be different from those in Drosophila. Indeed, the dynamics of ref. 8 with the anti-Eve antibody diluted to 1 20 or the anti-En ͞ pair-rule gene homolog expression in the spider Cupiennius (32) antibody diluted to 1 5. Germ bands were dissected from the suggest pair-rule gene functions that differ from those of their yolks of embryos, mounted in 80% glycerol, and photographed Drosophila counterparts. Third, if primary pair-rule genes func- using Nomarski optics on a BX50 compound microscope (Olym- tion in both elongation and segmentation in short-germ arthro- pus, Melville, NY). pods, they may produce dramatically stronger RNAi phenotypes than secondary pair-rule genes. RNAi analysis in more non- Phenotype Analysis. Cuticle preparations of RNAi embryos were performed as described in ref. 11. First-instar larvae were model arthropods is required to test these insights and provide observed and photographed under dark-field optics. a better understanding of the logic of the ancestral pair-rule patterning mechanism. We thank T. Shippy, Y. Tomoyasu, R. Denell, and M. Klingler for Materials and Methods valuable discussions and critical reading of the manuscript and the Baylor Human Genome Sequencing Center (Houston) for Tribolium genomic Molecular Analysis. Tc-odd, Tc-prd, and Tc-slp sequences were sequences. This work was supported by a grant from the National computationally identified in the Tribolium genome sequence by Institutes of Health (to S.J.B.).

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