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

Abstract

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.

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, labium. (b) This spherical, asegmental Tc-eve^RNAi cuticle contains labrum and antennae but no trunk segments. (c) In this severely affected Tc-run^RNAi 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-eve^RNAi embryo in b. (d) Preoral, mandibular, and maxillary segments developed normally in this severely affected Tc-odd^RNAi cuticle, but the absence of posterior segments produced a spherical body shape similar to the Tc-eve^RNAi and Tc-run^RNAi embryos. (e) This severely affected Tc-prd^RNAi cuticle contains maxillary, T1, T3, and four abdominal segments. (f) In this severely affected Tc-slp^RNAi cuticle, T2 and four abdominal segments formed, whereas all gnathal and even-numbered trunk segments are missing.

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-eve^RNAi (g–l), Tc-run^RNAi (m–r), and Tc-odd^RNAi (s-x) embryos is shown. (g) Antennal and intercalary Tc-En stripes formed in this severely affected Tc-eve^RNAi embryo. (h–l) In severely affected Tc-eve^RNAi embryos, expression of Tc-eve (h), Tc-run (i), and Tc-odd (j) were severely reduced or abolished, and Tc-prd (k) and Tc-slp (l) failed to resolve into stripes. (m) In this severely affected Tc-run^RNAi embryo, only antenna and mandibular Tc-En stripes formed. (n–r) In severely affected Tc-run^RNAi embryos, Tc-eve (n), Tc-prd (q), and Tc-slp (r) were ectopically expressed, but Tc-run (o) and Tc-odd (p) expression was strongly reduced. (s) Tc-Deformed (purple) and Tc-En are expressed normally in the mandibular and maxillary segments of this severely affected Tc-odd^RNAi embryo. (t–x) In severely affected Tc-odd^RNAi embryos, Tc-eve (t) and Tc-run (u) were expressed in broad continuous domains, but Tc-odd (v), Tc-prd (w), and Tc-slp (x) expression was abolished.

Fig. 3.

Tc-En staining reveals pair-rule defects in severely affected secondary T. castaneum pair-rule gene RNAi embryos. (a) Sixteen Tc-En stripes are visible in this fully elongated WT germ band. (b) Tc-run is transiently expressed in even-numbered parasegments in this elongating WT germ band. (c) There are only seven Tc-En stripes in this fully elongated Tc-prd^RNAi germ band. (d) The Tc-En stripes overlap Tc-run stripes, indicating that the odd-numbered Tc-En stripes are missing. (e and f) In this Tc-slp^RNAi embryo, all gnathal Tc-En stripes and every even-numbered Tc-En stripe in the trunk are missing.

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).