Summary wingless (wg)/Wnt family genes encode secreted glycoproteins essential for the development of virtually all metazoans. In short germ insects, including the red flour beetle, Tribolium castaneum, the segment-polarity function of wg is conserved [1]. Wnt signalling is also implicated in posterior patterning and germband elongation [2–4], but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions; [1–3]. Tribolium contains additional Wnt family genes of unknown function that are also expressed in the growth zone [5]. After depleting one of these, Tc-WntD/8, we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced this phenotype, suggesting functional redundancy. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls) and porcupine (porc), which process Wnt ligands, and pangolin (pan), which transduces the signal to the nucleus. Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1, but not Tc-WntD/8 RNAi, indicating that the effects of Tc-WntD/8 depletion are Tc-wls-independent. In contrast, depletion of Tc-porc and Tc-pan resulted in embryos resembling those of double Tc-Wnt1,Tc-WntD/8 RNAi, suggesting Tc-porc is essential for the function of both ligands and that they signal through the canonical pathway. Our results provide the first evidence of functional redundancy between Wnt ligands in posterior patterning in short germ insects. This Wnt function appears to be conserved in other arthropods [6] and vertebrates [7–9].

Canonical Wnt signaling has been implicated in an AP axis polarizing mechanism in most animals, despite limited evidence from arthropods. In the long-germ insect, Drosophila, Wnt signaling is not required for global AP patterning, but in short-germ insects including Tribolium castaneum, loss of Wnt signaling affects development of segments in the growth zone but not those defined in the blastoderm. To determine the effects of ectopic Wnt signaling, we analyzed the expression and function of axin, which encodes a highly conserved negative regulator of the pathway. We found Tc-axin transcripts maternally localized to the anterior pole in freshly laid eggs. Expression spread toward the posterior pole during the early cleavage stages, becoming ubiquitous by the time the germ rudiment formed. Tc-axin RNAi produced progeny phenotypes that ranged from mildly affected embryos with cuticles displaying a graded loss of anterior structures, to defective embryos that condensed at the posterior pole in the absence of serosa. Altered expression domains of several blastodermal markers indicated anterior expansion of posterior fates. Analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the effects of Tc-axin depletion are mediated through this pathway and that Wnt signaling must be inhibited for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt signaling must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt activity in defining AP polarity and patterning in metazoan development.A-P axis patterning | short-germ segmentation | anterior patterning | maternal determination

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