Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum

Abstract

wingless (wg)/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved [1]. Wnt signaling 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 that are also expressed in the growth zone [5]. After depleting Tc-WntD/8 we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is 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].

Figure 1. Cuticular and embryonic RNAi phenotypes

When possible to determine, embryos are oriented with anterior to the left. (A–B’) Wild-type embryos. (A) Wild-type embryonic cuticle. (B–B’) Developmental stage assessed by Tc-En staining during elongation (B) and retraction (B’). Wild type En expression is visible in all thoracic and abdominal segments. (C–D’) Tc-Wnt1 RNAi embryos. Cuticles are small spheres without signs of segmentation (C). During elongation all segments are formed although En expression is fading (D), after germband retraction the embryos are highly compacted but still contain Tc-En-expressing cell derivatives in all segments (D’). (E–F’) Tc-WntD/8 RNAi embryos. Cuticles have two pairs of legs but no abdominal segments. (E). In more severely affected embryos, abdominal segments failed to form (F) and three thoracic segments are observed after germband retraction (F’). (G–H’) Tc-Wnt1, Tc-WntD/8 double RNAi embryos. Cuticular phenotypes (G) are smaller than those produced by either single RNAi. Tc-En expression is visible in three thoracic segments (H). After germband retraction, the embryos are very compacted and show as few segments as Tc-WntD/8 RNAi embryos, as assessed by Tc-En (H’). (I–J’) Tc-wls RNAi embryos. Cuticles (I) resemble those from Tc-Wnt1 RNAi (C). Tc-En expression fades during germband elongation (J), and after germband retraction derivatives of all segments are highly compacted (J’), as in Tc-Wnt1 RNAi embryos. (K–L’) Tc-Wnt1,Tc-wls double RNAi. Cuticles (K) resemble those from Tc-Wnt1 or Tc-wls single RNAi. Tc-En expression fades during germband elongation (L), and after germband retraction derivatives of all segments are highly compacted (L’), as in Tc-Wnt1 or Tc-wls single RNAi germbands. (M–N’) Tc-wls,Tc-WntD/8 double RNAi embryos. Cuticular phenotypes (M) are more severe than those produced by either single RNAi. Tc-Eve (brown) and Tc-Wnt1 (purple) mRNA expression are visible in three thoracic segments (N). After germband retraction, the embryos are very small and show rudimentary heads and three thoracic segments as assessed by Tc-En (N’). (O–P’) Tc-porc RNAi embryos. Cuticular phenotypes (O) resemble those produced by Tc-wls/Tc-WntD/8 double RNAi (M). Abdominal segments do not form (P) and after germband retraction, the embryos are very small and show rudimentary heads and three thoracic segments as assessed by Tc-En (P’), similar to Tc-wls,Tc-WntD/8 double RNAi embryos. (Q–R’) Tc-pan RNAi embryos. Cuticles are small spheres (Q) that resemble Tc-Wnt1 embryos (C). Tc-En staining shows that abdominal segments failed to form during germband elongation (R) and after germband retraction Tc-En is expressed in the remaining segments (R’), like Tc-WntD/8 RNAi embryos. Scale bars represent 0.1mm.

Figure 2. Tc-Wnt1 and Tc-WntD/8 mRNA expression in wild-type and Tc-torso RNAi embryos

In these embryos, the anterior region is to the left and posterior is to the right. (A–B) During the blastoderm stage, Tc-Wnt1 is expressed as a ring around the posterior pole, leaving the pole itself free of expression. (C) During germband elongation Tc-Wnt1 is expressed, broadly in the posterior growth zone. (D–E) Tc-WntD/8 is expressed at the posterior pole complementary to Tc-Wnt1 expression. (G–H) Tc-WntD/8 expression resolves into intense spots on either side of the midline in the posterior growth zone of the condensing germ rudiment. (F) During germband elongation, two distinct spots of Tc-WntD/8 expression are visible in the posterior growth zone (*) (I) Tc-WntD/8 expression is abolished in Tc-torso RNAi embryos.