Fig. 4.

In situ hybridization of Wnt target candidates. Posterior Wnt targets: 23 candidate genes were selected for evaluation in the in situ screen. We obtained clones for 21 of them. These included seven genes with a Drosophila ortholog or homolog known to be involved in signaling and that are transcription factors. Six of these candidates showed specific posterior expression in the early germ band rudiment. In addition, 14 genes with no Drosophila homolog were added to the candidates for the in situ screen. Ten showed posterior expression. Three genes with highly similar sequences related to a retrotransposon gave ubiquitous staining and one gene did not stain at all. In summary, of the 28 candidates (seven already published, seven with Drosophila homologs and 14 unknown) that we examined, 23 (82%) are expressed at the posterior in Tribolium. Gene au2.g216 (asterisk) is an overlapping candidate from both the Wnt and Hh gene sets. Anterior Wnt targets: this gene set contains eight genes. We selected six for the in situ screen and obtained clones for five. Tc-notum and au2.g10282 are expressed exclusively in the head at this early stage. Tc-adenosine2 and au2.g1134 are expressed in the extraembryonic region at the anterior. Tc-amylase distal showed no expression. Tc-ey was known to be expressed in the head. Of the six genes (one published, three with Drosophila homologs and two unknown) that we examined, three are localized in the anterior germ band (50%) and two in the anterior extraembryonic region (33%). Tc-amylase distal (double asterisk) is an overlapping candidate from both the Wnt and Hh posterior gene sets. Insets show anterior expression at the blastoderm stage.