SRA

Hedgehog signaling plays pivotal roles in formation of the embryonic anterior-posterior axis in the spider Parasteatoda tepidariorum. In this work, using the combination of RNA-sequencing and parental RNA interference (pRNAi), we genome-widely identified genes that transcriptionally responded to altered states of Hedgehog signaling in spider embryos, which were caused by Pt-hedgehog (Pt-hh) pRNAi and Pt-patched (Pt-ptc) pRNAi. The identified genes were classified into four classes (Classes I to IV) based on the positive/negative responses to the Pt-hh pRNAi and Pt-ptc pRNAi states. The Class II genes were negatively regulated by Hh signaling (positive response to Pt-hh pRNAi and negative response to Pt-ptc pRNAi). Among the Class II genes, we identified Pt-msx1 as a key segmentation gene in the spider embryo. Moreover, we conducted a transcriptomic analysis of Pt-msx1 pRNAi embryos and identified additional genes whose expression showed patterns associated with segmentation. Overall design: We used RNA-sequencing to obtain transcriptomic data for comparison between untreated (normal) and Pt-hh pRNAi-treated embryos, between untreated (normal) and Pt-ptc pRNAi-treated embryos, and between untreated (normal) and gfp pRNAi-treated embryos. We obtained at least two replicates from independent parents for each comparison. Poly(A) RNA was extracted from early stage 3 and late stage 5 embryos derived from mated females before (normal) and after (pRNAi-treated) injection of dsRNA targeted for each of Pt-hh and Pt-ptc. The RNAs were sequenced using the Illumina Miseq, and differentially expressed genes (DEGs) were evaluated in each comparison using EdgeR. The comparisons between the untreated and the gfp pRNAi-treated embryos served as negative controls. Based on the analyses of the expression and function of the identified DEGs, our study was focused on the Pt-msx1 gene. An additional transcriptomic analysis of Pt-msx1 pRNAi embryos was carried out.