Genome-wide Identification of genes that transcriptionally respond to altered states of Hedgehog signaling in embryos of the spider Parasteatoda tepidariorum

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. 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.