GO enrichment analysis of clusters
GSEA were run on genes targeted by multiple miRNAs in a given cluster. When run on all identified targets, regardless of the number of targets per gene, the head tissue had between 1332-2035 targets, with no overlap each cluster. Abdomen tissue had fewer GO terms enriched than the head tissue, with 607-1571 per cluster. In general, the GSEA from these analyses were large and diffuse, which prevented easy biological interpretation (Table S5).
We next filtered our miRNA targets for the GSEA, with the number of miRNAs targeting a given gene that met our threshold criteria of majority of enriched GO terms having more than one gene, varied between 3 to 8 miRNAs. Once the thresholds were applied, the number of genes was greatly reduced in both the head and abdomen tissue to between 10-79 gene targets per cluster (Table S6). In the head, clusters H2 and H4 had the most miRNAs targeting single genes. Importantly, these targets all had at least 6 miRNA targets each, and this high degree of multiple miRNAs targeting these genes in these clusters makes them interesting candidates for coordinated gene expression regulation. Cluster H2 was enriched for genes targeted by multiple miRNAs related to “regulation of lipid metabolic processes” and “lipid metabolic processes” (Figure 5A), which are highly expressed in the first days of pupation and remain low through the remainder of the samples (Figure 3). Cluster H4, which is characterized by upregulation around day 24 that plateaus between day 114 and 144, then is downregulated after (Figure 3), is enriched for genes that are involved in “regulation of Wnt signaling pathway” and “imaginal disc pattern formation” (Figure 5A). In the abdomen tissue, cluster A4 was also enriched for “Regulation of Wnt signaling pathway”, A4, which has similar expression through diapause as cluster H4 (Figures 4 & 5B). Although there the amount of overlap in miRNAs targeting a given gene is lower than H2 and H4, H6 is enriched for genes involved in “glucocorticoid metabolic processes”, which is not hormone class present in insects (Figure 5A).