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).