ABSTRACT: Gene set and sub-network enrichment analysis provides an integrated approach for identifying gene regulatory networks underlying development. Anuran development is a coordinated process, and levels of mRNA are first dominated by maternally deposited genes, followed by active transcription of embryonic genes as the embryo undergoes morphogenesis and organ formation. The objectives of this study were to characterize early gene regulatory networks underlying Silurana tropicalis development. A custom Agilent 4 x 44 K microarray was developed to characterize networks during early development (1, 17, 36, 96 hpf). Cluster analysis revealed that each stage showed unique gene expression profiles and that 1 hpf was most different than the other three stages. There were > 8000 unique gene probes (p<0.01, FDR = 5%) that were differentially expressed between 1 hpf (2 cell stage) and 17 hpf and > 2000 gene probes differentially expressed between 36 hpf and 96 hpf. Genes higher in abundance (>100-fold) at 1 hpf compared to 17 hpf included oocyte-specific histone RNA stem-loop-binding protein 2, mitogen-activated protein kinase 14, and cyclin B5, suggesting these transcripts are maternally inherited or actively transcribed at fertilization. Gene ontology revealed that genes involved in nucleosome assembly, cell division, pattern specification, neurotransmission, and general metabolism were increasingly regulated throughout development, consistent with active development. In the period between 17-36 hpf, gene networks that play a role in organogenesis and organ function, including those related to the heart (heart morphogenesis, central nervous system (olfactory bulb development, dopamine metabolism), and kidney (renal reabsorption, water balance) were activated while between 36-96 hpf, networks involving in gut development, immune responses, lipid metabolism, hormone signaling, and brain development were prevalent. This study increases understanding of the spatiotemporal S. tropicalis embryonic development using gene regulatory networks. Gene expression analysis was performed with four biological replicates for four developmental stages (n = 16). Stages were NF stages 2, 16, 34, and 46.
