A developmental stage specific network approach for studying dynamic transcription factor-microRNA co-regulation during craniofacial development.

Craniofacial development is regulated through dynamic and complex mechanisms that involve various signaling cascades and gene regulations. Disruption of such regulations may result in craniofacial birth defects. Here, we propose the first developmental stage-specific network approach by integrating two critical regulators, transcription factor (TF) and microRNA (miRNA), to study their co-regulation during craniofacial development. Specifically, we used TFs, miRNAs, and non-TF genes to form Feed-forward Loops (FFLs) using genomic data covering mouse embryonic days E10.5 to E14.5. We identified key novel regulators (TFs: Foxm1, Hif1a, Zbtb16, Myog, Myod1, and Tcf7, and miRNAs: miR-340-5p and miR-129-5p) and target genes (Col1a1, Sgms2, and Slc8a3) whose expression changed in a developmental stage-dependent manner. We found Wnt-FoxO-Hippo pathway (from E10.5 to E11.5), tissue remodeling (from E12.5 to E13.5), and miR-129-5p-mediated Col1a1 regulation (from E10.5 to E14.5) might play crucial roles in craniofacial development. Enrichment analyses further suggested their functions. Our experiments validated the regulatory roles of miR-340-5p and Foxm1 in Wnt-FoxO-Hippo subnetwork, as well as the role of miR-129-5p in the miR-129-5p-Col1a1 subnetwork. Thus, our study helps understand comprehensive regulatory mechanisms for craniofacial development.