Central regulator genes integrate signaling pathways ensuring robust embryonic development

Abstract

During embryonic development, the activity of signaling pathways regulates precise gene expression patterns, ensuring proper development. The interaction between signaling pathways with the non-coding genome, particularly at cis-regulatory elements (CREs), is thought to coordinate gene transcription. However, how a transcriptional output is regulated by multiple signaling pathways and at the genome-wide level remains poorly understood. To address this, we combined in vivo perturbations of RA, Wnt, and FGF signaling pathways in zebrafish with genome-wide RNA- and ATAC-seq analysis to determine genes and CREs regulated by these signals. This approach identified both pathway-specific and multi-pathway responses. Using integrated ATAC-seq and RNA-seq data we identified so called integrator genes that mediate transcriptional responses to multiple pathways at different developmental stages. These genes function as transcriptional hubs within stage-specific gene regulatory networks (GRNs), ensuring precise gene expression throughout early development. Among known gene targets, the NET family paralogs znf503 and znf703 emerged as key regulatory factors. Functional analysis of znf503 and znf703 knockouts revealed the existence of a partial compensatory mechanism and functional redundancy between these genes. Detailed GRN analysis revealed that both genes operate within a shared gene network, largely mediated by transcription factors associated with multiple signaling pathways. Additionally, znf503 and znf703 mutants exhibited locomotor defects, resembling the phenotype previously described for insm1a, a critical regulator of motoneuron differentiation. The presence of insm1a as a central node in both mutant GRNs suggests a functional link between these genes in motor circuit development. Moreover, insm1a and the two paralogs znf503 and znf703 are implicated in cancer, suggesting that transcriptional programs governing embryogenesis may be co-opted in tumorigenesis. Together, our findings provide insights into how genomes integrate multiple signaling pathways at the transcriptional and cis-regulatory level to investigate GRNs controlled by them. The functional dissection of the integrator genes znf503 and znf703 highlight functional redundancy in these gene networks, potentially providing robustness during embryonic development. Furthermore, our results highlight the need to explore how znf503 and znf703 could engage in distinct regulatory mechanisms depending on the developmental stage and cell type, potentially diversifying their functions beyond their shared gene network and influencing gene regulation in both normal physiology and disease.