hlh-1 is a myogenic transcription factor required for body-wall muscle specification during embryogenesis in Caenorhabditis elegans. Despite its well-known role in muscle specification, comprehensive regulatory control upstream of hlh-1 remains poorly defined. Here, we first established a statistical reference for the spatiotemporal expression of hlh-1 at single-cell resolution up to the second last round of divisions for most of the cell lineages (from 4- to 350-cell stage) using 13 wild-type embryos. We next generated lineal expression of hlh-1 after RNA interference (RNAi) perturbation of 65 genes, which were selected based on their degree of conservation, mutant phenotypes, and known roles in development. We then compared the expression profiles between wild-type and RNAi embryos by clustering according to their lineal expression patterns using mean-shift and density-based clustering algorithms, which not only confirmed the roles of existing genes but also uncovered the potential functions of novel genes in muscle specification at multiple levels, including cellular, lineal, and embryonic levels. By combining the public data on protein-protein interactions, protein-DNA interactions, and genetic interactions with our RNAi data, we inferred regulatory pathways upstream of hlh-1 that function globally or locally. This work not only revealed diverse and multilevel regulatory mechanisms coordinating muscle differentiation during C. elegans embryogenesis but also laid a foundation for further characterizing the regulatory pathways controlling muscle specification at the cellular, lineal (local), or embryonic (global) level.
Keywords: Caenorhabditis elegans; Embryogenesis; Lineal expression; Multilevel regulation; Transcription factor; hlh-1.