The evolution of multicellular organisms was made possible by the evolution of underlying gene regulatory networks. In animals, the core of gene regulatory networks consists of kernels, stable subnetworks of transcription factors that are highly conserved in distantly related species. However, in plants it is not clear when and how kernels evolved. We show here that RSL (ROOT HAIR DEFECTIVE SIX-LIKE) transcription factors form an ancient land plant kernel controlling caulonema differentiation in the moss Physcomitrella patens and root hair development in the flowering plant Arabidopsis thaliana. Phylogenetic analyses suggest that RSL proteins evolved in aquatic charophyte algae or in early land plants, and have been conserved throughout land plant radiation. Genetic and transcriptional analyses in loss of function A. thaliana and P. patens mutants suggest that the transcriptional interactions in the RSL kernel were remodeled and became more hierarchical during the evolution of vascular plants. We predict that other gene regulatory networks that control development in derived groups of plants may have originated in the earliest land plants or in their ancestors, the Charophycean algae.T he development of multicellular organisms is controlled by gene regulatory networks (GRNs) and the reorganization of GRN architecture is thought to be a major factor underlying morphological evolution (1-5). GRNs are hierarchic and modular structures where four major component classes can be identified (3): at the periphery of GRNs are differentiation gene batteries encoding proteins that execute cell type-specific functions (such as building a pigmented cell); upstream of these batteries are switches that allow or prevent subcircuits to function in specific developmental contexts, and "plug-ins," small subcircuits that are flexibly and repeatedly used during development (such as signal transduction pathways); at the core of GRNs are kernels, small conserved subcircuits that execute specific developmental functions (such as defining spatial patterns in an embryo). Kernels are comprised of transcription factors that are highly conserved in distantly related species and are unusually stable components of GRNs.Ancient kernels that regulate body plan and organ development are highly conserved among diverse groups of metazoans (animals) (6-10). In contrast, the core components of plant GRNs are difficult to identify because of the dynamic nature of plant genome evolution and the plastic character of plant development. Floral homeotic genes form a relatively recent kernel controlling flower development (11). Homologs of KNOX and LEAFY transcription factors control shoot development in vascular plants and sporophyte development in mosses (12-15). KNOX/BEL genes also control the development of the diploid phase in unicellular chlorophytes (16) and the haploid-to-diploid transition in mosses (15), suggesting that KNOX and LEAFY genes may be core members of ancient GRNs that control diploid development in plants. Auxin signaling (17,18), et...