For example, Drosophila GRH regulates the levels of genes encoding enzymes involved in cuticle melanization and chitin metabolism, cell adhesion proteins, and protein components of the cuticle. In mice, Grhl3 regulates the levels of genes that encode structural-barrier proteins in keratinocytes and the enzymes that crosslink such proteins, as well as cell-adhesion proteins and proteins that modulate the lipid composition of the epidermis. We propose that the original functions of Grainy head-like proteins in the opisthokont last common ancestor predisposed GRH-like proteins to regulate many aspects of extracellular-barrier formation and wound healing in early animals, as well as to evolve the related ability of regulating cellcell adhesion genes in many epithelial tissues. In the metazoan lineage, many types of epidermal barriers have evolved over time, including epithelia with chitin-based extracellular barriers, and it is interesting that chitin is one of the few extracellular structural biopolymers common to both fungi and animals. While chitin synthase itself does not appear to be regulated by GRH-like proteins in any system yet studied, it appears that GRH and GRH-like proteins of the CP2 superfamily regulate the expression of many genes involved in the formation and remodeling of chitin-based barriers, at least in Neurospora and Drosophila. It is also intriguing that chitinase 1 in Neurospora and chitinase 3 in Drosophila both appear to be strongly regulated by GRHL and GRH, respectively, consistent with an ancestral transcriptional control of chitinase expression by GRH-like proteins in the opisthokont last common ancestor. We believe it is possible that components of the ancestral opisthokont cell wall were repurposed during the evolution of chitin-based apical extracellular barriers in some basal multicellular animals, with GRH proteins maintaining a role in barrier formation and remodeling during the process. A similar process may have occurred during the evolution of multicellular volvocine algae, as it has been proposed that the outer cell wall of unicellular algae evolved to become part of the apical extracellular barrier of multicellular algae. This would have been Bortezomib independent of control by CP2 superfamily proteins, as sequenced genomes in the algal lineage do not encode recognizable members of this superfamily. The evolution of multicellularity in fungi was presumably less complicated than in metazoans, as one can invoke incomplete cell division creating syncytial colonies of fungi. In this evolutionary scenario, the conservation of ancestral GRHL function with respect to barrier formation and remodeling would be straightforward, as the cell walls of the unicellular opisthokont last common ancestor and extant multicellular fungi would be very similar in structure and function. In addition to the greatly lowered expression of the chitinase 1 gene, we also found evidence that Neurospora GRHL plays a role in the expression of enzymes involved in the synthesis and remodeling of another key biopolymer of the fungal cell wall – beta-1,3-glucan. GRHL may turn out to have a more general role in promoting cell wall development, although we were unable to uncover phenotypic evidence for this, despite testing the growth of grhl mutant strains under several conditions shown elsewhere to inhibit the growth of S. cerevisiae strains with compromised cell walls.