Dorso-ventral pigment pattern differences are the most widespread pigmentary adaptations in vertebrates. In mammals, this pattern is controlled by regulating melanin chemistry in melanocytes using a protein, agouti-signalling peptide (ASIP). In fish, studies of pigment patterning have focused on stripe formation, identifying a core striping mechanism dependent upon interactions between different pigment cell types. In contrast, mechanisms driving the dorso-ventral countershading pattern have been overlooked. Here, we demonstrate that, in fact, zebrafish utilize two distinct adult pigment patterning mechanisms - an ancient dorso-ventral patterning mechanism, and a more recent striping mechanism based on cell-cell interactions; remarkably, the dorso-ventral patterning mechanism also utilizes ASIP. These two mechanisms function largely independently, with resultant patterns superimposed to give the full pattern.

To assess the hypothesis of central amino acid-sensing systems involved in the control of food intake in fish, we carried out two experiments in rainbow trout. In the first one, we injected intracerebroventricularly two different branched-chain amino acids (BCAAs), leucine and valine, and assessed food intake up to 48 h later. Leucine decreased and valine increased food intake. In a second experiment, 6 h after similar intracerebroventricular treatment we determined changes in parameters related to putative amino acid-sensing systems. Different areas of rainbow trout brain present amino acid-sensing systems responding to leucine (hypothalamus and telencephalon) and valine (telencephalon), while other areas (midbrain and hindbrain) do not respond to these treatments. The decreased food intake observed in fish treated intracerebroventricularly with leucine could relate to changes in mRNA abundance of hypothalamic neuropeptides [proopiomelanocortin (POMC), cocaine- and amphetamine-related transcript (CART), neuropeptide Y (NPY), and agouti-related peptide (AgRP)]. These in turn could relate to amino acid-sensing systems present in the same area, related to BCAA and glutamine metabolism, as well as mechanistic target of rapamycin (mTOR), taste receptors, and general control nonderepressible 2 (GCN2) kinase signaling. The treatment with valine did not affect amino acid-sensing parameters in the hypothalamus. These responses are comparable to those characterized in mammals. However, clear differences arise when comparing rainbow trout and mammals, in particular with respect to the clear orexigenic effect of valine, which could relate to the finding that valine partially stimulated two amino acid-sensing systems in the telencephalon. Another novel result is the clear effect of leucine on telencephalon, in which amino acid-sensing systems, but not neuropeptides, were activated as in the hypothalamus.

The circadian clock is a highly conserved cell-autonomous mechanism that directs daily rhythms in most aspects of biology. Daily entrainment by environmental signals, notably light, is essential for its function. However, our understanding of the mechanisms and the evolution of photic entrainment remains incomplete. Fish represent attractive models for exploring how light regulates the circadian clock due to the direct light sensitivity of their peripheral clocks. Central to this property is the light induced expression of clock genes that is mediated by D-box enhancer elements. Here, using zebrafish cells, we reveal that the light responsive D-box enhancer serves as a nuclear target for reactive oxygen species (ROS). We demonstrate that exposure to short wavelengths of visible light triggers increases in ROS levels via NADPH oxidase activity. Elevated ROS activates the JNK and p38 MAP kinases and in turn, induces clock gene expression via the D-box. In blind cavefish and mammals, where peripheral clocks are no longer entrained by direct illumination, ROS levels are still increased upon light exposure. However, in these species ROS no longer induces D-box driven clock gene transcription. Thus, during evolution, alterations in ROS-responsive signal transduction pathways underlie fundamental changes in peripheral clock photoentrainment.