Dept. of Cell and Developmental Biology
Oregon Health & Science University
The melanocortin system plays multiple physiological roles in vertebrates. Zebrafish is an excellent teleost model for genetic studies of melanocortin signaling because of the powerful genetic tools available in this system, such as the use of Morpholino Oligonucleotides (MO) for the temporary suppression of gene expression. While many aspects of the melanocortin system are highly conserved across vertebrates, interesting variations can be found in teleosts, including additional members of the agouti gene family. In this dissertation, I characterize the physiological functions of two agouti proteins, AgRP and AgRP2, in the zebrafish. Chapter Two reports on the characterization of the function of a new agouti gene in zebrafish, termed agrp2. Due to genomic duplication, another agouti related protein---AgRP2, is found in most teleosts examined. agrp2 transcripts were exclusively detected in pineal gland, and regulated by light/dark cycle, not by metabolic state. AgRP2 peptide showed highest antagonistic potency to MC1R, less to MC4R and none at MC3R. Larval fish injected with antisense MO against either the 5UTR or ATG site of agrp2 mRNA failed to contract their melanocytes in response to white background at 3-5 days post fertilization (dpf). Without altering pomc gene expression, failure of normal background adaptation in agrp2 morphants or floating head (flh) mutant are due to the defect of melanin concentrating hormones (MCH) in lateral hypothalamus. Further experiments in nrc mutants suggest that up-regulation of MCH synthesis through pineal AgRP2-hypothalamic projections is retina cone/rod cells independent. These studies expanded our view of melanocortin functions in lower vertebrates to include pineal gland regulation of teleost background adaptation and evidence for light-responsive pineal cells communicating with hypothalamus in an AgRP2-MC1R dependent manner. This project led to my first research article in PNAS. Chapter Three reports the hypophysiotropic nature of zebrafish AgRP and POMC neurons, a huge functional departure from mammals. MC4R dependent dose responsive suppression of somatic growth was clearly seen in wild type fish injected with MO against endogenous agrp. Hypothalamic AgRP and POMC fibers densely project to the pituitary by 5 dpf. Multiple pituitary endocrine axes were altered in agrp morphant fish. Increased somatic growth was observed in MC4R null juvenile and adult fish. MC4R suppression by agrp is required for the maximal rate of growth during embryonic growth, allowing rapid maturation and thus perhaps reducing predation. Unlike mammals, in which circulating leptin regulates hypothalamic-pituitary axis, zebrafish pituitary is regulated directly by hypothalamic AgRP/POMC neurons. These findings clarify how teleosts differ from mammals in terms of leptin dependent energy homeostasis and how a single gene (MC4R) can coordinately regulates somatic growth and other endocrine functions in multiple fish species. Novel findings from this project resulted in my second research article in Cell Metabolism.
School of Medicine
Zhang, Chao, "Elucidation of the roles of agouti related proteins in zebrafish" (2012). Scholar Archive. 755.