Characterization of the Kiss system in medaka during development. Does Kiss control neuron migration through the chemokine receptor cxcr4?

Kiss is a substance in the brain that regulates the onset of puberty in different organisms, from fish to mammals. This project aimed at elucidating the new idea that Kiss in addition plays an important role in brain development. This was investigated in medaka, a small freshwater fish from Japan that is widely used in biological research. The see-through embryos make this an excellent research animal for brain development. A recent finding in Finn-Arne Weltzien´s research group at NMBU, Oslo, Norway, showed that turning off (knocking down) the Kiss gene or its receptor during early development caused severe forebrain defects in medaka. The mechanism behind this is not known, but it has been suggested that the receptor Cxcr4 could be involved, as Kiss has been found to interact with this receptor in mammalian cells. Cxcr4 is a cytokine receptor and is involved in neuron migration during brain development in mammals. We hypothesized that Kiss and its receptors could interact with Cxcr4 signaling, and was therefore involved in control of neuron migration in the developing medaka brain. We also wanted to see which cells could be affected by Kiss in development. We exposed medaka embryo to Kiss peptide and Kiss receptor blocker, and measured the expression of cxcr4a and the presence of tyrosine hydroxylase (TH) protein, an enzyme involved in dopamine synthesis. We found that cxcr4a expression was unaltered, but the number of TH cells in the brain increased after 3 days. This shows that Kiss can affect brain development, but are probably affecting other pathways than migration through cxcr4a. Future studies will show if Kiss can have a role in brain development also in other species, as humans.

Kisspeptin signalling through its receptor Gpr54 has been recently identified as a central regulator of puberty, but its function in other physiological systems is poorly investigated. A recent publication from my host group showed that high dose Morpholi no knockdown of either Kiss or Gpr54 in early development caused developmental arrest around gastrulation, whereas low dose knockdown caused severe forebrain and eye developmental defects. The intracellular pathways of Kiss signaling are not well known, b ut in mammals Kiss has been found to interact with the chemokine receptor Cxcr4 pathway, which is important in neuron migration during brain development. I hypothesize that Kiss/Gpr54 are implicated with Cxcr4 during brain development and control neuron m igration in developing medaka. I will test this hypothesis by several different strategies. First, I will characterize the temporal and spatial distribution of Kiss and Gpr54 in developing medaka, using in situ hybridization and immunohistochemistry. This has never been done before and will give valuable insight into the distribution of the Kiss system in vertebrate early development. I will also map the mRNA and protein expression of Cxcr4 in the developing medaka, and using double-staining, test for neu ronal co-expression of Gpr54 and Cxcr4, which would support the hypothesis that Kiss can control Cxcr4 signaling. Second, to test my hypothesis further, I will examine the effect of Kiss and Gpr54 loss-of-function on Cxcr4-expressing neurons by using Morp holino knockdown. Third, I will compare brain development in an available medaka Cxcr4 mutant to that of the Kiss/Gpr54 knockdown. The proposed experiments will give valuable new insight in vertebrate brain development in general, and the developmental ro le of Kiss in particular.