Podlasz, P., Sallinen, V., Chen, Y.C., Kudo, H., Fedorowska, N., and Panula, P. (2012) Galanin gene expression and effects of its knock-down on the development of the nervous system in larval zebrafish. The Journal of comparative neurology. 520(17):3846-3862.
Despite the known importance of galanin in the nervous system of vertebrates, the galanin gene structure, expression and consequences of galanin deficiency in developing zebrafish are unknown. We cloned the galanin gene, analyzed its expression using in situ hybridization, PCR and immunocytochemistry throughout the early development of zebrafish until the end of the first week of life. The single zebrafish galanin gene encoded for a single amidated galanin peptide and a galanin message-associated peptide. Two forms resulting from alternative processing were identified. Galanin mRNA was maternally expressed and found in developing fish throughout the early development. In situ hybridization showed the first positive neurons in three groups in the brain at 28 hours post fertilization. At 2 days post fertilization, three prosencephalic neuron groups were seen in the preoptic area, and in rostral and caudal periventricular hypothalamus. In addition, two other groups of weakly stained neurons were visible, one in the midbrain and another one in the hindbrain. Translation inhibition of galanin mRNA with morpholino oligonucleotides caused complete disappearance of galanin immunoreactivity in the brain until 7 dpf and did not induce known cascades of non-specific pathways or morphological abnormalities. A minor disturbance of sensory ganglia was found. Galanin knockdown did not alter the expression of tyrosine hydroxylases 1 and 2, choline acetyltransferase, histidine decarboxylase, or orexin mRNA. The results suggest that galanin doesn't regulate the development of these key markers of specific neurons, although galanin-expressing fibers were in a close spatial proximity with several neurons of these neuronal populations.