A METHOD FOR IN-SITU HYBRIDIZATION IN WHOLEMOUNTED LAMPREY BRAIN - NEUROFILAMENT EXPRESSION IN LARVAE AND ADULTS

Nonisotopic in situ hybridization (NISH) using both cDNA and cRNA prob es is rapidly gaining favor over autoradiographic methods. Typically, either biotinylated or digoxigenin-labeled probes are used to detect m RNAs in sectioned tissue or in cultured cells. With a few exceptions, most applications of NISH in wholemount preparations have been limited 60 Drosophila embryos. A protocol developed for NISH in whole adult D rosophila CNS was extended to wholemounted larval and adult lamprey br ain preparations. Digoxigenin-labeled RNA probes were transcribed from cloned fragments of a lamprey neurofilament (NF180) cDNA. Hybridizati on with these probes, and comparisons with Nissl-stained wholemounts a nd wholemounts retrogradely labeled by injections of tracer into the s pinal cord, demonstrated that NF180 mRNA was expressed in only a subse t of neurons in the lamprey CNS. These included primarily neurons with long axons that project out of the brainstem, e.g., reticulospinal ne urons and cranial motor neurons. Metamorphosis from the larval to the adult form was accompanied by an increase in the number of neurons exp ressing NF180 and in the apparent level of NF expression as judged by the intensity of labeling. For example, in the oculomotor and trochlea r nuclei, expression of NF180 was seen in postmetamorphic young adult lampreys but not in larvae. In the trigeminal motor nucleus, both the number of neurons expressing NF180 and the intensity of the hybridizat ion labeling increased with metamorphosis. The ability to do NISH in l amprey brain wholemounts eliminates the need for serial reconstruction s and thus facilitates the study of selected gene expression during me tamorphosis and regeneration. (C) 1994 Academic Press,Inc.