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 probes is rapidly gaining favor over autoradiographic methods. Typically, either biotinylated or digoxigenin-labeled probes are used to detect mRNAs in sectioned tissue or in cultured cells. With a few exceptions, most applications of NISH in wholemount preparations have been limited to Drosophila embryos. A protocol developed for NISH in whole adult Drosophila CNS was extended to wholemounted larval and adult lamprey brain preparations. Digoxigenin-labeled RNA probes were transcribed from cloned fragments of a lamprey neurofilament (NF180) cDNA. Hybridization with these probes, and comparisons with Nissl-stained wholemounts and wholemounts retrogradely labeled by injections of tracer into the spinal cord, demonstrated that NF180 mRNA was expressed in only a subset of neurons in the lamprey CNS. These included primarily neurons with long axons that project out of the brainstem, e.g., reticulospinal neurons and cranial motor neurons. Metamorphosis from the larval to the adult form was accompanied by an increase in the number of neurons expressing NF180 and in the apparent level of NF expression as judged by the intensity of labeling. For example, in the oculomotor and trochlear 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 hybridization labeling increased with metamorphosis. The ability to do NISH in lamprey brain wholemounts eliminates the need for serial reconstructions and thus facilitates the study of selected gene expression during metamorphosis and regeneration.