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Neuropeptide Y (NPY) is the only member of its peptide family that has been isolated from the mammalian CNS. We have recently found that two different NPY-related molecules are present in the CNS of a cyclostome, the river lamprey (Lampetra fluviatilis) (Soderberg et al., 1991). Here we show that this is also true for the rat CNS, by demonstrating expression of peptide YY (PYY) mRNA in brainstem neurons distinct from those neurons that express NPY mRNA. Dissimilar oligonucleotide DNA probes complementary to 3' untranslated regions of the rat PYY, NPY, and pancreatic polypeptide (PP) mRNA were used in in situ hybridization experiments on sections of rat brain and spinal cord, visceral organs, and peripheral nerve ganglia. The PYY probe hybridized with two populations of neurons in the brainstem: one dispersed along the midline in the rostral medulla and another in the lateral caudal medulla (A1 region). No additional labeling was detected in the remainder of the neuraxis. In the periphery, PYY hybridization was seen only in endocrine cells of the colon, and not in sympathetic ganglia or the adrenal gland, suggesting that previous observations of PYY immunoreactivity in these latter structures were due to antibody cross- reactivity with NPY. The NPY probe did not hybridize with cells on the midline region that contains PYY neurons, but it did label large numbers of neurons throughout the neuraxis. No expression of PP mRNA was detected in the CNS. Northern blot analysis failed to detect PYY mRNA in the CNS, further supporting the observation that PYY is only expressed by a discrete collection of CNS neurons. The anatomy of PYY- and NPY-expressing cells in the CNS and gut shows a striking similarity between rat and lamprey (Brodin et al., 1989), vertebrates that diverged evolutionarily about 450 million years ago, suggesting that both peptide systems have been conserved throughout vertebrate evolution.

The evolution of the neuropeptide Y (NPY) family of peptides has been unclear despite sequence information from many vertebrates. We describe here two NPY-related peptides deduced from cDNA clones of the river lamprey (Lampetra fluviatilis), a cyclostome providing one of the best models of a primitive vertebrate brain. One peptide corresponds to NPY as it has 83% identity to human NPY and its mRNA is expressed in the lateral brainstem, dorsal spinal cord and retina. The second lamprey peptide corresponds anatomically to peptide YY (PYY) as its mRNA is found in gut cells and in medial brainstem neurons. Its sequence is 60-70% identical to both PYY and NPY of mammals. These data suggest that the gene duplication leading to NPY and PYY had already occurred in the ancestral vertebrate 450 million years ago. The expression of the presumed PYY homolog in both gut and central nervous system indicates that PYY has served the dual role as a hormone and a neuropeptide from an early stage in vertebrate evolution. The similarities in the location of NPY- and PYY-expressing cells between lamprey and mammals suggest that the functions of these peptides may have been conserved.

Multipotential stem cells in the neural tube give rise to the different neuronal cell types found in the brain. Abrupt changes in intermediate filament gene expression accompany this transition out of the precursor state: transcription of the intermediate filament nestin is replaced by that of the neurofilaments. In order to identify human neural precursor cells, and to learn more about the evolution of the intermediate filaments expressed in the central nervous system, we have isolated the human nestin gene. Despite considerable divergence between the human and rat nestin genes, in particular in the repetitive parts of the carboxy-terminal region, the positions of the introns are perfectly conserved. Two of the three intron positions are also shared by the neurofilaments, but not by other classes of intermediate filaments. This implies that nestin and the neurofilaments had a common ancestor after branching off from the other classes of intermediate filaments, and that nestin separated from the neurofilament branch before the different neurofilament genes diverged. The characterization of human nestin also facilitates the identification of human multipotential neural precursor cells. This will be of importance for central nervous system (CNS) tumor diagnosis and transplant-based clinical approaches to human neurodegenerative diseases.