BackgroundVertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L).ResultsSequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R.ConclusionsWe present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the other neuronal and neuroendocrine functions exerted by the proteins encoded by these gene families. In pouched lamprey all five visual opsin genes have previously been identified, suggesting that lampreys diverged from the jawed vertebrates after 2R.

Although brain organization in lampreys is of great interest for understanding evolution in vertebrates, knowledge of early development is very scarce. Here, the development of the forebrain and midbrain gamma-aminobutyric acid (GABA)-ergic systems was studied in embryos, prolarvae, and small larvae of the sea lamprey using an anti-GABA antibody. Ancillary immunochemical markers, such as proliferating cell nuclear antigen (PCNA), calretinin, and serotonin, as well as general staining methods and semithin sections were used to characterize the territories containing GABA-immunoreactive (GABAir) neurons. Differentiation of GABAir neurons in the diencephalon begins in late embryos, whereas differentiation in the telencephalon and midbrain was delayed to posthatching stages. In lamprey prolarvae, the GABAir populations appear either as compact GABAir cell groups or as neurons interspersed among GABA-negative cells. In the telencephalon of prolarvae, a band of cerebrospinal fluid-contacting (CSF-c) GABAir neurons (septum) was separated from the major GABAir telencephalic band, the striatum (ganglionic eminence) primordium. The striatal primordium appears to give rise to most GABAir neurons observed in the olfactory bulb and striatum of early larval stages. GABAir populations in the dorsal telencephalon appear later, in 15-30-mm-long larvae. In the diencephalon, GABAir neurons appear in embryos, and the larval pattern of GABAir populations is recognizable in prolarvae. A small GABAir cluster consisting mainly of CSF-c neurons was observed in the caudal preoptic area, and a wide band of scattered CSF-c GABAir neurons extended from the preoptic region to the caudal infundibular recess. A mammillary GABAir population was also distinguished. Two compact GABAir clusters, one consisting of CSF-c neurons, were observed in the rostral (ventral) thalamus. In the caudal (dorsal) thalamus, a long band extended throughout the ventral tier. The nucleus of the medial longitudinal fascicle contained an early-appearing GABAir population. The paracommissural pretectum of prolarvae and larvae contained a large group of non-CSF-c GABAir neurons, although it was less compact than those of the thalamus, and a further group was found in the dorsal pretectum. In the midbrain of larvae, several groups of GABAir neurons were observed in the dorsal and ventral tegmentum and in the torus semicircularis. The development of GABAergic populations in the lamprey forebrain was similar to that observed in teleosts and in mouse, suggesting that GABA is a very useful marker for understanding evolution of forebrain regions. The possible relation between early GABAergic cell groups and the regions of the prosomeric map of the lamprey forebrain (Pombal and Puelles [ 1999] J. Comp. Neurol. 414:391-422) is discussed in view of these results and information obtained with ancillary markers.

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and gamma-aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA-negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium-sized to large larvae, PCNA-immunoreactive (-ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late-proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA-ir perikaryon was observed in the retina of larval lampreys, although a few GABA-ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA-ir ganglion cells occur in the pineal organ of 15-17 mm larvae, and their number increases during the larval period. The only GABA-ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods.