DEVELOPMENTALBIOLOGY164, 402-408 (1994) Ancestral Hemoglobin Switching in Lampreys GEROLAMO LANFRANCHI, ALBERTO PALLAVICINI, PAOLO LAVEDER, AND GIORGIO VALLE Dipartimento di Biologia, Universitd degH Studi di Padova, Via Trieste 75, 35121 Padova, Italy AcceptedMarch 11, 1994 A very simple hemoglobin switching was discovered in the lamprey Lampetra zanandreai. A single larval globin cDNA and two adult globin cDNAs were fully sequenced and their differential expression during lamprey develop- ment was investigated. The evolutionary positions of these new globin sequences are also discussed. © 1994 Academic Press, Inc. INTRODUCTION Globins of higher vertebrates are believed to have originated from a common ancestral gene (Goodman et al., 1975). When gnathostome fish evolved from the more primitive agnathan, tetrameric hemoglobins with a and fl subunits replaced monomeric globins. Myoglobins also arose around that period (Czelusniak et al., 1982) con- firming the key position of Agnatha in the molecular evolution of globins. During vertebrate evolution new globin genes were established by gene duplications and their expression was restricted to specific stages of ani- mal development. In amphibians a complex mechanism of r~egulation was established to drive the temporal or- dered expression of several a- and fl-globin genes (Broyles et al., 1981; Hosbach et al., 1983). The situation is far more complicated in mammals where the globin gene families are clustered in different chromosomes. The mechanisms of globin regulation seem to involve different regulative DNA sequences and several trans- acting factors (Karlsson and Nienhuis, 1985; Orkin, 1990). Therefore studying hemoglobin switching in more primitive vertebrates would be useful in understanding the mechanism of globin regulation. We focused our attention on the globin genes of Lam- petra zanandreai (Agnatha, Cyclostomata). This is a non- parasitic freshwater lamprey inhabiting the rivers of Northern Italy (Vladykov, 1955; Zanandrea, 1958). This species spends the majority of its lifetime as a larval form deeply hidden in the sandy ground of rivers. The metamorphosis occurs at the end of winter, around the sixth year of life, and causes major changes in the mor- phology, physiology, and behavior of the animal. The adult can swim freely, undergoes sexual maturation, and dies a few months after mating, usually before the beginning of the next summer. The hemoglobin of this lamprey, like other members of the Agnatha group, is monomeric in the deoxygenated state with a single heine group (Riggs, 1972). So far, all the published globin se- quences are derived from adult forms of lampreys (Braunitzer and Fujiki, 1969; Li and Riggs, 1970). How- ever, several studies have found differences in the elec- trophoretic patterns between larval and adult hemoglo- bins in some species of agnathans such as Lampetra fluviatilis, Lampetra planeri, and Petromyzon marinus (Adinolfi and Chieffi, 1958; Wald and Riggs, 1961; Potter and Brown, 1975) and comparative biochemical analyses have established that important modifications occur in the hemoglobin molecule of lampreys after the meta- morphosis (Manwell, 1963; Bird et al., 1976). We recently reported that L. zanandreai has a larval globin polypep- tide that is not present in the adult (Lanfranchi et al., 1991). In this paper we demonstrate that the differential globin expression seen in lamprey is due to a primordial developmentally regulated switch of globin genes. We also report the cDNA sequences of the larval globin and of two adult globins of lamprey. Phylogenetic parsimo- nial analysis would indicate that the larval form of glo- bin originated very early during the evolution of Cyclo- stomes. MATERIALS AND METHODS Globin preparation and gel electrophoresis. Larval or adult lampreys were collected from freshwater rivers using an electric fish shocker. After a brief anesthetiza- tion with MS222 (Sandoz), lampreys were sacrificed by decapitation and blood was collected in heparinized phosphate-buffered saline (0.9 mM CaC12, 2.68 mM KC1, 1.46 mM KH2P04, 0.5 mM MgCl~-6H20, 137 mM NaC1, 8 mM Na2HPO4-7H20, and 50 U/ml heparin). Erythro- cytes were purified by Ficoll density centrifugation and lysed in distilled water. The hemoglobin content was de- termined by spectrophotometric absorption of the he- molysates at 414 nm (Kabat et al., 1975). Hemolysates were denatured with 3 vol of a buffer containing 8 M 0012-1606/94 $6.00 Copyright© 1994 by Academic Press,Inc. All rights of reproductionin any formreserved. 402