Evolutionary crossroads in developmental biology: cyclostomes (lamprey and hagfish)

Lampreys and hagfish, which together are known as the cyclostomes or 'agnathans', are the only surviving lineages of jawless fish. They diverged early in vertebrate evolution, before the origin of the hinged jaws that are characteristic of gnathostome (jawed) vertebrates and before the evolution of paired appendages. However, they do share numerous characteristics with jawed vertebrates. Studies of cyclostome development can thus help us to understand when, and how, key aspects of the vertebrate body evolved. Here, we summarise the development of cyclostomes, highlighting the key species studied and experimental methods available. We then discuss how studies of cyclostomes have provided important insight into the evolution of fins, jaws, skeleton and neural crest.     

The pineal organ as a folded retina: immunocytochemical localization of opsins

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina-like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a 'folded retina'. The cell membrane of several pineal photoreceptor outer-segments of vertebrates immunoreact with anti-retinal opsin antibodies supporting the view of retina-like organization of the pineal. Some other pineal outer segments do not react with retinal anti-opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin-negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green-blue light-sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.     

Impact of asymmetric gene repertoire between cyclostomes and gnathostomes

Extant vertebrates are divided into the two major groups, cyclostomes and gnathostomes (jawed vertebrates). The former includes jawless fishes, hagfishes and lampreys, and the latter includes all extant jawed vertebrates. In many research fields, the phenotypic traits of the cyclostomes have been considered crucial in understanding the evolutionary process from invertebrates to vertebrates. Recent studies have suggested that the common ancestor of the extant vertebrates including hagfishes and lampreys underwent two-round of whole genome duplications, and thus the genome expansion solely does not account for phenotypic differences between cyclostomes and gnathostomes. Emerging evidence from molecular phylogeny of individual gene families indicates that the gene repertoire expanded at the common ancestor of vertebrates were later reshaped asymmetrically between the two lineages, resulting in the retention of differential gene sets. This also confuses interpretation of conserved synteny which often serves as indicator of orthology and the ploidy level. In this review, current controversy and future perspectives of cyclostome genomics are discussed with reference to evolutionary developmental biology.     

Studies on the evolution of elastin--I. Phylogenetic distribution.

1. Aortae and other tissues from numerous animals were examined for the presence of the rubbery protein elastin by (a) chemical purification and amino acid analysis, (b) presence of the cross-linking amino acids desmosine and isodesmosine and (c) histological staining. 2. Elastin was found in all vertebrates examined (42 species) with the exception of cyclostomes (3 species). It was absent from all invertebrates tested (14 species). 3. The amino acid compositions of vertebrate elastins showed marked and interesting interspecies variations.     

Comparative physiology of the renin-angiotensin system.

Renin or a renin-like substance is found in the kidneys of many vertebrate species. It is absent from the kidneys of cyclostomes and elasmobranchs and first appears in holosteans and the bony fishes as well as in all higher vertebrate species. Juxtaglomerular cell granules also appear first in holosteans and the bony fishes while the macula densa first appears in amphibians. In telecost fishes, the corpuscles of Stannius contain Bowie-stainable granules and a renin-like pressor substance. Among classes and, in some cases, species of vertebrates, specificity in the reaction of renin with a substrate has been demonstrated. There is also some species and class variation in the angiotensin molecule since angiotensins of fishes, amphibians, reptiles and birds have chemical characteristics different from each other and from those of ammmals. A role for renin in stimulating interrenal gland steroid biosynthesis and in influencing water and ion regulation in nonmammalian vertebrates is discussed.     

High resolution transmission electron microscopy of developing enamel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The permanent tooth plates of the Australian lungfish, Neoceratodus forsteri, are covered by enamel that develops initially in a similar manner to that of other vertebrates. As the enamel layer matures, it acquires several unusual characteristics. It has radially oriented protoprismatic structures with the long axes of the protoprisms perpendicular to the enamel surface. Protoprisms can be defined as aggregations of hydroxyapatite crystals that lack the highly ordered arrangement of the rods of mammalian enamel but are not without a specific structure of their own. The protoprisms are arranged in layers of variable thickness that are deposited sequentially as the tooth plate grows. They may be confined to the separate layers, or may cross the boundary between each layer. Crystals within the protoprisms are long and thin with hydroxyapatite c-axis dimensions of between 30 and 350 nm, and with typical a-b axis dimensions of 5-10 nm. The hydroxyapatite crystals of lungfish enamel have no centre dark lines of octacalcium phosphate, an unusual character among vertebrates. As each crystal develops, arrays of atoms may change direction, and regions exist where dislocations and extra lattice planes are inserted into the long crystal. The resulting hydroxyapatite crystal is not straight, and has a rough surface. The crystals are arranged in tangled structures with their crystallographic c-axes closely aligned with the long axis of the protoprism. Lungfish enamel differs from the enamel of higher vertebrates in that the hydroxyapatite crystals are of different shape, and, in mature enamel, the protoprisms remain as protoprisms and do not develop into the conventional prismatic structures characteristic of mammalian enamel.     

Origin of mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate,amphioxus

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.     

Isolation of a hagfish gene that encodes a complement component.

It has been widely accepted that cyclostomes are the most primitive vertebrates extant with the ability to produce antibodies. We isolated cDNA clones that encode a putative 'antibody' from one of the cyclostomes, Eptatretus burgeri. The amino acid sequence predicted from the nucleotide sequences of the cDNA clones indicated that this gene does actually encode the proteins isolated as hagfish 'antibodies' by various investigators. However, these proteins are not similar to mammalian immunoglobulins but have some characteristics common to complements C3, C4 and C5 in higher vertebrates. We discuss the relationships of the isolated gene for hagfish complement with the mammalian genes for complements C3, C4 and C5. We also discuss the possibility of the presence of antibodies in cyclostomes.     

Brain – Hatschek's pit relationships in amphioxus species

The vertebrate hypothalamo-hypophyseal neurosecretory system is a complex anatomical device for central nervous control over secretion of pituitary hormones. Since it is present in the most primitive vertebrates, the cyclostomes, it is of interest to look for a possible invertebrate anatomical equivalent, or precursor, for clues as to its evolution. We have found in six species of amphioxus, members of an invertebrate group (cephalochordates), considered to be closest to the vertebrates, that there is a morphologically equivalent neuro-epithelial complex, that in many ways resembles the hypothalamo-hypophyseal system of vertebrates. In the six amphioxus species described here the nervous element is a ventral lobe of the brain, the infundibulum, that extends downward along the right side of the notochord, and ends near the dorsal surface of a Rathke's pouch-like structure known as Hatschek's pit. This part of Hatschek's pit has been found earlier to contain a vertebrate LH-like gonadotropin. Therefore, the infundibulum-Hatschek's pit system of amphioxus may be involved in regulating the seasonal reproductive cycle, and it appears to be a direct homologue of the vertebrate hypothalamo-hypophyseal neurosecretory system functionally as well as morphologically.     

Electron diffraction studies of the calcareous skeletons of bryozoans

Electron microscopy and electron diffraction were used to investigate mineral crystallites dissociated from the skeletal walls of six species belonging to the Bryozoa, a phylum of predominantly marine colony-forming invertebrate animals. Four cheilostome bryozoans (Flustra foliacea, Membranipora membranacea, Thalamoporella novaehollandiae and Cellarinella foveolata) and two cyclostomes (Fasciculipora ramosa and Hornera robusta) were analysed. In each case, an attempt was made to relate the crystal morphology imaged in situ by scanning electron microscopy with the crystallographic orientation of isolated crystals determined by electron diffraction analysis in the transmission electron microscope. The results showed that the calcitic cheilostome and cyclostome skeletons consisted of closely packed arrays of plate-like Mg-containing calcite crystallites, and that the crystallographic a-axis was preferentially aligned perpendicular to the top and bottom surfaces of the flattened particles. The results suggest that calcite biomineralization occurs under similar crystallographic constraints in the five species studied even though the origins of cheilostomes and cyclostomes are separated by over 300 million years in the fossil record of the bryozoans. Similar studies for the aragonite crystallites in skeletons of M. membranacea indicated that the crystallographic b-axis was preferentially oriented perpendicular to the basal surfaces of irregular plate-like particles.     

Influence of lipids on ice formation in cryoprotective media

Cryomicroscopic analysis demonstrated that two lipid preparations from marine vertebrates (<0.1%) and liposomes prepared from rainbow trout sperm lipids (<0.5%) efficiently hindered the growth of ice crystals during freezing of multicomponent cryoprotective media used for trout sperm cryopreservation. At higher lipid concentrations, crystals either did not form at all or had altered shape and blurred boundaries. Addition of egg yolk (10%) together with these lipids increased the size of crystal structures and markedly changed their shape.     

On the origin of the adaptative immune system (AIS): the hypothesis

The adaptive immune system (AIS) appears exclusively at the vertebrate ones with jaw. In parallel, the lymphoid tissu associated with the digestive tract, or GALT (gut associated lympho?d tissu), seems to play an essential part in the development of this response immune with memory. That one could find its origin in the innate immune system of the invertebrates and closer the cyclostomes (vertebrates without jaws). But the transition is brutal since the chondrychtyens (lines, sharks) do have the AIS but the cyclostomes not. Moreover, it is still enigmatic and source of speculations. The gnathostomes (vertebrate with jaw) raise ancestral and adaptive innate defences of which acquisition will be discussed here. We will also discuss the consequences of integration in the genome by rag1 and rag2 genes (recombination activating genes).     

Crystal structures of deoxy- and carbonmonoxyhemoglobin F1 from the hagfish Eptatretus burgeri

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.     

Impact of oceanic upwelling on morphometric and molecular indices of an intertidal fish Scartichthys viridis (Blenniidae)

Upwelling is a determinant factor for intertidal community structure on all coasts where it occurs. Most of the evidence has been obtained from sessile species or those with limited mobility and it is still unknown whether nutrients are transferred from primary producers to upper trophic levels in upwelling systems. We studied a fish species from two localities in Central Chile, Quintay, and Las Cruces, the former affected by upwelling and the latter not as evidenced by sea surface temperatures. Specimens of the herbivorous fish Scartichthys viridis, from the two sites were compared for weight–length relationships and RNA?:?DNA ratio in muscle tissue. The results showed that in the upwelling zone, fish increase their weight faster and have greater RNA?:?DNA ratios than those from the non-upwelling zone. This suggests that nutrient subsidies can alter the performance of key intertidal vertebrates such as S. viridis. The consequences of this effect on community structure and dynamics are not known.     

Hagfish (cyclostomata, vertebrata): searching for the ancestral developmental plan of vertebrates

The phylogenetic position of the hagfish remains enigmatic. In contrast to molecular data that suggest monophyly of the cyclostomes, several morphological features imply a more ancestral state of this animal compared with the lampreys. To resolve this question requires an understanding of the embryology of the hagfish, especially of the neural crest. The early development of the hagfish has long remained a mystery. We collected a shallow-water-dwelling hagfish, Eptatretus burgeri, set up an aquarium tank designed to resemble its habitat, and successfully obtained several embryos. By observing the histology and expression of genes known to play fundamental roles in the neural crest, we found that the hagfish crest develops as delaminating migratory cells, as in other vertebrates. We conclude that the delaminating neural crest is a vertebrate synapomorphy that seems to have appeared from the beginning of their evolutionary history, before the splitting away of the hagfish lineage.     

Genomics reveal ancient forms of stanniocalcin in amphioxus and tunicate

Stanniocalcin (STC) is present throughout vertebrates, including humans, but a structure for STC has not been identified in animals that evolved before bony fish. The origin of this pleiotropic hormone known to regulate calcium is not clear. In the present study, we have cloned three stanniocalcins from two invertebrates, the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae. Both species are protochordates with the tunicates as the closest living relatives to vertebrates. Amphioxus are basal to both tunicates and vertebrates. The genes and predicted proteins of tunicate and amphioxus share several key structural features found in all previously described homologs. Both the invertebrate and vertebrate genes have four conserved exons. The predicted length of the single pro-STC in Ciona is 237 amino acids and the two pro-hormones in amphioxus are 207 and 210 residues, which is shorter than human pro-STCs at 247 and 302 residues due to expansion of the C-terminal region in vertebrate forms. The conserved pattern of 10 cysteines in all chordate STCs is crucial for identification as amphioxus and tunicate amino acids are only 14-23% identical with human STC1 and STC2. The 11th cysteine, which is the cysteine shown to form a homodimer in vertebrates, is present only in amphioxus STCa, but not in amphioxus STCb or tunicate STC, suggesting the latter two are monomers. The expression of stanniocalcin in Ciona is widespread as shown by RT-PCR and by quantitative PCR. The latter method shows that the highest amount of STC mRNA is in the heart with lower amounts in the neural complex, branchial basket, and endostyle. A widespread distribution is present also in mammals and fish for both STC1 and STC2. Stanniocalcin is a presumptive regulator of calcium in both Ciona and amphioxus, although the structure of a STC receptor remains to be identified in any organism. Our data suggest that amphioxus STCa is most similar to the common ancestor of vertebrate STCs because it has an 11th cysteine necessary for dimerization, an N-glycosylation motif, although not the canonical one in vertebrate STCs, and similar gene organization. Tunicate and amphioxus STCs are more similar in structure to vertebrate STC1 than to vertebrate STC2. The unique features of STC2, including 14 instead of 11 cysteines and a cluster of histidines in the C-terminal region, appear to be found exclusively in vertebrates.     

Serotonin-like immunoreactive cells in the pulmonary epithelium of ancient fish species

The pulmonary mucosa of three species of ancient fish was studied immunohistochemically to show the distribution of serotonin, regarded as the main monoamine of mammalian bronchopulmonary paraneurons. Serotonin-like immunoreactive cells, dispersed through the airway epithelium as single cells, were found in all the fish species studied. They are presumably equivalent to the neuroendocrine cells reported in the lungs of mammalian and submammalian vertebrates. However, the precise role and the function of these cells remain unknown. Since the species studied belong to the most primitive extant groups of ancient fish, the present investigation suggests that serotonin is widely distributed in the lungs of the vertebrates. Several peptides, known to be specific cytochemical markers for the identification of the pulmonary neuroendocrine cells of mammals, are being investigated in the lungs of the fish species studied. They may help to trace the phylogeny of the pulmonary neuroendocrine cell system and to elucidate its function in lower vertebrates.     

Serotonin-like immunoreactive cells in the pulmonary epithelium of ancient fish species

Summary The pulmonary mucosa of three species of ancient fish was studied immunohistochemically to show the distribution of serotonin, regarded as the main monoamine of mammalian bronchopulmonary paraneurons. Serotonin-like immunoreactive cells, dispersed through the airway epithelium as single cells, were found in all the fish species studied. They are presumably equivalent to the neuroendocrine cells reported in the lungs of mammalian and submammalian vertebrates. However, the precise role and the function of these cells remain unknown. Since the species studied belong to the most primitive extant groups of ancient fish, the present investigation suggests that serotonin is widely distributed in the lungs of the vertebrates. Several peptides, known to be specific cytochemical markers for the identification of the pulmonary neuroendocrine cells of mammals, are being investigated in the lungs of the fish species studied. They may help to trace the phylogeny of the pulmonary neuroendocrine cell system and to elucidate its function in lower vertebrates.     

Perspective: Research Activity of Enteropancreatic and Brain/Central Nervous System Hormones Across Invertebrates and Vertebrates

During the past two decades there have been rapid advances inour knowledge of the structure and function of the protein hormonesin the brain and gastroenteropancreatic system (GEP). Many publishedarticles have highlighted the superfamily of hormonal peptides,specifically, the mechanisms and control of peptide synthesisin neural and non-neural tissues, and gene structure. Here wepresent an analysis of the annual trends, between 1980 and 1997,of research emphasis on six protein/peptide hormones, as reflectedby their individual frequency of publication per year. Althoughthis symposium is focused on the GEP hormones, we provide hereina perspective on the level of research activity of the hormonesinsulin, glucagon, cholecystokinin, insulin-like growth factor-Iand -II, neuropeptide Y and somatostatin in the brain/gut systemsthroughout the vertebrates and invertebrates. Many publicationsdeal with the evolution of these peptides and their superfamilies,yet as noted in this review, there are relatively few referencesto these peptides in invertebrates and non-mammalian species.Typically in invertebrates, the number of citations is low andmostly focused on three phyla, the arthropods, mollusks andhelminths. Generally, in the vertebrates the smallest numberof citations is in the cyclostomes and elasmobranchs. Becausemost groups of invertebrates and vertebrates have received scantattention, phylogenetic comparisons are limited. Evolutionaryinformation concerning important groups of animals, such ashelminths, mollusks, protochordates and cyclostomes, is essentialto establish the phylogenetic histories of the hormonal peptides.The challenge to comparative endocrinologists is to examinespecies in key evolutionary positions in order to gain an understandingof the diversity and function of the hormones and to determinethe molecular features that form clues to their phyletic interrelationshipsand progression.     

Diversity and distribution of idioblasts producing calcium oxalate crystals in Dieffenbachia seguine (Araceae)

Although cells that synthesize crystals are known throughout the plant kingdom, their functional significance is still unknown. Mechanical support, mineral balance, waste sequestration, and protection against herbivores have all been proposed as crystal functions. To seek clues to their role(s), I systematically examined all organs except fruit of Dieffenbachia seguine (Araceae) for crystals. Crystals were found in nearly every organ. Raphides (long, slim, pointed crystals) were most common, but druses (crystal aggregates) and prisms were also found. Raphides varied in size by a factor of 10 and also in organization from tightly bundled to loosely organized. Biforines, a type of cell capable of expelling raphides, or biforine-like cells, were found in nearly all organs, but especially in leaves, spathes, and anthers. Different organs had different crystal complements, and characteristic crystals were found at specific locations, such as among pollen, along the undersides of leaf veins, and at root branch points. All crystals appeared to be composed of calcium oxalate, based on acid solubility. Possible roles of the crystals are discussed in light of these findings.