The Lympho-Hemopoietic Organs of the Anadromous Sea Lamprey,Petromyzon marinus. A Comparative Study throughout its Life Span

We have analyzed morphological changes affecting the lympho-hemopoietic organs of the anadromous sea lamprey, Petromyzon marinus throughout its life span. For this analysis, ammocoetes (2–4 years), premetamorphosing lampreys (nearly 5 years), metamorphosing lampreys, macrophtalmia stages (young adults) and parasitic adults (nearly 7 years) were used. The principal lympho-hemopoietic organs in the ammocoete are typhlosole, larval opisthonephros and nephros-associated adipose tissue. After metamorphosis, these organs degenerate, and their lympho-hemopoietic tissue is replaced by dense connective tissue. The supraneural body and to a lesser degree, the definitive opisthonephros, are the main blood-forming organs in adult lampreys. During larval life, lympho-hemopoietic cells appear in the branchial area, associated with pharyngeal epithelium. These loci are not morphologically homologous to the thymus gland of jawed vertebrates. These results are discussed, with special emphasis on the importance of cell microenvironments in eluciding changes in different blood-forming loci throughout the life cycle and their significance for the lamprey's immune capacity.     

Changes in haemopoietic sites during the metamorphosis of the lampreys Lampetra fluviatilis and Lampetra planeri

An investigation has been made in both the parasitic lamprey Lampetra fluviatilis and in its non-parasitic derivative Lampetra planeri of the rate at which the fat column replaces the typhlosole and nephric fold as the principal site of haemopoiesis. In the typhlosole, blood cell formation started to decline prior to the onset of external metamorphosis and had ceased within four weeks of the commencement of transformation. In the nephric fold haemopoiesis continued for several weeks in the region where the larval opisthonephros persisted but was never observed in the newly developing adult kidney. Soon after the onset of external metamorphosis the fat column started to become haemopoietic and later became the main site of blood cell formation. The rate at which the haemopoietic function was transferred from the nephric fold and typhlosole to the fat column was greater in L. fluviatilis than in L. planeri. Since a similar more rapid change in L. fluviatilis has also been found in the switch from larval to adult haemoglobin, the former type of haemoglobins may be produced only in erythrocytes originating in the nephric fold and typhlosole, whereas the latter type may be restricted to cells developed in the fat column. It is also suggested that the functional significance of the alteration in haemopoietic sites is related to changes at metamorphosis in the three regions where blood cell formation occurs.     

Connective tissue is involved in adult epithelial development of the small intestine during anuran metamorphosis in vitro

Summary The role of connective tissue in metamorphic changes of the small intestinal epithelium inXenopus laevis tadpoles was investigated by using organ culture techniques and electron microscopy. Tissue fragments isolated from various parts of the small intestine at stage 57 were cultivated. Larval cell death of the epithelium was induced by thyroid hormone in all fragments, whereas adult epithelial development was observed only in fragments isolated from the anterior intestinal region containing the typhlosole where most of the larval connective tissue was localized. The epithelium was then cultivated in recombination with homologous or heterologous non-epithelial components. The adult epithelium developed only in recombinants containing a thick connective tissue layer from the typhlosole. There was no regional difference in the developmental potency of the epithelium itself. In all explants where adult epithelium developed, the connective tissue increased in cell density just beneath the epithelium, which was rapidly proliferating and forming typical islets. At the same time, fibroblasts possessing well-developed rough endoplasmic reticulum differentiated close to epithelial cells and often made contact with them. These results indicate that the connective tissue originating from the typhlosole plays an important role in adult epithelial development of the anuran small intestine, probably via direct cell-to-cell contacts or some factor(s) synthesized by the fibroblasts.     

Cell renewal in the epithelium of the alimentary tract of the larval lamprey,Petromyzon marinus L.

Light microscopic autoradiography with ³H-thymidine demonstrates that the three regions of the alimentary tract in the larval (ammocoete) lamprey, Petromyzon marinus L., possess different patterns for renewing their epithelium. In the oesophagus, columnar and mucous cells originate from stem cells located at the bases of folds and migrate to the tops of the folds where they are apparently extruded. Ciliated cells, located only at the tops of the folds, seem to differentiate from migrating columnar cells. In the anterior intestine, stem cells are present throughout the epithelium so that there is limited migration of cells and their extrusion occurs randomly. In the posterior intestine, the stem cells located at the bases of the typhlosole provide a continuous population that differentiates and migrates to the top of the typhlosole and to the opposite epithelial wall where they are presumably extruded. The rates of cell renewal in all three epithelial regions of the alimentary tract are slower in animals maintained at 10 ± 1°C compared with those kept at 21 ± 1°C. Comparatively, ammocoetes have the least specialized system for cell renewal known in the alimentary tract of a vertebrate.     

Of blood cells and the nervous system: Hematopoiesis in the Drosophila larva

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.     

Of blood cells and the nervous system

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.     

Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys, Petromyzon marinus L

Immunocytochemistry with protein A-gold and routine electron microscopy were used to identify cell types within the endocrine pancreas of larvae, juvenile adults, and upstream-migrant adults of the sea lamprey, Petromyzon marinus. The larval pancreatic islets are composed only of insulin-immunoreactive B-cells, which are uniform in their fine structure. The cranial and caudal pancreatic tissue in both adult periods contains three cell types: B-cells, somatostatin-immunoreactive D-cells, and a third cell type of unknown content. No glucagon-immunoreactive cells are present in lampreys, but B- and D-cells exist in equal numbers in the pancreatic tissue of adults. The B-cells of adults have a fine structure similar to those in larvae. D-cells have secretory granules that are distinctly different from those both in B-cells and in the third cell type. Although B- and D-cells in lamprey pancreatic tissues have a basic morphological similarity to these cells in other vertebrates, their granules are generally of smaller dimensions. The inclusion of granules within large pleomorphic bodies in many D-cells indicates that granule turnover is common. Immunocytochemistry will be a useful tool for showing the relationship between the cells in the degenerating bile ducts and those of the developing adult pancreas.     

Occurrence and Structure of Iron Inclusions in Adipocytes of Larval Lampreys

The occurrence and structure of adipocytes in the larvae of two lamprey species, Geotria australis and Petromyzon marinus, were examined by electron microscopy. Adipocytes from both species possessed large electron-dense inclusions which histochemical and energy dispersal X-ray analyses show as containing iron. The greatest concentration of inclusions in adipocytes was found in the nephric fold of G. australis. While some iron is present in the cytoplasmic matrix as ferritin, the majority is seen in large ammocoetes in membrane-bound dense aggregations of haemosiderin. The wide variety of inclusion types seen in smaller larvae may reflect on the method of formation of these inclusions within the cell. Because of the high level of iron loading in the larval lamprey nephric fold, this readily accessible tissue may provide a valuable model for studies of iron metabolism in vertebrates.     

Radioautography of Presumptive Interrenal Cells in the Sea Lamprey After 3H-cholesterol Injection

Abstract Radioautography demonstrates that the injection of ³H-cholesterol into larval and adult sea lamprey, Petromyzon marinus L., results in labelling of their presumptive interrenal cells. This localization of cholesterol and/or its metabolites is: (1) further evidence for the homology of these cells with adrenocortical cells of higher vertebrates and (2) a suggestion of the possibility that these cells in lampreys are able to absorb cholesterol for utilization in corticosteroid synthesis.     

Transformation of the endostyle of the anadromous sea lamprey,Petromyzon marinus L., during metamorphosis. II. Electron microscopy

Electron microscopy was used to follow the transformation of the endostyle to a thyroid gland in the anadromous sea lamprey, Petromyzon marinus L., throughout metamorphosis (stages 1–7). Transformation of the larval (ammocoete) endostyle begins at the first signs of external change (stages 1–2), and the adult form of the gland is reached by stage 5. Only slight modifications of the gland accompany further development to the end of metamorphosis. Development of the thyroid gland involves degeneration, proliferation, and reorganization of the cells in the endostyle, and changes in their fine structure. Ultrastructural changes during early stages are most obvious in the type 1 cells that make up the shrinking glandular tracts, and involves the accumulation of cytoplasmic microfilaments and a variety of cytoplasmic inclusions. The glandular tracts and their cells gradually disappear through autolysis and, apparently, through phagocytosis by neighboring epithelial cells and macrophages. Although the fine structure of the type 2, 3, 4, and 5 cells is not altered in the early stages, by stage 3, many of these cells become either vacuolated, undergo autolysis, or are extruded. Phagocytosis of some of each of these cell types likely occurs. Thyroid follicles are first observed during stage 4. Some of their lumina seem to arise from the accumulation of material in intercellular spaces and from vacuoles among cell clusters. Other lumina may represent a portion of the original lumen of the endostyle. Many follicles appear to be comprised of cells with cytological characteristics similar to those of larval cell types 3 and 2c. Some of the other larval cell types, such as type 5, may also be involved. In young adult lampreys follicles are composed of cuboidal to columnar cells that lack the dilated cisternae of rough endoplasmic reticulum seen in follicular cells of higher vertebrates. Dense collagenous connective tissue surrounding the follicles contains relatively few blood vessels. The transformation process described may have some relevance to our understanding of the development and evolution of the vertebrate thyroid gland.     

Evidence for a membrane-bound carbonic anhydrase in the heart of an ancient vertebrate,the sea lamprey (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

In order to gain insight into the early evolution of carbonic-anhydrase (CA) isozymes in vertebrates, the main objective of the present study was to determine whether the hearts of an ancient vertebrate species, Petromyzon marinus, possess a membrane-bound CA isozyme. Since a significant amount of CA activity appeared to be strongly associated with the heart membrane fraction after differential centrifugation and washing, further experiments were conducted to examine the inhibitor properties of the CA from the membrane fraction in comparison with lamprey cytoplasmic CA from the red blood cell (rbc) fraction. These experiments showed that the inhibitor properties of the CA from the heart membranes were significantly different from those of the cytoplasmic CA from lamprey rbcs. A final series of experiments showed that the membrane-bound CA in the lamprey heart is not anchored via a glycosylphosphatidylinositol (GPI) linkage. Taken together, the results of these studies indicate that a membrane-bound CA does appear to be present in the hearts of lamprey, but the properties of the membrane-bound CA isozyme in these ancient vertebrates appear to differ from those in more recently evolved groups.Abbreviations Az acetazolamide - CA carbonic anhydrase - GPI glycosylphosphatidylinositol - PI-PLC phosphatidylinositol specific phospholipase C - Rbc red blood cell     

Morphology of the epithelium in the alimentary tract of the larval lamprey,Petromyzon marinus L.

The alimentary tract of the ammocoete of the lamprey, Petromyzon marinus L., is divisible into three morphologically distinct regions: the oesophagus, the anterior intestine, and the posterior intestine. The epithelium of the oesophagus possesses mucous, ciliated, and columnar cells and appears to be specialized for movement of food particles. The epithelium of the anterior intestine possesses secretory cells with numerous zymogen granules, ciliated cells, and columnar-absorptive cells. Although some absorption occurs in the anterior intestine, the main function of this region seems to be the release of digestive enzymes and the continued movement of food particles. The epithelium of the posterior intestine is entirely comprised of columnar absorptive cells, namely tall (light and dark) columnar and low columnar, and the primary function of this region is one of absorption. The epithelium of the hindgut resembles that of the archinephric duct (Youson and McMillan, '71). The morphology of the alimentary tract of ammocoetes suggests that some differentiation and renewal of cell types may occur in the epithelium of the three regions. Comparison of the alimentary tract of larval lamprey with that of other vertebrates indicates that the gut of the ammocoete represents a less specialized level of vertebrate development.     

Photoreceptor cells and neural elements with long axonal processes in the pineal organ of the lamprey,Lampetra japonica,identified by use of the horseradish peroxidase method

Summary Horseradish peroxidase (HRP) was applied to the transected end of the pineal tract of the lamprey, Lampetra japonica. Distinct reaction products of HRP were observed in 2 types of cell other than ganglion cells. The first type of cell protrudes a knob-like process into the pineal lumen. This type of cell was clearly identified by electron microscopy as a photoreceptor cell; its outer segment was connected to the ellipsoid through a sensory cilium. The other type of cell was located among photoreceptor and supporting cells. The processes of these cells were thin and slender, and they obviously did not represent photoreceptor, supporting, or conventional ganglion cells. The present results indicate that, in the lamprey, some of the photoreceptor cells of the pineal organ project their axon-like processes toward the posterior commissure, but that there is also another type of cell displaying long axonal projections. HRP-containing cells were distributed randomly over the pineal organ and were occasionally also observed in the parapineal organ.     

Development of the renal corpuscle during metamorphosis in the lamprey.

The renal corpuscle of the adult lamprey, Petromyzon marinus L., is formed during the programmed period of metamorphosis. Development is initiated early in this metamorphic period and is marked by the synchronous formation and growth of rudimentary nephron units (RNU) from longitudinal cord of nephrogenictissue extending from the posterior tip of the degenerating larval kidney to the cloaca and connected to the peritoneal epithelium. Detachment of the RNU from the peritoneum involves autolysis and cell death and is accompanied by their branching into five or six hexagonally-arranged nephrons which radiate from the original point of attachment. Differentiation of the epithelial cells at the proximal ends of the nephrons is preceded by the widening of lateral intercellular spaces, the formation of tubular lumina (primitive urinary spaces), the loss of apical cell junctions, and the development of a capillary network with its associated mesangium. With the extension of the capillaries and mesangium between the proximal ends of adjacent undifferentiated nephrons, visceral epithelial cells (podocytes), with long cell processes (trabeculae) and slit membranes, make their appearance. The urinary spaces resulting from this form of development are lined by the epithelium of the dilated ends of the nephrons (nephric capsules). The cells of these capsules differentiate mainly into podocytes, but a few parietal cells connect to the draining tubule. This method of development explains the unique form of the renal corpuscle in the adult lamprey. Despite the type of morphogenesis, this renal corpuscle possesses the fine-structural features seen in the renal corpuscles of other vertebrates.     

Crystal Structure of the Lamprey Variable Lymphocyte Receptor C Reveals an Unusual Feature in Its N-Terminal Capping Module

Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.     

In vitro studies of hematopoiesis in the silkworm: cell proliferation in and hemocyte discharge from the hematopoietic organ

The lepidopteran hematopoietic process is poorly understood. We therefore examined the fundamental properties of hematopoiesis in the silkworm Bombyx mori using hematopoietic organ culture. In a medium containing larval plasma taken from the fourth day of the final larval stadium, over 50,000 hemocytes per hematopoietic organ were discharged within 48 h, with the number of cells comprising the hematopoietic organ simultaneously increasing from approximately 20,000 to 40,000. However, in the absence of plasma, cell numbers comprising the hematopoietic organ were unchanged and the number of discharged cells was much less. Hematopoietic organs cultured with plasma showed strong mitotic indices in a BrdU incorporation assay, but did not when cultured without plasma, indicating that plasma contains hematopoietic factor(s). The hematopoietic stimulation ability of larval plasma was observed from the last day of the penultimate larval stadium to the prepupal stage. The response of the hematopoietic organs to larval plasma was highest at the beginning of the final larval stadium and decreased with aging. Most cells discharged from the hematopoietic organ were plasmatocytes and prohemocytes, irrespective of location and developmental stage. Using this in vitro culture method, we tested the effects of 20-hydroxyecdysone (20E) and juvenile hormone-I (JH-I) on B. mori hematopoiesis. 20E showed a weak, but significant, hematopoietic activity, whereas JH-I did not, suggesting that a part of larval hematopoiesis is endocrinally regulated.     

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus)

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.     

A histological and electron-microscopic study of the cell types involved in rejection of skin allografts in ammocoetes

The rejection of skin allografts by the larval lamprey, Lampetra reissneri, was studied by light- and electron-microscopy, with particular attention to the cell types involved in the reaction. In all allografts, melanophores were destroyed within 20-60 days (the mean survival time, 36 +/- 12 days). Neither the epidermis nor the underlying collagenous lamella was invaded by host cells until the 60th day. A heavy infiltration of host leucocytes was observed in the allografts in melanophore and adipose layers and in the bundles of muscles. Throughout all stages from 10 to 60 days after the grafting, the cells of the polymorphonuclear leucocyte (PMN) series and eosinophilic granulocytes predominated, but macrophages were not observed at any stages examined. Plasma cells occurred occasionally at later stages (40-60 days) of allograft rejection, but lymphocytes were rarely found at any stages of graft rejection. These observations, combined with the recent finding of the antibody-enhanced phagocytic activity of granulocyte-series cells in the lamprey, indicate that PMNs, but not lymphocytes, function as the major effector cells in allograft rejection in this phylogenetically oldest class of contemporary vertebrates.