Electron microscopical studies on the thyroid of a cyclostome,Lampetra japonica,during its upstream migration

Summary Electron microscopical studies were made of the thyroid gland of an adult lamprey, Lampetra japonica, in the upstream migration period.The thyroid consists of many usual follicles containing the colloid in their lumina, and a large parafollicle without colloid. The paper concerns only the usual follicle.The follicle cells found in the usual follicle wall are classified into three types; 1. a non-ciliated taller cell, 2. a ciliated taller one, and 3. a non-ciliated cuboidal one. From their cytoplasmic fine structure, it is considered that all these cells are essentially identical and differences among them are due to their functional state.All these type cells are characterized by irregularly developed interdigitations and aggregates of tonofilaments throughout the cytoplasm, especially in the perinuclear region. Although the rough-surfaced endoplasmic reticulum and the Golgi apparatus are fairly well developed in the first and second type cells, the cisternae are not so large-vacuolated but flattened, and the cytoplasm is more compact as compared with that of the higher vertebrate. In the third type cell, the cytomembranes are poorly developed.Large dense inclusion-bodies consisting of heterogeneously dense materials, of lamellar structures, and of less dense vacuoles, which are found often in taller follicle cells, are also characteristic for the lamprey thyroid. The body which might be intimately related to the Golgi apparatus is considered to be a kind of lysosomes and it perhaps corresponds to the yellow pigment observed by light microscopy.In the apical part of the cytoplasm in taller cells, there are three kinds of granules or vesicles; numerous small vesicles considered to be derived from the Golgi apparatus, a few small dense granules which seem to originate from the Golgi region, and a few large less-dense granules.In the third type cell, the cytomembranes are not so well developed as those of the first and second type cells. The large heterogeneously dense bodies and the cytoplasmic granules are very few in number.Around the follicle of the lamprey thyroid, there are a dense basement membrane and a relatively compact connective tissue with few blood capillaries. Characteristic fat cells are found in the connective tissue.     

Effect of Insulin and Insulin-Like Growth Factor 1 (IGF-1) on Activity of Glycogen Synthase in Skeletal Muscles of the Lamprey Lampetra fluviatilis

Activity of glycogen synthase (GS) in the muscle tissue of the lamprey Lampetra fluviatilis is studied in dynamics of the pre-spawning period as well as under effects of insulin and IGF-1. It is shown that GS exists in the muscles in two forms, the active (I-form) and inactive (D-form), the I-form prevailing during all studied time periods. With approaching the spawning, the GS activity fell 1.5–2 times due to a decrease of the I-form activity. From October to January, both insulin and IGF-1 stimulated GS at concentrations of 10^–10–10^–8 M and 10^–9–10^–8 M, respectively. The maximally effective concentrations (10^–9 M insulin, 10^–8 M IGF-1) produced a 2.5–3-fold rise of the I-form activity of GS at the period from October to December. In January the stimulating effect of these peptides decreased. In March the GS was insensitive both to insulin and to IGF-1. The obtained data indicate participation of insulin and IGF-1 in regulation of glycogen synthesis in lamprey skeletal muscles, the ability of IGF-1 to stimulate the enzyme activity being shown in the lower vertebrates for the first time. It is concluded that IGF-1 takes part in regulation of the carbohydrate metabolism already at early stages of evolution of vertebrates.     

Development of cephalic neural crest cells in embryos of Lampetra japonica, with special reference to the evolution of the jaw

Neural crest cells contribute extensively to vertebrate head morphogenesis and their origin is an important question to address in understanding the evolution of the craniate head. The distribution pattern of cephalic crest cells was examined in embryos of one of the living agnathan vertebrates, Lampetra japonica. The initial appearance of putative crest cells was observed on the dorsal aspect of the neural rod at stage 20.5 and ventral expansion of these cells was first seen at the level of rostral somites. As in gnathostomes, cephalic crest cells migrate beneath the surface ectoderm and form three major cell populations, each being separated at the levels of rhombomeres (r) 3 and r5. The neural crest seems initially to be produced at all neuraxial levels except for the rostral-most area, and cephalic crest cells are secondarily excluded from levels r3 and r5. Such a pattern of crest cell distribution prefigures the morphology of the cranial nerve anlage. The second or middle crest cell population passes medial to the otocyst, implying that the otocyst does not serve as a barrier to separate the crest cell populations. The three cephalic crest cell populations fill the pharyngeal arch ventrally, covering the pharyngeal mesoderm laterally with the rostral-most population covering the premandibular region and mandibular arch. The third cell population is equivalent to the circumpharyngeal crest cells in the chick, and its influx into the pharyngeal region precedes the formation of postotic pharyngeal arches. Focal injection of DiI revealed the existence of an anteroposterior organization in the neural crest at the neurular stage, destined for each pharyngeal region. The crest cells derived from the posterior midbrain that express the LjOtxA gene, the Otx2 cognate, were shown to migrate into the mandibular arch, a pattern which is identical to gnathostome embryos. It was concluded that the head region of the lamprey embryo shares a common set of morphological characters with gnathostome embryos and that the morphological deviation of the mandibular arch between the gnathostomes and the lamprey is not based on the early embryonic patterning.     

A Fluorescence Microscopic Study of the Retina of the River Lamprey (Lampetra fluviatilis) and the Atlantic Hagfish, (Myxine glutinosa)

During the last few years many facts have been revealed about the neural structure in the retina of several vertebrate groups, particularly with regard to the monoamine containing neurons. A number of investigations have been made on mammals, birds and teleost fishes by the Falck-Hillarp method for the cellular demonstration of such amines. The present study has paid attention to two cyclostomes, the river lamprey, Lampetra fluviatilis, and the atlantic hagfish, Myxine glutinosa.     

An electron microscopic study on the innervation of the gill filaments of a lamprey,Lampetra japonica

This paper reports observations on the innervation of gill filaments of the lamprey, Lampetra japonica. Nerve fibers run on each side of the afferent filament artery (AFA nerve) and in the connective tissue compartment along the efferent filament artery (EFA nerve). The AFA nerve supplies vasomotor fibers to the afferent filament artery and arteriovenous anastomoses and special visceral motor fibers to branchial muscle fibers (musculus compressor branchialis circularis). Nerve endings of the vasomotor fibers contain large, cored vesicles (60–180 nm in diameter) with a variable number of small, clear vesicles (30–70 μm in diameter), whereas those of the visceral motor fibers have many small, clear vesicles with few large, cored vesicles. The EFA nerve supplies vasomotor fibers to the efferent filament artery. Their endings, containing mixtures of predominantly large, cored vesicles and small, clear vesicles make close synaptic contacts with reticular cells. The latter in turn are connected with each other or with smooth muscle cells in the wall of the efferent filament artery by nexuses. No nerves are found in the axial plate between the afferent and efferent filament arteries nor in the secondary lamellae of individual gill filaments. No afferent nerve supply to the gill filament has been found.     

Homeostasis of Sodium and Potassium Ions in Erythrocytes of the Lamprey Lampetra fluviatilis: Effect of Ion Transport and Metabolic Blockers, and Ionophores

After incubation of lamprey Lampetra fluviatilis erythrocytes in the standard medium for 90–120 min, intracellular Na⁺ and K⁺ content remained unchanged (28.7 ± 1.1 and 66.3 ± 1.5 mmol/l cells, respectively, n = 33). The erythrocyte ion content also did not change after treatment of the cells with ion transport inhibitors, Ba^{2 +} and amiloride. Addition of 0.1 mM ouabain to the incubation medium led to a decrease of K⁺ content by 8.4 ± 1.2 and to an increase of Na⁺ content by 2.4 ± 0.8 mmol/l/2 h. Similar reciprocal changes in the cellular ion composition were observed after treatment of the erythrocytes by oxidative metabolism inhibitors (rotenone and CCCP—carbonyl cyanide m-chlorophenyl-hydrazone). The metabolic blockers produced more significant ion composition changes in comparison with ouabain. An increase of intracellular Na⁺ content under effect of CCCP was completely inhibited by amiloride. It can be suggested that inhibition of oxidative metabolism is accompanied by a cell acidification and Na⁺/H⁺ exchange activation. Erythrocyte acidification by a K⁺/H⁺ ionophore led to a rapid cellular Na⁺ accumulation, which indicates the presence of a Na⁺/H⁺ exchanger with high activity. The K⁺ ionophore valinomycin produced a relatively small K⁺ loss from the lamprey erythrocytes to indicate a low anion conductance of the cells. The data obtained indicate an important role of oxidative metabolism in the monovalent ion homeostasis in the lamprey red blood cells.     

X-ray microanalysis on the thyroid follicle of the hagfish, Eptatretus burgeri and lamprey, Lampetra japonica.

Intracellular dense bodies, cytoplasmic matrices, and luminal colloids in the thyroid follicle of cyclostomes, hagfish and lamprey were examined to identify the distribution of iodine using an energy dispersive X-ray microanalyzer attached to a scanning transmission electron microscope. High level of iodine was detected only in the large dense body of the hagfishes, while it was too slight in quantity to detect by this method in the cytoplasmic matrix as well as in the luminal colloid. In the adult lamprey thyroid, an appreciable amount of iodine was detected in a few large dense bodies. In mice and rats, it is very hard to detect iodine in the luminal colloid, intracellular colloid droplet, and in the lysosomal dense granule by this method, though these structures have been well known to contain a fairly large amount of iodine which is combined with thyroglobulin. These facts mean that intracellular dense bodies in the thyroid follicular epithelium of the cytoclostome, especially of the hagfish have an extremely larger amount of iodine. These bodies are considered to be secondary lysosomes or residual bodies containing reabsorbed colloid materials which are highly condensed.     

Iodine distribution in the thyroid follicles of the hagfish,Eptatretus burgeri and lamprey,Lampetra japonica: Electron-probe X-ray microanalysis

Summary In the thyroid follicles of species of cyclostomes, a hagfish and a lamprey, the distribution of stable iodine was examined by electron-probe X-ray microanalysis. A high concentration of stable iodine, heterogeneously distributed, was observed in the follicular cells of hagfish thyroid follicles. In the lamprey a low concentration of iodine was seen in the follicular lumina. The relative values for stable iodine determined in this way corresponded to values obtained by a chemical analytical method.     

Profiles of the female-specific serum protein in the Japanese river lamprey, Lampetra japonica (Martens), and the sand lamprey, Lampetra reissneri (Dybowski), in relation to sexual maturation

The occurrence of female-specific serum protein (FSSP) was examined in the anadromous lamprey, Lampetra japonica, and a freshwater lamprey, Lampetra reissneri, by an immunodiffusion technique using antiserum raised against the serum of sexually mature female lamprey. A considerable amount of FSSP was consistently detected in the blood of vitellogenic, but not in that of nonvitellogenic, females of both species of lampreys. The relative amount of the FSSP tended to become larger in female lampreys at the spawning period. The FSSP may correspond to the yolk precursor protein which is incorporated into ovarian oocytes during the period of exogenous vitellogenesis in the lampreys.     

Drawing a Stripe in Drosophila Imaginal Disks: Negative Regulation of Decapentaplegic and Patched Expression by Engrailed

During development of the Drosophila adult appendage precursors, the larval imaginal disks, the decapentaplegic (dpp) gene is expressed in a stripe just anterior to the anterior/posterior (A/P) compartment boundary. Here, we investigate the genetic controls that lead to production of this stripe. We extend previous observations on leaky engrailed (en) mutations by showing that mutant clones completely lacking both en and invected (inv) activity ectopically express dpp-lacZ reporter genes in the posterior compartment, where dpp activity ordinarily is repressed. Similarly, patched (ptc) is also ectopically expressed in such posterior compartment en(-)inv(-) null clones. In contrast, these en(-)inv(-) clones exhibit loss of hedgehog (hh) expression. We suggest that the absence of dpp expression in the posterior compartment is due to direct repression by en. Ubiquitious expression of en in imaginal disks, produced by a hs-en construct, eliminates the expression of dpp-lacZ in its normal A/P boundary stripe. We identify three in vitro Engrailed binding sites in one of our dpp-lacZ reporter gene. Mutagenesis of these Engrailed binding sites results in ectopic expression of this reporter gene, but does not alter the normal stripe of expression at the A/P boundary. We propose that the en-hh-ptc regulatory loop that is responsible for segmental expression of wingless in the embryo is reutilized in imaginal disks to create a stripe of dpp expression along the A/P compartment boundary.     

Effects of cGMP and cAMP on light responses of the photosensory pineal neurons in the lamprey, Lampetra japonica.

We examined in this study how external cyclic nucleotides affect the light response mechanism of the pineal photoreceptors and explored the existence of parietal eye type of photoreceptor of which the internal cGMP concentration increased during the light response. Pineal organs of river lampreys, Lampetra japonica, were treated with 8-bromo guanosine 3',5'-cyclic monophosphate (8Br-cGMP) or 8-bromo adenosine 3',5'-cyclic monophosphate (8Br-cAMP) before light stimuli, and the light responses were recorded from the second order neurons, chromatic or achromatic-type neurons. Excitatory and inhibitory light responses of the chromatic-type neuron became obscure by 9 and 3 mM 8Br-cGMP without changing the spontaneous spike discharge in the dark. 8Br-cAMP (3 mM) increased the frequency of spontaneous spike discharge, though it did not inhibit the light responses themselves. The inhibitory light response of the achromatic-type neuron decreased after adding 3 mM 8Br-cGMP, and it was unchanged by 3 mM 8Br-cAMP. The spontaneous spike discharge of the neurons in the dark was not affected by the cyclic nucleotides. The mechanism of these results can be explained if cGMP is an intracellular second messenger of light responses in the pineal photoreceptors and the blocking effect on photoresponses by externally applied 8Br-cGMP is caused by compensating for the reduction in intracellular cGMP by light. However, it does not indicate that the parietal eye type of photoreceptor found in lizard participates in the chromatic and achromatic-type responses in the lamprey pineal organ.     

Expression, purification, and human antibody response to a 67 kDa vaccine candidate for schistosomiasis japonica

Schistosomiasis remains a leading cause of morbidity and mortality in the developing tropical world, and vaccines to prevent these infections remain a scientific and public health priority. Sj67 is a 67 kDa Schistosoma japonicum surface membrane protein homologous to a family of actin-binding proteins. Sj67 is recognized by a mouse monoclonal antibody (mAb 6) that confers resistance to challenge infection in passive transfer experiments. These data support Sj67 as a potential vaccine candidate for schistosomiasis japonica. In the present study, we report the ligation-independent cloning of a cDNA encoding thioredoxin/elastin-like polypeptide (ELP)/rSj67 into a pET-32 Xa/LIC vector. Soluble recombinant fusion protein (Thio-ELP-rSj67) was expressed and purified using anion-exchange and size exclusion chromatography. rSj67 was cleaved from the Thio-ELP fusion partner by digestion with Factor Xa protease and purified using hydroxyapatite column chromatography. Endotoxin was reduced by absorption to a polymyxin support. Purified rSj67 had a molecular weight of 67 kDa and N-terminal sequencing confirmed that the first five amino acids of the recombinant protein matched the predicted sequence for the Sj67 gene. In Western blot analysis, rSj67 was recognized by the Sj67 specific mAb 6 antibody. IgG antibodies in sera from schistosomiasis infected volunteers living in an endemic area of the Philippines (n = 13) recognized rSj67 with 4.7-fold greater median fluorescence compared to uninfected North American controls (n = 5) (p < 0.009). Together, these data confirm the expression and purification of recombinant Sj67 and its immuno-reactivity with sera from S. japonicum infected humans.     

Immunocytochemical localization of serotonin and photoreceptor-specific proteins (rod-opsin,S-antigen) in the pineal complex of the river lamprey,Lampetra japonica,with special reference to photoneuroendocrine cells

Summary The pineal complex of the river lamprey, Lampetra japonica, was examined by means of immunocytochemistry with antisera against serotonin, the precursor of melatonin, and two photoreceptor proteins, rod-opsin (the apoprotein of the photopigment rhodopsin) and S-antigen. Serotonin-immunoreactive cells were observed in both the pineal and the parapineal organ. The proximal portion of the pineal organ (atrium) comprised numerous serotonin-immunoreactive cells displaying spherical somata. In the distal end-vesicle of the pineal organ, the serotonin-immunoreactive elements resembled photoreceptors in their size and shape. These cells projecting into the pineal lumen and toward the basal lamina were especially conspicuous in the ventral portion of the end-vesicle. In addition, single serotonin-immunoreactive nerve cells were found in this location. Retinal photoreceptors were never seen to contain immunoreactive serotonin; amacrine cells were the only retinal elements exhibiting serotonin immunoreaction. Strong S-antigen immunoreactivity was found in numerous photoreceptors located in the pineal end-vesicle. In contrast, the S-antigen immunoreactivity was weak in the spherical cells of the atrium. Thus, the pattern of S-antigen immunoreactivity was roughly opposite to that of serotonin. Similar findings were obtained in the parapineal organ. The rod-opsin immunoreaction was restricted to the outer segments of photoreceptors in the pineal end-vesicle and parapineal organ. No rodopsin immunoreactive outer segments occurred in the proximal portion of the atrium. Double immunostaining was employed to investigate whether immunoreactive opsin and serotonin are colocalized in one and the same cell. This approach revealed that (i) most of the rodopsin-immunoreactive outer segments in the end-vesicle belonged to serotonin-immunonegative photoreceptors; (ii) nearly all serotonin-immunoreactive cells in the end-vesicle bore short rod-opsin-immunoreactive outer segments protruding into the pineal lumen; and (iii) the spherical serotonin-immunoreactive cells in the pineal stalk lacked rod-opsin immunoreaction and an outer segment. These results support the concept that multiple cell lines of the photoreceptor type exist in the pineal complex at an early evolutionary stage.     

The Development of Teeth and Associated Feeding Structures During the Metamorphosis of the Lamprey,Geotria australis

The pattern of development of the teeth, laminae, piston, muscles, cartilages and fimbriae associated with the suctorial disc of lampreys has been investigated histologically during the seven stages of metamorphosis in the Southern Hemisphere species, Geotria australis. The cirrhi-bearing hood of ammocoetes and the earliest stage of metamorphosis (S1) were indistinguishable. In stages S2, S3, S4, these cirrhi regressed and the supraoral lamina, piston and infraoral lamina primordial regions began to differentiate. The fifth stage (S5) was characterised by an elaboration of the annular cartilage and disc musculature, deposition of the tectal cartilage, initiation of tooth development, formation of oral fimbriae, and eruption of the keratin cone of the transverse lingual lamina. Subsequently (S6), the keratin cusps of the supraoral and infraoral laminae became exposed at the surface, and distinct retractor and protractor muscles formed around the lingual cartilage. In the latter part of the terminal stage in metamorphosis (S7), just prior to the time when the animal migrates downstream, the primary tooth cones and the keratin cusp of the longitudinal lingual laminae began breaking through the epithelial surface of the disc.     

Light- and electron-microscopic observations of the tail bud of the larval lamprey (Lampetra japonica), with special reference to neural tube formation

Serial transverse and horizontal sections of the tail of the 26-day larval lamprey, Lampetra japonica, were observed by light and electron microscopy. The axial structures in the tail of the larval lamprey seem to differentiate from the prospective materials derived individually from the tail bud. The latter consists of two closely adjoined cell populations (C1 and C2). C1 is a small cell cluster located posterior to the other group (C2) and consists of loosely arranged polymorphous cells. The cell cluster extends cranially as a cell sheet on the ventral surface of C2; somites differentiate from this cell sheet. C2 is composed of cells elongated mediolaterally and stacked horizontally to form a compact cell mass which is covered on each lateral surface by a basal lamina. The upper one-third of C2 seems to differentiate into the neural tube, anteceding other axial structures. The middle one-third of C2 seems to develop into the notochord, and the lower one-third into the subchord and the undefined cell cord. The central canal develops in the upper one-third of C2 through the following events: 1) appearance of cilia and a small cavity between adjoining cells; 2) appearance of microvilli in the cavity, in addition to cilia; and 3) development of junctional complexes along the luminal borders of cells surrounding the cavity. Together with these events, cells surrounding the cavity increase in number, acquiring apicobasal polarity and radial arrangement. The cavity itself enlarges by incorporation of periciliary clefts and fusion of cavities with each other to establish the central canal. Near the caudal end of the neural tube, the central canal is directly confluent with the connective-tissue space through an opening in the dorsal wall of the neural tube.     

Anatomy and ultrastructure of epidermal cells in the haustorium of a parasitic flowering plant,Cuscuta japonica, during attachment to the host

Changes of epidermal cells in the haustorium of the parasiticCuscuta japonica during its attachment to the host plantimpatiens balsamina were studied with light and electron microscopy. In the transverse sections of dodder stems not in contact with the host, epidermal cells had rounded outlines. However, when haustorial initials developed in the cortex of the parasite stem at the contact site, the epidermal cells had more dense cytoplasm and conspicuous nuclei than before, and their outline was flat in the longitudinal section. As meristem cells developed from those initials, the epidermal cells became more elongated. When the haustorium was fully matured, the apical tips of the elongated epidermal cells at the contact site branched like toes, producing numerous projections via cell wall invaginations. This event caused spaces to form between the projections; coincidently, the surface area of the apical ends of the epidermal cells increased. The dense cytoplasm at those projections contained prominent nuclei and abundant other organelles, suggesting a active metabolism. Osmiophilic particles, releasing into the cell walls from the cytoplasm, were though to be associated with the loosening and elongating of the epidermal cell walls. Dense and homogeneous materials were secreted within the spaces between the projections. These materials could play an important role in cementing the haustorium onto the surface of the host organ.