Developmental patterns and cell lineages of vermiform embryos in dicyemid mesozoans.

Patterns of cell division and cell lineages of the vermiform embryos of dicyemid mesozoans were studied in four species belonging to four genera: Conocyema polymorpha, Dicyema apalachiensis, Microcyema vespa, and Pseudicyema nakaoi. During early development, the following common features were apparent: (1) the first cell division produces prospective cells that generate the anterior peripheral region of the embryo; (2) the second cell division produces prospective cells that generate the posterior peripheral region plus the internal cells of the embryo; (3) in the lineage of prospective internal cells, several divisions ultimately result in cell death of one of the daughter cells. Early developmental processes are almost identical in the vermiform embryos of all four dicyemid genera. The cell lineages appear to be invariant among embryos and are highly conserved among species. Species-specific differences appear during later stages of embryogenesis. The number of terminal divisions determines variations in peripheral cell numbers among genera and species. Thus, the numbers of peripheral cells are fixed and hence species-specific.

Expression patterns of HNK-1 carbohydrate and serotonin in sea urchin, amphioxus, and lamprey, with reference to the possible evolutionary origin of the neural crest.

We examined deuterostome invertebrates, the sea urchin and amphioxus, and an extant primitive vertebrate, the lamprey, for the presence of structures expressing the HNK-1 carbohydrate and serotonin. In sea urchin embryos and larvae, HNK-1 positive cells were localized in the ciliary bands and in their precursor ectoderm. Serotonergic cells were exclusively observed in the apical organs. In juvenile amphioxus, primary sensory neurons in the dorsal nerve cords were HNK-1 immunoreactive. The juvenile amphioxus nerve cords contained anti-serotonin immunoreactive nerve fibers that seem to be the Rohde axons extending from amphioxus interneurons, the Rohde cells. In lamprey embryos, migrating neural crest cells and primary sensory neurons, including Rohon-Beard cells, expressed the HNK-1 carbohydrate. Lamprey larvae (ammocoetes) contained cell aggregates expressing both the HNK-1 carbohydrate and serotonin in the pronephros and in the regions adjacent to the gut epithelium. Some of these cell aggregates were present in the anti-serotonin positive visceral motor nerve net. Motor neurons and Müller fibers were serotonergic in ammocoetes. Comparison of the expression patterns of HNK-1 carbohydrate among sea urchins, amphioxus and lampreys seem to suggest the possible evolutionary origin of the neural crest, that is, ciliary bands in dipleurula-type ancestors evolved into primary sensory neurons in chordate ancestors, as inferred from Garstang's auricularia hypothesis, and the neural crest originated from the primary sensory neurons.

Development of the Infusoriform Embryo of Dicyema japonicum (Mesozoa: Dicyemidae).

The cleavage pattern and cell lineage of the infusoriform embryo of the dicyemid mesozoan Dicyema japonicum were studied in fixed material with the aid of a light microscope. The early cleavages are holoblastic and spiral. At the 16-cell stage, the animal pole consists of four mesomeres, the equatorial region consists of four macromeres with four alternating sub-macromeres, and the vegetal pole is composed of four micromeres. At around the 20- to 24-cell stage, cleavage becomes asynchronous and its pattern changes from spiral to bilateral. The four micromeres, namely, the presumptive germinal cells, do not divide further and are finally incorporated into the cytoplasm of four urn cells, which are generated after divisions of the sub-macromeres. The blastomeres situated in the animal hemisphere give rise to ciliated cells that cover the posterior part of the embryo. Two blastomeres (2a2 and 2d2) undergo extremely unequal divisions and the much smaller sister blastomeres degenerate and ultimately disappear during embryogenesis. The fully formed embryo consists of 37 cells. These cells are produced after only four to eight rounds of cell division. The cell lineage appears to be invariant among embryos, apart from the derivation of the lateral cells.

A survey of occurrence of about seventeen circumventricular organs in brains of various vertebrates with special reference to lower groups.

The occurrence of about 17 circumventricular organs is described in 31 species belonging to various groups of vertebrates from cyclostomes to mammals and the phylogeny of each organ is discussed briefly. The neurohypophysis (median eminence and neural lobe) and the subcommissural organ are observed in all species studied. The pineal is also ubiquitous except for the hagfish (Eptatretus) and the caiman (Caiman). These four circumventricular organs are phylogenetically the oldest organs. The subfornical organ and the area postrema may be phylogenetically the youngest organs, although the subfornical organ is found in the lungfish (Lepidosiren) and the area postrema is observed in the dogfish (Mustelus) and the brachiopterygians (Polypterus and Erpetoichthys) among the piscine vertebrates studied. The other significant findings may be summarized as follows. Osteoglossomorphous teleosts (Osteoglossum and Gnathonemus) possess a distinct median eminence. The median eminence of the caiman (Caiman) is divided into the anterior and posterior parts as in birds. The saccus vasculosus is observed in the freshwater ray (Potamotrygon), but not in the freshwater teleost (Osteoglossum). The organum vasculosum laminae terminalis of the dogfish (Etmopterus) contains fuchsinophilic neurosecretory fiber terminals and is considered as the neurohemal organ. The paraventricular organ is not differentiated in the lungfish (Lepidosiren) and the caecilian (Typhlonectes). The paraphysis is found in the dogfish (Mustelus) and the osteoglossomorphous teleosts (Osteoglossum and Gnathonemus). The so-called paraphysis of the lungfish is not a paraphysis, but a dorsal sac. The development of the subfornical organ is variable among amphibians. The area postrema is well developed in the caecilian (Typhlonectes).

Electron microscopic study of the innervation of the renal tubules and urinary bladder epithelium in Rana catesbeiana and Necturus maculosus.

The fine structure of the kidney and the bladder of the bullfrog (Rana catesbeiana), the bullfrog tadpole, and the mudpuppy (Necturus maculosus) were studied with special attention to the innervation of renal tubule cells and bladder epithelial cells. In the bullfrog kidney, nerve terminals and varicosities were frequently associated with the tubule cells, apparently in an increasing order from the proximal tubule to the connecting tubule. Although these terminals and varicosities did not directly contact the tubular cell membrane, an aggregation of synaptic vesicles on the side facing the tubule was considered as morphological evidence that neurotransmitter can be released here and can affect the transport activity of the tubule cells. The association of nerve varicosities with canaliculi cells in the connecting tubule was also demonstrated. In the bullfrog tadpoles, renal tubule cells were occasionally innervated. In the mudpuppy, renal tubule cells were only poorly innervated. The epithelium of the bullfrog bladder was commonly innervated. Nerve terminals with synaptic vesicles were located very near basal cells and even contacted them directly on rare occasions. In the mudpuppy, the innervation of the bladder epithelium was observed infrequently. The bullfrog tadpoles did not possess an apparent bladder. In all materials studied, renal arterioles and bladder smooth muscle cells were innervated.

Electron-microscopic study of innervation of smooth muscle cells surrounding collecting tubules of the fish kidney.

The fine structure of the collecting tubules of the trout and killifish kidney was studied. These tubules are surrounded by layers of smooth muscle cells which are commonly innervated. The nerve terminals contain synaptic vesicles and, occasionally, a few dense-cored granules as well. Capillaries occur in the connective tissue space between these smooth muscle cells and the collecting tubule. Epithelial cells of the collecting tubules contain abundant mitochondria and a well developed membrane system displaying parallel arrays, and were considered to be actively involved in the transport of materials. In the trout, the collecting tubules contain peculiar cells in addition to regular tubule cells. The fine structure of these peculiar cells is highly reminiscent of that of gill chloride cells. The significance of these findings may be summarized as follows: If the smooth muscles around the collecting tubule contract under neural influence, intratubular pressure may be increased and, thus affect glomerular filtration rate. The contraction of these muscles may also cause the collapse of peritubular capillaries, affecting the transport activity of tubule cells.

Histochemical distribution of peroxidase in ascidians with special reference to the endostyle and the branchial sac.

The histochemical distribution of peroxidase was studied in 10 species of ascidians. In the endostyle, strong peroxidase activity was found in zone 7 in Ciona intestinalis, Ascidia zara, Ascidia sydneiensis samea, Cnemidocarpa areolata, Styela clava, and Pyrura vittata. The activity in zone 7 was weak in Styela plicata and Halocynthia hilgendorfi. Pyura michaelseni and Halocynthia roretzi showed only faint activity in zone 7, but showed strong activity in zone 9 and in the transitional zone, respectively. Pyura vittata exhibited peroxidase activity in zone 5 as well as in zone 7. Zone 8 was negative for peroxidase, but the cilia of zone 8 cells were distinctly stained for peroxidase in Ciona intestinalis and Cnemidocarpa areolata. These results show that wide species differences exist in the distribution of peroxidase in the ascidian endostyle. Peroxidase activity was also detected in the branchial sac, although here again wide species differences were noted in terms of peroxidase-positive sites. Peroxidase activity was also found in the postpharyngeal alimentary canal, but not in the tunic.

Histochemical distribution of peroxidase in amphioxus and cyclostomes with special reference to the endostyle.

The histochemical distribution of peroxidase was studied in amphioxus, ammocoetes larvae, adult lampreys, and hagfish. The endostyle of amphioxus displayed peroxidase activity in zone 5 and, in some individuals, in zone 1 as well. The endostyle of ammocoetes exhibited strong peroxidase activity in type 2c and type 3 cells. These peroxidase-positive regions coincide well with radioiodine-binding regions previously described. Thyroid follicles of adult lampreys stained strongly for peroxidase, but those of the hagfish did not. The branchial sac of amphioxus showed peroxidase activity, but the gill sac of cyclostomes did not. The intestine did not show peroxidase activity in amphioxus or in cyclostomes.

Electron microscope study of vertebrate liver innervation.

Liver fine structure was studied in various groups of vertebrates to reveal intrahepatic nerves. Nerve fibers were found in the connective tissue of the liver in all mammals, birds, and reptiles studied (Japanese monkey, crab-eating monkey, rabbit, guinea pig, rat, golden hamster, pigeon, Japanese quail, and turtle, Pseudemys scripta). Nerve fibers also made direct contact with hepatocytes in these animals except for the rat and the golden hamster. Intrahepatic nerves were rare or absent in amphibians (Rana catesbeiana and Cynops pyrrhogaster pyrrhogaster) and fishes (Anguilla japonica and Misgurnus anguillicaudatus). The livers of mammals and birds consisted of hepatic lobules and interlobular connective tissue carrying a portal triad. The liver of lower vertebrates was a simple mass of hepatic cell cords and contained relatively small amounts of connective tissue. The increased number of intrahepatic nerves appears to be correlated with the development of higher organization of liver structure and a concomitant increase in the amount of connective tissue.

Surface fine structure of the subfornical organ in the Japanese quail, Coturnix coturnix japonica.

The surface ultrastructure of the subfornical organ (SFO) was investigated in the Japanese quail. The SFO consists of a body and a stalk. The body of the SFO can be divided into rostral and caudal parts. On the rostral part, each ependymal cell possesses a short central solitary cilium; clustered cilia are also occasionally seen. Microvilli are abundant. On the caudal part, cells with a solitary cilium are fewer in number, and clustered cilia are rarely found. Microvilli are not as abundant as on the rostral part. In addition, large bulbous protrusions, tufts of small protrusions, deep funnel-shaped hollows, small pinocytotic invaginations and possible cerebrospinal fluid-contacting axons are sporadically observed on the surface of various regions of the body. Each ependymal cell of the stalk has a wide apical surface. A central solitary cilium, microvilli and other structures are observed more rarely on the stalk than on the body, while clustered cilia are not seen on the stalk. These structures are compared with those of the mammalian SFO and further discussed in relation to the possible dipsogenic receptor function for angiotensin II.

Parenchymal fine structure of the subfornical organ in the Japanese quail, Coturnix coturnix japonica.

The parenchyma of the subfornical organ (SFO) of the Japanese quail was studied by light and electron microscopy. The SFO consists of ependymal, intermediate, and basal (perimeningeal) layers. In the intermediate layer, neurons, glial cells, and their processes are found. Axons containing dense core granules approximately 80 nm in diameter are numerous, some of which make synaptic contact with the neuronal perikarya or dendrites. Synaptic vesicles in some axons contain a dense dot in the interior after treatment with 5-hydroxydopamine. The activity of the SFO, which is probably concerned with elicitation of drinking by angiotensin II, may be regulated at least partly by afferent monoaminergic axons. Capillaries with a non-fenestrated endothelium are occasionally found in the parenchyma. The basal layer is occupied by glial processes abutting on the digitating layer of perivascular connective tissue of meningeal vessels. The endothelium of these vessels is occasionally fenestrated. Trypan blue injected systemically accumulated in the SFO, but not in the deeper areas of the brain. The absence of a blood-brain barrier is suggested in the SFO.

Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish, Eptatretus burgeri.

Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. Thes types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.

Neurosecretory axo-axonic synapses in the median eminence of the turtle, Geoclemys reevesii.

Neurosecretory axo-axonic synapses were found in the median eminence of the turtle. Most of the presynaptic axons contain granules approximately 95 nm in diameter, and the postsynaptic elements have granules approximately 110 nm in diameter. The functional significance of axo-axonic synapses in the median eminence is discussed in relation to the discharge of releasing hormones from the axon terminals.

Ultrastructure of the neurohemal hypothalamic floor of the frog, Rana catesbeiana.

The fine structure of the hypothalamic floor was studied in the frog, Rana catesbeiana. The regions slightly anterior and posterior to thw swollen hypothalamic floor part, which has been classically designated as the median eminence, contained neurosecretory axon terminals abutting on the capillary walls. The region relatively far anterior to the swollen part did not show neurosecretory axons terminating on capillary walls. The neural stalk contiguous to the rostral border of the pars intermedia had few neurosecretory axon terminals which end on the terminal portions of the ependymal processes covering the capillary wall. The functional significance of the regional differentiation of the frog neurohypophysis is discussed in connection with the regional differentiation of various secretory cells in the adenohypophysis.

Histochemical distribution of monoamines in the hypothalamo-hypophysial region of the lamprey, Lampetra japonica.

The distribution of monoamine fluorescence was studied in the hypothalamohypophysial region of the lamprey. Groups of intensely fluorescent cells were observed in the lateral walls of the caudal part of the third ventricle. The anterior part of the neurohypophysis which is situated over the pars distalis showed weak fluorescence. The posterior part of the neurohypophysis which is contiguous to the pars intermedia contained highly fluorescent material in its rostral part. The distribution of monoamines in the lamprey neurohypophysis is compared with that in the higher vertebrates and their functional significance is discussed.

Ultrastructure of pars nervosa and pars intermedia of the Lamprey, Lampetra tridentata.

The pars intermedia of the adult lamprey is separated by perivascular spaces and a capillary plexus from the pars nervosa. No penetration of nerve fibers into the pars intermedia was found. The pars nervosa, which constitutes the posterior wall of the infundibulum, consists of an ependymal layer and a fuchsinophilic fiber layer; the latter contains at least four different types of axonal endings. The pars intermedia is avascular and is composed of a small proportion of non-secretory cells and a large proportion of secretory cells. The secretory granules in the cells of the pars intermedia seem to be discharged toward the capillaries that separate the pars intermedia from the pars nervosa. Although no direct nervous or vascular connections were found between the pars nervosa and pars intermedia, a mechanism of control of secretory activity in the pars intermedia cells by the central nervous system appears likely.