Kinya Yasui

An anterograde and retrograde tract-tracing study on the projections from the thalamic gustatory area in the rat: distribution of neurons projecting to the insular cortex and amygdaloid complex.

Projections from the thalamic gustatory nucleus, i.e. the parvicellular part of the posteromedial ventral thalamic nucleus (VPMpc) to the forebrain regions were studied in the rat by the tract-tracing methods with anterograde tracer (biotinylated dextran amine, BDA) and anterograde/retrograde tracer (wheat-germ agglutinin-horseradish peroxidase, WGA-HRP). After BDA injection into the VPMpc, terminal labeling was observed in the insular cortex, amygdaloid complex, and fundus striati. The terminal labeling in the amygdaloid complex was distributed in dorsolateral area of the rostral part of the lateral amygdaloid nucleus and the rostral part of the lateral subdivision of the central amygdaloid nucleus. The terminal labeling in the central amygdaloid nucleus extended to the fundus striati. The retrograde tracing study with WGA-HRP revealed that the projection fibers from the VPMpc to the amygdaloid complex originated from the medial part of the VPMpc and also from the thalamic area medial to the VPMpc. In the rats injected with Fluoro-Gold and WGA-HRP, respectively into the insular cortex and amygdaloid complex, no double-labeled neuronal cell bodies were found in the VPMpc, although neurons labeled singly with Fluoro-Gold were intermingled with those singly labeled with WGA-HRP in the medial part of the VPMpc. The results indicated that VPMpc neurons projecting to the amygdaloid complex constituted a population different from VPMpc neurons projecting to the insular cortex.

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and se-quenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

Early development of the peripheral nervous system in a lancelet species

The developmental pattern of the lancelet (amphioxus) peripheral nervous system from embryos to larvae has been studied by using wholemount immunostaining and transmission electron microscopy. The peripheral nerves first appeared on the anterior dorsal surface of the medulla at the middle neurula stage, when the anterior nerve cord was just closing. A single axon with a large growth cone was the progenitor of each nerve. The nerve roots adopted an asymmetric arrangement soon after. The first nerve, likely a pair of pure sensory nerves, sprouted from the anterior tip of the nerve cord. This nerve may be comparable topographically to the preoptic nerve (the posterior branch of the terminal nerve) in lungfishes. However, the neuron that first extends its axon was located in the medulla, as in the other posterior nerves. One of the extramedullary primary sensory neurons, the corpuscles of de Quatrefages, appeared in larvae with the mouth and two anterior gill pores. Their axons were seemingly fasciculated with the efferent axon of the first nerve. The second nerve, the most complex one to appear during embryonic and early larval development, innervated the preoral pit and the buccal region. The third and fourth nerves on the left side also innervated the buccal region. The larval innervation patterns in the anterior region differed from the adult organization, suggesting a segmental rearrangement of the nerve supply during development. There was no evidence to dichotomize the peripheral nerves into cranial and spinal nerves, as exist in vertebrates. These characteristics of the peripheral nervous system in the lancelet indicate that this animal has a rather derived or primitive developmental system of peripheral nerves, making the analysis of homology with vertebrates difficult. J. Comp. Neurol. 393:415–425, 1998.

Establishment of left-right asymmetric innervation in the lancelet oral region

Lancelets (amphioxus) exhibit a remarkable asymmetric development in the anterior body region, which is reflected in the peripheral nervous system even at adulthood. Not all of the anterior nerves are involved, but the left third to fifth nerves are clearly asymmetric. To trace the developmental process responsible for asymmetric innervation, the peripheral nerves in the anterior region were studied in pre‐ and mid‐metamorphic larvae, 1‐cm‐long juveniles, and in adults by using whole‐mount immunostaining. The mouth changes in size and location during larval life before moving ventrally and, in conjunction with this change, nerves in the oral region are also modified. The left second nerve initially innervates the oral region, but this connection is secondarily lost. As the mouth expands and shifts posteriorly, the left fifth to ninth nerves join the left third and fourth in the innervation of the oral region. The left third to sixth nerves anastomose with the oral nerve ring, which encircles the mouth on the left side. In the juveniles and adults, there are two nerve plexuses that run parallel to the margin of the oral hood. The innermost of these, the “inner oral‐hood nerve plexus”, is asymmetrically connected with the left third to fifth nerves on both sides. The other, the “outer oral‐hood nerve plexus”, is ipsilaterally connected with the third to seventh nerves on both sides. The velar nerve ring is also innervated asymmetrically by the left fourth and fifth nerves. From these observations, we suggest that the oral nerve ring is the precursor of both the inner oral‐hood nerve plexus and the velar nerve ring, and that the asymmetric innervation retained in adult lancelets is related to the early anastomosis of the left nerves with the oral nerve ring. We also show that, contrary to the persistent asymmetric innervation, the axonal patterns of the anterior peripheral nervous system in developing lancelets can change. J. Comp. Neurol. 435:394–405, 2001.

Collagen Fibrils in the Odontoblast Layer in the Teeth of the Rat and the House Shrew, Suncus murinus by Scanning Electron Microscopy Using a Maceration Method

It is not well known whether there are gaps in the tight junctions between odontoblasts and whether the fluid flows from the pulp to the predentin through these gaps. The collagen fibrils in the odontoblast layer were investigated using a maceration method in order to show the existence of the gaps between tight junctions of the odontoblasts. The mandibles containing teeth of the rat and the house shrew were digested by NaOH maceration and revealed the architecture of the collagen fibrils under scanning electron microscopy. The collagen fibrils went from the pulp, through the odontoblast layer, and were woven into the collagen network of the predentin in all teeth used in this study. Thick bundles of collagen were seen in the odontoblast layer at the pulp horn of the rat molars. Because there are many collagen fibrils in the odontoblast layer, it is considered that the tight junction of the odontoblast is of the discontinuous type.

The Lancelet and Ammocoete Mouths

Abstract The evolutionary history of the vertebrate mouth has long been an intriguing issue in comparative zoology. When the prevertebrate state was considered, the oral structure in adult lancelets (amphioxus) was traditionally referred to because of its general similarity to that of the ammocoete larva of lampreys. The larval mouth in lancelets, however, shows a peculiar developmental mode. Reflecting this, the affinity of the lancelet mouth has long been argued, but is still far from a consensus. The increase in available data from molecular biology, comparative developmental biology, paleontology, and other related fields makes it prudent to discuss morphological homology and homoplasy. Here, we review how the lancelet mouth has been interpreted in the study of evolution of the vertebrate mouth, as well as recent advances in chordate studies. With this background of increased knowledge, our innervation analysis supports the interpretation that the morphological similarity in the oral apparatus between ammocoetes and lancelets is a homoplasy caused by their similar food habits.

DISTRIBUTION PATTERN OF HNF-3BETA PROTEINS IN DEVELOPING EMBRYOS OF TWO MAMMALIAN SPECIES, THE HOUSE SHREW AND THE MOUSE

The pattern of expression of HNF‐3β in organizing centers and axial structures during early vertebrate development suggests an important role for this protein in the establishment of the vertebrate body plan. To establish whether the pattern of expression during embryogenesis is species specific, a comparative immunohistochemical study of two mammalian species, the house shrew, insectivore, and the mouse was carried out; it is difficult to obtain accurate morphological differences from the study of remotely related animals. The earliest expression of HNF‐3β appeared in the node and hypoblast (or endoderm) in both species, where the presumptive foregut endoderm showed intense immunoreactivity prior to the formation of the axial mesoderm, suggesting a role different from that in axial formation. The anterior extension of immunopositive axial mesoderm and the median band of the neural plate varied between the two species, and was delayed in the house shrew. HNF‐3β in the anterior end of the foregut disappeared transiently in the house shrew but persisted in the mouse embryo. An asymmetric pattern of distribution in the primitive streak was also observed in the mouse but not in the house shrew. The present immunohistochemical study elucidated that the distribution of HNF‐3β is conserved initially but soon manifests species specificities in development even between mammalian species.

Embryonic development of the house shrew (Suncus murinus). I. Embryos at stages 9 and 10 with 1 to 12 pairs of somites.

SummaryThe embryonic development during the period from 1 to 12 pairs of somites was observed in an insectivore species, the house shrew (Suncus murinus), which has been bred within a closed colony. Embryos were staged by the number of somite pairs. Each stage was punctuated at every addition of three pairs of somites and numbered after the Carnegie system. The first somite became apparent between 8 and 9.0 days after fertilization, and the 12th somite appeared between 9.5 and 10.0 days. The rate of somite formation was one pair in every 3–4 h on average. The embryonic events during this period were as follows: 1. From the beginning of stage 9, the embryonic body consistently displayed a kyphosis, and as development progressed, the caudal portion of the embryo spiralled clockwise. 2. The first and second pharyngeal arches formed; their development was precocious among mammalian embryos in relation to somitic count. 3. The segmental pattern of the neural fold was similar to that of laboratory rodents and primates. The first fusion of the cranial neural folds took place in the occipital somite region, the second fusion in the diencephalic region, and the third at the end of the neural plate, thus leaving two neuropores in the cephalic region. 4. The timing of appearance of the optic sulcus was similar to that of human embryos but was delayed in comparison with that of laboratory rodents. 5. The heart always showed a more advanced state than that of other mammalian embryos. From the beginning of stage 9, an unpaired endocardial tube was seen in the bulbo-ventricular region, and deflection from a symmetrical appearance soon took place. 6. The differentiation of foregut was also precocious, and the thyroid and respiratory primordia appeared earlier than in other mammals. The present study emphasizes that there are considerable variations in timing and manner of morphogenesis among early mammalian embryos.

Apical cell escape from the neuroepithelium and cell transformation during terminal lip fusion in the house shrew embryo

The house shrew embryo has many cells in the ventricular lumen and on the luminal surface of the fusing terminal lip of the cephalic neural tube. The origin and fate of these cells were studied by means of light and electron microscopy, and by DiI labeling in a whole-embryo culture system. The cells appeared at stage 11A and persisted until stage 12A. Most of the cells seemed to originate from the neuroepithelium, as shown by frequent observations of epithelial cell escape and DiI labeling analysis. The cells on the luminal surface sometimes showed apoptotic features, but were not subjected to phagocytosis. Some of the escaping cells seemed to migrate to the ventral part of the prosencephalic neuropore and insert themselves into it. Others separated from the luminal surface and floated into the lumen. It seems likely that the floating cells either become autolyzed, or else change into macrophage-like cells, the latter alternative being supported by the results of DiI labeling. The macrophage-like cells actively phagocytosed the other degenerating cells and apoptotic bodies. These observations suggest that the apical escape of cells may play an important role in the remodeling of the neural fold during the terminal lip fusion, and that early neuroepitheial cells may have the potential to become cells with vigorous phagocytic activity, like macrophages.

Dynamic Modification of Oral Innervation During Metamorphosis in Branchiostoma belcheri, the Oriental Lancelet

The oral apparatus in lancelets undergoes a remarkable modification during larval development, especially during metamorphosis, when the oral innervation is radically altered. The larval mouth opens on the left side at the early larval stage, and a peripheral nerve network, the oral nerve ring (ONR), develops around it. The ONR enlarges as the mouth expands caudally, eventually receiving fibers from nerves as far back as the tenth on the left side. The mouth shrinks during metamorphosis, and with this change the ONR regresses; the posterior sixth to tenth nerves become freed from the connection with the ONR, whereas the fourth and fifth nerves retain their connections. This modification is the basis for the asymmetric innervation to the velum. There is no mesodermal or mesenchymal restriction for guiding nerve patterning as typically found in vertebrate cranial nerves. Rather, it seems to be the ONR, which has no counterpart in vertebrates, that plays pivotal roles for patterning the nervous system in the oral region. The oral innervation pattern in lancelets represents a derived character state that may be related to the asymmetry of the ancestral body and head.