Harukazu Nakamura

Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order

The retinotectal projection is organized in a precise retinotopic manner. We find, though, that during development the growth and arborization of temporal retinal axons within the optic tectum of chick embryos is initially imprecise. Axonal targeting errors occur along the rostral-caudal and medial-lateral tectal axes, and arbors are formed at topographically inappropriate positions. Subsequent course corrections along both tectal axes and large-scale axonal remodeling lead to the retinotopic ordering of terminal arborizations characteristic of the mature projection. The trajectories and branching patterns of temporal retinal axons labeled with Dil or DiO were determined in whole mounts of retina and tectum from chicks ranging in age from embryonic day 9 to posthatching. Within the retina, labeled retinofugal axons travel in a compact bundle but do not maintain strict neighbor relations, as they course to the optic fissure. The axons enter the contralateral tectum at its rostral edge and grow caudally. Many extend well past their appropriate terminal zone within rostral tectum; a proportion of these later reverse their direction of growth. Many axons grow onto the tectum at incorrect positions along the medial-lateral tectal axis. Some correct this error in a directed manner by altering their trajectory or extending collateral branches at right angles. About 80% of the positional changes of this type are made in the direction appropriate to correct axon position, and thus are likely a response to tectal positional cues. After maturation of retinotopic order, about half of the axons that project to a mature terminal zone have made abrupt course corrections along one or both tectal axes, indicating that initially mistargeted axons can establish appropriately positioned arbors and survive. The development of temporal axons within the tectum is characterized by 3 phases: elongation, branch and arbor formation, and remodeling. After considerable rostrocaudal elongation, an axon typically develops numerous side branches and arbors, many at inappropriate locations. Most arbors are formed by side branches that develop as interstitial collaterals; few axons grow directly to their appropriate terminal zone and arborize. Aberrant arbors, and axons and axon segments that fail to form arbors in the appropriate terminal zone, are rapidly eliminated over about a 2 d period. Axon degeneration appears to play a role in this remodeling process.

Plasticity and rigidity of differentiation of brain vesicles studied in quail-chick chimeras

Transplantation of a piece of the alar plate of the prosencephalon or of the rhombencephalon of quail embryos into the roof of the mesencephalon of chick embryos was carried out at 7-10 somite stage. Results obtained were: the transplanted alar plate of the prosencephalon differentiated into tissue closely resembling the tectum when the transplants were integrated into the host mesencephalon; in all the cases, the alar plate of the rhombencephalon did not differentiate into tectum-like structure, but into rhombencephalic descendants. We conclude that the alar plate of the prosencephalon at 7-10 stage is not definitively determined and may retain an ability to differentiate into the optic tectum, whereas the prospective fate of the rhombencephalon has already been determined at 7-10 stage.

Distribution of the neural crest cells in the heart of birds: a three dimensional analysis.

SummaryThe distribution of neural crest derived cells (NC) in the heart of quail-chick chimeric embryos was analyzed three-dimensionally after computer reconstruction. During the division of the truncus arteriosus into the aorta and the pulmonary trunk, ventral and dorsal columns of NC-derived cells were found in the truncal swellings. These columns were elongations from the aorticopulmonary (AP) septum. The dorsal column extended more proximally than did the ventral column. Around hatching, NC-derived cells located between the proximal aorta and the pulmonary trunk, differentiated into cartilage and connective tissue. They formed a part of the cardiac skeleton. A small number of NC-derived cells were scattered in the cusps of the arterial valves. Cells derived from the right NC were located around the aorta and the right arch arteries but not around the distal pulmonary trunk and the left arch arteries. At the proximal level, cells derived from the rigth NC were located in both the dorsal and ventral columns. These results suggest that the AP septum is mainly formed by NC-derived cells, right and left NC cells migrating into assigned areas in the heart. Location of two columns of NC-derived cells may support a translocation hypothesis for the AP septum during truncal division.

Disturbance of refinement of retinotectal projection in chick embryos by tetrodotoxin and grayanotoxin

The role of neuronal activity in the refinement of the retinotectal projection in chick embryos was studied using tetrodotoxin (TTX) which blocks sodium channels and grayanotoxin I (GTX) which opens the channels. Optic nerve fibers were traced by the fluorescent dye DiI. The toxins were injected into the eyeball during the refinement phase of retinotectal projection (either the 13th, 14th or 15th day of incubation). A tiny crystal of DiI was placed in the temporal part of the retina. In the control embryos, fibers caudally overshooting and arborizations outside the terminal zone were found before maturation of retinotectal projection. Overshooting fibers regressed linearly, and aberrant arborizations reduced quadratically, and then the precise retinotopic map is formed by stage 44 (18th day of incubation). Both TTX and GTX interfered with the regression of overshooting fibers and arborizations outside the terminal zone. The initial action of the toxins are reverse, but their final effect may be comparable, and they may interfere with synchronous firing of the neighboring fibers. Our results emphasize a role of neuronal activity in the refinement of retinotectal projection.

Do CNS anlagen have plasticity in differentiation? Analysis in quail-chick chimera.

Heterotopic transplantations of brain vesicles of a quail embryo into a chick embryo were carried out in order to elucidate if CNS anlagen have plasticity in differentiation at the 7-10 somite stage. Quail cells are distinguished from chick cells due to the difference in nuclear morphology. The prosencephalon did not differentiate into the cerebellum when transplanted into the metencephalon, although previous study showed that the prosencephalon has the capacity to differentiate into the optic tectum. The mesencephalon differentiated as an optic tectum when transplanted into the prosencephalon or into the rhombencephalon. The metencephalon differentiated as a cerebellum in the telencephalon. It is concluded that only the prosencephalon has limited plasticity, but the mesencephalon and rhombencephalon are determined by the 7-10 somite stage.

Changes in the arrangement of actin bundles during heart looping in the chick embryo

SummaryWe assessed the arrangement of actin bundles in the looping chick heart. Actin filaments were stained with rhodamine-labeled phalloidin, and their total arrangement was observed in whole mount specimens. Before the straight heart tube was formed, actin bundles were in a net-like arrangement as if to indicate the cell borders. With progress of the heart tube formation, actin bundles were gradually arranged in a circumferential direction. In the looped heart, regional differences in actin arrangements were observed. In the truncus arteriosus, actin bundles ran in a net-like arrangement. In the bulbus cordis, actin bundles ran in random directions. In the ventricle, actin bundles were roughly arranged in a circumferential direction. Between these three regions, actin bundles ran in a circumferential direction especially on the concave side. Near the right contour on the ventral face, some actin bundles ran in a longitudinal direction along the axis of the tubular heart. In the bulbus cordis and the ventricle at the looped stage, there was another group of actin bundles in the inner layer of the myocardium which ran in a circumferential direction. We presume that the arrangement of actin bundles is related to heart looping.

The Prosencephalon Has the Capacity to Differentiate into the Optic Tectum: Analysis by Chick‐Specific Monoclonal Antibodies in Quail‐Chick‐Chimeric Brains

The alar plate of the prosencephalon of the quail embryo was heterotopically transplanted into the alar plate of the mesencephalon of the chick embryo at the 7–10 somite stage. Chick and quail cells in chimeric brains were distinguished after Feulgen‐Rossenbeck staining and/or immunohistochemical staining with a species specific monoclonal antibody MAb‐37F5 which recognized cytoplasmic components of chick brain cells. Neural connections between the transplant and the host were studied by monoclonal antibodies, MAb39‐B11, which recognizes a species specific antigen on chick nerve fibers, and MAb‐29B8, which reacts to 160 kD neurofilaments of both chick and quail.

An electron microscopic study of periderm cell development in mouse limb buds.

SummaryDevelopment of periderm cells covering fore-and hindlimb buds of mouse em`ryos was observed by scanning and transmission electron microscopy at half day intervals from day 9.5 to 12.5 of gestation (vaginal plug=day 0).At day 9.5, the epidermis is single layered. Occasional periderm cells are present at day 10.5. By day 11.5 a complete layer of periderm cells has covered the entire limb bud.By scanning electron microscopic observation, periderm cells covering the apical ectodermal ridge (AER) are characterized by a small surface size and an elongated polygonal shape with the long axis parallel to the antero-posterior contour of the apical rim. Periderm cells covering the dorsal and ventral surfaces of the limb bud are relatively large and have a polygonal surface shape.The periderm covering the apical tip reflects well the developmental state of the AER. Hence, it is possible to estimate the development of the AER by observing the surface features of the apical periderm by scanning electron microscopy.

Bis(dicholroacetyl)diamine-induced Craniofacial Anomalies in Jcl: ICR and A/J Mice

Abstract Craniofacial anomalies induced by bis(dichloroacetyl)diamine (bisdiamine) were studied in two strains of mice, Jcl:ICR and A/J. Bisdiamine was given by oral intubation at a dose of 3,200 mg/kg/day at days 7.5 and 8.5, 8.5 and 9.5, 9.5 and 10.5 or 10.5 and 11.5 of pregnancy (VP = day 0). A/J was more susceptible to embryolethal effects of bisdiamine and had a longer sensitive period to teratogenic effects of bisdiamine. The most susceptible period for craniofacial anomalies induced by bisdiamine in A/J was one day later than that in Jcl:ICR. In both strains, median facial anomalies, cleft palate, protuberance(s) on the forehead and open eyelids were commonly induced craniofacial anomalies. Median facial anomalies were more frequent and also more severe in Jcl:ICR, while lateral cleft lip was observed only in A/J.

The Ontogeny of Thymic Myoid Cells in the Chicken

The ontogeny of thymic myoid cells in the chick was studied electron microscopically and immunohistochemically. An anticreatine kinase antibody which reacts specifically to skeletal muscle cells was used. This antibody reacts only to myoid cells in the thymus. Myoid cells were found in the medulla or in the interlobular region, though the number of the myoid cells was small. Immunohistochemically, myoid cells were detected on the 18th day of incubation. Mature myoid cells showed clear cross striations after immunohistochemical staining around the time of hatching. Electron microscopically, myoid cells were detectable on the 19th day of incubation. The discrepancy between immunohistochemical and electron microscopical detection may be due to the low number of myoid cells.