Hajime Fujisawa

Spatial and temporal expression pattern of N-cadherin cell adhesion molecules correlated with morphogenetic processes of chicken embryos☆

N-cadherin is a Ca2+-dependent cell-cell adhesion molecule, which was identified in brain cells of mouse and chicken. In the present study, we have determined the pattern of expression of N-cadherin in chicken embryos at various stages by means of immunohistochemistry. N-cadherin was expressed in cells derived from all three primary germ layers. Its expression was transient in many tissues but permanent in others. The transient expression occurred in nephric tubules, skeletal muscles, mesenchymal tissues, endodermal organs, and epidermis, while the permanent expression occurred in nervous systems, lens, and myocardiac cells. Appearance or disappearance of N-cadherin could be generally correlated with morphogenetic events, such as rearrangement, segregation, or association of cells. Comparison of the expression pattern of N-cadherin with that of L-CAM and N-CAM determined by other workers suggests that there is some mechanism controlling expression of multiple classes of adhesion molecules. The pattern of expression of N-cadherin was generally complementary to that of L-CAM; that is, if N-cadherin appeared, L-CAM disappeared or vice versa. We also found cases in which N-cadherin was expressed in the same local regions as L-CAM. The distribution of N-cadherin was similar to that of N-CAM with some exceptions. Thus, N-cadherin and other cell-cell adhesion molecules seem to be expressed under a precise spatial and temporal control so as to be associated with a variety of morphogenetic events during development.

Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies

Monoclonal antibodies (MAbs) against the optic tectum of Xenopus tadpoles were generated and screened by the immunofluorescent staining of frozen sections of tadpole brains. MAb-A5 stains the 8th and 9th plexiform layers of the optic tectum, whereas MAb-B2 stains all but the eighth and ninth plexiform layers of the optic tectum. MAb-A5 antigen is also detectable in the nucleus of Belonci, the corpus geniculatum thalamicum, the pretectal area, and the basal optic nucleus, all targets of the optic nerve, but is not detectable in the optic nerve or the optic tract. On the other hand, MAb-B2 does not stain any of these visual centers, though many fibers surrounding them are stained. Eye-enucleation experiments showed that MAb-A5 antigen is expressed in the optic tectum even when it is not innervated by optic nerves. Staining of viable brains with these MAbs indicates that these antigens are cell surface molecules. Immunoadsorption followed by SDS-PAGE suggests that proteins are constituents of these antigens. The MAb-A5 antigen in the diencephalon and the mesencephalon is not detectable at stage 35/36, but is detectable at stage 39 when the optic nerves begin to innervate the optic tectum. The spatial as well as the temporal patterns of the expression of the MAb-A5 antigen suggest that this molecule may be involved in the target recognition of optic nerve fibers.

Branching of regenerating retinal axons and preferential selection of appropriate branches for specific neuronal connection in the newt

Abstract Exact trajectory and pattern of branching of individual regenerating retinal axons derived from different retinal quadrants was detected within the tectum by labeling selected retinal axons with horseradish peroxidase (HRP) at the 4th, 6th, and 10th weeks after transection of the optic nerve in adult newt Cynops pyrrhogaster . In the early phase of regeneration, each regenerating retinal axon sprouted several branches, in random directions within the tectum. As the regeneration proceeded, only branches directed toward the site of normal innervation were maintained, while the sproutings toward the ectopic part of the tectum were retracted or became atrophic. The surviving branches then sprouted several new branches. Sprouting of new branches and the disappearance of old branches occurred simultaneously throughout the process of regeneration. At the 10th week of regeneration, most regenerating retinal axons possessed branches only at the distal part within the site of normal innervation. These observations strongly suggest that the retinotopic central connection of regenerating retinal axons may be reestablished by means of a stepwise selection of widespread axonal branches.

Retinotopic analysis of fiber pathways in the regenerating retinotectal system of the adult newt cynops Pyrrhogaster.

Retinotopic analysis of the pathways of regenerating retinal fibers within the optic tract and in the tectum of an adult newt was performed by selective labeling of the retinal fibers with horseradish peroxidase. At the tenth week of regeneration, all the regenerating retinal fibers from different retinal quadrants had terminal arbors nearly at the parts of the tectum innervated normally by those quadrants. The pathways for individual retinal fibers, however, were greatly disorganized within the optic tract and did not show any retinotopic ordered geography. The most rostral segregation of pathways of regenerating fibers was observed at the diencephalo-tectal junction. THe temporal retinal fibers invaded the tectum directly, while the dorsal, ventral and nasal retinal fibers generally shifted toward the dorsomedial or the lateral direction, as if they traced the dorsomedial or the lateral tracts formed in normal newt. The direction of the shifting of fiber pathways, however, did not depend on the origins of retinal fibers within retinal circumference, but depended on the location of fibers with in the optic tract. As a result, a large number of regenerating fibers reached their normal sites of innervation within the tectum via anomalous routes. These mis-routed fibers did not form branches or terminal arbors at ectopic parts within the tectum.

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.

Retinotopic analysis of fiber pathways in amphibians. I. The adult newt Cynops pyrrhogaster

The possibility that pathways of retinal fibers within the optic tract and the tectum of the adult newt are retinotopic was examined by selective labeling of the retinal fibers with horseradish peroxidase. Within the optic tract fibers from the ventral, temporal and dorsal retinal quadrants were ordered from the dorsal to ventral edges of th optic tract. The nasal retinal fibers exhibited two different pathways. The fibers from the dorsonasal retina ran along the ventral edge of the optic tract, while the fibers from the ventronasal retina ran along the dorsal edge of the optic tract. Segregation of pathways within the optic tract was incomplete between the nasal and other retinal fibers. The dorsonasal retinal fibers were mixed completely with the dorsal retinal fibers, and the ventronasal retinal fibers were mixed partly with the ventral retinal fibers. Both the dorsal and dorsonasal retinal fibers preferentially entered the lateral tract, and finally projected onto the ventrolateral parts of the middle tectum and of the caudal tectum, respectively. The ventral and ventronasal retinal fibers entered the dorsomedial tract, and projected onto the dorsomedial parts of the middle tectum and of the caudal tectum, respectively. The temporal retinal fibers invaded the nasal tectum directly. Most dorsal, ventral, and nasal retinal fibers ran along the sub-tracts as far as to the level of their terminals, then sharply turned in a direction to the tectum.

Retinotopic analysis of fiber pathways in amphibians. II. The frog Rana nigromaculata

The possibility of retinotopic organization of pathways of retinal fibers within the optic tract and the tectum of the frog was studied by selective labeling of the retinal fibers with horseradish peroxidase. Within the optic tract the pathways of the ventral, temporal and dorsal retinal fibers were ordered from the dorsal to ventral edges of the optic tract. The nasal retinal fibers ran along both the dorsal and ventral edges of the optic tract. The dorsal retinal fibers and the nasal retinal fibers which were located along the ventral edge of the optic tract entered the ventrolateral perimeter of the tectum and formed the lateral tract. The ventral retinal fibers and the nasal retinal fibers which were located along the dorsal edge of the optic tract entered the dorsomedial perimeter of the tectum and formed the dorsomedial tract. The temporal retinal fibers invaded the tectum directly at the diencephalo-tectal junction. The topography of fiber pathway observed for the frog was exactly the same as that seen in the newt, and seemed to be common to all amphibian species.

Specific expression of the chicken δ-crystallin gene in the lens and the pyramidal neurons of the piriform cortex in transgenic mice☆

Two transgenic mice, 5-8 and 7-5, carrying the chicken delta-crystallin gene were produced by microinjecting cloned genes into male pronuclei. The mice were analyzed at 8 weeks of age with respect to gene integration and expression by means of blotting techniques and immunohistochemistry. Southern blot analysis indicated that both mice carried, on average, 50 copies of intact delta-crystallin gene per cell. Histological analysis of the mice using DNA-DNA in situ hybridization indicated that mouse 5-8 carried the delta-crystallin gene in every cell while mouse 7-5 was mosaic, with 20-40% of the cells of various tissues carrying the gene. Western blot analysis indicated that in both mice delta-crystallin is expressed in the lens and the cerebrum, but not in any other tissue examined. Immunohistological analysis revealed that, in the cerebrum of the mice, delta-crystallin was expressed specifically in pyramidal neurons located in layer IIb of the anterior piriform cortex. Thus, our results with transgenic mice not only demonstrate the primary specificity of delta-crystallin gene expression in authentic lens tissue, but reveal the unexpected specificity of this chicken gene in the central nervous system of the mouse.

Involvement of neuronal cell surface molecule B2 in the formation of retinal plexiform layers

The B2 molecule is a 220 kd neuronal cell surface protein of Xenopus, recognized by monoclonal antibody B2 (MAb B2). Immunohistochemistry using MAb B2 revealed that the B2 molecule was expressed in both the inner and outer plexiform layers within the neural retina. During development of the neural retina, the B2 molecule first appeared at stages 35/36 in the newly formed plexiform layers. When embryonic eyes were cultured in the presence of anti-B2 antiserum (Fab fragments), the formation of the retinal plexiform layers was impeded. These data suggest that the cell surface molecule B2 plays a role in the development of retinal plexiform layers.

Cell dynamics of the blastulation process in the starfish, Asterina pectinifera

Abstract Embryonic cells of the starfish, Asterina pectinifera, both enclosed in and deprived of the fertilization membrane, were observed through the process of blastula formation by light and electron microscopy. The first few divisions of the denuded egg produce blastomeres which are virtually unconnected. However, these cells start to pack closely together after the eighth cleavage (28-cell stage) and soon arrange themselves into a tightly packed sheet. This cell sheet, then, turns its free edges up at the late 29- or very early 210-cell stage and forms itself into an irregularly shaped, hollow blastula, usually before another cell division. Cleavage of these cells takes place in perfect synchrony with the cells of the normal embryo. Ultrastructural studies of the contact area between these cells and cells of a normal embryo reveal a good correlation between the dynamic closing movement of blastulating cells and the developmental appearance of septate desmosomes. Basement membrane was laid down only after the cell sheet has closed itself into a hollow blastula, at the early 211-cell stage. Cellular activities, mentioned above, are discussed in connection with a process in which “epithelium” is organized from relatively independent morular cells.