Norio Nakatsuji

Cloning of inv, a gene that controls left/right asymmetry and kidney development

Most vertebrate internal organs show a distinctive left/right asymmetry. The inv (inversion of embryonic turning) mutation in mice was created previously by random insertional mutagenesis; it produces both a constant reversal of left/right polarity (situs inversus) and cyst formation in the kidneys. Asymmetric expression patterns of the genes nodal and lefty are reversed in the inv mutant, indicating that inv may act early in left/right determination. Here we identify a new gene located at the inv locus. The encoded protein contains 15 consecutive repeats of an Ank/Swi6 motif, at its amino terminus. Expression of the gene is the highest in the kidneys and liver among adult tissues, and is seen in presomite-stage embryos. Analysis of the transgenic genome and the structure of the candidate gene indicate that the candidate gene is the only gene that is disrupted in inv mutants. Transgenic introduction of a minigene encoding the candidate protein restores normal left/right asymmetry and kidney development in the inv mutant, confirming the identity of the candidate gene.

Granule cell behavior on laminin in cerebellar microexplant cultures

In order to study roles of the extracellular matrix (ECM) in the cerebellar granule cell migration, cerebellar microexplants of neonatal to postnatal 11-day-old mice were cultured on 3 kinds of substrata, poly-L-lysine (PL), PL/fibronectin and PL/laminin. A prominent outgrowth of small granule cells, which did not uptake GABA, was observed only on the PL/laminin substratum. The granule cells showed the following sequence of events: (1) Many polygonal undifferentiated cells migrated out from the microexplants. These blast cells differentiated into small bipolar neurons with long fine neurites which extended radially from the explants. (2) These cells then changed their orientation perpendicular to their radial neurites, by protruding a short process from the cell body at right angles. (3) Finally, cell bodies of these granule cells adhered to each other to form cell aggregates. Quantitative labelings by bromodeoxyuridine revealed that there were less mitotic cells in explants from the later postnatal cerebellar compared to the earlier postnatal ones. Anti-MAP2 immunoreactivity was localized in short perpendicular processes of the aggregated granule cells. Thus, this unique cell behavior exhibited on the PL/laminin substratum provides the first defined experimental system for studying the granule cell differentiation in vitro.

Proto‐oncogene of int‐3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis

Background: Notch and its homologues are key regulatory receptors of the cell fate decision in various developmental processes. The int‐3 oncogene was originally identified as a frequent target in Mouse Mammary Tumour Virus (MMTV)‐induced mammary tumours and has been regarded as a Notch homologue, based on its similarity to the intracellular domain of Notch. Studies with int‐3 transgenic mice have suggested that the int‐3 transgene affects the differentiation capacity of stem cells and leads to neoplastic proliferation in epithelial cells. However, the exact nature and the in vivo expression pattern of the int‐3 proto‐oncogene are unknown. The function of gene products in embryogenesis is also not clear.

Filopodia and growth cones in the vertically migrating granule cells of the postnatal mouse cerebellum

Abstractu2002The details of the morphology of vertically migrating granule cells were examined semiquantitatively in the postnatal mouse cerebellum by a Golgi method, with special reference to the growth cone-related structures such as filopodia and lamellipodia. The first sign of inward migration was extension of short, vertical filopodium-like processes from the sides of the perikarya of tangentially oriented granule cells, followed by a change of orientation of cell bodies to the vertical axis showing a T-shaped morphology. The T-shaped migratory cells formed sprouted filopodia (side spikes) from their vertical leading processes and perikarya at right angles to the vertical axis. More than three-quarters of the migratory cells extended the side spikes. The presence of such side spikes was confirmed with laser scanning confocal microscopy of granule cells labeled with 1,1′, dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchlorate and also with transmission electron microscopy (TEM). In addition, about one-fourth of migratory cells extended lamellipodia of web-like forms along the stem or at the tip of the leading process, some of which showed a typical growth cone. Several morphological variations of vertical granule cells were also observed. Furthermore, TEM observation confirmed that side spikes from migratory cells made direct contact with parallel fibers. The present results suggest that, during vertical migration, growth cone-related structures of the leading processes of granule cells adhere to and probably recognize tangentially oriented parallel fibers. Therefore, the mechanisms of the vertical guidance and migration of granule cells in the cerebellar cortex seem to be multiple, involving not only parallel contact guidance by the Bergmann glia fibers but also perpendicular contact guidance by the parallel fibers. These parallel and perpendicular geometric cues surrounding the granule cells seem to have produced the varying morphology of vertically migrating granule cells.

Stages of Embryonic Development of the Ice Goby (Shiro-uo), Leucopsarion petersii

Abstract A series of normal stages for the embryonic development of the ice goby (shiro-uo), Leucopsarion petersii, which belongs to the Perciformes, is described. Stages are based on morphological features, by utilizing the optical transparency of live embryos from the first cleavage to the hatching stage. Fertilized eggs were obtained by artificial insemination and normal embryogenesis was accomplished in a defined medium in plastic petri dishes at 19°C. Shiro-uo eggs were surrounded by a very thin and clear chorion and could be dechorionated with forceps very easily. Developmental stages were mostly comparable to those of other fish embryos described so far, but several differences were indicated, such as the third cleavage plane being horizontal, and that the length of the cleavage cycle increased gradually from the very early stages. Also, there were differences in the relative rates of organogenesis of the brain, eyes, otic vesicles, and somites when compared to the zebrafish and medaka.

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro. Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo, suggesting the presenge of other growth factor(s) in vivo. We previously reported that tumor necrosis factor‐α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4‐m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo. Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo; however, it did stop afterwards. Such proliferating PGC continue to express c‐kit and Oct‐3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES‐like EG cells.

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study

We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance.

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures I. A PKH26 Labeling Study in Microexplant and Organotypic Cultures

Granule cells were dissociated from early postnatal mouse cerebella and labeled with a fluorescent dye probe PKH26. Small number of the labeled cells were mixed with cerebellar cortical microexplant cultures or transplanted into cerebellar cortical organotypic explants, and their time‐dependent morphological changes during cultures were examined with fluorescence microscopy. Granule cell neurons first extended asymmetrical short bipolar processes in both cultures, and migrated actively in microexplant cultures. After elongation of symmetrically bipolar long and thin neurites, they sprouted short thick processes from cell bodies and migrated perpendicular to neurite bundles that were devoid of glia in microexplant cultures, or migrated vertically inward into the internal granular layer in the organotypic explant. During such migrations, they extended short thick processes in front and thin processes behind the cell body. The latter processes were connected to thin long neurites with T‐ or Y‐shaped junctions in both cultures. Finally, they extended many short thick processes from cell bodies in both cultures. Such behaviors of granule cell neurons in microexplant cultures were, thus, similar to those in organotypic explant cultures despite of the absence of Bergmann glial cells. These migration patterns may be closely related to migration of granule cells in histogenesis of the cerebellar cortex.

Development of Postimplantation Mouse Embryos: Unexplored Field Rich in Unanswered Questions

In mammalian development, many embryologically important events occur during the early postimplantation period. These include determination and formation of the embryonic axis and germ layers, the central nervous system (CNS), neural crest cells, and the heart and circulatory system. The primordial germ cells (PGCs) also appear and migrate to the genital ridges during this period. Difficulties in culture and experimental manipulation of postimplantation embryos have, however, hindered detailed study of these important events in mammalian embryos, leaving many important questions unanswered. In this review, I describe many important but still unexplored developmental events during the postimplantation period by reviewing studies in recent reports, which are relatively limited in number but strongly encourage further investigation. I also summarize various methods to circumvent the problem of i naccessi bi I ity of post i m plan t at ion embryos inside the uterus and enable experimental manipulation of living embryos. Mammalian embryos have unique tissues that differ from those of other chordate embryos. Two embryonic tissues receive particular attention here, because they need renaming judging from the results of recent lineage studies (Fig. 1). One is embryonic ectoderm. In this review, I use epiblast in place of it, because as this tissue produces not only ectodermal tissues but also mesodermal and endodermal tissues of the fetus, epiblast, which implies only the topological location of the tissue, seems more appropriate. The second tissue that needs renaming is mesoderm, which is formed in embryos at the primitive streak stage. Again, it includes not only embryonic and extra-embryonic mesoderm cells but also definitive endoderm cells of the fetus. In

Culture of Embryonic Cells for Analysis of Amphibian and Mammalian Early Embryogenesis

Culture of embryonic cells isolated from early embryos allows detailed analysis of the cell motility, interactions between cells, and between cells and the extracellular matrix (ECM). There is always the risk that cell behavior in culture might be artifacts not related to the behavior inside embryos. In many situations, one must use cells immediately after isolation from embryos, and try to prepare culture conditions similar to those inside the embryo.