Isao Hori

The C. elegans unc-18 gene encodes a protein expressed in motor neurons.

The C. elegans unc-18 gene is required to maintain normal acetylcholine levels. We determined the complete structure of an unc-18 cDNA that encodes a protein of 591 highly charged and hydrophilic amino acids. The protein shows sequence similarity with elements of the secretory pathway in the yeast S. cerevisiae. Antibodies raised against a portion of the unc-18-encoded protein (UNC-18) detected a 68 kd soluble antigen on immunoblots and intensely stained all vertical cord motor neurons in situ. These findings suggest that UNC-18 participates in the axonal transport system and influences the acetylcholine flow in motor neurons.

Characterization and categorization of fluorescence activated cell sorted planarian stem cells by ultrastructural analysis.

Planarians have regenerative ability made possible by pluripotent stem cells referred to as neoblasts. Classical ultrastructural studies have indicated that stem cells can be distinguished by a unique cytoplasmic structure known as the chromatoid body and their undifferentiated features, and they are specifically eliminated by X‐ray irradiation. Recently, by using fluorescence activated cell sorting (FACS), planarian cells were separated into two X‐ray‐sensitive fractions (X1 and X2) and an X‐ray‐insensitive fraction (XIS) according to DNA content and cytoplasmic size. Here we analyzed the fractionated cells by transmission electron microscopy (TEM). First, we found that both undifferentiated cells (stem cells) and regenerative cells (differentiating cells) were concentrated in the X1 fraction containing the S/G2/M phase cells. The regenerative cells were considered to be committed stem cells or progenitor cells, suggesting that some stem cells may maintain proliferative ability even after cell fate‐commitment. Second, we succeeded in identifying a new type of stem cells, which were small in size with few chromatoid bodies and a heterochromatin‐rich nucleus. Interestingly, they were concentrated in the X2 fraction, containing G0/G1 phase cells. These results suggest that planarian stem cells are not homogeneous, but may consist of heterogeneous populations, like mammalian stem cells.

Metamorphosis of Coeloblastula Performed by Multipotential Larval Flagellated Cells in the Calcareous Sponge Leucosolenia laxa

The calcareous sponge Leucosolenia laxa releases free-swimming hollow larvae called coeloblastulae that are the characteristic larvae of the subclass Calcinea. Although the coeloblastula is a major type of sponge larva, our knowledge about its development is scanty. Detailed electron microscopic studies on the metamorphosis of the coeloblastula revealed that the larva consists of four types of cells: flagellated cells, bottle cells, vesicular cells, and free cells in a central cavity. The flagellated cells, the principal cell type of the larva, are arranged in a pseudostratified layer around a large central cavity. The larval flagellated cells characteristically have glutinous granules that are used as internal markers during metamorphosis. After a free-swimming period the larva settles on the substratum, and settlement apparently triggers the initiation of metamorphosis. The larval flagellated cells soon lose their flagellum and begin the process of dedifferentiation. Then the larva becomes a mass of dedifferentiated cells in which many autophagosomes are found. Within 18 h after settlement, the cells at the surface of the cell mass differentiate to pinacocytes. The cells beneath the pinacoderm differentiate to scleroblasts that form triradiate spicules. Finally, the cells of the inner cell mass differentiate to choanocytes and are arranged in a choanoderm that surrounds a newly formed large gastral cavity. We found glutinous granules in these three principal cell types of juvenile sponges, thus indicating the multipotency of the flagellated cells of the coeloblastula.

Metamorphosis of calcareous sponges I. Ultrastructure of free-swimming larvae

Summary Free-swimming larvae of two calcareous sponges were studied by electron microscopy. The larvae are composed of four kinds of cells, namely flagellated cells, granular cells, four cruciform cells, and several yolk-containing cells. In the anterior hemisphere of the larvae, the columnar flagellated cells are arranged in a single layer. Their nucleus and Golgi apparatus are located in close proximity to the flagellar rootlets. There are granular cells in the posterior hemisphere of the larvae. They have a nucleus with a nucleolus, large phagosomes, well-developed Golgi apparatus, and numerous RER cisternae. There is a cruciform cell in each quadrant of the larvae. From its characteristic arrangement of organelles, it is suggested but not concluded that the cruciform cells participate in photoreception. The yolk-containing cells are, most probably, nutritive cells derived from the mother sponge. The roles of these four kinds of cells in habitat selection and metamorphosis are discussed.

Metamorphosis of calcareous sponges II. Cell rearrangement and differentiation in metamorphosis

Summary The free-swimming larva of the calcareous sponge turns into a sessile juvenile during metamorphosis. Electron microscopic observations of metamorphosing larvae reveal the rearrangement and differentiation of larval cells. About 12 h after the larvae were released from a mother sponge, the settled larvae without flagella consist of an inner cell mass and an enveloping layer of pinacocytes. The inner mass cells have residual flagellar rootlets which clearly show the origin of the cells. On the other hand, the pinacocytes still show the intracellular profile characteristic of the granular cells of the swimming larva. One day after release, scleroblasts and other mesohyl cells differentiate in the peripheral region of the inner cell mass. Two days after release, the central cells of the inner cell mass differentiate into choanocytes. Three days after release, a large gastral cavity is formed and lined by a layer of choanocytes. These results demonstrate the cell lineage in the metamorphosis of the cal...

A fine structural study of regeneration after fission in the planarian Dugesia japonica

We examined morphologically the process of regeneration before and after fission in a sexual strain of the freshwater planarian Dugesia japonica. Usually fission takes place in the post-pharyngeal region. Decapitation significantly accelerates the rate of fissioning. When decapitated worms were treated with substance P and neuropeptide K separately, the rate of fission markedly decreased in both cases. Before the onset of fission, a presumptive region of fission was recognized in the post-pharyngeal portion where undifferentiated cells, regenerative cells and newly differentiated cells were localized. Moreover a functional network of fixed parenchyma cells was noted in this region. After fission, cell distribution in the blastema became quite different from that of artificially amputated worms. This difference seems to be due to the process that occurs in the presumptive region of fission.

Localization of newly synthesized precursors of basal lamina in the regenerating planarian as revealed by autoradiography

Autoradiography has been carried out to investigate the site of synthesis of the basal lamina in the regenerating planarian, Dugesia japonica. Since the basic collagenous structures of the basal lamina rose from RR-positive amorphous precursor, [3H]proline, [3H]glucose and [35S]sodium sulphate were used as radioactive precursors of collagen, unsulphated and sulphated GAG respectively. Cytoplasm of the most regenerating epidermal cells was heavily labeled with [3H]proline during epithelization. A quantitative uptake analysis of [3H]proline indicates a progressive decline in the amount of labeled precursor in the epidermis with a corresponding increase in deposition of the labeled collagen at the presumptive basal lamina. Several myoblasts at the subepidermal region were highly labeled with both [3H]glucose and [35S]sodium sulphate. Silver grains of these labeled precursors were also present in the presumptive portion of basal lamina. These observations suggest that the regenerating epidermal cell is the only site of synthesis of the basal lamina collagen while the myoblast exclusively secretes extracellular GAG. Some of the GAG may be closely associated with the amorphous zone.

Structure and regeneration of the planarian basal lamina: An ultrastructural study

The structure and regeneration of the planarian subepidermal basement membrane or basal lamina have been electron microscopically examined, particularly in relation to the changes of extracellular products at the wounded area. The intact basal lamina consists of three structural elements; namely, an electron-lucent zone, a limiting layer and a microfibrillar layer. Ultrastructural changes during wound healing have suggested that the amorphous material secreted in the interspace between the epidermal cells and blastema contains precursors of the basal lamina. Within the amorphous zone two distinct phases of the basal lamina regeneration are observed: one is a reconstitution of the limiting layer and the other is a polymerization of the microfibrils. The limiting layer arises from areas subjacent to newly developed hemidesmosomes of epidermal cells. The unit microfibrils are formed from an accumulation of the precursors through transitional smaller microfibrils. At the late stage, individual mature microfibrils are regularly lined with the limiting layer and cell membranes of the newly differentiated muscle fibres. On the basis of these observations we suggest that the planarian basal lamina is regenerated by the interaction between epidermal cells and myoblasts.

Establishment of a metastatic murine cell line carrying the human c-Ha-ras

Dear Editor: Development of new modalities to control metastasis is an urgent requirement of cancer therapy. However, the available methods have an inherent limitation in detecting and quantifying micro-metastases. It is possible to experimentally detect metastasis if genes of a species are detected in the tissues of another animal. Therefore, a new system needs to be established which consists of: 1) ceils that grow and metastasize in immunocompetent syngeneic animals, a n d 2) cells that have genes which are distinguishable from those of the animal tissues. The combination of r/mHM-SFME-1 cells and Balb/c mice provides a good model system for this application. The r/mHM-SFME1 cells are derived from ras/myc SFME cells transformed by activated human c-Ha-ras and mouse c-myc genes (5,7). Since original serum-free mouse embryo (SFME) cells were established from a Balb/c mouse embryo (3), immunocompetent Balb/c mice are syngeneic for both ras/myc SFME and r/mHM-SFME-1 cells. Here we describe the establishment of the r/mHM-SFME-1 cell line in vitro. One million of G418-resistant ras/myc SFME cells, which were transfected with pSV2-neo by calcium-phosphate co-precipitation (7), were injected subcutaneously into the backs of Balb/c mice. All of mice, which developed solid tumors within 2 months, were sacrificed to check metastases. One of them had metastases in the subaxillary and submaxillary lymph nodes, the lung and the liver. The metastases were excised from each organ and transplanted subcutaneously again into the mice. Only mice that received the tung metastases developed solid tumors and pulmonary metastases. Thereafter, these pulmonary metastases were serially transplanted subcutaneously into Balb/c mice. The solid tumors of the 7th passage were excised under sterile conditions and the cells were then cultured in serum-free medium (3) followed by colonization in soft agar. One of the clones derived from a colony was designated as r/mHM-SFME1. The r/mHM-SFME-1 cells were no longer resistant to G418. The profiles of the two cell lines in culture are shown in Figure 1. The r/mHM-SFME-1 cells tended to aggregate in culture whereas the ras/myc SFME cells did not. Aggregates always appeared 3 4 days after plating whether in serum-free or in serum-supplemented media and did not disappear upon adding fresh media. The aggregates sometimes detached from cells on dishes so that freely floating cells were observable in aged cultures even maintained by frequent media change. In order to confirm that the r/mI-LM-SFME-1 cells were derived from the ras/myc SFME cells, we determined whether the r/mHMSFME-1 cells had human ras genes. Hind III-digested fragments of DNA from r/mHM-SFME-1 and ras/myc SFME ceils were hybridized with the human c-Ha-ras exon-2 (6). A plasmid pUCC-H-ras, which contains normal human ras gene from placenta, for probe was obtained from the Japanese Cancer Research Resources Bank (JCRB). Six bands were detected in each of the fragments and no significant differences in the band profiles of either fragment were observed (Fig. 2). A faint band detectable in the DNA fragments from non-transformed SFME cells may be due to endogenous

Differentiation of myoblasts in the regenerating planarian Dugesia japonica

Abstract The regenerative cells mainly derived from undifferentiated cells developed into myoblasts in the regeneration blastema. The subepidermal region of the blastemas was the primary or only site of myoblast development. Synthesis of thin myofilaments took place predominantly in the cytoplasmic location between the plasma membrane and the ribosome-rich portions of the myoblasts. There was intracellular accumulation of dense materials adjacent to hemidesmosome-like structures, suggestive of the formation center of Z-lines. The initial alignment of the myofiber was invariably accomplished from the epidermal side. The significance of deposit of extracellular substances including basal lamina precursors is discussed with respect to myoblast regeneration.