Atsumi Shimozawa

INDUCTION OF METAMORPHOSIS BY THYROID HORMONE IN ANURAN SMALL INTESTINE CULTURED ORGANOTYPICALLY IN VITRO

SummaryWe have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T3) at a concentration higher than 10−9M to the medium could induce cell death of larval epithelial cells, but T3 alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 µg/ml) and cortisol (0.5 µg/ml) besides T3 were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.

Connective tissue is involved in adult epithelial development of the small intestine during anuran metamorphosis in vitro

SummaryThe role of connective tissue in metamorphic changes of the small intestinal epithelium inXenopus laevis tadpoles was investigated by using organ culture techniques and electron microscopy. Tissue fragments isolated from various parts of the small intestine at stage 57 were cultivated. Larval cell death of the epithelium was induced by thyroid hormone in all fragments, whereas adult epithelial development was observed only in fragments isolated from the anterior intestinal region containing the typhlosole where most of the larval connective tissue was localized. The epithelium was then cultivated in recombination with homologous or heterologous non-epithelial components. The adult epithelium developed only in recombinants containing a thick connective tissue layer from the typhlosole. There was no regional difference in the developmental potency of the epithelium itself. In all explants where adult epithelium developed, the connective tissue increased in cell density just beneath the epithelium, which was rapidly proliferating and forming typical islets. At the same time, fibroblasts possessing well-developed rough endoplasmic reticulum differentiated close to epithelial cells and often made contact with them. These results indicate that the connective tissue originating from the typhlosole plays an important role in adult epithelial development of the anuran small intestine, probably via direct cell-to-cell contacts or some factor(s) synthesized by the fibroblasts.

Inductive action of epithelium on differentiation of intestinal connective tissue of Xenopus laevis tadpoles during metamorphosis in vitro.

The action of the epithelium on differentiation of connective tissue cells of Xenopus small intestine during metamorphosis was investigated by using culture and morphological techniques. Connective tissue fragments isolated from the small intestine at stage 57 were cultivated in the presence or absence of homologous epithelium. In the presence of the epithelium, metamorphic changes in the connective tissue were fully induced by hormones including thyroid hormone (T3), as during spontaneous metamorphosis, whereas they were partially induced in the absence of the epithelium. Macrophage-like cells showing non-specific esterase activity in the connective tissue were much fewer in the absence of the epithelium than in the presence of it, and aggregates of fibroblasts possessing well-developed rough endoplasmic reticulum developed only in the presence of the epithelium. Just before the aggregation of the fibroblasts, the connective tissue close to the epithelium became intensely stained with concanavalin A (ConA) and wheat germ agglutinin (WGA). The present results indicate that the epithelium plays important roles in the differentiation of intestinal connective tissue cells, which in turn affect the epithelial transformation from larval to adult form during anuran metamorphosis. Thus, the tissue interaction between the epithelium and the connective tissue in the anuran small intestine is truly bidirectional.

Cell-specific and spatio-temporal expression of intestinal fatty acid-binding protein gene during amphibian metamorphosis

Intestinal fatty acid-binding protein (IFABP) gene is known to be regulated during Xenopus metamorphosis. To determine the relationship between its regulation and cellular differentiation during metamorphosis, we have examined the distribution of IFABP mRNA in the Xenopus digestive tract by in situ hybridization techniques. Throughout all stages examined, transcripts of IFABP gene were observed exclusively in absorptive epithelial cells of the small intestine, and they decreased in amount towards the posterior intestine. Around stage 58, just before metamorphic climax, IFABP mRNA level began to decrease in larval absorptive cells that still remained intact morphologically. Thereafter, IFABP mRNA was no longer detected among larval cells. In turn, at stage 62, IFABP mRNA became detectable in some of the newly formed adult epithelium that had not yet developed a brush border, but not in the remaining larval cells. By the end of metamorphosis, IFABP mRNA became more abundant towards the crest of intestinal folds. These results suggest that IFABP gene expression is specific for absorptive epithelial cells of the small intestine and is regionally regulated along the intestinal anterior-posterior axis in both tadpoles and frogs and also along the trough-crest axis of frog intestinal folds. In addition, our present study directly shows that IFABP mRNA level decreases in larval absorptive cells but increases in adult ones during metamorphosis, preceding morphological changes of both types of cells. Therefore, the regulation of IFABP gene is an early event during both larval epithelial cell death and adult epithelial cell differentiation.

Quantitative studies on the motor root of the mouse facial nerve

Fiber count analysis was performed with the electron microscope on the motor root of the facial nerve in six mice. On an average, 3,433 (84.9%) of the total nerve fibers (4,046) were myelinated and 592 (14.6%) unmyelinated. The motor root consisted mostly of large myelinated fibers (large fiber group), but a part of the root consisted of small myelinated and unmyelinated fibers (small fiber group). The nerve fibers of the small fiber group appear to correspond with those of the intermediate nerve, and to pass through the motor root and enter the intermediate nerve near the geniculate ganglion.

The multiple eyes of Polycelis. 1. Relation between the number of eyes and body length

Species of the freshwater planarian genus Polycelis have a variable number of eyes in the head, typically more than a hundred. To elucidate the mechanisms determining the number of eyes, we investigated the relation between eye number and body length in Polycelis sapporo (Ijima & Kaburaki), a non-fissioning species, and P. auriculata Ijima & Kaburaki, a fissioning species. In P. sapporo reared at 7–8 °C, a positive correlation existed between number of eyes and body length. Eye number decreased with starvation. A similar correlation was true of P. auriculata. In specimens of P. auriculata undergoing regeneration, the rate of eye formation was higher in newly formed heads originating from larger tail-pieces than in those from smaller pieces. As a head regenerated from a tail piece or as the body size increased with feeding, the number of eyes increased. These results suggest that the number of eyes in an individual of Polycelis is determined by body length. The fine structure of the photoreceptor and pigment cells in the eyes of P. auriculata is similar to that of comparable cells in the pair of eyes in Dugesia despite the difference in the number of cells comprising an eye.

Anatomical Observations on the Musculi suprahyoidei and Apparatus hyoideus with Special Reference to the Musculus jugulohyoideus and Os stylohyoideum of the Mouse, Hamster and Rat

The musculi suprahyoidei and apparatus hyoideus were investigated using a combination of gross dissection and histological techniques in the mouse, hamster and rat. The new findings with regard to these are as follows: (1) the musculus jugulohyoideus, which runs from the processus jugularis to the os stylohyoideum, exists in the mouse and hamster, but is absent in the rat; (2) the m. jugulohyoideus is innervated with the ramus jugulohyoideus from the nervus facialis; (3) the os stylohyoideum and the os tympanohyoideum exist on the lateral surface of the bulla tympanica in the mouse, hamster and rat, and (4) four kinds of muscle (m. stylohyoideus, m. styloglossus, m. stylopharyngeus, m. jugulohyoideus) attach to the os stylohyoideum in the mouse and hamster; however, no muscles attach to the os stylohyoideum in the rat.

Quantitative studies on the mouse facial nerve trunk distal to the geniculate ganglion

A fiber count analysis was performed with the electron microscope on the facial nerve trunk distal to the geniculate ganglion in seven mice. On an average, 4,836 (93%) of the total nerve fibers (5,201) were myelinated and 328 (6.3%) unmyelinated. The large part of the nerve consisted mostly of large myelinated fibers (large-fiber group). The small part of the nerve consisted of small myelinated and unmyelinated fibers (small-fiber group). The fiber constitution of the small-fiber group in the nerve was more like that in the intermediate nerve than that of the small-fiber group in the motor root of the facial nerve. Thus, it is postulated that the nerve fibers of the small-fiber group in the facial nerve trunk distal to the geniculate ganglion may be derived from the intermediate nerve, and that the nerve fibers of the small-fiber group in the motor root of facial nerve may course to the greater petrosal nerve.

Melano-macrophage centre-like structure in the heart of the Medaka, Oryzias latipes

Endocardial lining cells in the heart of certain teleosts are phagocytic against foreign materials. In the medaka Oryzias latipes, in addition to the endocardial lining cells, cells in the subendocardial spaces are also phagocytic. Most subendocardial cells contain yellowish-brown pigments and are often aggregated to form nodules resembling splenic and kidney melano-macrophage centres. Subendocardial cells are positive to PAS and Schmorls reaction. Macrophages containing large heterogeneous inclusions together with lymphocytes, were observed with the TEM. The definitive characteristics of reticular cells, including the cell sheaths, were not observed. Because of these two characteristics they were regarded as more primitive aggregates of macrophages than the ordinary melano-macrophage centres. The possible analogy between these structures and MMCs is discussed.

Electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion

An electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion shows that there are, on the everage, 603 more nerve fibers in the portion of the nerve distal to the geniculate ganglion than there are in the part proximal to the ganglion. The average distal increase in the number of unmyelinated fibers is 444 and that in the myelinated fibers is 165. The somatic motor nerve fibers and the parasympathetic fibers in the mouse facial nerve may not contribute to the distal excess. It is possible that the increase in the number of unmyelinated fibers distal to the geniculate ganglion is mainly due to the presence of postganglionic sympathetic fibers in the facial trunk distal to the geniculate ganglion and the greater petrosal nerve. The distal increase in the number of myelinated fibers may be mainly contributed by the sensory fibers.