Requirement for matrix metalloproteinase stromelysin-3 in cell migration and apoptosis during tissue remodeling in Xenopus laevis.

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) was originally discovered as a gene whose expression was associated with human breast cancer carcinomas and with apoptosis during organogenesis and tissue remodeling. It has been shown previously, in our studies as well as those by others, that ST3 mRNA is highly upregulated during apoptotic tissue remodeling during Xenopus laevis metamorphosis. Using a function-blocking antibody against the catalytic domain of Xenopus ST3, we demonstrate here that ST3 protein is specifically expressed in the cells adjacent to the remodeling extracellular matrix (ECM) that lies beneath the apoptotic larval intestinal epithelium in X. laevis in vivo, and during thyroid hormone-induced intestinal remodeling in organ cultures. More importantly, addition of this antibody, but not the preimmune antiserum or unrelated antibodies, to the medium of intestinal organ cultures leads to an inhibition of thyroid hormone-induced ECM remodeling, apoptosis of the larval epithelium, and the invasion of the adult intestinal primodia into the connective tissue, a process critical for adult epithelial morphogenesis. On the other hand, the antibody has little effect on adult epithelial cell proliferation. Furthermore, a known MMP inhibitor can also inhibit epithelial transformation in vitro. These results indicate that ST3 is required for cell fate determination and cell migration during morphogenesis, most likely through ECM remodeling.

Thyroid hormone regulation of apoptotic tissue remodeling: implications from molecular analysis of amphibian metamorphosis.

Organogenesis and tissue remodeling are critical processes during postembryonic animal development. Anuran metamorphosis has for nearly a century served as an excellent model to study these processes in vertebrates. Frogs not only have essentially the same organs with the same functions as higher vertebrates such as humans, but also employ similar organogenic processes involving highly conserved genes. Development of frog organs takes place during metamorphosis, which is free of any maternal influences but absolutely dependent on the presence of thyroid hormone. Furthermore, this process can be easily manipulated both in intact tadpoles and in organ cultures by controlling the availability of thyroid hormone. These interesting properties have led to extensive morphological, cellular, and biochemical studies on amphibian metamorphosis. More recently, the cloning of thyroid hormone receptors and the demonstration that they are transcription factors have encouraged enormous interest in the molecular pathways controlling tissue remodeling induced by thyroid hormone during metamorphosis. This article summarizes some of the recent studies on the mechanisms of gene regulation by thyroid hormone receptors and isolation and functional characterization of genes induced by thyroid hormone during Xenopus metamorphosis. Particular focus is placed on the remodeling of the animal intestine, which involves both apoptosis (programmed cell death) of larval cells and de novo development of adult tissues, and the roles of thyroid hormone-induced genes that encode matrix metalloproteinases during this process.

Spatial and temporal regulation of collagenases-3, -4, and stromelysin -3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis.

Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix (ECM). They have been implicated to play important roles in a number of developmental and pathological processes, such as tumor metastasis and inflammation. Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development. Using Xenopus laevis development as a model system, we demonstrate here that three MMPs, stromelysin-3 (ST3), collagenases-3 (Col3), and Col4, have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog. In situ hybridizations reveal a tight, but distinct, association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis. In particular, ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling) for apoptosis, respectively. On the other hand, Col3 and Col4 are present in regions where extensive connective tissue remodeling take place. These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development.

Age-related dopamine deficiency in the mesostriatal dopamine system of zitter mutant rats: regional fiber vulnerability in the striatum and the olfactory tubercle.

Oxidant stress has been implicated in the pathogenesis of Parkinson's disease. To test the oxidant stress hypothesis of dopaminergic degeneration, age-related changes in the mesostriatal dopamine neuron system were compared between zitter mutant rats which have abnormal metabolism of oxygen species in the brain and Sprague-Dawley rat as a control using the neurochemistry and immunohistochemistry. Dopamine content in the caudate-putamen, nucleus accumbens and olfactory tubercle of zitter rats decreased significantly with age, and was lower than that found in corresponding age-matched controls. In the zitter rats, the reduction of dopamine was more prominent in the caudate-putamen than in the nucleus accumbens and olfactory tubercle. A characteristic decline of tyrosine hydroxylase-immunoreactive fibers in the caudate-putamen of the zitter rat was also observed. In the dorsolateral caudate-putamen, reduction of tyrosine hydroxylase-immunoreactive fibers was observed in the matrix-like area, whereas in the ventromedial caudate-putamen the reduction occurred in the patch-like areas. Degeneration of tyrosine hydroxylase-immunoreactive fibers which was characterized by swollen varicosities and clustered fibers was observed in the caudate-putamen and nucleus accumbens and preceded loss of normal tyrosine hydroxylase-immunoreactive fibers in the caudate-putamen. Thus, the depletion of dopamine in the terminal areas is related to axonal degeneration. However, there was no degenerative tyrosine hydroxylase-immunoreactive fibers in the olfactory tubercle at any examined age, but reductions of tyrosine hydroxylase-immunoreactive fibers and dopamine contents were noted in the olfactory tubercle after four months-of-age. Since the zitter rats have an abnormal oxygen metabolism, the degeneration of tyrosine hydroxylase-immunoreactive fibers could result from an accumulation of superoxide species. The present results provide support for the oxidant stress hypothesis of dopaminergic neuronal degeneration and further indicate the region-specific vulnerability of the nigrostriatal dopamine system.

Role of ECM remodeling in thyroid hormone-dependent apoptosis during anuran metamorphosis.

Programmed cell death or apoptosis is an important aspect in organogenesis and tissue remodeling. It is precisely controlled both temporally and spatially during development. Amphibian metamorphosis is an excellent model to study developmental control of apoptosis in vertebrates. This process involves the transformation of essentially every organ/tissue as tadpoles change to frogs, yet is controlled by a single hormone, thyroid hormone (TH). Although different organs and tissues undergo vastly different developmental changes, including de novo development and total resorption, most require apoptotic elimination of at least some cell types. Such properties and the dependence on TH make frog metamorphosis a unique model to isolate and functionally characterize genes participating in the regulation of tissue specific cell death during organ development in vertebrates. Indeed, molecular studies of the TH-dependent gene regulation cascade have led to the discovery of a group of genes encoding matrix metalloproteinases (MMPs) participating in metamorphosis. In vivo and in vitro studies have provided strong evidence to support a role of MMP-mediated remodeling of the extracellular matrix in regulating apoptotic tissue remodeling during metamorphosis.

Apoptosis of larval cells during amphibian metamorphosis.

Programmed cell death occurs in a variety of organs during amphibian metamorphosis and is usually identified by electron microscopy as apoptosis or its modifications. Because of the massive cell death that occurs during a short period, amphibian organs serve as an ideal model system for the study of mechanisms underlying programmed cell death. In this article, a series of morphological changes in apoptosis from their nuclear changes to removal by phagocytic macrophages is reviewed, mainly in the small intestine of metamorphosing Xenopus laevis tadpoles. It is well known that cell death during amphibian metamorphosis is under the control of thyroid hormone (TH), and changes in gene expression induced by TH have been recently analyzed in a few Xenopus organs. On the other hand, there is a growing body of evidence that cell death is regulated by various kinds of local factors. For example, roles of interactions with other tissue cells and/or participation of immunocompetent cells in cell death have been experimentally shown. Therefore, to clarify the mechanisms of this complicated process, it is important at present that TH-induced changes in gene expression of each cell type comprising the organ are chronologically examined by combining morphological and molecular biological techniques.

Autoactivation of Xenopus Thyroid Hormone Receptor beta Genes Correlates with Larval Epithelial Apoptosis and Adult Cell Proliferation.

The thyroid hormone (T(3))-dependent amphibian metamorphosis involves degeneration of larval tissues through programmed cell death (apoptosis) and concurrent proliferation and differentiation of adult cell types. As the mediators of the causative effects of T(3) on metamorphosis, both thyroid hormone receptor (TR) alpha and beta genes have been found to be expressed in different tissues during this process. In particular, the Xenopus TRbeta genes have been shown to be regulated by T(3) at the transcriptional level and their expression correlates with organ-specific metamorphosis. We demonstrate here by in situ hybridization that the Xenopus TRbeta genes are regulated in a cell-type specific manner that correlates with tissue transformation. In particular, they are found to be expressed in the larval intestinal epithelial cells prior to their apoptotic degeneration and in the proliferating cells of the adult epithelium, connective tissue, and muscles. However, they are repressed again upon the differentiation of these adult cells. These results implicate that TRbeta participates both in inducing apoptosis and stimulating cell proliferation during development. Copyright 1997 S. Karger AG, Basel

Alteration of serotonergic innervation in the suprachiasmatic nucleus of the rat following removal of input fibers from retina and lateral geniculate nucleus.

To examine the influence of afferent input to the suprachiasmatic nucleus (SCN) on the development of serotonergic fibers in the SCN, afferent fibers from the retina and lateral geniculate nucleus (LGN) were eliminated in neonatal rats. Eight weeks after lesion, the distribution pattern of serotonergic fibers in the SCN was examined immunohistochemically. Neither bilateral enucleation nor LGN ablation altered the serotonergic fiber distribution in the SCN as compared to the normal adult rat. However, following combined lesions of bilateral enucleation and bilateral LGN ablation, the density of serotonergic fibers decreased throughout the SCN. The present results indicate that both retino-hypothalamic and geniculo-hypothalamic fibers may play an important role in the development of serotonergic innervation in the SCN in vivo.

Organization of regenerating serotonergic fibers in the hippocampal formation.

To evaluate the capacity of fiber outgrowth of serotonergic and dopaminergic neurons from the dorsal raphe tissue, the following three experiments were performed; (1) fetal mesencephalic raphe tissue was transplanted into the ventricle near the denervated hippocampal formation of adult rats, (2) fetal mesencephalic raphe and neonatal hippocampal tissues were transplanted into the anterior eye chamber of adult rats, and (3) fetal mesencephalic raphe tissue was explanted together with the neonatal hippocampal tissue. The extent of the fiber outgrowth was examined immunohistochemically using serotonin and tyrosine hydroxylase (TH) antisera. Three months after transplantation into the host brain, serotonin-immunoreactive (ir) fibers from raphe graft were densely distributed throughout the graft and in the host hippocampal formation, and TH-ir fibers were restricted to an area near the somata of TH-ir neurons. In particular, hyperinnervation of serotonin-ir fibers was observed in the molecular layer of the dentate gyrus. Two months after intraocular transplantation, mesencephalic raphe tissue contained a large number of serotonin- and TH-ir neurons and fibers. The distribution pattern of outgrowing serotonin-ir fibers in the hippocampal tissue was similar to that observed following intraventricular transplantation. Two weeks after explantation, the raphe tissue contained numerous serotonin-ir neurons and their fibers. These fibers extended into the hippocampal tissue in the same manner as the intraventricular and intraocular transplants. These results indicate that the intrinsic factors of hippocampal tissue influence the organization of serotonergic fibers in the hippocampal formation.

Regulation of apoptosis during development: input from the extracellular matrix (review).

Programmed cell death or apoptosis is an important aspect in organogenesis and tissue remodeling during development. Extensive investigations have led to the identification of many genes that participate in the regulation of cell death execution. These include the caspases and nucleases, which are involved in the degradation of cellular proteins and nuclear DNA to initiate the irreversible death process. In addition, several families of proteins like Bcl-2 superfamily can either prevent or promote cell death. The function of these proteins are getting to be understood. On the other hand, how these proteins are regulated remains to be investigated. This is in part due to the presence of diverse upstream signals that can influence cell fate. One such signal is the remodeling of the extracellular matrix (ECM), which is largely due to the action of matrix metalloproteinases (MMPs). The regulation of MMPs and ECM remodeling has been shown to affect apoptosis in different systems, including the apoptotic remodeling of the intestine during Xenopus laevis metamorphosis and post-lactation involution of the mouse mammary gland. Current evidence suggests that ECM regulates cell fate at least in part through its membrane receptors, the integrins, which in turn send the signal through yet poorly understood pathways to the nucleus to regulate gene expression.

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis.

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis have been studied. Throughout the larval period to stage 60, the connective tissue consists of a few immature fibroblasts surrounded by a sparse extracellular matrix: few collagen fibrils are visible except close to the thin basal lamina. At the beginning of the transition from larval to adult epithelial form around stage 60, extensive changes are observed in connective tissue. The cells become more numerous and different types appear as the collagen fibrils increase in number and density. Through gaps in the thickened and extensively folded basal lamina, frequent contacts between epithelial and connective tissue cells are established. Thereafter, with the progression of fold formation, the connective tissue cells become oriented according to their position relative to the fold structure. The basal lamina beneath the adult epithelium becomes thin after stage 62, while that beneath the larval epithelium remains thick. Upon the completion of metamorphosis, the connective tissue consists mainly of typical fibroblasts with definite orientation and numerous collagen fibrils. These observations indicate that developmental changes in the connective tissue, especially in the region close to the epithelium, are closely related spatiotemporarily to the transition from the larval to the adult epithelial form. This suggests that tissue interactions between the connective tissue and the epithelium play important roles in controlling the epithelial degeneration, proliferation, and differentiation during metamorphic climax.

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. I. Gastric region.

The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. II. Small intestine.

The binding of seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin, UEA-I; and wheat germ agglutinin, WGA) to the small intestine in metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) method. The staining pattern of the epithelium with all lectins except for UEA-I and Con A changed gradually during metamorphic climax; the main component of the epithelium, absorptive cells, gradually became positive for DBA, PNA, and SBA and the scattered goblet cells for RCA-I and WGA. On the other hand, the change of the staining pattern in the connective tissue occurred only for Con A, RCA-I, and WGA, and this change took place rapidly at the beginning of climax (stage 60). Increased staining for Con A and WGA at stage 60 was observed only in a group of connective tissue cells close to the epithelium and in the basement membrane. As metamorphosis progressed, this localization of the staining intensity became less clear. At the completion of metamorphosis (stage 66), the absorptive cells were stained with all lectins except for UEA-I, whereas the goblet cells stained only with RCA-I and WGA. These results indicate that lectin histochemistry can distinguish between larval and adult cells of both two epithelial types (absorptive and goblet cells). The technique may also identify a group of connective tissue cells, close to the epithelium, that possibly induce the metamorphic epithelial changes.

Programmed cell death and heterolysis of larval epithelial cells by macrophage-like cells in the anuran small intestine in vivo and in vitro.

The degenerative processes in the larval small intestine of Xenopus laevis tadpoles during spontaneous metamorphosis and during thyroid hormone-induced metamorphosis in vitro were examined by electron microscopy. Around the beginning of spontaneous metamorphic climax (stages 59-61), both apoptotic bodies derived from larval epithelial cells and intraepithelial macrophage-like cells suddenly increase in number. The macrophage-like cells become rounded and enlarged because of numerous vacuoles containing the apoptotic bodies. Mitotic profiles of the macrophage-like cells, however, are localized in the connective tissue where different developmental stages of macrophage-like cells are present. After stage 62, the intraepithelial macrophage-like cells decrease in number, while large macrophage-like cells which include the apoptotic bodies and retain intact cell membranes and nuclei appear in the lumen. Degenerative changes similar to those during spontaneous metamorphosis described above could be reproduced in vitro. In tissue fragments isolated from the small intestine of stage 57 tadpoles and cultured in the presence of thyroid hormone, the number of intraepithelial macrophage-like cells reaches its maximum around the 3rd day of cultivation when the larval epithelial cells most rapidly decrease in number. These results suggest that the rapid degeneration of larval epithelial cells occurs not only because of apoptosis of the epithelial cells themselves but also from heterolysis by macrophages. The macrophages probably originate in the connective tissue, actively proliferate, migrate into the larval epithelium around the beginning of metamorphic climax, and are finally extruded into the lumen.

Dual functions of thyroid hormone receptors during Xenopus development.

Thyroid hormone (TH) plays a causative role in anuran metamorphosis. This effect is presumed to be manifested through the regulation of gene expression by TH receptors (TRs). TRs can act as both activators and repressors of a TH-inducible gene depending upon the presence and absence of TH, respectively. We have been investigating the roles of TRs during Xenopus laevis development, including premetamorphic and metamorphosing stages. In this review, we summarize some of the studies on the TRs by others and us. These studies reveal that TRs have dual functions in frog development as reflected in the following two aspects. First, TRs function initially as repressors of TH-inducible genes in premetamorphic tadpoles to prevent precocious metamorphosis, thus ensuring a proper period of tadpole growth, and later as activators of these genes to activate the metamorphic process. Second, TRs can promote both cell proliferation and apoptosis during metamorphosis, depending upon the cell type in which they are expressed.

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.

Apoptosis and cell proliferation in the Xenopus small intestine during metamorphosis.

In the amphibian small intestine, the epithelial transformation from the larval to adult type is mainly the result of degeneration of the larval epithelium and development of the new (adult) epithelium. In this analysis at the cellular level, we chronologically examined apoptosis and cell proliferation in the Xenopus intestine by using in situ nick end-labeling of genomic DNA (TUNEL) and bromodeoxyuridine (BrdU) immunohistochemistry. During pre- and prometamorphosis, few apoptotic cells were detected by TUNEL, and a small number of proliferating cells randomly distributed in the larval epithelium were labeled by BrdU. At the beginning of the metamorphic climax, when primordia of the adult epithelium were first detected, numbers of apoptotic cells suddenly increased in the larval epithelium, whereas numbers of proliferating cells increased only in the adult epithelium. Subsequently, a dramatic cell loss of the larval epithelium and a rapid growth of the adult epithelium occurred. Following complete epithelial replacement, the adult epithelium became differentiated into a simple columnar epithelium possessing a cell renewal system similar to that of mammalian intestinal epithelium. These results indicate that larval epithelial apoptosis progresses simultaneously with active proliferation of the adult epithelium during the early period of metamorphic climax, which coincides with the modification of the basement membrane lining both types of epithelia.

Biphasic intestinal development in amphibians: embryogenesis and remodeling during metamorphosis.

Intestinal development in anurans is a biphasic process. The embryogenesis of intestine resembles that in higher vertebrates. The subsequent remodeling process during metamorphosis to produce an adult organ is controlled by TH. Recent progress in studying TH action and its application to amphibian metamorphosis has provided considerable insights into the remodeling process. One possible model for the TH-induced gene regulation cascade of intestinal remodeling is presented in Fig. 9. It is assumed that TRs function as heterodimers with RXRs. In the absence of TH, the TR-RXR heterodimers can bind to TH response elements present in the target genes and repress the basal transcription of these genes (Fondell et al., 1993; Damm et al., 1989; Sap et al., 1989; Baniahmad et al., 1992; Ranjan et al., 1994). The binding of TH leads to conformational changes in the receptor complexes that in turn activate gene transcription. The products of these early response genes then participate in the activation of the remaining gene regulation cascade. Exactly how this occurs remains unknown. Interestingly, the early response genes include not only transcription factors but also other proteins such as metalloproteinases. The transcription factors could activate or repress downstream TH response genes directly. Other proteins are likely to assert their effect indirectly. For example, they could modify the ECM or cell surface. In addition, they could regulate and/or participate in signal transduction by growth factors. The cooperation between these complex intra- and extracellular processes eventually results in the degeneration of the larval organ and formation of the adult tissue. This simplified scheme immediately raises many questions. Although the mRNAs for TRs and RXRs are present in the intestine and the other tissues during metamorphosis (Yaoita and Brown, 1990; Kawahara et al., 1991; Y.-B. Shi, unpublished observations), it is unknown whether the mRNA levels reflect the protein levels. It also remains to be tested whether TR-RXR is indeed the functional complex in vivo and whether the heterodimer is responsible for the activation of the early response genes isolated to date. The majority of the early response genes are ubiquitous. Of the few intestine-specific genes, none of them have yet been identified by sequence analysis. It is of great interest to understand how the same genes expressed in tissues undergoing drastically different changes can exert their biological effects. It is likely that together with existing proteins in the intestine, these early genes regulate tissue-specific downstream genes, which in turn determine the tissue-specific transformation. An important issue is to establish the identity of these downstream genes.

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro.

We 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 of Xenopus 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(-9) M 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 micrograms/ml) and cortisol (0.5 microgram/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.

Muscle fiber type analysis in the mouse m. digastricus, m. stylohyoideus, m. zygomaticus and m. buccinator.

It is generally accepted in vertebrates that large motor nerve fibers innervate the white (fast) muscle fibers and small motor nerve fibers innervate the red (slow) muscle fibers. In the previous study (SHIMOZAWA, NAKAMURA 1985), we showed that nerve branches to the venter caudalis m. digastrici and m. stylohyoideus of the mouse consist of considerably larger myelinated nerve fibers compared with the truncus facialis distal to the foramen stylomastoideum innervating the other skeletal muscles in the face. To examine the relationship between the size of nerve fibers and the type of muscle fibers, we performed, in this investigation, quantitative analysis of muscle fiber types (white, intermediate and red) in the venter rostralis (RD) et venter caudalis (CD) m. digastrici, m. stylohyoideus (St), m. zygomaticus (Zy) and m. buccinator (Bu) of the mouse with the Sudan Black B stain preparations. Ratios of the number of muscle fibers of 3 types were RD (28.1%), CD (24.6%), St (27.5%), Bu (28.5%) less than Zy (40.8%) for the white muscle fibers, the RD (58.5%), CD (62.9%), St (53.9%), Bu (60.3%) greater than Zy (47.3%) for the red muscle fibers, while differences between the ratios for these muscles were not significant for the intermediate muscle fibers. Mean major and minor diameters and average transverse areas of the muscle fibers of 3 types were white greater than intermediate greater than red muscle fiber in all muscles examined in this study. When compared between these muscles, they were RD, CD, St greater than Zy, Bu for any type of muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)