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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.

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.

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.

To determine whether the remodeling of the well-organized intestinal epithelium during amphibian metamorphosis is regionally regulated along the anteroposterior axis of the intestine, we raised a polyclonal antibody against the Xenopus laevis intestinal fatty acid-binding protein (IFABP), which is known to be specifically expressed in intestinal absorptive cells, and examined immunohistochemically the differentiation, proliferation, and apoptosis of the epithelial cells throughout X. laevis small intestine. During pre- and prometamorphosis, IFABP-immunoreactive (ir) epithelial cells were localized only in the anterior half of the larval intestine. At the beginning of metamorphic climax, apoptotic cells detected by nick end-labeling (TUNEL) suddenly increased in number in the entire larval epithelium, concurrently with the appearance of adult epithelial primordia. Subsequently, the adult primordia in the anterior part of the intestine developed more rapidly by active cell proliferation than those in the posterior part, and replaced the larval epithelial cells earlier than those in the posterior part. IFABP-ir cells in the adult epithelium were first detectable at the tips of newly formed folds in the proximal part of the intestine. Thereafter, IFABP expression gradually progressed both in the anteroposterior direction and in the crest-trough direction of the folds. These results suggest that developmental processes of the adult epithelium in the X. laevis intestine are regionally regulated along the anteroposterior axis of the intestine, which is maintained throughout metamorphosis, and along the trough-crest axis of the epithelial folds, which is newly established during metamorphosis. Furthermore, the regional differences in IFABP expression along the anteroposterior axis of the intestine were reproduced in organ cultures in vitro. In addition, IFABP expression was first down-regulated and then reactivated in vitro when the anterior part, but not the posterior part, of the larval intestine was treated with thyroid hormone (TH) for extended periods. Therefore, it seems that, in addition to TH, an endogenous factor(s) localized in the intestine itself with an anteroposterior gradient participates in the development of the adult epithelium during amphibian metamorphosis., (Copyright 1997 Academic Press.)

Homozygotes of a mouse strain with genetic polydactyly (Pdn) show disrupted cortical lamination and a significant decrease of S-100beta-immunoreactive elements in a particular area of the brain. In order to understand the abnormal cortical formation at the cellular level, the migration of cortical neurons and the development of glial cells were studied using bromodeoxyuridine (BrdU), S-100beta, and glial fibrillary acidic protein (GFAP) immunohistochemistry. Homozygous mice (Pdn/Pdn) displayed a variable pattern of abnormalities. Irregular GFAP-positive radial glial cells and disturbance of neuronal migration were found in a circumscribed area of the caudo-dorsal cortex of newborn Pdn mouse. The number of S-100beta-positive cells was reduced in this area. The present results suggest that abnormal cortical lamination closely correlates with disturbance of neuronal migration and abnormalities of glial cells, especially a significant decrease of S-100beta-immunoreactive cells.

The amphibian intestine has two morphologically distinct structures during development. Early embryogenesis generates a simple, tube-like intestine in the tadpole whereas after thyroid hormone (T3)-dependent metamorphosis a newly remodeled adult intestine is formed similar to that of higher vertebrates. This change requires a drastic transformation of the epithelial layer We have isolated a Na+/PO4 3- cotransporter gene that may contribute to this transformation. The deduced amino acid sequence of this gene shows a high degree of homology to the mammalian renal Na+/PO4 3- cotransporters, which have little or no expression in organs other than the kidney. The frog gene is highly expressed and regulated by T3 in the intestine with little expression and/or regulation by T3 in most other organs. Its mRNA is restricted to the differentiated epithelial cells both in tadpoles and postmetamorphic frogs. Interestingly, its expression is low in premetamorphic tadpoles, but up-regulated when metamorphosis is initiated by endogenous T3. As the larval epithelium undergoes programmed cell death (apoptosis), the mRNa level drops to a minimum. Subsequently, the gene is reactivated at the tip region of the newly formed adult intestinal folds and a crest-trough polarity of expression is established by the end of metamorphosis. This temporal regulation profile is also reproduced when premetamorphic tadpoles are treated with T3 to induce precocious intestinal remodeling. These results suggest a possible role of the Na+/PO 4 3- cotransporter during metamorphosis and demonstrate that the adult epithelial cell differentiation pattern is established in the direction of crest-to-trough of the intestinal fold, concurrent with the epithelial morphogenic process.

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

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.

It has been suggested that a matrix metalloproteinase, stromelysin-3 (ST3), is an important enzyme for epithelial transformation; the ST3 gene is known to be regulated by thyroid hormone during Xenopus metamorphosis. In this study, we have examined the distribution of ST3 mRNA in the small intestine of Xenopus during metamorphosis by using in situ hybridization. Around stage 58, ST3 mRNA is first detectable in larval fibroblasts near the muscular layer, and then increases in amount throughout the entire region of connective tissue. By stage 61, when connective tissue cells are rapidly increasing in number, ST3 mRNA is localized in fibroblasts just beneath the epithelium. This localization of ST3 mRNA is in good temporal accordance with modification of the basement membrane and epithelial transformation from the larval to adult form. Thereafter, ST3 mRNA gradually decreases and is no longer detected after stage 63 when the adult epithelium has completely replaced the larval type. The transient expression of ST3 mRNA in the fibroblasts of connective tissue during metamorphosis indicates that it plays an important role in epithelial transformation by changing the basement membrane.

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.

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.

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.

When stomach endoderm of chick embryos was recombined and cultured with duodenal mesenchyme, the endoderm developed a brush border structure over a large area and also differentiated into mucous cells in a small area according to its own developmental fate. In the present investigation, we examined whether the induced brush border structure expressed sucrase antigen by immunoelectron microscopy using the antiserum raised against chicken sucrase. Sucrase immunoreactivity could be detected as ferritin particles in the region where the brush border was induced, whereas it was never detected on microvilli of endodermal cells which differentiated into the mucous cells. Thus, almost all of the endodermal cells could be identified as either small intestine-type cells possessing the sucrase antigen or stomach-type cells possessing mucous granules but not the sucrase antigen. The results indicate that stomach endodermal cells of chick embryos can differentiate not only morphologically but also functionally into typical intestinal epithelial cells under the inductive influence of the duodenal mesenchyme.

The 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.

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.

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.

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.

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.

The self-differentiation potency of the endoderm of the chick embryo was investigated mainly by transmission electron microscopy. Endodermal fragments isolated from 4- to 6-day stomach or small intestine were cultured in the absence of mesenchyme and were able to differentiate in vitro into organ-specific epithelia. Endodermal fragments isolated from the stomach region differentiated into a pseudo-stratified epithelium with periodic acid Schiff-positive mucous granules in the apical cytoplasm, while those from the small intestinal region differentiated into a simple columnar epithelium with a striated border which was positive in alkaline phosphatase activity. These features are comparable with those of the mucous secretory epithelium of the normal embryonic stomach and the absorptive epithelium of normal embryonic small intestine, respectively. Next, the self-differentiation potencies were investigated of the upper and lower layers of the blastoderms, at stages 1-5 of Hamburger and Hamilton (H. and H.). Both stomach-type and small-intestine-type epithelia developed only when fragments of the lower layer isolated from the blastoderms older than stage 3 of H. and H. were cultured, suggesting that cells possessing the potency to differentiate into the stomach- and small-intestine-type epithelia exist in the definitive endoderm at the beginning of its formation.

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)

The inductive action of duodenal mesenchyme on the cytodifferentiation of stomach endoderm in chick embryos was investigated in vitro with electron microscopy and immunofluorescence. Morphologically undifferentiated endoderm of the stomach of a 4-day embryo could differentiate only into a mucous secretory epithelium when cultured in the absence of mesenchyme. However, when cultivated in recombination with 6-day duodenal mesenchyme, most cells of 4-day stomach endoderm differentiated into intestinal absorptive cells possessing striated border and sucrase, and goblet cells, but not into stomach-type mucous secretory cells. In contrast, when 4-day stomach endoderm was cultured recombined with mesenchyme of embryonic digestive organs other than intestine, none of the stomach endoderm cells differentiated into intestinal epithelial cells. The competence of stomach endoderm for intestinal cytodifferentiation decreased rapidly with development, but remained until relatively later stages in the gizzard region. The present investigation demonstrates that duodenal mesenchyme can induce stomach endoderm, which has acquired the potency for self-differentiation into stomach-type epithelium, to cytodifferentiate into intestinal epithelium.

Developmental changes, until the completion of metamorphosis, of the connective tissue of the digestive tract in the South African clawed frog Xenopus laevis were examined by light and scanning electron microscopy. During the pre- and prometamorphosis (stages 50 through 59) the layer of connective tissue was prominent in the typhlosole, while that in the rest of the intestine and stomach remained very thin. Most of the connective tissue cells were fibroblasts and were concentrated mainly in the typhlosole, the boundaries of which were distinct. In the anterior part of the typhlosole just behind the stomach, the layers of connective tissue and muscle were the thickest and the boundary between these 2 layers was obscure. Although the mitotic cells of the connective tissue were rare, if any, and no migrating cells were observed on the surface of the connective tissue, the connective tissue of the intestine gradually increased in cell number during the pre- and prometamorphosis. In the stomach, on the other hand, there was no such increase. At the beginning of the metamorphic climax (stages 60 and/or 61), the layer of connective tissue suddenly became thick in both the stomach and the intestine. The boundaries of the typhlosole became less distinct. Mitotic index of the connective tissue became high, and its cells dramatically increased in number and contained many undifferentiated mesenchyme-like cells. In the stomach, these connective tissue cells invaded the glands of the larval epithelium into just below the surface epithelium and mitotic index of the entire stomach epithelium increased. In the intestine, in contrast, mitotic epithelial cells were localized in the islets which invaginated into the connective tissue and later formed the adult epithelium. With the growth of islets, the intestinal folds were formed as straight rows and, after stage 63, were modified into a zigzag pattern and had mitotic cells localized in the troughs of folds. The present results indicate that dramatic changes of the connective tissue of both the stomach and the intestine occur at the beginning of the metamorphic climax, concomitantly with those of the epithelium. Moreover, the results also show that, before the climax, even when there were no indications of metamorphic changes in the epithelium, cells of the connective tissue gradually increased in number in the intestine (but not in the stomach). It is also suggested that the muscle cells of the typhlosole just behind the stomach may participate in this gradual increase of connective tissue cells.