Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with adult epithelial development during amphibian intestinal remodeling.

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal 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.

Thyroid hormone-induced expression of sonic hedgehog correlates with adult epithelial development during remodeling of the Xenopus stomach and intestine.

Sonic hedgehog (Shh) was isolated from the Xenopus laevis intestine as an early thyroid hormone (TH) response gene. To investigate possible roles of TH-upregulated expression of Shh during metamorphosis, we raised a polyclonal antibody against Xenopus Shh and immunohistochemically examined the relationship between Shh expression and the larval-to-adult intestinal remodeling at the cellular level. Our results indicate that the epithelial-specific expression of Shh in the intestine spatiotemporally correlates well with active proliferation and/or initial differentiation of the secondary (adult) epithelial primordia that originate from stem cells, but not with apoptosis of the primary (larval) epithelium. Given the similar transformations of the stomach during metamorphosis, we also analyzed Shh expression in this organ and found similar correlations in the stomach, although the position of the adult epithelial primordia and their final differentiation in the stomach are different from those in the intestine. Furthermore, we show here that Shh expression is organ-autonomously induced by TH and its correlation with the adult epithelial development is reproduced in vitro in both the intestine and the stomach. More importantly, addition of recombinant Shh protein to the culture medium results in developmental anomalies of both organs. However, differentiation of the adult epithelium is more severely inhibited by exogenous Shh in the intestine than in the stomach. These results suggest that TH-upregulated expression of Shh plays important roles in the postembryonic gastrointestinal remodeling, but its roles are at least partially different between the intestine and the stomach.

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.

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.

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.

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.

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.

Thyroid hormone regulation of Xenopus laevis metamorphosis: functions of thyroid hormone receptors and roles of extracellular matrix remodeling.

The regulatory effects of the thyroid hormone on amphibian metamorphosis is mediated by thyroid hormone receptors. Using Xenopus laevis as a model system, we and others have shown that the mRNA levels of thyroid hormone receptors and 9-cis retinoic acid receptors, which form the functional heterodimers with thyroid hormone receptors, are regulated temporally in a tissue-dependent manner so that high levels of their mRNAs are present in an organ when metamorphosis is occurring. By overexpressing thyroid hormone receptors, 9-cis retinoic acid receptors, or both into developing Xenopus embryos, we have shown that both thyroid hormone receptors and 9-cis retinoic acid receptors are required for mediating the effects of thyroid hormone on embryo development and precocious but specific regulation of the genes, which are normally regulated by thyroid hormone during metamorphosis. Analyses of the developmental expression of one class of thyroid hormone response genes, which encode extracellular matrix-degrading metalloproteinases, suggest that extra cellular remodeling plays an important role during tissue remodeling, including cell death (apoptosis) and cell proliferation and differentiation. This effect of extracellular matrix on cell behavior has been supported directly by in vitro primary cell culture experiments, in which intestinal epithelial cells undergo thyroid hormone-induced apoptosis, just like that during natural metamorphosis.

Age-related degeneration of the serotoninergic fibers in the zitter rat brain.

The serotonin neuron system was studied using immunohistochemical and neurochemical techniques in zitter mutant rats aged 1-14 months, which are characterized by abnormal metabolism of superoxides. The morphology of the serotoninergic neuron system and the serotonin level in the zitter rat were compared to those of age-matched Sprague-Dawley (SD) rats. Up to age 4 month, the density and distribution of serotoninergic fibers in the zitter rat brain were similar to those of control rats. However, several serotoninergic fibers with abnormal morphology, characterized by swollen varicosities, were observed in the cerebral cortex and caudate putamen of 6-month-old zitter rats. The density and distribution of these fibers in other regions of the brain were similar to those of control rats. The abnormal serotoninergic fibers increased in number and extended into other regions of the brain such as the thalamus, hippocampus, and vestibular nucleus. On the other hand, the density of normal serotoninergic fibers decreased throughout the brain of the 14-month-old zitter rat. Abnormal serotoninergic fibers have also been reported in the brain of normal older (24 months) SD rats. Neurochemical analysis revealed lower levels of serotonin, and 5-hydroxyindoleacetic acid, in all cortical areas (prefrontal, parietal, and occipital cortices), the caudate putamen, and the hippocampus of 12-month-old zitter rats. Levels differed significantly in the parietal cortex and hippocampus between the zitter and SD rats. Based on the morphological and neurochemical similarities, the present results suggest that age-related degeneration of serotoninergic fibers occurs in the zitter rat brain. Furthermore, degeneration of serotoninergic fibers appears to be induced by superoxide species. Thus, the zitter rat may provide a good model for studying the neurotoxic effects of superoxide species on the serotoninergic neuron system.

Thyroid hormone-induced apoptosis of larval cells and differentiation of pepsinogen-producing cells in the stomach of Xenopus laevis in vitro.

It is generally known that the anuran stomach begins to express pepsinogens (Pg) during metamorphosis. To clarify the mechanisms of differentiation of Pg-producing cells, we examined immunohistochemically the epithelial transformation from larval to adult form in Xenopus laevis stomach at the cellular level. At the beginning of metamorphic climax, concomitantly with the modification of the basement membrane, apoptotic cells labelled by TUNEL suddenly increased in number in the entire epithelium except for the primordia of adult epithelial cells in the basal region of larval glands. Subsequently, with the development of connective tissue, the adult epithelial cells actively proliferated and replaced the larval cells from the basal to the luminal region. Following the start of morphogenesis of adult glands, Pg-producing cells became differentiated in newly formed adult glands, but not in the adult surface epithelium. We then developed an organ culture system and examined effects of thyroid hormone (TH) on the differentiation of Pg-producing cells in X. laevis stomach in vitro. In the presence of TH, just as in spontaneous metamorphosis, Pg-producing cells differentiated from the adult epithelial primordia after the apoptosis of larval epithelial cells. In contrast, in the absence of TH, neither apoptotic larval cells no Pg-producing cells were detected. Therefore, we conclude that TH triggers organ-autonomously the entire process leading to the differentiation of Pg-producing cells in X. laevis stomach. In addition, the strict localization of Pg-producing cells in the adult glands both in vivo and in vitro suggests the correlation between the differentiation of Pg-producing cells and morphogenesis of the glands surrounded by the developed connective tissue.

Local disturbance of neuronal migration in the S-100beta-retarded mutant mouse.

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.

Temporal and spatial regulation of a putative transcriptional repressor implicates it as playing a role in thyroid hormone-dependent organ transformation.

Thyroid hormone (T3) induces both larval cell death and adult cell proliferation and differentiation during amphibian metamorphosis. We have previously isolated a bZip transcription factor (TH/bZip) as a T3 response gene in the metamorphosing Xenopus intestine. We demonstrate that the Xenopus TH/bZip gene is a direct T3-response gene and ubiquitously regulated by T3 in tadpoles. Developmental in situ hybridization analyses have shown that TH/bZip gene is regulated in a cell-type-specific manner that correlates with tissue transformation. In particular, it is found to be expressed in the larval intestinal epithelial cells prior to their apoptotic degeneration and in the proliferating adult cell types. However, the gene is repressed again upon adult cell differentiation. This regulation pattern mimics that of the thyroid hormone receptor (TR)beta genes. Since the TH/bZip gene is a direct T3-response gene, such a correlation suggests that TR beta may be involved in the regulation of the TH/bZip gene. More importantly, in situ hybridization reveals a strong spatiotemporal correlation of TH/bZip expression with the tissue-specific remodeling in the intestine, suggesting that TH/bZip gene may participate, depending on the cell types, in both inducing apoptosis and stimulating cell proliferation. A similar role has been reported for the proto-oncogene c-myc, another leucine-zipper-containing transcription factor, in tissue culture cell systems.

Temporal and spatial expression of an intestinal Na+/PO4 3- cotransporter correlates with epithelial transformation during thyroid hormone-dependent frog metamorphosis.

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.

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

Anteroposterior gradient of epithelial transformation during amphibian intestinal remodeling: immunohistochemical detection of intestinal fatty acid-binding protein.

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.

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.

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.

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.