No abstract

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.

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.

No abstract

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.

During amphibian metamorphosis, the larval tissues/organs rapidly degenerate to adapt from the aquatic to the terrestrial life. At the cellular level, a large quantity of apoptosis occurs in a spatiotemporally-regulated fashion in different organs to ensure timely removal of larval organs/tissues and the development of adult ones for the survival of the individuals. Thus, amphibian metamorphosis provides us a good opportunity to understand the mechanisms regulating apoptosis. To investigate this process at the molecular level, a number of thyroid hormone (TH) response genes have been isolated from several organs of Xenopus laevis tadpoles and their expression and functional analyses are now in progress using modern molecular and genetic technologies. In this review, we will first summarize when and where apoptosis occurs in typical larva-specific and larval-to-adult remodeling amphibian organs to highlight that the timing of apoptosis is different in different tissues/organs, even though all are induced by the same circulating TH. Next, to discuss how TH spatiotemporally regulates the apoptosis, we will focus on apoptosis of the X. laevis small intestine, one of the best characterized remodeling organs. Functional studies of TH response genes using transgenic frogs and culture techniques have shown that apoptosis of larval epithelial cells can be induced by TH either cell-autonomously or indirectly through interactions with extracellular matrix (ECM) components of the underlying basal lamina. Here, we propose that multiple intra- and extracellular apoptotic pathways are coordinately controlled by TH to ensure massive but well-organized apoptosis, which is essential for the proper progression of amphibian metamorphosis.

No abstract

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