Identification and characterization of a novel collagenase in Xenopus laevis: possible roles during frog development.

Matrix metalloproteinases (MMPs) participate in extracellular matrix remodeling and degradation and have been implicated in playing important roles during organ development and pathological processes. Although it has been hypothesized for > 30 years that collagenase activities are responsible for collagen degradation during tadpole tail resorption, none of the previously cloned amphibian MMPs have been biochemically demonstrated to be collagenases. Here, we report a novel matrix metalloproteinase gene from metamorphosing Xenopus laevis tadpoles. In vitro biochemical studies demonstrate that this Xenopus enzyme is an interstitial collagenase and has an essentially identical enzymatic activity toward a collagen substrate as the human interstitial collagenase. Sequence comparison of this enzyme to other known MMPs suggests that the Xenopus collagenase is not a homologue of any known collagenases but instead represents a novel collagenase, Xenopus collagenase-4 (xCol4, MMP-18). Interestingly, during development, xCol4 is highly expressed only transiently in whole animals, at approximately the time when tadpole feeding begins, suggesting a role during the maturation of the digestive tract. More importantly, during metamorphosis, xCol4 is regulated in a tissue-dependent manner. High levels of its mRNA are present as the tadpole tail resorbs. Similarly, its expression is elevated during hindlimb morphogenesis and intestinal remodeling. In addition, when premetamorphic tadpoles are treated with thyroid hormone, the causative agent of metamorphosis, xCol4 expression is induced in the tail. These results suggest that xCol4 may facilitate larval tissue degeneration and adult organogenesis during amphibian metamorphosis.

Cloning and characterization of the ribosomal protein L8 gene from Xenopus laevis.

A full-length cDNA clone encoding the Xenopus laevis ribosomal protein L8 has been isolated and sequenced. The cDNA has an open reading frame of 257 amino acids. The deduced amino acid sequence is rich in basic residues and extremely homologous to the rat L8 protein. The Xenopus gene is ubiquitously expressed at fairly constant levels during development, consistent with its role as a house-keeping gene.

Tissue-dependent developmental expression of a cytosolic thyroid hormone protein gene in Xenopus: its role in the regulation of amphibian metamorphosis.

We have cloned the cDNA encoding the Xenopus laevis homolog of mammalian cytosolic thyroid hormone binding protein (CTHBP). We found that while its mRNA level varies little in whole animals during development, the expression of CTHBP is inversely correlated with tissue-specific transformations during metamorphosis. A high level of its mRNA was observed in the tail of premetamorphic tadpoles. However, the expression is dramatically repressed with the onset of rapid tail resorption. In the hindlimb, the expression of CTHBP is very low during morphogenesis. Subsequently, its expression continuously increases during the period of limb growth. In contrast, a low level of CTHBP expression was detected in the intestine throughout metamorphosis. These results suggest that CTHBP could function to modulate the metamorphic process by regulating the level of intracellular thyroid hormones.

Characterization of the Xenopus homolog of an immediate early gene associated with cell activation: sequence analysis and regulation of its expression by thyroid hormone during amphibian metamorphosis.

The complex transformation of a tadpole to a frog during amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previously isolated many genes which are up-regulated by T3 in the intestine of Xenopus laevis tadpoles. We have now cloned a full-length cDNA for one such gene (IU12). Sequence analysis shows that the IU12 cDNA encodes a plasma membrane protein with 12 transmembrane domains and homologous to a mammalian gene associated with cell activation and organ development. Similarly, we have found that IU12 is activated during intestinal remodeling when both cell death and proliferation take place. Furthermore, IU12 is an early T3-response gene and its expression in the intestine during T3-induced metamorphosis mimics that during normal development. These results argue for a role of IU12 in the signal transduction pathways leading to intestinal metamorphosis.

Unique organization and involvement of GAGA factors in transcriptional regulation of the Xenopus stromelysin-3 gene.

Expression of the matrix metalloproteinase (MMP) gene stromelysin-3 ( ST3 ) has been shown to be tightly associated with cell migration and apoptosis inmammals and amphibians. This contrasts with most other MMP genes. We demonstrate here that the Xenopus ST3 gene also has a structure distinct from other MMP genes, with its C-terminal half (the hemopexin domain) encoded by 4 instead of 6 exons, as in other MMP genes. Our primer extension analysis reveals the existence of two transcription start sites and at least one is needed for transcription of the promoter in transient transfection assays. Furthermore, our deletion analysis has demonstrated a requirement for at least one GAGA factor binding site for promoter function. In vitro DNA binding and mutational studies have provided strong evidence for the participation of GAGA or GAGA-like factors in transcriptional regulation of the frog ST3 gene. This contrasts with regulation of the human ST3 promoter. These results suggest that the ST3 gene evolved prior to most other metalloproteinase genes and uses distinct regulation pathways to achieve similar expression profiles and serve similar functions in mammals and amphibians.

Transcription from the thyroid hormone-dependent promoter of the Xenopus laevis thyroid hormone receptor betaA gene requires a novel upstream element and the initiator, but not a TATA Box.

The thyroid hormone receptor (TR) beta genes in Xenopus laevis are regulated by thyroid hormone in all organs of an animal during metamorphosis. This autoregulation appears to be critical for systematic transformations of different organs as a tadpole is transformed into a frog. To understand this autoregulation, we have previously identified a thyroid hormone response element in the hormone-dependent promoter of the X. laevis TRbetaA gene. We report here the detailed characterization of the promoter. We have now mapped the transcription start site and demonstrated the existence of an initiator element at the start site critical for promoter function. More important, our deletion and mutational experiments revealed a novel upstream DNA element that is located 125 base pairs upstream of the start site and that is essential for active transcription from the promoter. Promoter reconstitution experiments showed that this novel element does not function as an enhancer, but acts as a core promoter element, which, together with the initiator, directs accurate transcription from the promoter. Finally, we provide evidence for the existence of a protein(s) that specifically recognizes this element. Our studies thus demonstrate that the TRbetaA promoter has a unique organization consisting of an initiator and a novel upstream promoter element. Such an organization may be important for the ubiquitous but tissue-dependent temporal regulation of the gene by thyroid hormone during amphibian metamorphosis.

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.