The research in the Section on Molecular Morphogenesis focuses on the understanding of the molecular mechanism of amphibian metamorphosis. The control of this developmental process by thyroid hormone (TH) (Fig. 1) offers a unique paradigm in which to study genes that are important for postembryonic organ development. During metamorphosis, different organs undergo vastly different changes. Some, like the tail, undergoes complete resorption, while others, such as the limb, are developed de novo. Most of the larval organs persist through metamorphosis but are dramatically remodeled to function in a frog. For example, tadpole intestine in Xenopus laevis is a simple tubular structure consisting of primarily a single layer of primary epithelial cells. During metamorphosis, it is transformed into a multiply folded adult epithelium with elaborate connective tissue and muscles through specific cell death and selective cell proliferation and differentiation.
Figure 1. TH and metamorphosis- Thyroid hormone controls the transformation of the tadpole to a frog.
The wealth of knowledge from past research and the ability to manipulate amphibian metamorphosis both in vivo and in vitro in organ culture offer an excellent opportunity to 1) study the developmental function of thyroid hormone receptors (TRs) and the underlying mechanisms in vivo and 2) identify and functionally characterize genes which are critical for postembryonic organ development in vertebrates.
In the first area, we have demonstrated earlier in a reconstituted oocyte system and in developing embryos that heterodimers between TR and RXR (9-cis retinoic acid receptor) can function within a chromatin context to repress or activate gene expression in the absence or presence of TH, respectively (Fig. 2).
Figure 2. A model for transcriptional regulation by TRs- A model for transcriptional regulation by TRs. TR functions as a heterodimer with RXR. In the absence of TH, the heterodimer represses gene transcription through the recruitment of corepressor complexes containing the corepressor such as N-CoR or SMRT and histone deacetylase (HDAC). This leads to histone deacetylation and transcriptional repression. When TH is present, the corepressor complexes are released and coactivator complexes containing coactivators such as SRC, p300, and/or the DRIP/TRAP coactivator complex are recruited. The DRIP/TRAP complex may contact RNA polymerase directly to activate gene transcription. On the other hand, the SRC and p300 complexes may function through chromatin modification as they possess histone acetylase activity. In addition, transcriptional activation is associated with chromatin disruption, which may be due to the recruitment of chromatin remodeling machinery by TR/RXR.
Our studies have also revealed a role of histone acetylation changes in transcriptional regulation by TR/RXR. These have led us to hypothesize that TRs have dual functions during frog development. They activate gene expression during metamorphosis when TH is present. In premetamorphic tadpoles, they repress gene expression to prevent metamorphosis, thus ensuring a tadpole growth period. Our studies since have provided strong support and mechanistic insights for such a model. First, chromatin immunoprecipitation (ChIP) assay shows for the first time that TR/RXR are bound to thyroid hormone response elements (TREs) in the target genes in premetamorphic tadpoles. In support of a role of histone acetylation in gene regulation by TR/RXR, blocking deacetylase function with the drug trichostatin A (TSA) can activate TH response genes in the intestine of premetamorphic tadpoles, just like TH. Furthermore, treatment of premetamorphic tadpole with TH leads to increased histone acetylation at the TRE regions as the target genes are being activated. In addition, TH treatment causes a reduction in the association of corepressors with and an increase in the recruitment of coactivators to TH response genes. Together, these studies suggest a role of deacetylase complexes in gene repression by unliganded TR in premetamorphic tadpoles and acetyltransferase complexes in gene activation when TH is present.
To directly investigate the roles and mechanisms of TR function in development, we are employing the transgenesis technology in Xenopus to analyze TR function directly during metamorphosis. We have developed a double promoter construct approach (Fig. 3), where the gama-crystalline gene promoter drives the expression of GPF marker gene in the eyes of transgenic animals and another promoter drives the expression of the desired transgene. This allows us to rear and treat wild type and transgenic animals together throughout experiments and later easily identify transgenic animals by examining their eyes under a fluorescent microscope. Using such an approach, we have shown that over-expressing a dominant negative TR under the constitutive CMV promoter had no effect on tadpole development but inhibited TH-induced metamorphosis by inhibiting TH-response gene regulation through competition for target gene binding against endogenous TRs (Fig. 4).
Figure 3. Transgenic animals (Tg)- Thransgenic animals (Tg) generated with a double promoter construct (g- crystalline promoter-GFP and heat inducible promoter- GFP) had green eyes and upon heat shock, expressed GFP throughout the animals. In contrast, non-transgenic animasl (nTg), which had no GFP in the eyes, had no GFP expression anywhere either before or after heat shock.
Figure 4. Transgenic expression of a dominant negative TR (dnTR)- Transgenic expression of a dominant negative TR (dnTR) inhibits TH-induced morphological changes. Wild type (WT) or transgenic tadpoles expressing dnTR under control of the CMV promoter (dnTR) were treated at premetamorphic stage 54 with or without TH for 3 days and were examined for changes in external morphology.
Since the dnTR does not bind TH, this leads to the retention of corepressors at the target genes even when TH is present, thereby repressing TH-inducible genes. More recently, we have shown overexpression of a dominant negative coactivator (dnSRC3) also inhibits both TH-induced as well as natural metamorphosis (Fig. 5), demonstrating a critical role of coactivators in gene activation and metamorphosis in vivo. Our current work continues on these studies and makes use of cDNA arrays to analyze gene expression profiles induced by TH during metamorphosis.
Figure 5. Transgenic tadpoles (Tg1 and Tg2)Transgenic tadpoles (Tg1 and Tg2) overexpressing a dominant negative form of the steroid receptor coactivator 3 (SRC3) failed undergo the tail resorption during natural development even after 4 months while wild type animals (WT) completed metamorphosis in about 2 months.
In the second area, we have identified many TH-response genes during intestinal remodeling. Several of them encode matrix metalloproteinases (MMPs), which are extracellular enzymes capable of digesting various ECM components. Our earlier studies have led us to propose that the MMP stomelysin-3 (ST3) is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior. By using intestinal organ cultures and a function-blocking antibody against the catalytic domain of ST3, we have demonstrated that blocking ST3 function inhibits TH-induced apoptosis of larval intestinal epithelial cells and the invasion of the proliferating adult epithelial cells into the connective tissue. These effects are accompanied by an inhibition in the remodeling of the basal lamina or basement membrane, the ECM that separate the connective tissue and the epithelium, supporting the argument that ST3 is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior. The importance of MMP function in larval epithelial cell death is also supported by the ability of a synthetic MMP inhibitor to inhibit TH-induced apoptosis in vitro. To directly investigate the roles of MMPs in developing animals, we have again employed the transgenic approach to express the wild type ST3 and catalytically inactive ST3 (ST3m) under a heat shock-inducible promoter in Xenopus tadpoles since ubiquitous expression of MMPs leads to embryonic death. When tadpoles reach premetamorphic stages, they are subjected to heat shock treatment to induce the transgene expression. While no gross morphological changes have been observed due to transgene expression, analysis of the intestine revealed that larval epithelial cell death was prematurely induced by ST3 but not by ST3m (Fig. 6), accompanied by ECM remodeling. These results complement our in vitro studies to show the ability of ST3 to regulate cell fate during metamorphosis.
Figure 6. Transgenic overexpression of stromelysin-3 (ST3)Transgenic overexpression of stromelysin-3 (ST3) but not catalytically inactive mutant ST3 (ST3m) leads to intestinal epithelial cell death. Transgenic tadpoles containing ST3 or ST3m transgene and wild type tadpoles were reared to premetamorphic stage 54 and subjected to daily heat shock to induce transgene expression. Four days after the initial heat shock treatment, the intestine was isolated for TUNEL assay to detect apoptotic cells (brown). Ty, typhlosole; Lu, lumen; Ep, epithelium. Bar: 100 mm.
Toward understanding the molecular basis of ST3 action, we have recently identified laminin receptor (LR) as a ST3 substrate by using yeast-two-hybrid screening. We have mapped the two cleavage sites of ST3 within LR to be located in between the transmembrane domain and the laminin binding sequence, suggesting that LR cleavage by ST3 may alter cell-ECM interaction. More importantly, LR fragments of sizes expected from cleavage by ST3 are present in the tadpole intestine during metamorphic climax when ST3 is highly expressed and transgenic expression of ST3 in premetamorphic tadpoles leads to the formation of such products. These results together suggest that ST3 cleaves LR in vivo during metamorphosis, which in turn may affect cell-ECM interaction in cell fate determination and tissue morphogenesis. Currently, we are continuing to investigate the role of ST3 in metamorphosis and its associated mechanisms. We are also investigating other MMPs in an effort to determine if different MMPs have similar or different roles during metamorphosis.
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