The Unit on Molecular Morphogenesis is exploring molecular mechanisms in amphibian metamorphosis. The control of metamorphosis by thyroid hormone (TH) 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, undergo complete resorption while others, such as the limb, are developed de novo. Most larval organs persist through metamorphosis, but, for the frog, they are dramatically remodeled. For example, tadpole intestine in Xenopus laevis is a simple tubular structure consisting primarily of a single layer of primary epithelial cells. During metamorphosis, specific cell death and selective cell proliferation and differentiation transform the intestine into a multiply folded adult epithelium with elaborate connective tissue and muscles. 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 study the developmental function of thyroid hormone receptors (TRs) and the underlying mechanisms in vivo and to identify and functionally characterize genes that are critical for postembryonic organ development in vertebrates.

Function and Mechanism of Gene Regulation by TR during Frog Development
Amano, Jones, Rouse, Sachs,a Shi in collaboration with Wadeb
We have proposed a dual TR function model that is based on our earlier studies of the oocyte and developing embryos and tadpoles. According to the model, the heterodimers between TR and RXR (9-cis retinoic acid receptor) activate gene expression during metamorphosis when TH is present, while, in premetamorphic tadpoles, they repress gene expression to prevent metamorphosis, thus ensuring a tadpole growth period. Our studies have since provided strong support for and mechanistic insights into 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, we have demonstrated that blocking the 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 the histone deacetylase Rpd3 with TH response genes. Together, these studies suggest a role for the deacetylase complex in gene repression by unliganded TR in premetamorphic tadpoles (Sachs et al., 2000, 2001).

Interestingly, our studies with TSA also showed that, during metamorphosis, histone deacetylases are required at a step or steps downstream of gene activation by TH-bound TR. Thus, histone deacetylases plays a role at two distinct steps during frog development (Sachs et al., 2001). To investigate how histone deacetylases participate in development, we have begun to characterize frog histone deacetylase complexes. To isolate TR complexes that contain the TR-interaction corepressor N-CoR, we made use of the fact that the frog egg contains all necessary components for gene regulation. We were able to purify three N-CoR complexes (Jones et al., 2001), two of which possess histone deacetylase activity, and one of which contains Sin3, Rpd3, and RbAp48. They are the first such complexes to be purified, even though interactions among Sin3, Rpd3, and RbAp48 have been known for some time. The second complex contains a Sin3-independent histone deacetylase while the third lacks histone deacetylase activity. Our complexes differ from similar mammalian complexes reported by a few other laboratories, demonstrating the complexity of gene repression mediated by N-CoR. Immunoprecipitation studies show that N-CoR binds to unliganded TR expressed in the frog oocyte, confirming that N-CoR complexes are involved in repression by unliganded TR. The failure to bring precipitate Sin-3 by anti-TR antibody suggests that the first complex is unlikely to be involved in gene repression by unliganded TR. Our current studies aim at identifying the components of the complexes and investigating which complex is involved in gene repression by TR during frog development. We are also employing the recently developed transgenesis technology in Xenopus to analyze TR function directly during metamorphosis. Preliminary data showed that overexpressing a dominant negative TR in the tadpole intestine had no effect on tadpole development but interfered with adult epithelial morphogenesis, consistent with the role of TR in mediating the effects of TH during tissue transformation.

Roles of Matrix Metalloproteinases during TH-Induced Tissue Remodeling
Amano, Damjanovski,c Shi in collaboration with Ishizuya-Okad
In a second area of research, we have been focusing on TH-response genes encoding matrix metalloproteinases (MMPs), which are extracellular enzymes capable of digesting various extracellular matric (ECM) components. Our earlier studies 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, we have now shown that ST3 expression is associated with ECM remodeling and cell death in TH-induced intestinal remodeling in vitro. Studies with primary cell cultures of the intestine have shown that ECM inhibits TH-induced larval epithelial cell death in vitro. These observations prompted us to investigate whether ST3 is required for epithelial transformation in organ cultures. By using 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, which is the ECM that separates the connective tissue from the epithelium. The results support the argument that ST3 is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior (Ishizuya-Oka et al., 2000).

To investigate directly the roles of MMPs in developing animals, we are employing the transgenic approach to express wild-type and mutant MMPs in Xenopus embryos and tadpoles. In our initial study, we overexpressed Xenopus MMPs stromelysin-3 (ST3) and collagenases-4 (Col4) under the control of a ubiquitous promoter and observed that embryos with overexpressed ST3 or Col4, but not the control green fluorescent protein (GFP), died in a dose-dependent manner during late embryogenesis. The specificity of this embryonic lethal phenotype was confirmed by the failure of a catalytically inactive mutant of ST3 to affect development. Finally, overexpression of a MMP of the mammalian membrane type also led to late embryonic lethality in Xenopus embryos, suggesting that membrane type-MMPs have functions in vivo for ECM remodeling, in addition to being activators of other proMMPs. These data, together with the developmental expression of several MMPs during Xenopus development, suggest that MMPs play important roles during mid- to late embryogenesis and that proper regulation of MMP genes is critical for tissue morphogenesis and organogenesis (Damjanovski et al., 2001). The data also suggest that alteration in MMP expression during metamorphosis may alter TH-dependent tissue remodeling process. We have therefore begun to investigate MMP function during metamorphosis by using tissue-specific promoters to drive the expression of MMPs during metamorphosis.

Mechanism of Transcriptional Regulation of HIV LTR by TR
Hsia, Shi
In a separate study supported by an NIH Intramural AIDS Targeted Antiviral Program (IATAP) grant, we have investigated the roles of TR/RXR in the regulation of HIV LTR, which controls the transcription of the AIDS virus. Using the frog oocyte as an in vivo system that allows assembly of the LTR into chromatin through a replication-coupled chromatin assembly process, thus mimicking somatic cells, we have demonstrated that unliganded TR/RXR heterodimers repress LTR in the context of chromatin, whereas the addition of T3 relieves the repression and further activates the promoter (Hsia et al., 2001). To investigate how TR regulates HIV LTR, we have determined the binding of TR to the putative TREs in the LTR in vivo by using ChIP assay. The results demonstrated for the first time that both TR, most likely as a homodimer, and TR/RXR heterodimers are bound constitutively to the putative TREs in the LTR in chromatin and that transcriptional repression by unliganded TR leads to a decrease in histone acetylation levels at the LTR. Furthermore, we have found that the activation of the promoter by TH causes chromatin disruption. The results suggest that chromatin remodeling plays an important role in LTR regulation by TH in vivo.