Sashko Damjanovski

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.

Spatial and temporal regulation of collagenases-3, -4, and stromelysin -3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis

ABSTRACTMatrix 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.

Differential regulation of three thyroid hormone-responsive matrix metalloproteinase genes implicates distinct functions during frog embryogenesis

Matrix metalloproteinases (MMPs) are a family of Zn2+‐dependent extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix (ECM). They are expressed in developmental and pathological processes such as postlactation mammary gland involution and tumor metastasis. Relatively few studies have been carried out to investigate the function of MMPs during embryogenesis and postembryonic organ development. Using Xenopus development as a model system, we and others have previously isolated three MMP genes as thyroid hormone response genes. They have distinct temporal and organ‐specific regulations during thyroid hormone‐dependent metamorphosis. We demonstrate here that three MMPs—stromelysin‐3 (ST3), collagenases‐3 (Col3), and collagenases‐4 (Col4)—also have distinct spatial and temporal expression profiles during embryogenesis. Consistent with earlier suggestions that ST3 is a direct thyroid hormone response gene whereas Col3 and Col4 are not, we show that precocious overexpression of thyroid hormone receptors in the presence of thyroid hormone lead to increased expression of ST3, but not Col3. Furthermore, our whole‐mount in situ hybridizations reveal a tight but distinct association of individual MMPs with tissue remodeling in different regions of the animal during embryogenesis. These results suggest that ST3 is likely to play a role in ECM remodeling that facilitate apoptotic tissue remodeling or resorption, whereas Col3 and Col4 appear to participate in connective tissue degradation during development.—Differential regulation of three thyroid hormone‐responsive matrix metalloproteinase genes implicates distinct functions during frog embryogenesis. Damjanovski, S., Puzianowska‐Kuznicka, M., Ishuzuya‐Oka, A., Shi, Y.‐B. FASEB J. 14, 503–510 (2000)

Overexpression of matrix metalloproteinases leads to lethality in transgenic Xenopus laevis: implications for tissue-dependent functions of matrix metalloproteinases during late embryonic development.

The extracellular matrix (ECM) functions as the structural support of cells and as a medium for cell–cell interactions. It is understood to play critical roles in development. ECM remodeling is mediated largely through the action of matrix metalloproteinases (MMPs), a family of Zn2+‐dependent proteases capable of degrading various proteinaceous components of the ECM. MMPs are expressed in many developmental and pathologic processes. However, few studies have been carried out to investigate the function of MMPs during embryogenesis and postembryonic organogenesis. By using Xenopus development as a model system, we have previously shown that several MMP genes are expressed from neurulation to the completion of embryogenesis in distinct tissues/organs, suggesting that ECM remodeling during mid‐ to late embryogenesis occurs in an organ‐specific manner. By using the recently developed transgenic technology for Xenopus laevis, we overexpressed Xenopus MMPs stromelysin‐3 (ST3) and collagenase‐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 mammalian membrane type‐MMP 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 pro‐MMPs. 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.

Transcriptional repression by XPc1, a new Polycomb homolog in Xenopus laevis embryos, is independent of histone deacetylase.

ABSTRACT The Polycomb group (Pc-G) genes encode proteins that assemble into complexes implicated in the epigenetic maintenance of heritable patterns of expression of developmental genes, a function largely conserved from Drosophila to mammals and plants. The Pc-G is thought to act at the chromatin level to silence expression of target genes; however, little is known about the molecular basis of this repression. In keeping with the evidence that Pc-G homologs in higher vertebrates exist in related pairs, we report here the isolation of XPc1, a second Polycomb homolog in Xenopus laevis. We show that XPc1 message is maternally deposited in a translationally masked form in Xenopus oocytes, with XPc1 protein first appearing in embryonic nuclei shortly after the blastula stage. XPc1 acts as a transcriptional repressor in vivo when tethered to a promoter in Xenopus embryos. We find that XPc1-mediated repression can be only partially alleviated by an increase in transcription factor dosage and that inhibition of deacetylase activity by trichostatin A treatment has no effect on XPc1 repression, suggesting that histone deacetylation does not form the basis for Pc-G-mediated repression in our assay.

Ectopic Expression of SPARC in Xenopus Embryos Interferes with Tissue Morphogenesis: Identification of a Bioactive Sequence in the C-terminal EF Hand

SPARC is a matricellular Ca2+-binding glycoprotein that exhibits both counteradhesive and antiproliferative effects on cultured cells. It is secreted by cells of various tissues as a consequence of morphogenesis, response to injury, and cyclic renewal and/or repair. In an earlier study with Xenopus embryos we had shown a highly specific and regulated pattern of SPARC expression. We now show that ectopic expression of SPARC before its normal embryonic activation produces severe anomalies, some of which are consistent with the functions of SPARC proposed from studies in vitro. Microinjection of SPARC RNA, protein, and peptides into Xenopus embryos before endogenous embryonic expression generated different but overlapping phenotypes. (a) Injection of SPARC RNA into one cell of a two-cell embryo resulted in a range of unilateral defects. (b) Precocious exposure of embryos to SPARC by microinjection of protein into the blastocoel cavity was associated with certain axial defects comparable to those obtained with SPARC RNA. (c) SPARC peptides containing follistatin-like and copper-binding sequences were without obvious effect, whereas SPARC peptide 4.2, corresponding to a disulfide-bonded, Ca2+-binding domain, was associated with a reduction in axial structures that led eventually to complete ventralization of the embryos. Histological analysis of ventralized embryos indicated that the morphogenetic events associated with gastrulation might have been inhibited. Microinjection of other Ca2+-binding glycoproteins, such as osteopontin and bone sialoprotein, resulted in phenotypes that were unique. We probed further the structural correlates of this region of SPARC in the context of tissue development. Co-injection of peptide 4.2 with Ca2+ or EGTA, and injection of peptide 4.2K (containing a mutated consensus Ca2+-binding sequence), demonstrated that the developmental defects associated with peptide 4.2 were independent of Ca2+. However, the disulfide bridge in this region of SPARC was found to be critical, as injection of peptide 4.2AA, a mutant lacking the cystine, generated no axial defects. We have therefore shown for the first time in vivo that the temporally inappropriate presence of SPARC is associated with perturbations in tissue morphogenesis. Moreover, we have identified at least one bioactive region of SPARC as the C-terminal disulfide-bonded, Ca2+-binding loop that was previously shown to be both counteradhesive and growth-inhibitory.

Role of ECM Remodeling in Thyroid Hormone-Dependent Apoptosis during Anuran Metamorphosis

Abstract: 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.

Overexpression of the tissue inhibitor of metalloproteinase-3 during Xenopus embryogenesis affects head and axial tissue formation

ABSTRACTTissue inhibitors of metalloproteinases (TIMPs) modulate extracellular matrix remodeling during embryonic development and disease. TIMP-3 expression was examined during Xenopus laevis embryogenesis: TIMP-3 transcripts detected in the maternal pool of RNA increased at the mid-blastula transition, decreased dramatically during gastrulation and increased again during neurulation and axis elongation. Interestingly, the decrease during gastrulation was not seen in LiCl treated (dorsalized) embryos. Whole mount in situ hybridization of TIMP-3 using DIG-labeled RNA probes demonstrated that the transcripts were present in all dorsal tissues during embryogenesis, but were prominent only in head structures starting at stage 35. Overexpression of TIMP-3 through transgenesis and RNA injections led to developmental abnormalities and death. Both overexpression strategies resulted in post-gastrulation perturbation including those to neural and head structures, as well as truncated axes. However, RNA injections resulted in more severe early defects such as failure of neural tube closure, and transgenesis caused truncated axes and head abnormalities. No transgenic embryo expressing TIMP-3 survived past stage 40.