Roles of Matrix Metalloproteinases and ECM Remodeling during Thyroid Hormone-Dependent Intestinal Metamorphosis in Xenopus laevis

Abstract

Intestinal metamorphosis in anurans is an excellent model system for studying post-embryonic tissue remodeling and organ development in vertebrates. This process involves degeneration of the larval or tadpole form of its primary functional tissue, the simple tubular epithelium through apoptosis or programmed cell death. Concurrently, adult epithelial stem cells, whose origin remains to be determined, proliferate and differentiate to form a multiply folded, complex adult epithelium. The connective tissue and muscles also develop extensively during this period. Like all other changes during amphibian metamorphosis, intestinal remodeling is controlled by thyroid hormone (TH). Isolation and characterization of genes that are regulated by TH has implicated the involvement of matrix metalloproteinases (MMPs) in the remodeling of the extracellular matrix (ECM) during intestinal metamorphosis. Here we will review some studies, almost exclusively in Xenopus laevis, that support a role of MMPs, particularly stromelysin 3, and ECM remodeling in regulating cell fate and tissue morphogenesis.

Introduction

Anuran development takes place in two phases. Its embryogenesis produces a free living, aquatic, and often herbivorous tadpole. After a growth period, the tadpole undergoes metamorphosis that changes essentially every organ of the animal to produce, in the vast majority of cases, a terrestrial carnivorous frog.¹ During this period, some organs degenerate completely while others develop de novo. The majority of the organs are partially, yet dramatically, transformed in order to function in the adult frog. One such organ is the intestine. The tadpole intestine is a simple tubular organ made of mostly a monolayer of larval epithelial cells surrounded by little connective tissue or muscles. In Xenopus laevis, a single epithelial fold, the typhlosole, exists in the anterior half, where connective tissue is abundant (Fig. 1).² As metamorphosis takes place, the tadpole stops feeding and the larval epithelial cells of the intestine undergo apoptosis or programmed cell death. Adult epithelial cells, which are probably derived from the larval epithelial cells, begin to appear as islets or nests of small cell clusters and proliferate rapidly,^2,3,4 although a recent study on limited stages failed to detect such islets.⁵ Concurrently, cells of the connective tissue and muscles also proliferate. Toward the end of metamorphosis, these adult cells differentiate to form a multiply folded intestinal epithelium surrounded by elaborate connective tissue and thick muscle layers. In the adult epithelium, the stem cells are located in the troughs of the folds, equivalent to the crypts in mammalian intestine, while fully differentiated epithelial cells are present in the crests of the folds, equivalent to the villi in mammals.

Figure 1.

Remodeling of the intestine during Xenopus laevis metamorphosis. Cross sections of the intestine from animals at premetamorphic (stage 54), metamorphic climax (stage 60), and the end of metamorphosis (stage 66) ⁶⁸ were stained with pyronin Y and methyl green to show the morphology. The strong pyronin Y signals observed at metamorphic climax indicate islets of proliferating adult epithelial cells. Scale bars are 100 µm.

While these changes are complex, they are controlled by essentially a single hormone, the thyroid hormone (TH), just like all other processes during metamorphosis.^1,6–8,10 The lack of endogenous synthesis of TH or exogenously added TH will enable the tadpole to grow into a giant animal without metamorphosis, while addition of TH to premetamorphic tadpoles can induces precocious metamorphosis. TH can function through nongenomic pathways and regulate gene transcription through thyroid hormone receptors (TRs). Recent studies by us and others have shown that TR is both necessary and sufficient for the effects of TH during amphibian metamorphosis, at least under the experimental conditions.^11–13 Thus, isolation and characterization of genes regulated by TR during metamorphosis offers an effective means to understand the molecular mechanisms governing postembryonic organ remodelling. Through this and other approaches, a number of genes encoding matrix metalloproteinases (MMPs) have been found to be regulated either directly or indirectly by TR during metamorphosis.^14,15–24 Here, we will review some of the expression and function studies that support the involvement of MMPs in extracellular matrix (ECM) remodelling and tissue morphorgenesis, with a particular focus on the role of the MMP stromelysin 3 (ST3) during intestinal remodelling.

Cell-ECM and Cell-Cell Interactions During Intestinal Metamorphosis

There are four major tissue types in the intestine: the epithelium, connective tissue, muscles, and the nerves. The first three are physically separated but adjacent to each other (Fig. 1) while the nerves are mostly localized in the connective tissue near the circular muscle and between the circular and longitudinal muscles. As indicated above, in the tadpoles, the epithelium is the predominant tissue of the intestine while the connective tissue and muscle layers are thin except for the typhlosole. The epithelial cells are separated from the connective tissue by a special ECM, the basement membrane or basal lamina. In the intestine of premetamorphic tadpole and postmetamorphic frogs, this ECM is a thin but continuous layer. During metamorphosis, as the intestine shortens (by as much as 10 fold in the small intestine), it becomes much thicker but appears to be more permeable.^2,25,26 (also see Fig. 4 below). This permeability is reflected by (1) the migration of macrophages from the connective tissue across the basal lamina to the epithelium, where they engulf the apoptotic bodies from the dying epithelial cells,²⁷ and (2) frequently observed contacts between proliferating progenitor cells of the adult epithelium and fibroblasts of the connective tissue.²⁶ These findings suggest that ECM remodelling plays a role in intestinal remodelling. Such a role is also supported by studies using primary cultures of tadpole intestinal cells. When the epithelial and fibroblastic cells were isolated from premetamorphic tadpole intestine and cultured in vitro on plastic dishes, TH treatment led to enhanced DNA synthesis in both cell types and at same time caused the epithelial cells, but not the fibroblasts, to undergo apoptosis.^28,29 This resulted in reduction in epithelial cells but enhanced proliferation of the fibroblasts, mimicking the events during metamorphosis. Interestingly, coating the dishes with ECM proteins such as laminin, collagen, and fibronectin inhibited TH-induced epithelial cell death in vitro.²⁸ While these results do not necessarily indicate that these ECM proteins play a protective role against TH induced cell death in vivo, they do suggest that ECM remodelling can influence epithelial cell response to TH.

Figure 4.

ST3 is required for ECM remodeling during TH-induced intestinal remodeling. Intestinal fragments from tadpoles at stage 57 were cultured in vitro in the presence or absence of TH and in presence or absence of indicated serum. After three days of culturing, cross sections were analyzed with an electron microscope to examine the interface between the epithelium (E) and connective tissue. Note that the basal lamina (BL) became multiply folded in the presence of TH (right panel) whereas the basal lamina remained as a thin continuous layer in the absence of TH or in the presence of both TH and anti-ST3 antibody (see ref. 59 for details).

ECM can influence cell fate through direct interactions with cells through cell surface receptors such as integrins or by altering the availability of extracellular signalling molecules such as growth factors.^{30 33} In addition, it can also affect cells indirectly by regulating cell-cell interactions. Cell-cell interactions, in particular, those between the epithelial cells and fibroblasts, are known to be important for intestinal metamorphosis. During metamorphosis, extensive contacts exist between the fibroblasts and proliferating adult epithelial cells prior to their differentiation to form the multiply folded epithelium.²⁶ These contacts extend from the epithelium to the connective tissue across the basal lamina that has been modified, i.e., thicker but apparently more permeable compared to the intestinal basal lamina in pre or post metamorphic animals. More importantly, in vitro organ culture studies have demonstrated an interdependence of epithelium and connective tissue for the connective tissue remodelling and adult epithelium development in the presence of TH.^34,35 The molecular basis for this is yet unknown but a few factors may contribute. First, both tissues contribute to the synthesis of the ECM that separates them. Second, both are induced by TH to synthesize signalling molecules, e.g., the sonic hedgehog gene in the epithelial cells and BMP 4 in fibroblasts.^36,37 Finally, as described below, the induction of MMPs by TH in the fibroblasts will lead to modifications of the ECM.

Regulation of MMP Genes

MMPs have long been implicated in amphibian metamorphosis. In fact, the first MMP, collagenase, was isolated as a collagen degradation enzyme from the resorbing tadpole tail.³⁸ Since then, a large family of MMPs have been cloned and characterized in vertebrates, especially in mammals. MMPs are membrane bound or extracellular, Zn²⁺ dependent proteases that are capable of cleaving proteinaceous components of the ECM.^39–44 They are normally synthesized as preenzymes, secreted as proenzymes, and eventually activated extracellularly into mature enzymes.^{45 49} The exceptions are membrane type-MMPs (MT-MMP) and ST3, which are matured intracellularly and transported to the plasma membrane (MT-MMPs) or secreted (ST3) as mature enzymes.^{50 53} In addition to ECM proteins, the mature MMPs can also cleave a number of other proteins including cell surface receptors and growth factors, etc.^{42,44,52,54,55}

A role of MMPs in intestinal remodelling was suggested first from a subtractive screening for genes regulated by TH, which isolated ST3 as an early TH response gene in the intestine of tadpoles.¹⁴ Subsequently, several other MMPs, including collagenase 3 and 4, gelatinase A (GelA), and membrane type 1-MMP (MT1-MMP), have also been shown to be upregulated during intestinal remodelling in Xenopus laevis.^15,24,21,56 Interestingly, among these MMPs, only ST3 has been shown directly upregulated by TH through TRs as its upregulation by TH is independent of new protein synthesis.^14,15,21 On the other hand, all the other MMPs are upregulated by TH relatively slowly, requiring more than 1 days of TH treatment of premetamorphic tadpoles (Fig. 2). Consistently, ST3 is the first one to be upregulated during natural intestinal remodelling with its mRNA levels upregulated before the onset of epithelial cell death in the intestine (by stage 58), while the other MMPs are upregulated at or after stage 60 when cell death has begun. These results suggest that ST3 may play a role in cell fate regulation by facilitating TH-induced larval epithelial cell death, while the other MMPs may play a role after cell death has begun, such as removal or remodelling the ECM near the dying cells.

Figure 2.

Regulation of MMP expression by TH in Xenopus laevis . Tadpoles at premetamorphic stage, i.e., stage 54, were treated with 5 nM T3 (3, 5, 3′ triiodothyronine, the more active form of the two naturally occurring TH) to induce metamorphosis. The animals were sacrificed and RNA was isolated from the intestines after 0 to 4 days of T3 treatment and used for total RNA isolation with TRIzol reagent. The RNA was made DNA free and subjected to RT-PCR to analyze the expression of the MMPs with the ribosome protein L8 (rpL8) gene as the control.

Spatially, ST3 mRNA and protein have been shown to be expressed in all fibroblasts of the connective tissue underlying the larval epithelium,^15,16 again supporting a role in ECM remodelling and/or cell death. In situ hybridization has shown that the mRNAs of the other MMPs are also expressed in the connective tissue of the intestine during metamorphosis, although the expression of collagenase 3 and 4 appears to be more sporadic, probably due to lower levels of expression.^17,56 Interestingly, MT1-MMP has been shown to participate in the in vivo activation of pro-GelA in mammalian tissue culture cells and during mouse development.^53,57 During Xenopus laevis metamorphosis, MT1-MMP is coexpressed with GelA developmentally and spatially in the metamorphosing intestine and tail.⁵⁶ The only exception is that MT1-MMP but not GelA is also expressed in the longitudinal muscle of the metamorphosing intestine (Fig. 3). Thus, different MMPs may play distinct roles during intestinal metamorphosis. Some, like GelA and collagenases, may function mainly by modifying the ECM. MT1-MMP may function in part by modifying ECM and in part through activation of GelA in the connective tissue. In addition, it functions independently of GelA in the longitudinal muscle.

Figure 3.

Spatial localization of MT1-MMP and GelA mRNAs in the intestine at metamorphic climax. Cross sections of intestine from animals at metamorphic climax (stage 62) were examined for MT1-MMP (A and B) and GelA (c and d) expression by in situ hybridization. Dark blue deposits indicate the sites of probe binding. (b and d) Higher magnification images of a boxed area in (A and C), respectively. MT1-MMP is expressed in the connective tissue (CT) and longitudinal muscle layer (LM) but not in epithelium (EP), circular muscle layer (CM) and serosa (S), while GelA is exclusively expressed in CT. Ty, typhlosole. Scale bars are 100 µm (A and C) and 50 µm (B and D) (see ref. 56 for details).

Role of the MMP ST3 in ECM Remodelling and Cell Fate Determination

ST3 was the first MMP to be isolated in the remodelling intestine and has best association with TH-induced cell death in this process. To investigate the function of ST3 during intestinal remodelling, we have taken two complementary approaches. First, fragments of tadpole intestine cultured in vitro can be induced to undergo remodelling as in intact tadpoles with TH treatment.⁵⁸ When a functional blocking polyclonal antibody against Xenopus laevis ST3 is added to the organ culture medium, it inhibits TH-induced ECM remodelling as well as epithelial cell death after three days (Fig. 4).⁵⁹ After five days of culturing, adult epithelial cells proliferate as clusters of cells or islets that expand three dimensionally in TH-treated organ cultures in the absence of the anti ST3 antibody. In the presence of the antibody, the TH induced cell proliferation occurs but the growth of the adult epithelial islets expands only laterally along the epithelium connective tissue interface but they fail to invade the connective tissue, a process probably important for adult epithelial fold formation during intestinal metamorphosis. These results suggest that ST3 is important, not only for ECM remodelling and larval cell death, but also for adult cell migration (the migration of the epithelial cells or fibroblasts relative to each other).⁵⁹

Our second approach takes advantage of the ability to overexpress MMPs in developing Xenopus laevis animals through transgenesis. Transgenic expression of ST3, collagenase 4, and MT1-MMP under the control of the constitutive CMV promoter leads to lethality of Xenopus laevis animals during late embryonic and early tadpole stages,⁶⁰ making it impossible to study the effects of the MMPs on metamorphosis. Thus, we have used a double promoter construct to express ST3 under the control of a heat shock-inducible promoter.⁶¹ In addition, a second promoter, the γ-crystallin promoter, is also present to drive the expression of green fluorescent protein (GFP) in the tadpoles' eyes. This allows us to identify transgenic tadpoles by simply checking for GFP fluorescence in the eyes under a fluorescence microscope, thereby enabling us to keep both wild type and transgenic animals, generated from the transgenic procedure, together to ensure no variation in animal growth and treatment conditions between the two groups. The expression of transgenic ST3 can be induced by simply treating the animals with heat shock. Induction of ST3 expression upon heat shock during early embryonic stages leads to animal lethality,⁶¹ similar to that observed with constitutive overexpression of ST3 through transgenesis. On the other hand, induction of ST3 at tadpole stages (between stage 45 to 54) for up to a few weeks has little effect on overall growth and development.⁶² However, analysis of the intestine of tadpoles at stage 54 subjected to 4 days of heat shock (about 1 hr heat shock treatment per day) shows that transgenic overexpression of wild type but not catalytically inactive ST3 causes larval epithelial cells to undergo apoptosis (Fig. 5), and induces the activation of fibroblasts and cell-cell contacts between epithelial cells and fibroblasts.⁶² These changes are similar to those during natural metamorphosis. Such changes are not present in nontransgenic animals treated with heat shock or in any animals without heat shock treatments. The basal lamina separating the epithelium and connective tissue is also altered by transgenic expression of ST3 in premetamorphic tadpoles. In some areas, including areas where epithelial cells and fibroblasts contact, the basal lamina is thicker and resembles that during metamorphosis. In other areas, ECM appears to be absent between the epithelium and connective tissue, a phenomenon differs from that during metamorphosis. In addition, adult epithelial cell islets are not induced by ST3 expression. Such differences indicate that ST3 expression alone is sufficient to induce some but not all TH induced metamorphic program in the intestine, a conclusion also supported by the alterations in the expression of a few TH-response genes in transgenic animals.⁶²

Figure 5.

Precocious expression of ST3 leads to epithelial cell death in premetamorphic tadpole intestine. Transgenic tadpoles expressing wild type (ST3) or catalytically inactive ST3 (ST3m) were reared to premetamorphic stage 54 and then subjected to daily heat shock for four days. Cross section of the intestine were isolated and subjected to TUNEL labeling to detect apoptotic cells, which were found in the epithelium (EP) of animals expression ST3 but not ST3m. Lu, intestinal lumen (see ref. 62 for details).

How ST3 causes ECM remodelling and cell death remains to be investigated. Compared to other MMPs, ST3 has much weaker activities toward known ECM proteins but much higher activities toward a few non-ECM proteins such as a1-protease inhibitor, at least in vitro.^{63 65} While it is possible that ST3 may cleave ECM proteins well in vivo, the in vitro studies raise the possibility that ST3 may function at least in part by cleaving non-ECM proteins. To search for potential substrates of ST3 during intestinal remodelling, we have undertaken a yeast-two-hybrid approach to isolate proteins that bind to ST3. This led to the identification of the 67 kd laminin receptor as a likely in vivo substrate of ST3.^66,67 The 67 kd laminin receptor is derived from its 37 kd precursor LR protein, although the exact mechanism is yet to be determined. LR is highly conserved from frog to human. Both human and Xenopus LR can be cleaved by Xenopus ST3 catalytic domain at two conserved sites located between the transmembrane domain and laminin binding sequence in the extracellular half of the protein,⁶⁶ while several other MMPs cleaves LR outside of the region between the transmembrane domain and laminin binding sequence. More importantly, LR fragments of the sizes expected from ST3 cleavage are present in the intestine at the climax of metamorphosis but hardly any are found in pre- or post-metamorphic intestine.⁶⁷ Furthermore, transgenic expression of ST3 leads to the formation of these fragments in premetamorphic tadpoles.⁶⁷ These results suggest that LR is an in vivo substrate of ST3, at least during intestinal remodelling. LR is highly expressed in the differentiated epithelial cells in pre- and post-metamorphic intestine. During intestinal metamorphosis, LR is, however, expressed in the connective tissue. Thus, one mechanism of ST3 function may be that the cleavage of LR on larval epithelial cells by ST3 reduces epithelial cell-ECM interaction, thus facilitating larval epithelial cell death during intestinal metamorphosis.

Conclusions

Amphibian metamorphosis is a postembryonic process that changes essentially every organ/tissue of the animal. Its dependence on TH has made it a model system of choice to study postembryonic tissue remodeling and organogenesis. Because of its simple organization and its similarity to mammalian intestine (with the tadpole and frog intestine mimicking embryonic and mature intestine, respectively, in mammals), the remodeling of the intestine during metamorphosis has been studied extensively, first at morphological and cellular levels and more recently at the molecular level. The molecular and cellular studies in Xenopus laevis have provided strong evidence that cell-cell and cell-ECM interactions play critical roles in the development of the adult intestine and that MMPs are likely key players in regulating these interactions. In particular, the MMP ST3 is both necessary and sufficient at least for some aspects of intestinal metamorphosis, especially larval epithelial cell death, although in vivo, ST3 is likely to function together with other genes to coordinate both spatially and temporally tissue specific changes in the intestine. One potential mechanism by which ST3 exerts its effects on intestinal remodeling may be through its cleavage of laminin receptor on the larval epithelial cells. Clearly, further analyses of LR and identification of additional ST3 substrates are needed to understand the mechanism of ST3 action. In addition, investigations on the roles of other MMPs and on ECM proteins are needed to determine how ECM remodeling contributes to the development of adult intestine in frogs.

Abbreviations

TH

thyroid hormone

TR

thyroid hormone receptor

MMP

matrix metalloproteinase

MT-MMP

membrane type-MMP

ECM

extracellular matrix