Matrix metalloproteinase expression and function during fin regeneration in zebrafish: Analysis of MT1-MMP, MMP2 and TIMP2☆
Introduction
Although published studies related to fin development and regeneration can be traced back some 200 years (for early vertebrate studies see: Broussant, 1786, Morgan, 1900, Nabrit, 1929), relatively little is known about the role of cell–extracellular matrix (ECM) interactions in these processes. Based on the fact that ECM functions as a structural entity and a signaling entity (Colognato and Yurchenco, 2000, Darribere et al., 2000, Relan and Schuger, 1999, Van der Flier and Sonnenberg, 2001), our current understanding of cell–ECM interactions indicates that they encompass a broad spectrum of underlying mechanisms. In regard to signaling, ECM contains endogenous receptor binding motifs, such as the RGD sequence, that can be either openly exposed or cryptic within any given matrix molecule (Davis et al., 2000). In addition, ECM is known to be a scaffold for growth factors and other signaling molecules that can be released from the matrix for subsequent binding to their cell surface receptors (McCawley and Matrisian, 2001). The latency of cryptic endogenous signaling motifs or bound signaling molecules has been shown to be regulated by proteinases, such as matrix metalloproteinases (MMPs) (Davis et al., 2000, Nagase and Woessner, 1999). Because MMPs are important regulators of cell–ECM interactions, their expression provides us with an index of when and where these interactions may be occurring during development and regeneration. With this in mind, the current study has focused on the expression and possible function of MMPs during fin regeneration in zebrafish, with the objective of establishing whether cell–ECM interactions have an important role in this process.
Because muscles are restricted to the girdle (base) region of fins, the limbs of fish are mainly connective tissue structures containing bone, mesenchymal cells, blood vessels, nerves, and an overlying layer of epidermis. Fins are, therefore, a more simplified anatomical structure as compared to the limbs of amphibians, birds or mammals. Similar to Urodelan amphibians (Nye et al., 2003), however, zebrafish have the ability to regenerate their limbs (i.e. fins) (Poss et al., 2003, Misof and Wagner, 1992). Experimentally induced partial amputation of fins leads to formation of: 1) an epithelial wound layer and 2) a blastema. These two intermediate structures work in concert to regenerate a new functional fin in the adult zebrafish. Regeneration involves a complex array of cellular events such as 1) cell migration, 2) cell proliferation, 3) cell apoptosis, and 4) cell differentiation, orchestrated by a multitude of signaling pathways that somewhat mimic, but are not identical to those seen in normal limb/fin development (Grandel and Schulte-Merker, 1998, Poss et al., 2000, Poss et al., 2003). In the case of fin regeneration, cellular events are spatially and temporally well defined and this provides one with an ideal model to approach the fundamental mechanisms controlling this regenerative process.
The role of cell–ECM interactions in limb development and regeneration, particularly as related to fin regeneration, is not understood. Only a handful of studies have addressed this problem in terms of MMPs and these mainly have been restricted to amphibian systems such as newts (Miyazaki et al., 1996), salamanders (Park and Kim, 1999, Yang et al., 1999), and Xenopus (Yang et al., 1997). These studies established that a number of MMPs are expressed during limb regeneration, but provided little evidence that these enzymes had a functional role. For example, Yang et al. (1999) have provided well controlled in situ studies in axolotl indicating that Mmp-9 is expressed in a biphasic manner in which the transcript is first localized to the wound epithelium but is later localized to the mesenchyme. Specific inhibition of Mmp-9 mRNA expression was not possible in this system. In fact, to date, the only functional regeneration studies in amphibians have been reported in axolotl and newt. In the case of axolotl, morpholino-mediated knockdowns of MSX1 and PAX7 (Schnapp and Tamaka, 2005) during limb regeneration were recently reported and Vinarsky et al. (2005) reported that the MMP inhibitor, GM6001 inhibits limb regeneration in newts. Likewise, Sanano et al. (2002) recently reported the expression of both Mmp-8 and Mmp-13 during rat hind limb formation but were unable to perform functional studies. While knockout studies for a number of MMPs in mice have not yielded a clear role for these enzymes in early development or limb development (Sternlicht and Werb, 2001), these negative results can be attributed to compensatory mechanisms in which the function of one MMP is substituted for another in the null mutation (Sternlicht and Werb, 2001). Taken in total, the precise role of MMPs in limb regeneration is unclear. To extend these studies, the current work has utilized zebrafish as an experimental model and has focused on the expression of a subset of MMPs (MT1-MMP and MMP-2) and a modulator of MMP activity (TIMP-2) during fin regeneration. These three molecules were selected because they can act 1) independently of one another (Nagase and Woessner, 1999) or 2) together as a unit during development (Strongin et al., 1995, Butler et al., 1998, D'Angelo et al., 2000). When functioning as a unit, they are involved in the activation of pro-MMP-2 to its active form (Strongin et al., 1995, Butler et al., 1998). In the current study, functional studies using the MMP inhibitor GM6001 and the hemopexin domain of MT1-MMP provided evidence that matrix metalloproteinase activity is required for fin regeneration.
Section snippets
Expression of zmt1-mmp, zmmp-2, and ztimp-2 mRNA is associated with both the blastema and wound epithelium during caudal fin regeneration
Experiments were conducted to determine if MMP expression is associated with adult fin regeneration. Initial studies on adult caudal fin regeneration were limited to analyzing MT1-MMP, MMP-2, TIMP-2, and collagen type IV (a matrix component associated with the basement membrane of the caudal fin epithelium, unpublished observation). Whole mount in situ analysis of zmt1-mmp and zcol-IV(α5) at 3, 5 and 7 days post amputation (maintained at 28.5 °C) is shown in Fig. 1. A signal for mt1-mmp mRNA
Discussion
This is the first study to investigate the relationship of MMPs to fin regeneration. Our results indicate that MMPs have an important role in adult fin regeneration; however, the precise substrates for MT1-MMP and MMP-2 remain to be determined because of the diversity of substrates that have been shown associated with these enzymes under in vitro conditions (McCawley and Matrisian, 2001). These substrates involve not only matrix components, but also a large variety of matrix-associated and
Fish maintenance
Wild-type zebrafish (Danio rerio) were maintained under standard laboratory conditions (Westerfield, 1995, Detrich et al., 1999) using a light schedule of 14 h on and 10 h off at a temperature of 28.5 °C. Fish were fed daily using a combination of dry food and brine shrimp.
Zebrafish cDNA clones utilized in these studies
All zebrafish MMP and TIMP cDNAs used in this study have been previously characterized by our laboratory and include: zmt1-mmp (Zhang et al., 2003a), zmmp-2 (Zhang et al., 2003b) and ztimp-2 (Zhang et al., 2003c). The alpha
Acknowledgements
The authors wish to thank Eileen Roach for assistance with preparation of the figures used in this article. The work was supported by NIH Grants AR39189, DK61373, AR47233, American Heart Grant 0051346Z, and The Welcome Trust Grant 057508. RT was supported from the KUMC Biomedical Training Grant.
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Resubmission of article (Matrix Biology number: ATBIO_michaelsarras_20050207.1/1).
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Current address: Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, KS, 66160, USA.