Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membraneassociated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-␤3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-␤3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-␤3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion. INTRODUCTIONThe formation of the palate is of critical importance to separate the oropharynx from the nasopharynx. A dysfunction in one of the regulators of this developmental process can lead to a cleft palate, one of the most common birth defects in humans (Chenevix-Trench et al., 1992). In the mouse embryo, the entire process of palatal formation takes place between day 12 and 15 (E12 and E15) of development (Ferguson, 1988). The fusion itself occurs over a relatively short period of time during which the medial edge epithelia (MEE) of the shelves form a midline seam, which is then disrupted to allow mesenchymal continuity (Pourtois, 1966;Smiley and Koch, 1971). Complete fusion of the secondary palate requires disappearance of the MEE from the midline, as well as the breakdown of their basement membrane.The molecular mechanisms controlling palatal fusion are complex and not fully understood. However, studies in the mouse have pointed to primary and secondary causes of defective palatogenesis. In mice deficient for the epidermal growth factor receptor or the platelet-derived growth factor receptor, a cleft palate is often associated with a primary defect in the development of the first branchial arch (Shiota et al., 1990;Brunet et al., 1993;Robbins et al., 1999). In these cases, delayed development of the lower jaw interferes with forward displacement of the tongue and prevents the elevation and subsequent fusion of the shelves (Robbins et al., 1999). In transforming growth factor (TGF)-␤3-deficient mice a cleft palate develops in all mice due to ...

The Wnt signaling transduction pathway plays a critical role in the pathogenesis of several murine and human epithelial cancers. Here, we have used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop spontaneous mammary adenocarcinoma, to examine whether matrix metalloproteinases (MMPs)-a family of extracellular proteases implicated in multiple steps of cancer progressioncontributed to Wnt1-induced tumorigenesis. An analysis of the expression of several MMPs by RT-PCR and in situ hybridization revealed an increase in the expression of MMP-2, MMP-3, MMP-9, MMP-13, and MT1-MMP (MMP-14) in hyperplastic glands and in mammary tumors of MMTV-Wnt1 transgenic mice. Interestingly, whereas MMP-2, MMP-3, and MMP-9 were exclusively expressed by stromal cells in mammary tumors, MMP-13 and MT1-MMP were expressed by transformed epithelial cells in addition to the tumor stroma. To determine whether these MMPs contributed to tumorigenesis, MMTVWnt1 mice were crossed with transgenic mice overexpressing tissue inhibitor of metalloproteinase-2-a natural MMP inhibitor-in the mammary gland. In the double MMTV-Wnt1/tissue inhibitor of metalloproteinases-2 transgenic mice, we observed an increase in tumor latency and a 26.3% reduction in tumor formation. Furthermore, these tumors grew at a slower rate, exhibited an 18% decrease in proliferative rate, and a 12.2% increase in apoptotic rate of the tumor cells in association with a deficit in angiogenesis when compared with tumors from MMTV-Wnt1 mice. Thus, for the first time, the data provides evidence for the active role of MMPs in Wnt1-induced mammary tumorigenesis. (Cancer Res 2006; 66(5): 2691-9)

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Neuroblastoma is the second most common solid tumor in children. So far few tumor models for this cancer have been reported in mice. We have created a murine tumor model for studying human neuroblastoma based on surgical orthotopic implantation in scid mice. Small fragments of subcutaneous tumors of SK-N-BE(2) human neuroblastoma cells expressing enhanced green fluorescent protein were surgically implanted near the left adrenal gland of scid mice. One hundred percent of the animals (n = 21) successfully implanted developed a large retroperitoneal tumor and became moribund between 22 and 57 days after implantation (mean survival time = 41 days). At the time of sacrifice the presence of bone marrow metastasis was detected by RT-PCR for green fluorescent protein in 95% of the cases. The growth of small tumor implants could be easily visualized and quantified by surveillance MR imaging, with a resolution of 117 x 117 x 750 microm in two orthogonal planes allowing accurate volume measurements, as well as assessment of necrosis and tissue invasion. This novel model should be a valuable tool to study the biology and therapeutic approaches to neuroblastoma.