TGF-beta3-induced palatogenesis requires matrix metalloproteinases

Abstract

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 membrane-associated 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)-beta3-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-beta3 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-beta3-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.

Figure 1

(A) Histological analysis of a coronal section performed on a 14.5-d p.c. embryonic head, showing the palatal shelves in the process of fusion. (B) Enlargement of the same photomicrograph showing the interrupted medial edge epithelium (arrow) undergoing epithelial-mesenchymal transdifferentiation. n, nasopharynx; o, oropharynx; t, tongue; ps, palatal shelf. Bars, A, 300 μm; B, 100 μm.

Figure 2

Localization of mRNA expression of TIMPs during palatal fusion. Coronal sections of 14.5-d p.c. embryonic heads were analyzed by in situ hybridization with antisense riboprobes for TIMP-1 (A), TIMP-3 (B), TIMP-2 (C), and TIMP-4 (D). Photomicrographs were taken under dark-field illumination, the transcripts are seen as bright grains. The arrow points to the hybridization signal at the midline seam (C). (Note: the pigmented epithelium of the eye appears bright under dark-field, this is not a positive signal.) t, tongue; ps, palatal shelf. Bar, A, 200 μm.

Figure 3

Localization of mRNA expression of MMPs during palatal fusion. Coronal sections of 14.5-d p.c. embryonic heads were analyzed by in situ hybridization with antisense riboprobes for MMP-9 (A), MT1-MMP (B), MMP-2 (C), and MMP-13 (D). The arrows point to the positive hybridization signal at the midline seam (B–D). Photomicrographs were taken under dark-field illumination. t, tongue; ps, palatal shelf. Bar, A, 200 μm.

Figure 4

Comparison of cellular expression of TIMP-2, MMP-2, MT1-MMP, and MMP-13 at the site of palatal fusion. High magnification of the midline seam showing TIMP-2 (A) and MT1-MMP (C) expression by the MEE cells (arrows in A and C), MMP-2 (B) expression by the adjacent mesenchymal cells (arrow, B), and MMP-13 expression both in the MEE cells and adjacent mesenchymal cells (arrows, D). Photographs were taken under bright-field illumination, the transcripts are seen as dark grains. Bar, A, 10 μm.

Figure 5

Comparison of TGF-β3, TIMP-2, MMP-2, MT1-MMP, and MMP-13 expression by in situ hybridization in wild-type (A, C, E, G, and I) and TGF-β3-deficient embryos (B, D, F, H, and J). Arrows point to the hybridization signal in the midline seam. Photomicrographs were taken under dark-field illumination. n, nasopharynx; o, oropharynx; t, tongue; ps, palatal shelf. Dotted line in D and J indicates the surface of the tongue. Bar, A, 100 μm

Figure 6

Expression of MMP-13 in isolated palatal fibroblasts is modulated by TGF-β3. TGF-β3 induces MMP-13 expression both on an RNA (A) and a protein level (B) in palatal fibroblasts, whereas TIMP-2, MMP-2, and MT1-MMP do not display a similar response (C). Serum-starved cells were induced with 10 ng/ml TGF-β3 and cells and medium were harvested for Northern (10 μg of total RNA per lane) and Western (20 μg of total protein per lane) analyses, respectively.

Figure 7

Synthetic inhibitor of MMPs prevents the fusion of palatal shelves in culture. Palatal shelves from wild-type E14.0 embryos were dissected and cultured as pairs at the air–medium interface in organ culture dishes as described in MATERIALS AND METHODS. This figure shows representative sections of the palatal shelves after 63 h of culture under control conditions (A and B), and in the presence of 1 μM (C and D) and 10 μM (E and F) BB-3103. Bars, A, C, and E, 50 μm; B, D, and F, 10 μm.

Figure 8

Inhibition of palatal fusion and EMT in vitro by BB-3103 and TIMP-2. At the end of the organ culture, serial sections were examined for the presence of MEE cells in the midline (as shown in Figure 7). Sections were scored 0 for the absence of MEE cells, 1 for discontinuous MEE, and 2 for the presence of a continuous double layer of MEE (see MATERIALS AND METHODS). Each dot represents the mean score of 20 sections examined for each organ cultured under the indicated conditions. The number of organ cultures examined was 17 for control, eight in the presence of 1 μM BB-3103, nine in the presence of 10 μM BB-3103, and five in the presence of 10 μg/ml TIMP-2.