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Site-specific expression of gelatinolytic activity during morphogenesis of the secondary palate in the mouse embryo.

Gkantidis N, Blumer S, Katsaros C, Graf D, Chiquet M - PLoS ONE (2012)

Bottom Line: Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed.In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation.Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx.

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Double labeling for gelatinolytic activity and MMP-2 and MT1-MMP in the palatal region of the mouse embryo.Frontal cryosections of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for MMPs on the same section. Serial sections were used; only one zymography image (double labeling for MMP-2) per stage is shown (A, B). Note that despite of differences in distribution, both MMP-2 (C, D) and MT1-MMP (E, F) substantially overlap with gelatinolytic activity, most notably in developing cartilage and bone but also around the expanding nasopharyngeal cavity (arrowheads). For more details, see Results. n, nasal cartilage; p, palatal shelf; t, tongue; m, maxillary bone. Bar, 200 μm.
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pone-0047762-g008: Double labeling for gelatinolytic activity and MMP-2 and MT1-MMP in the palatal region of the mouse embryo.Frontal cryosections of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for MMPs on the same section. Serial sections were used; only one zymography image (double labeling for MMP-2) per stage is shown (A, B). Note that despite of differences in distribution, both MMP-2 (C, D) and MT1-MMP (E, F) substantially overlap with gelatinolytic activity, most notably in developing cartilage and bone but also around the expanding nasopharyngeal cavity (arrowheads). For more details, see Results. n, nasal cartilage; p, palatal shelf; t, tongue; m, maxillary bone. Bar, 200 μm.

Mentions: MMPs are secreted as latent proenzymes that require enzymatic processing for activation [19]; thus the distributions of latent and active gelatinases might differ. The membrane-bound MT1-MMP is known to activate other MMPs, particularly MMP-2, and itself possesses gelatinolytic activity [20]. Since these were the two MMPs found to be prominently expressed in the palatal region, we employed ISZ followed by immunohistochemistry on sections of E13.5, E14.5, and E15.5 wildtype mouse heads, in order to investigate the relationship between gelatinolytic activity and enzyme distribution (Fig. 8).


Site-specific expression of gelatinolytic activity during morphogenesis of the secondary palate in the mouse embryo.

Gkantidis N, Blumer S, Katsaros C, Graf D, Chiquet M - PLoS ONE (2012)

Double labeling for gelatinolytic activity and MMP-2 and MT1-MMP in the palatal region of the mouse embryo.Frontal cryosections of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for MMPs on the same section. Serial sections were used; only one zymography image (double labeling for MMP-2) per stage is shown (A, B). Note that despite of differences in distribution, both MMP-2 (C, D) and MT1-MMP (E, F) substantially overlap with gelatinolytic activity, most notably in developing cartilage and bone but also around the expanding nasopharyngeal cavity (arrowheads). For more details, see Results. n, nasal cartilage; p, palatal shelf; t, tongue; m, maxillary bone. Bar, 200 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472992&req=5

pone-0047762-g008: Double labeling for gelatinolytic activity and MMP-2 and MT1-MMP in the palatal region of the mouse embryo.Frontal cryosections of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for MMPs on the same section. Serial sections were used; only one zymography image (double labeling for MMP-2) per stage is shown (A, B). Note that despite of differences in distribution, both MMP-2 (C, D) and MT1-MMP (E, F) substantially overlap with gelatinolytic activity, most notably in developing cartilage and bone but also around the expanding nasopharyngeal cavity (arrowheads). For more details, see Results. n, nasal cartilage; p, palatal shelf; t, tongue; m, maxillary bone. Bar, 200 μm.
Mentions: MMPs are secreted as latent proenzymes that require enzymatic processing for activation [19]; thus the distributions of latent and active gelatinases might differ. The membrane-bound MT1-MMP is known to activate other MMPs, particularly MMP-2, and itself possesses gelatinolytic activity [20]. Since these were the two MMPs found to be prominently expressed in the palatal region, we employed ISZ followed by immunohistochemistry on sections of E13.5, E14.5, and E15.5 wildtype mouse heads, in order to investigate the relationship between gelatinolytic activity and enzyme distribution (Fig. 8).

Bottom Line: Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed.In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation.Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx.

Show MeSH