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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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Enlarged images of the palatal-nasopharyngeal fold after double labeling for gelatinolytic activity and laminin.Frontal cryosections at the middle anteroposterior level of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin in situ zymography (ISZ; A, B), followed by immunofluorescence labeling for laminin-111 (LN; C, D) on the same section. Merged images are shown in E and F. At E14.5, gelatinolytic activity colocalizes with the epithelial basement membrane at the lateral and palatal side of the fold (arrows). The pattern of gelatinolysis is similar at E15.5, although activity extends into the adjacent mesenchyme and its intensity is considerably increased. p, palatal shelf; n, nasal floor; np, nasopharynx. Bar, 50 μm.
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pone-0047762-g004: Enlarged images of the palatal-nasopharyngeal fold after double labeling for gelatinolytic activity and laminin.Frontal cryosections at the middle anteroposterior level of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin in situ zymography (ISZ; A, B), followed by immunofluorescence labeling for laminin-111 (LN; C, D) on the same section. Merged images are shown in E and F. At E14.5, gelatinolytic activity colocalizes with the epithelial basement membrane at the lateral and palatal side of the fold (arrows). The pattern of gelatinolysis is similar at E15.5, although activity extends into the adjacent mesenchyme and its intensity is considerably increased. p, palatal shelf; n, nasal floor; np, nasopharynx. Bar, 50 μm.

Mentions: ISZ/laminin double labeling also revealed extensive colocalization of gelatinolysis with the basement membranes of the nasopharyngeal epithelial folds that are generated upon palatal shelf elevation (Fig. 4). At stage E15.5 (Fig. 4B, D, F), gelatinolytic activity was also associated with palatal and nasal epithelial cells, as well as with the mesenchyme adjacent to the strongly labeled epithelial basement membranes. Interestingly, activity was strongest at the lateral bend where the oral epithelium folds back on itself, but faded out towards more central regions of the nasopharyngeal cavity. Gelatinolysis was asymmetric and more prominent on the palatal aspect of the folds, in epithelial cells as well as basement membranes and adjacent palatal mesenchyme (Fig. 4A, B).


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)

Enlarged images of the palatal-nasopharyngeal fold after double labeling for gelatinolytic activity and laminin.Frontal cryosections at the middle anteroposterior level of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin in situ zymography (ISZ; A, B), followed by immunofluorescence labeling for laminin-111 (LN; C, D) on the same section. Merged images are shown in E and F. At E14.5, gelatinolytic activity colocalizes with the epithelial basement membrane at the lateral and palatal side of the fold (arrows). The pattern of gelatinolysis is similar at E15.5, although activity extends into the adjacent mesenchyme and its intensity is considerably increased. p, palatal shelf; n, nasal floor; np, nasopharynx. Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472992&req=5

pone-0047762-g004: Enlarged images of the palatal-nasopharyngeal fold after double labeling for gelatinolytic activity and laminin.Frontal cryosections at the middle anteroposterior level of E14.5 (A, C, E) and E15.5 (B, D, F) wild type mouse heads were subjected to DQ-gelatin in situ zymography (ISZ; A, B), followed by immunofluorescence labeling for laminin-111 (LN; C, D) on the same section. Merged images are shown in E and F. At E14.5, gelatinolytic activity colocalizes with the epithelial basement membrane at the lateral and palatal side of the fold (arrows). The pattern of gelatinolysis is similar at E15.5, although activity extends into the adjacent mesenchyme and its intensity is considerably increased. p, palatal shelf; n, nasal floor; np, nasopharynx. Bar, 50 μm.
Mentions: ISZ/laminin double labeling also revealed extensive colocalization of gelatinolysis with the basement membranes of the nasopharyngeal epithelial folds that are generated upon palatal shelf elevation (Fig. 4). At stage E15.5 (Fig. 4B, D, F), gelatinolytic activity was also associated with palatal and nasal epithelial cells, as well as with the mesenchyme adjacent to the strongly labeled epithelial basement membranes. Interestingly, activity was strongest at the lateral bend where the oral epithelium folds back on itself, but faded out towards more central regions of the nasopharyngeal cavity. Gelatinolysis was asymmetric and more prominent on the palatal aspect of the folds, in epithelial cells as well as basement membranes and adjacent palatal mesenchyme (Fig. 4A, B).

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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