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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 laminin in the palatal region of a Bmp7Δ/Δ mouse embryo.A frontal cryosection of a E14.5 Bmp7Δ/Δ mouse head was subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin-111 on the same section. Note that in the knockout embryo, both palatal shelves are still vertically oriented at this stage. (A) Overview pointing to the regions that are shown in (B–E); laminin staining. The yellow box corresponds to the region that is depicted at higher magnification in (B, C), and the red box to the region in (D, E). (B) Labeling for laminin and (C) in situ zymography, respectively, shows gelatinolytic activity colocalizing with the basement membrane of the epithelial fold that is created at the onset of palatal shelf elevation (arrows); (D) Labeling for laminin and (E) in situ zymography, respectively, did not reveal gelatinolytic activity in the corresponding region of the opposite shelf where no epithelial invagination is visible yet (arrows). n, nasal cartilage; p, palatal shelf; t, tongue. Bar, 250 μm in A, 100 μm in B and E.
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pone-0047762-g006: Double labeling for gelatinolytic activity and laminin in the palatal region of a Bmp7Δ/Δ mouse embryo.A frontal cryosection of a E14.5 Bmp7Δ/Δ mouse head was subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin-111 on the same section. Note that in the knockout embryo, both palatal shelves are still vertically oriented at this stage. (A) Overview pointing to the regions that are shown in (B–E); laminin staining. The yellow box corresponds to the region that is depicted at higher magnification in (B, C), and the red box to the region in (D, E). (B) Labeling for laminin and (C) in situ zymography, respectively, shows gelatinolytic activity colocalizing with the basement membrane of the epithelial fold that is created at the onset of palatal shelf elevation (arrows); (D) Labeling for laminin and (E) in situ zymography, respectively, did not reveal gelatinolytic activity in the corresponding region of the opposite shelf where no epithelial invagination is visible yet (arrows). n, nasal cartilage; p, palatal shelf; t, tongue. Bar, 250 μm in A, 100 μm in B and E.

Mentions: In Bmp7Δ/Δ mouse embryos, both palatal shelves are still vertical at E14.5. The pattern of gelatinolytic activity was comparable to Bmp7wt/Δ embryos of the same stage. However, the even longer delay in elevation revealed that gelatinolysis started already in vertically oriented shelves at the onset of the process. In the example shown in Fig. 6, early formation of a nasopharyngeal fold is visible as a notch in the epithelial basement membrane of the left palatal shelf, and this site was clearly positive for gelatinolytic activity (Fig. 6B, C). The opposing shelf, where the elevation process had not started yet, did not present any activity at the corresponding location (Fig. 6D, E).


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 laminin in the palatal region of a Bmp7Δ/Δ mouse embryo.A frontal cryosection of a E14.5 Bmp7Δ/Δ mouse head was subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin-111 on the same section. Note that in the knockout embryo, both palatal shelves are still vertically oriented at this stage. (A) Overview pointing to the regions that are shown in (B–E); laminin staining. The yellow box corresponds to the region that is depicted at higher magnification in (B, C), and the red box to the region in (D, E). (B) Labeling for laminin and (C) in situ zymography, respectively, shows gelatinolytic activity colocalizing with the basement membrane of the epithelial fold that is created at the onset of palatal shelf elevation (arrows); (D) Labeling for laminin and (E) in situ zymography, respectively, did not reveal gelatinolytic activity in the corresponding region of the opposite shelf where no epithelial invagination is visible yet (arrows). n, nasal cartilage; p, palatal shelf; t, tongue. Bar, 250 μm in A, 100 μm in B and E.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472992&req=5

pone-0047762-g006: Double labeling for gelatinolytic activity and laminin in the palatal region of a Bmp7Δ/Δ mouse embryo.A frontal cryosection of a E14.5 Bmp7Δ/Δ mouse head was subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin-111 on the same section. Note that in the knockout embryo, both palatal shelves are still vertically oriented at this stage. (A) Overview pointing to the regions that are shown in (B–E); laminin staining. The yellow box corresponds to the region that is depicted at higher magnification in (B, C), and the red box to the region in (D, E). (B) Labeling for laminin and (C) in situ zymography, respectively, shows gelatinolytic activity colocalizing with the basement membrane of the epithelial fold that is created at the onset of palatal shelf elevation (arrows); (D) Labeling for laminin and (E) in situ zymography, respectively, did not reveal gelatinolytic activity in the corresponding region of the opposite shelf where no epithelial invagination is visible yet (arrows). n, nasal cartilage; p, palatal shelf; t, tongue. Bar, 250 μm in A, 100 μm in B and E.
Mentions: In Bmp7Δ/Δ mouse embryos, both palatal shelves are still vertical at E14.5. The pattern of gelatinolytic activity was comparable to Bmp7wt/Δ embryos of the same stage. However, the even longer delay in elevation revealed that gelatinolysis started already in vertically oriented shelves at the onset of the process. In the example shown in Fig. 6, early formation of a nasopharyngeal fold is visible as a notch in the epithelial basement membrane of the left palatal shelf, and this site was clearly positive for gelatinolytic activity (Fig. 6B, C). The opposing shelf, where the elevation process had not started yet, did not present any activity at the corresponding location (Fig. 6D, E).

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
Related in: MedlinePlus