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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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Close-up images of double labeling for gelatinolytic activity and laminin around medial edge epithelial cells of palatal shelves.Frontal cryosections (middle anteroposterior level) from E13.5 (A, B), E14.5 (C, D), and E15.5 (E, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin on the same section. (A) In situ zymography and (B) laminin labeling, respectively, in the distal-medial region of a E13.5 palatal shelf, where epithelial fusion will occur after shelf elevation. No sign of activity was detected at that site (arrows), while gelatinolysis was evident in the tongue mesenchyme (asterisks). (C) In situ zymography and (D) laminin labeling, respectively, immediately prior to midline fusion of the palatal shelves at E14.5. Note weak gelatinolytic activity at the site where epithelial fusion will occur (arrows). (E) In situ zymography and (F) laminin labeling, respectively, during midline fusion of the palate at E15.5. Prominent gelatinolytic activity is evident at sites of midline epithelial fusion (arrows), and at the epithelial remnants of the palatal shelves at the midline (arrowheads). p, palatal shelf; t, tongue. Bars, 50 μm.
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pone-0047762-g003: Close-up images of double labeling for gelatinolytic activity and laminin around medial edge epithelial cells of palatal shelves.Frontal cryosections (middle anteroposterior level) from E13.5 (A, B), E14.5 (C, D), and E15.5 (E, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin on the same section. (A) In situ zymography and (B) laminin labeling, respectively, in the distal-medial region of a E13.5 palatal shelf, where epithelial fusion will occur after shelf elevation. No sign of activity was detected at that site (arrows), while gelatinolysis was evident in the tongue mesenchyme (asterisks). (C) In situ zymography and (D) laminin labeling, respectively, immediately prior to midline fusion of the palatal shelves at E14.5. Note weak gelatinolytic activity at the site where epithelial fusion will occur (arrows). (E) In situ zymography and (F) laminin labeling, respectively, during midline fusion of the palate at E15.5. Prominent gelatinolytic activity is evident at sites of midline epithelial fusion (arrows), and at the epithelial remnants of the palatal shelves at the midline (arrowheads). p, palatal shelf; t, tongue. Bars, 50 μm.

Mentions: Higher magnification images of the midline epithelial seam (MES) region demonstrated that the appearance of gelatinolytic activity was tightly linked to the process of palatal shelf fusion (Fig. 3). At E13.5, when the palatal shelves were still vertically oriented, no sign of gelatinolysis was detected along the entire oral epithelium, including the prospective fusion sites on the distal-medial aspect of the shelves. Traces of gelatinolysis were evident only within the tongue mesenchyme (Fig. 3A, B). At E14.5, immediately prior to fusion of the palatal shelves, gelatinolytic activity was present at the MES and was located mainly in the pericellular space surrounding a subset of epithelial cells (Fig. 3C, D). At E15.5 during palatal shelf fusion, prominent gelatinolytic activity was associated mainly with remnants of basement membranes of the disorganizing MES (Fig. 3E, F).


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)

Close-up images of double labeling for gelatinolytic activity and laminin around medial edge epithelial cells of palatal shelves.Frontal cryosections (middle anteroposterior level) from E13.5 (A, B), E14.5 (C, D), and E15.5 (E, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin on the same section. (A) In situ zymography and (B) laminin labeling, respectively, in the distal-medial region of a E13.5 palatal shelf, where epithelial fusion will occur after shelf elevation. No sign of activity was detected at that site (arrows), while gelatinolysis was evident in the tongue mesenchyme (asterisks). (C) In situ zymography and (D) laminin labeling, respectively, immediately prior to midline fusion of the palatal shelves at E14.5. Note weak gelatinolytic activity at the site where epithelial fusion will occur (arrows). (E) In situ zymography and (F) laminin labeling, respectively, during midline fusion of the palate at E15.5. Prominent gelatinolytic activity is evident at sites of midline epithelial fusion (arrows), and at the epithelial remnants of the palatal shelves at the midline (arrowheads). p, palatal shelf; t, tongue. Bars, 50 μm.
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Related In: Results  -  Collection

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pone-0047762-g003: Close-up images of double labeling for gelatinolytic activity and laminin around medial edge epithelial cells of palatal shelves.Frontal cryosections (middle anteroposterior level) from E13.5 (A, B), E14.5 (C, D), and E15.5 (E, F) wild type mouse heads were subjected to DQ-gelatin zymography, followed by immunofluorescence labeling for laminin on the same section. (A) In situ zymography and (B) laminin labeling, respectively, in the distal-medial region of a E13.5 palatal shelf, where epithelial fusion will occur after shelf elevation. No sign of activity was detected at that site (arrows), while gelatinolysis was evident in the tongue mesenchyme (asterisks). (C) In situ zymography and (D) laminin labeling, respectively, immediately prior to midline fusion of the palatal shelves at E14.5. Note weak gelatinolytic activity at the site where epithelial fusion will occur (arrows). (E) In situ zymography and (F) laminin labeling, respectively, during midline fusion of the palate at E15.5. Prominent gelatinolytic activity is evident at sites of midline epithelial fusion (arrows), and at the epithelial remnants of the palatal shelves at the midline (arrowheads). p, palatal shelf; t, tongue. Bars, 50 μm.
Mentions: Higher magnification images of the midline epithelial seam (MES) region demonstrated that the appearance of gelatinolytic activity was tightly linked to the process of palatal shelf fusion (Fig. 3). At E13.5, when the palatal shelves were still vertically oriented, no sign of gelatinolysis was detected along the entire oral epithelium, including the prospective fusion sites on the distal-medial aspect of the shelves. Traces of gelatinolysis were evident only within the tongue mesenchyme (Fig. 3A, B). At E14.5, immediately prior to fusion of the palatal shelves, gelatinolytic activity was present at the MES and was located mainly in the pericellular space surrounding a subset of epithelial cells (Fig. 3C, D). At E15.5 during palatal shelf fusion, prominent gelatinolytic activity was associated mainly with remnants of basement membranes of the disorganizing MES (Fig. 3E, F).

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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