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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 the mouse embryo.Frontal cryosections of E13.5 (A–C), E14.5 (D–F), and E15.5 (G–I) wild type mouse heads were subjected to DQ-gelatin zymography (green panels), followed by immunofluorescence labeling for laminin-111 on the same section (red panels). The images show representative sections from the anterior (A, D, G), the middle (B, E, H), and the posterior (C, F, I) level of the palate. No site with significant gelatinolytic activity, as manifested by increased fluorescence, is detected at E13.5 prior to palatal shelf elevation. At E14.5, gelatinolytic activity is evident in the nasal cartilage and in the main palatal arteries (arrows). In addition, signs of gelatinolysis are visible at the midline epithelial seam (asterisks), whereas prominent gelatinolytic activity is detected around the folds of the elevated palatal shelves, where the nasopharynx will form (arrowheads). A similar pattern of gelatinolysis is observed at E15.5, although activity is increased compared to E14.5. Furthermore, prominent activity was evident for maxillary bone. p, palatal shelf; t, tongue; n, nasal cartilage; m, maxillary bone. Bar, 200 μm.
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pone-0047762-g002: Double labeling for gelatinolytic activity and laminin in the palatal region of the mouse embryo.Frontal cryosections of E13.5 (A–C), E14.5 (D–F), and E15.5 (G–I) wild type mouse heads were subjected to DQ-gelatin zymography (green panels), followed by immunofluorescence labeling for laminin-111 on the same section (red panels). The images show representative sections from the anterior (A, D, G), the middle (B, E, H), and the posterior (C, F, I) level of the palate. No site with significant gelatinolytic activity, as manifested by increased fluorescence, is detected at E13.5 prior to palatal shelf elevation. At E14.5, gelatinolytic activity is evident in the nasal cartilage and in the main palatal arteries (arrows). In addition, signs of gelatinolysis are visible at the midline epithelial seam (asterisks), whereas prominent gelatinolytic activity is detected around the folds of the elevated palatal shelves, where the nasopharynx will form (arrowheads). A similar pattern of gelatinolysis is observed at E15.5, although activity is increased compared to E14.5. Furthermore, prominent activity was evident for maxillary bone. p, palatal shelf; t, tongue; n, nasal cartilage; m, maxillary bone. Bar, 200 μm.

Mentions: A double-labeling technique comprising in situ zymography (ISZ) with dye-quenched (DQ-)gelatin, followed by brief acetone fixation and immunofluorescence labeling, was performed to associate gelatinolytic activity in situ with laminin-111 as a marker for embryonic basement membranes (Fig. 2). In the region of the developing secondary palate, no site with prominent gelatinolytic activity was identified prior to palatal shelf elevation at E13.5 at three anteroposterior levels (Fig. 2A–C). In contrast, after palatal shelf elevation at E14.5, gelatinolytic activity was evident in the nasal cartilage and in the two main palatal arteries (Fig. 2D–F). Interestingly, at this embryonic stage signs of gelatinolysis were evident at the midline epithelial seam (MES) formed prior to palatal shelf fusion, and in addition prominent activity was detected as sharp profiles surrounding distinct epithelial structures, such as the folds of the elevated palatal shelves, which contribute to morphogenesis of the nasal cavities in the front and the nasopharynx more posteriorly. In the posterior region, gelatinolytic activity faded out at the dorsal side of the palate. This pattern was also observed at E15.5 stage (Fig. 2G–I), although activity was increased compared to E14.5. Furthermore, prominent activity was evident for maxillary bone, as has been shown before for mandibular bone [33]. The present findings by ISZ of a temporal increase in gelatinolytic activity during embryonic development of the craniofacial region confirm previous results obtained with extracts from mouse embryo heads, which were analyzed for MMP activity (by SDS-gel zymography), protein (by immunoblotting), and mRNA (by RT-PCR), respectively [38].


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 the mouse embryo.Frontal cryosections of E13.5 (A–C), E14.5 (D–F), and E15.5 (G–I) wild type mouse heads were subjected to DQ-gelatin zymography (green panels), followed by immunofluorescence labeling for laminin-111 on the same section (red panels). The images show representative sections from the anterior (A, D, G), the middle (B, E, H), and the posterior (C, F, I) level of the palate. No site with significant gelatinolytic activity, as manifested by increased fluorescence, is detected at E13.5 prior to palatal shelf elevation. At E14.5, gelatinolytic activity is evident in the nasal cartilage and in the main palatal arteries (arrows). In addition, signs of gelatinolysis are visible at the midline epithelial seam (asterisks), whereas prominent gelatinolytic activity is detected around the folds of the elevated palatal shelves, where the nasopharynx will form (arrowheads). A similar pattern of gelatinolysis is observed at E15.5, although activity is increased compared to E14.5. Furthermore, prominent activity was evident for maxillary bone. p, palatal shelf; t, tongue; n, nasal cartilage; m, maxillary bone. Bar, 200 μm.
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Related In: Results  -  Collection

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pone-0047762-g002: Double labeling for gelatinolytic activity and laminin in the palatal region of the mouse embryo.Frontal cryosections of E13.5 (A–C), E14.5 (D–F), and E15.5 (G–I) wild type mouse heads were subjected to DQ-gelatin zymography (green panels), followed by immunofluorescence labeling for laminin-111 on the same section (red panels). The images show representative sections from the anterior (A, D, G), the middle (B, E, H), and the posterior (C, F, I) level of the palate. No site with significant gelatinolytic activity, as manifested by increased fluorescence, is detected at E13.5 prior to palatal shelf elevation. At E14.5, gelatinolytic activity is evident in the nasal cartilage and in the main palatal arteries (arrows). In addition, signs of gelatinolysis are visible at the midline epithelial seam (asterisks), whereas prominent gelatinolytic activity is detected around the folds of the elevated palatal shelves, where the nasopharynx will form (arrowheads). A similar pattern of gelatinolysis is observed at E15.5, although activity is increased compared to E14.5. Furthermore, prominent activity was evident for maxillary bone. p, palatal shelf; t, tongue; n, nasal cartilage; m, maxillary bone. Bar, 200 μm.
Mentions: A double-labeling technique comprising in situ zymography (ISZ) with dye-quenched (DQ-)gelatin, followed by brief acetone fixation and immunofluorescence labeling, was performed to associate gelatinolytic activity in situ with laminin-111 as a marker for embryonic basement membranes (Fig. 2). In the region of the developing secondary palate, no site with prominent gelatinolytic activity was identified prior to palatal shelf elevation at E13.5 at three anteroposterior levels (Fig. 2A–C). In contrast, after palatal shelf elevation at E14.5, gelatinolytic activity was evident in the nasal cartilage and in the two main palatal arteries (Fig. 2D–F). Interestingly, at this embryonic stage signs of gelatinolysis were evident at the midline epithelial seam (MES) formed prior to palatal shelf fusion, and in addition prominent activity was detected as sharp profiles surrounding distinct epithelial structures, such as the folds of the elevated palatal shelves, which contribute to morphogenesis of the nasal cavities in the front and the nasopharynx more posteriorly. In the posterior region, gelatinolytic activity faded out at the dorsal side of the palate. This pattern was also observed at E15.5 stage (Fig. 2G–I), although activity was increased compared to E14.5. Furthermore, prominent activity was evident for maxillary bone, as has been shown before for mandibular bone [33]. The present findings by ISZ of a temporal increase in gelatinolytic activity during embryonic development of the craniofacial region confirm previous results obtained with extracts from mouse embryo heads, which were analyzed for MMP activity (by SDS-gel zymography), protein (by immunoblotting), and mRNA (by RT-PCR), respectively [38].

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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