Mesenchymal Remodeling during Palatal Shelf Elevation Revealed by Extracellular Matrix and F-Actin Expression Patterns

Abstract

During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis.

Keywords: actin; extracellular matrix; mouse embryo; palatal shelf elevation; palate morphogenesis; tissue remodeling.

Figure 1

Periostin expression as a marker for palatal shelves during elevation. In situ hybridization for Postn mRNA on frontal sections of palatal shelves from wild-type mouse embryos at the anterior (A,B), medial (C,D), and posterior (E,F) level. Serial sections were obtained from an E13.5 (A,C,E) and an E14.5 (B,D,F) embryo, respectively. For description, see text. Abbreviations: palatal shelf (p), tongue (t). Bar, 400 μm.

Figure 2

Tenascin-C expression primarily in the lingual/medial half of palatal shelves both before and after shelf elevation. In situ hybridization for Tnc mRNA on frontal sections of palatal shelves from wild-type mouse embryos at the anterior (A,B), medial (C,D), and posterior (E,F) level. Serial sections were obtained from an E13.5 (A,C,E) and an E14.5 (B,D,F) embryo, respectively. For description, see text. Abbreviations: palatal shelf (p), tongue (t). Bar, 400 μm.

Figure 3

Tenascin-W expression in the dorsomedial quadrant of palatal shelves both before and after elevation. In situ hybridization for tenascin-W/Tnn mRNA on frontal sections of palatal shelves from wild-type mouse embryos at the anterior (A,B), medial (C,D), and posterior (E,F) level. Serial sections were obtained from an E13.5 (A,C,E) and an E14.5 (B,D,F) embryo, respectively. For description, see text. Abbreviations: palatal shelf (p), tongue (t). Bar, 400 μm.

Figure 4

Smoc2 expression in palatal tip epithelium before, but not midline epithelium after elevation. In situ hybridization for Smoc2 (A,B) or Tgfβ3 (C,D) mRNA, respectively, on frontal sections at the medial level of palatal shelves from wild-type mouse embryos. Serial sections were obtained from an E13.5 (A,C,E) and an E14.5 (B,D,F) embryo, respectively. For description, see text. Abbreviations: palatal shelf (p), tongue (t). Bar, 400 μm.

Figure 5

Scheme of the combined expression patterns of ECM genes in palatal shelves before and after elevation. The schematic drawings represent palatal shelves of wild-type mouse embryos at the medial level before (E13.5; above) and after (E14.5; below) elevation. The approximate areas of mesenchymal expression of periostin, tenascin-C, tenascin-W, and epithelial expression of Smoc2 and Tgfβ3 are indicated. Note that tenascin-C, tenascin-W, and Tgfβ3 expressing areas all end up at the midline after elevation, although their center of expression does not localize to the shelf tip before elevation. Conversely, Smoc2 mRNA is epithelially expressed at the shelf tip before, but excluded from midline epithelium after elevation.

Figure 6

Differences in tissue rearrangements between anterior, medial, and posterior palatal shelves during elevation. Serial sections through E14.5 palatal shelves at the anterior (A,B), medial (C,D), and posterior (E,F) level were double-stained with rhodamine-phalloidin for actin (red) and antibody to tenascin-C (green), and viewed in a fluorescence microscope. Sections were obtained from two embryos of the same E14.5 litter; one in which the shelves had just started to elevate (A,C,E), and one in which they had already risen above the tongue (B,D,F). Abbreviations: palatal shelf (p), tongue (t), nasal septum (n). The palatine artery is marked with an arrow in (C,D), and shown enlarged in the inserts. Bar, 400 μm.

Figure 7

Actin fiber and nuclear orientation in the mesenchyme of the posterior palate during elevation. Representative sections through E14.5 palatal shelves were triple-stained with phalloidin for actin (A,B), DAPI for nuclei (C,D), and antibody to tenascin-C (E,F), respectively, and viewed in a fluorescence microscope. On the left (A,C,E), a shelf just before elevation is shown, and on the right (B,D,F) an already elevated shelf from an embryo of the same litter. The images (C,D) were processed by ImageJ, such that all elongated nuclei with an aspect ratio above 2.5 are false-colored in red, those with 2.0–2.5 in pink. Roundish nuclei (aspect ratio < 2.0) are blue. Note that before elevation (C), many elongated (red/pink) nuclei are present between the original tip and the medial protrusion of the elevating shelf (arrowheads in A,C,E), and that their direction parallels that of the actin (A) and ECM (E) meshwork. The palatine artery is marked with an arrow in (A,B). Abbreviations: palatal shelf (p), tongue (t), nasal septum (n). Bar, 200 μm.