Dynamic activation of Wnt, Fgf, and Hh signaling during soft palate development - PubMed (original) (raw)

Dynamic activation of Wnt, Fgf, and Hh signaling during soft palate development

Eva Janečková et al. PLoS One. 2019.

Abstract

The soft palate is a key component of the oropharyngeal complex that is critical for swallowing, breathing, hearing and speech. However, complete functional restoration in patients with cleft soft palate remains a challenging task. New insights into the molecular signaling network governing the development of soft palate will help to overcome these clinical challenges. In this study, we investigated whether key signaling pathways required for hard palate development are also involved in soft palate development in mice. We described the dynamic expression patterns of signaling molecules from well-known pathways, such as Wnt, Hh, and Fgf, during the development of the soft palate. We found that Wnt signaling is active throughout the development of soft palate myogenic sites, predominantly in cells of cranial neural crest (CNC) origin neighboring the myogenic cells, suggesting that Wnt signaling may play a significant role in CNC-myogenic cell-cell communication during myogenic differentiation in the soft palate. Hh signaling is abundantly active in early palatal epithelium, some myogenic cells, and the CNC-derived cells adjacent to the myogenic cells. Hh signaling gradually diminishes during the later stages of soft palate development, indicating its involvement mainly in early embryonic soft palate development. Fgf signaling is expressed most prominently in CNC-derived cells in the myogenic sites and persists until later stages of embryonic soft palate development. Collectively, our results highlight a network of Wnt, Hh, and Fgf signaling that may be involved in the development of the soft palate, particularly soft palate myogenesis. These findings provide a foundation for future studies on the functional significance of these signaling pathways individually and collectively in regulating soft palate development.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Axin2 is expressed throughout the embryonic development of the murine soft palate region.

(A-D) Hematoxylin and eosin staining of frontal sections of a mouse head at E14.5. Schematic drawings of the mouse head in the top panel depict the position and angle of the sections. Boxed areas in A-D respectively indicate approximate locations of higher magnification images (E-P) of TVP (E,I,M), PLG (F,J,N), LVP (G,K,O) and PLP (H,L,P). (E-P) Visualization of Axin2-LacZ reporter mice at E14.5 (E-H), E16.5 (I-L) and E18.5 (M-P). Insets in E-P show expression of Axin2 mRNA detected by RNAscope (pink) and MHC protein in comparable regions to E-P visualized by immunohistochemistry (brown). Arrows indicate _Axin2_-positive cells. Co, cochlea; ET, Eustachian tube; GH, greater horns of the hyoid bone; HB, hyoid bone; H&E, hematoxylin and eosin staining; LVP, levator veli palatini; PLG, palatoglossus; PLP, palatopharyngeus; PP, pterygoid plate; SPC, superior pharyngeal constrictor; T, tongue; TVP tensor veli palatini. Scale bar in A = 100 μm (A-D), scale bar in E = 50 μm (E-P), 27 μm (insets in E-P).

Fig 2

Fig 2. Gli1 is dynamically expressed in the soft palate region.

(A-L) Visualization of Gli1-LacZ reporter mice at E14.5 (A-D), E16.5 (E-H), and E18.5 (I-L). Insets in A-L show expression of Gli1 by RNAscope (pink) and MHC immunohistochemistry (brown) in comparable regions to A-L. Arrows indicate Gli1 positive cells. Schematic drawings of the mouse head in the top panel depict the position and angle of the sections. LVP, levator veli palatini; PLG, palatoglossus; PLP. palatopharyngeus; TVP, tensor veli palatini. Scale bars = 50 μm (A-L), 27 μm (A-L).

Fig 3

Fig 3. Etv5 and Etv4, downstream targets of Fgf signaling, are abundantly expressed throughout embryonic soft palate muscle development.

(A-L) Etv5 and (M-X) Etv4 RNAscope (pink) and MHC immunohistochemistry (brown) in the soft palate region of C57BL/6J mice at E14.5 (A-D,M-P), E16.5 (E-H,Q-T), and E18.5 (I-L,U-X). Insets (A-X) show magnified views of Etv5 (A-L) and Etv4 (M-X) signal detected by RNAscope (pink) and MHC immunohistochemistry (brown). Arrows indicate Etv5 and Etv4 positive cells. Schematic drawings of the mouse head in the top panel depict the position and angle of the sections. LVP, levator veli palatini; MHC; myosin heavy chain; PLG, palatoglossus; PLP, palatopharyngeus; TVP, tensor veli palatini. Scale bars = 50 μm (A-X), 12 μm (insets in A-X).

Fig 4

Fig 4. Expression of Fgfr1, Fgfr2, Fgfr3, and Fgfr4 in the soft palate myogenic sites.

(A-P) Co-staining of MHC immunohistochemistry (brown) and Fgf receptor RNAscope (pink) in the soft palate myogenic sites of C57BL/6J mice at E16.5: Fgfr1 (A-D), Fgfr2 (E-H), Fgfr3 (I-L), Fgfr4 (M-P). Arrows indicate positive signal. Insets (A-P) show magnified views of Fgfr1 (A-D), Fgfr2 (E-H), Fgfr3 (I-L) and Fgfr4 (M-P) signal detected by RNAscope (pink) and MHC immunohistochemistry (brown). Schematic drawings of the mouse head in the top panel depict the position and angle of the sections. LVP, levator veli palatini; MHC; myosin heavy chain; PLG, palatoglossus; PLP, palatopharyngeus; TVP, tensor veli palatini. Scale bars = 50 μm (A-P), 13.5 μm (insets in A-P).

Fig 5

Fig 5. Schematic drawing summarizing the expression of Wnt, Hh, and Fgf signaling in the LVP region.

(A-B) Visualization of myogenic fibers by MHC immunofluorescence at E14.5 (A) and E18.5 (B). (C-D) Schematic drawings summarizing Wnt signaling activity (purple dots) at E14.5 (C) and E18.5 (D); (E-F) Hh signaling activity (yellow dots) at E14.5 (E) and E18.5 (F); and (G-H) Fgf signaling activity (dark blue dots) at E14.5 (G) and E18.5 (H). Light blue indicates the CNC-derived mesenchymal cells and green indicates soft palatal epithelium. MHC, myosin heavy chain.

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