Modulating Wnt Signaling Rescues Palate Morphogenesis in Pax9 Mutant Mice

Abstract

Cleft palate is a common birth defect caused by disruption of palatogenesis during embryonic development. Although mutations disrupting components of the Wnt signaling pathway have been associated with cleft lip and palate in humans and mice, the mechanisms involving canonical Wnt signaling and its regulation in secondary palate development are not well understood. Here, we report that canonical Wnt signaling plays an important role in Pax9-mediated regulation of secondary palate development. We found that cleft palate pathogenesis in Pax9-deficient embryos is accompanied by significantly reduced expression of Axin2, an endogenous target of canonical Wnt signaling, in the developing palatal mesenchyme, particularly in the posterior regions of the palatal shelves. We found that expression of Dkk2, encoding a secreted Wnt antagonist, is significantly increased whereas the levels of active β-catenin protein, the essential transcriptional coactivator of canonical Wnt signaling, is significantly decreased in the posterior regions of the palatal shelves in embryonic day 13.5 Pax9-deficent embryos in comparison with control littermates. We show that small molecule-mediated inhibition of Dickkopf (DKK) activity in utero during palatal shelf morphogenesis partly rescued secondary palate development in Pax9-deficient embryos. Moreover, we found that genetic inactivation of Wise, which is expressed in the developing palatal shelves and encodes another secreted antagonist of canonical Wnt signaling, also rescued palate morphogenesis in Pax9-deficient mice. Furthermore, whereas Pax9^del/del embryos exhibit defects in palatal shelf elevation/reorientation and significant reduction in accumulation of hyaluronic acid-a high molecular extracellular matrix glycosaminoglycan implicated in playing an important role in palatal shelf elevation-80% of Pax9^del/del;Wise^-/- double-mutant mouse embryos exhibit rescued palatal shelf elevation/reorientation, accompanied by restored hyaluronic acid accumulation in the palatal mesenchyme. Together, these data identify a crucial role for canonical Wnt signaling in acting downstream of Pax9 to regulate palate morphogenesis.

Keywords: Sostdc1; Wnt antagonist; cell signaling; cleft palate; craniofacial biology; transcription factors.

Figure 1.

Changes in expression of Wnt signaling components in the developing palatal shelves of Pax9^del/del mutant embryos. (A–D) Whole mount view of patterns of Wise mRNA expression in the palatal shelves of Pax9^del/+ control and Pax9^del/del mutant embryos at E12.5 and E13.5. Arrows point to the medial edge of the palatal shelves. (E–H) Frontal sections showing Wise mRNA expression patterns in the anterior and posterior regions of palate shelves in E13.5 Pax9^del/+ control and Pax9^del/del mutant embryos. (I) RT-qPCR analysis of the levels of expression of Wise mRNAs in E13.5 palatal shelves in Pax9^del/+ control and Pax9^del/del mutant embryos (n = 5). (J–M) Frontal sections showing Axin2 mRNA expression patterns in the anterior and posterior regions of palate shelves in E13.5 Pax9^del/+ control and Pax9^del/del mutant embryos. Green arrows point to the domains of Axin2 mRNA expression in the palatal mesenchyme. Black arrowheads point to Axin2 mRNA expression in the tooth germs. (N) RT-qPCR analysis of the levels of expression of Axin2 mRNAs in the anterior and posterior regions, respectively, of the palatal shelves in E13.5 Pax9^del/+ control and Pax9^del/del mutant embryos (n = 3). (O) Western blot analysis of the levels of active β-catenin proteins in the posterior halves of palatal shelves from E13.5 Pax9^del/+ control and Pax9^del/del mutant embryos. The levels of β-actin in each sample were detected as an internal loading control. (P) Relative active β-catenin band intensity on Western blots normalized against β-actin (n = 3 samples each for Pax9^del/+ and Pax9^del/del embryos). (Q, T) Whole mount view of patterns of Dkk2 mRNA expression in the palate of E12.5 Pax9^del/+ control and Pax9^del/del mutant embryos. (R, S, U, V) Frontal sections showing Dkk2 mRNA expression patterns in the anterior and posterior regions of palate shelves of E13.5 Pax9^del/+ control and Pax9^del/del mutant embryos. Black arrowheads point to Dkk2 mRNA expression in the tooth germs. (W) RT-qPCR analysis of the levels of Dkk2 mRNAs in the anterior and posterior regions, respectively, of the palatal shelves from E13.5 Pax9^del/+ control and Pax9^del/del mutant embryos (n = 3). E, embryonic day; ns, not significant; p, palatal shelf; t, tongue. Error bars represent SD. *P < 0.05. **P < 0.01.

Figure 2.

Inhibition of DKK activity in utero partly rescued palate development in Pax9^del/del mutant embryos. (A, E, I) Whole mount view of the palate in WT, DMSO-treated Pax9^del/del, and IIIC3a-treated Pax9^del/del pups at P0. Arrowhead in panel I points to cleft between the primary and anterior secondary palates. (B–D, F–H, J–L) Representative frontal sections from the anterior, middle, and posterior regions of the secondary palate in WT, DMSO-treated Pax9^del/del, and IIIC3a-treated Pax9^del/del embryos at E16.5. Of 11 IIIC3a-treated Pax9^del/del pups, 7 exhibited secondary palate that was fused in the middle and posterior regions, whereas the other 4 still had cleft palate. Asterisk indicates cleft palate in panels E–H. Arrows point to molar tooth germs. E, embryonic day; P0, postnatal day; p, palatal shelf; t, tongue; WT, wild type.

Figure 3.

Genetic inactivation of Wise rescued palate morphogenesis in Pax9^del/del mice. (A, E, I) Whole mount view of the palate in WT, Pax9^del/del, and Pax9^del/del;Wise^-/- pups at P0. (B–D, F–H, J–L) Representative frontal sections from anterior, middle, and posterior regions of the secondary palate in E16.5 WT, Pax9^del/del, and Pax9^del/del;Wise^-/- embryos. Of 20 Pax9^del/del;Wise^-/- pups examined after E16.5, 14 exhibited fused palate, whereas the other 6 Pax9^del/del;Wise^-/- pups had cleft palate. Asterisk indicates the cleft palate in panels E–H. Arrows point to the molar tooth germs. E, embryonic day; P0, postnatal day; p, palatal shelf; t, tongue; WT, wild type.

Figure 4.

Genetic inactivation of Wise restored canonical Wnt signaling activity in the developing palatal shelves in Pax9^del/del embryos. (A–D) Frontal sections of E13.5 palatal shelves showing Axin2 mRNA expression pattern in the anterior and posterior regions of palate shelves in Pax9^del/+;Wise^+/- control and Pax9^del/del;Wise^-/- double-mutant embryos. Green arrows point to the domains of Axin2 mRNA expression in the anterior palatal mesenchyme. Black arrowheads point to Axin2 mRNA expression in the tooth germs. (E) RT-qPCR analysis of the levels of expression of Axin2 mRNAs in the anterior and posterior regions of the palatal shelves in E13.5 Pax9^del/+;Wise^+/- control and Pax9^del/del;Wise^-/- double-mutant embryos (n = 3). (F) Western blot detection of active β-catenin in the posterior palatal shelves in E13.5 Pax9^del/+;Wise^+/- control and Pax9^del/del;Wise^-/- mutant embryos. The levels of β-actin were detected as an internal loading control. (G) Relative band intensity of active β-catenin protein on Western blots was analyzed by ImageJ (n = 3) and normalized to that of β-actin. (H–M) Frontal sections showing Bmp4 mRNA expression patterns in the anterior and posterior regions of palatal shelves in E13.5 WT, Pax9^del/del, and Pax9^del/del;Wise^-/- embryos. (N) RT-qPCR analysis of the levels of expression of Bmp4 mRNAs in E13.5 palatal shelves in Pax9^del/+ control and Pax9^del/del mutant embryos, as well as Pax9^del/+;Wise^+/- control and Pax9^del/del;Wise^-/- mutant embryos (n = 5 for each genotype). (O–T) Immunofluorescence detection of pSmad1/5/9 proteins in the posterior and anterior regions of the palatal shelves from E13.5 WT, Pax9^del/del, and Pax9^del/del;Wise^-/- embryos. Yellow arrows point to comparable regions of the posterior palatal mesenchyme, whereas white arrows point to comparable regions of anterior palatal mesenchyme. Error bars represent SD. E, embryonic day; ns, not significant; p, palatal shelf; t, tongue; WT, wild type. ***P < 0.001.

Figure 5.

Inactivation of Wise rescued palatal shelf elevation/reorientation in Pax9^del/del embryos. (A–I) Representative frontal sections from anterior, middle, and posterior regions of the secondary palate in E14.5 WT, Pax9^del/del, and Pax9^del/del;Wise^-/- embryos. Arrows point to the molar tooth germs. (J–L) Representative frontal sections from the posterior region of the palatal shelves of E13.5 WT, Pax9^del/del, and Pax9^del/del;Wise^-/- embryos showing fluorescent staining of hyaluronic acid (red color). White dashed line marks the proximal boundary of the palatal shelf area used for quantification of fluorescence intensity. (M) Quantification of mean fluorescence intensity of hyaluronic acid staining in the WT, Pax9^del/del, and Pax9^del/del;Wise^-/- samples (n = 4 for each genotype). Fluorescence intensity of hyaluronic acid staining was normalized against the area of palatal shelves. Error bars represent SD. p, palatal shelf; t, tongue; WT, wild type. *P < 0.05. **P < 0.01.