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Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis

Zunyi Zhang¹, Yiqiang Song¹, Xiang Zhao^1,*, Xiaoyun Zhang¹, Cesar Fermin² and YiPing Chen^1,

¹ Department of Cell and Molecular Biology and Center for Bioenvironmental Research, Tulane University, New Orleans, LA 70118, USA
² Department of Pathology, Tulane University, New Orleans, LA 70118, USA
^* Present address: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada

View larger version (80K): [in a new window]   Fig. 1. Ectopic Bmp4 expression in the palatal mesenchyme rescued the cleft palate in Msx1–/– mice. (A-C) Expression of the human Bmp4 transgene (arrows) was detected in the anterior palatal mesenchyme of E12.5 (A) and E13.5 (B) Msx1-Bmp4 transgenic embryos using a transgene-specific probe, as described previously (Zhao et al., 2000). No signals were detected in an E13.5 wild-type control palate using the same transgene-specific probe (C). (D) A coronal section through an E14.5 wild-type embryonic head showing fusion of the palatal shelves. (E) An E14.5 Msx1–/– embryo showing cleft secondary palate (arrows). (F) A coronal section of an E14.5 Msx1–/–/Tg embryo showing fusion of the palatal shelves. (G,H) A wild-type newborn mouse displaying a closed palate (G) when compared with the cleft palate (arrows) shown in an Msx1–/– newborn (H). (I) Rescue of cleft palate in an Msx1–/–/Tg newborn mouse. (Insert) Rugae did not fuse at the midline (arrow) of the palate in a three-month-old Msx1–/–/Tg mouse. (J-L) Skeletal staining showing cleft palate in a newborn Msx1–/– mouse (K) and the rescue of cleft palate in a newborn Msx1–/–/Tg mouse (L), when compared with a newborn wild type mouse (J). The sphenoid bone (white arrow in K), which could be directly viewed in the Msx1 mutant, was not apparent in the Msx1–/–/Tg mouse (L). The sharp appearance of the premaxilla (black arrow in K) found in Msx1 mutant was also seen in a Msx1–/–/Tg mouse (L), when compared with the normal rounded morphology of premaxilla in a wild-type mouse (black arrow, J), indicating that the rescue seen in Msx1–/–/Tg mouse was specific to the cleft palate phenotype. (M) Scanning electron microscopic image of the oral view of an E13.5 wild-type embryonic head showing the overall shape of the developing palate, the planes of sections shown in Figs 2, 3, 6, and the regions excised for the explantation studies shown in Figs 4, 5. The yellow line indicates cuts made during dissection to separate the anterior region (top to the line) from the posterior region (bottom from the line) of the palate in this study. The red line indicates the anterior section plane, and green line indicates the posterior section plane used in the section in situ hybridization studies and BrdU labeling experiments shown in Figs 2, 3, 6. The white-lined box labeled ‘A’ represents the anterior region of palatal tissue, and the black-lined box labeled ‘P’ represents the posterior region of palatal tissue used for the explantation experiments shown in Figs 4, 5. (N) A coronal section through the oral region of an E13.5 embryo indicating the precise location (boxed) of palatal shelf shown in Fig. 1A-C, Figs 2, 3, Fig. 6D-F. I, incisor; M, molar; T, tongue; NS, nasal septum; PS, palatal shelf. Scale bars: in D,E, 100 µm; in N, 300 µm.

View larger version (140K): [in a new window]   Fig. 2. Expression of Msx1, Bmp2, Bmp4 and Shh in the developing palatal shelves. (A-C) Msx1 transcripts were detected in the mesenchyme of the anterior region of developing palatal shelf at E12.5 (A) and E13.5 (B), but was not detected in the posterior region of palatal shelf (C). Strong Msx1 expression was seen in the maxillary molar dental mesenchyme (dm) (C). (D-F) Bmp4 expression was found in the palatal epithelium (arrow) and mesenchyme (arrowheads) at E12.5 (D), and was restricted to the mesenchyme (arrow) at E13.5 (E). Bmp4 expression was absent in the posterior region of E13.5 palatal shelf (F). (G-I) Bmp2 expression was detected in both the epithelium and mesenchyme of the anterior region of developing palatal shelves at E12.5 (G) and E13.5 (H), but was absent in the posterior palate (I). (J,K) Shh expression was detected in the MEE (arrows) of the anterior palatal shelves at E12.5 (J) and E13.5 (K), but was not detected in the MEE of the E13.5 posterior palatal shelf (L), where Shh expression in the rugae was seen (arrows). Scale bar, 100 µm.

View larger version (124K): [in a new window]   Fig. 3. Expression of Bmp4, Shh and Bmp2 was downregulated in the anterior region of the Msx1–/– palatal shelves, but was restored in the Msx1–/–/Tg palatal shelves. (A) Bmp4 expression was downregulated in the Msx1–/– palatal mesenchyme, but remained in the epithelium (arrow) at E12.5. (B) At E13.5, Bmp4 expression was not detected in the Msx1–/– palatal mesenchyme. (C) Endogenous Bmp4 expression was not detected in the Msx1–/–/Tg palatal mesenchyme at E13.5, indicating that transgenic Bmp4 did not induce endogenous Bmp4 expression in the absence of Msx1. (D-F) Shh expression remained unaltered in the MEE (arrow) of the E12.5 Msx1–/– palatal shelf (D), but was not detectable in the MEE (arrow) of the E13.5 palatal shelf (E). (F) However, Shh expression was restored in the MEE (arrow) of the E13.5 Msx1–/–/Tg palatal shelf. (G-I) Bmp2 expression was detected in the E12.5 Msx1–/– palatal shelf (G), but was downregulated in the palate at E13.5 (H). In the E13.5 Msx1–/–/Tg palatal shelf, Bmp2 expression was again detected (I). Scale bar, 100 µm.

View larger version (81K): [in a new window]   Fig. 4. Gene expression induced by protein soaked beads in the anterior palatal mesenchyme. (A-E) BMP4-soaked beads induced the expression of Msx1 (A) and Bmp4 (C) in E12.5 wild-type palatal mesenchyme, but failed to induce Bmp4 expression in the Msx1–/– palatal mesenchyme (D). Control BSA beads failed to induce Msx1 (B) and Bmp4 (E). (F) Shh-soaked beads failed to induce Bmp4 expression in wild-type palatal mesenchyme. (G,H) Endogenous Bmp4 expression was not affected by an anti-Shh antibody bead implanted in the palatal explant containing both the epithelium and mesenchyme (G), when compared with the BSA control (H). (I-K) Similar to the BSA-soaked control beads (K), BMP4-soaked bead failed to induce Bmp2 expression in the Msx1–/– palatal mesenchyme (I) and wild-type palatal mesenchyme (J). (L,M) Shh-soaked beads induced Bmp2 expression in the palatal mesenchyme of both Msx1 mutant (L) and wild-type (M) palatal mesenchyme. (See Fig. 1M for indication of cuts made during dissection to isolate the regions of palatal tissue used in this figure.) b, bead.

View larger version (87K): [in a new window]   Fig. 5. Gene expression induced by protein-soaked beads in the posterior palatal mesenchyme of wild-type embryos. (A,B) BMP4-soaked beads failed to induce the expression of Msx1 (A) and Bmp4 (B) itself in the posterior palatal mesenchyme. (C) Shh-soaked beads also failed to induce Bmp2 expression in the posterior palatal mesenchyme. (D-F) FGF8-soaked beads induced Pax9 expression in the posterior palatal mesenchyme (D), but failed to do so in the anterior palatal mesenchyme (E), when compared with controls in which BSA-soaked beads were implanted into the posterior palatal mesenchyme (F).

View larger version (81K): [in a new window]   Fig. 6. Cleft palate in Msx1 mutants is caused by defective cell proliferation in the anterior portion of palatal shelves. (A-C) Palatal shelves from E13.5 Msx1–/– embryo (B), like those from wild type (A) and Msx1–/–/Tg embryos (C), fused and exhibited a disruption of the midline seam when placed in contact in vitro. Arrows indicate the remainder of the midline seam. (D-F) Cell proliferation, indicated by BrdU labeling, was significantly reduced in the E13.5 Msx1–/– palatal mesenchyme (E), particularly in the mesenchyme immediately beneath the MEE (arrow), when compared with mesenchyme in wild-type palates (D). Cell proliferation was restored to normal levels in Msx1–/–/Tg palatal shelves (F). (G) A summary of BrdU-labeling studies in the anterior and posterior portions of the palatal shelves of the E13.5 wild type, Msx1–/– and Msx1–/–/Tg embryos. The box in D represents the position and size of counting area (22.5 mm2), as determined by use of an ocular grid. Using Student’s t-test, P values were obtained by comparing numbers from Msx1–/– or Msx1–/–/Tg with those from wild type. T, tongue. Scale bars: 100 µm.

View larger version (83K): [in a new window]   Fig. 7. Shh and BMP2 induced cell proliferation in the anterior palatal mesenchyme determined by BudU labeling. (A,B) BMP2-soaked beads induced cell proliferation in E13.5 palatal mesenchyme 8 hours after bead implantation, in both wild-type (A) and Msx1mutant tissues (B). Note that BrdU-labeled cells asymmetrically localized closer to the MEE (arrows). (C,D) BSA-soaked control beads failed to induce cell proliferation in the palatal mesenchyme of E13.5 wild-type (C) and Msx1 mutant embryos (D). (E,F,H) Shh-soaked beads induced cell proliferation (arrows) around the beads in E13.5 wild-type (E) and Msx1–/– palatal mesenchyme (F) 24 hours after bead implantation. However, Shh-soaked beads failed to induce cell proliferation in both wild-type (data not shown) and Msx1–/– palatal mesenchyme (H) 8 hours after implantation. (G) A bead loaded with both Shh and Noggin proteins failed to induce cell proliferation 24 hours after bead implantation. (I) Beads soaked with an anti-Shh antibody inhibited cell proliferation in palatal tissue explants containing both the epithelium and mesenchyme of E13.5 wild-type embryo. (J) A BSA-soaked control bead did not affect cell proliferation (arrow) when implanted into E13.5 wild-type palatal tissue explants containing both the epithelium and mesenchyme. (K) Cell proliferation (arrows) was induced in E13.5 wild-type palatal tissue explants containing both the epithelium and mesenchyme 24 hours after implantation of a bead soaked with both an anti-Shh antibody and BMP2 protein. In all panels, the MEE aspect is towards the left. All palatal tissues used in this figure were from the anterior region of palatal shelves. b, bead.

View larger version (70K): [in a new window]   Fig. 8. A model for a genetic pathway integrating Msx1, Bmp4, Shh and Bmp2 in the epithelial-mesenchymal interactions that regulate mammalian palatogenesis. In this model, it is proposed that in the anterior palatal shelves, mesenchymally expressed Msx1, which can be induced by Bmp4, is required for Bmp4 expression in the palatal mesenchyme. Mesenchymally expressed BMP4 maintains Shh expression in the MEE and Shh in turn induces Bmp2 expression in the mesenchyme. BMP2 functions to induce cell proliferation in the palatal mesenchyme, which leads to palatal growth.