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Conditional deletion of Bmp2 in cranial neural crest cells recapitulates Pierre Robin sequence in mice

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Abstract

Bone morphogenetic protein (BMP) signaling plays a crucial role in the development of craniofacial organs. Mutations in numerous members of the BMP signaling pathway lead to several severe human syndromes, including Pierre Robin sequence (PRS) caused by heterozygous loss of BMP2. In this study, we generate mice carrying Bmp2-specific deletion in cranial neural crest cells using floxed Bmp2 and Wnt1-Cre alleles to mimic PRS in humans. Mutant mice exhibit severe PRS with a significantly reduced size of craniofacial bones, cleft palate, malformed tongue and micrognathia. Palate clefting is caused by the undescended tongue that prevents palatal shelf elevation. However, the tongue in Wnt1-Cre;Bmp2f/f mice does not exhibit altered rates of cell proliferation and apoptosis, suggesting contribution of extrinsic defects to the failure of tongue descent. Further studies revealed obvious reduction in cell proliferation and differentiation of osteogenic progenitors in the mandible of the mutants, attributing to the micrognathia phenotype. Our study illustrates the pathogenesis of PRS caused by Bmp2 mutation, highlights the crucial role of BMP2 in the development of craniofacial bones and emphasizes precise coordination in the morphogenesis of palate, tongue and mandible during embryonic development.

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Funding

This study was supported by grant from the National Natural Science Foundation of China (81870739), and by NIH grant (DE026482) to YPC.

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Authors and Affiliations

  1. Southern Center for Biomedical Research, Fujian Key Laboratory of Developmental and Neural Biology, College of Life Sciences, Fujian Normal University, Fuzhou, 350108, China

    Yixuan Chen, Zhengsen Wang, YiPing Chen & Yanding Zhang

  2. Department of Cell and Molecular Biology, Tulane University, New Orleans, LA, 70118, USA

    YiPing Chen

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  1. Yixuan Chen
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  2. Zhengsen Wang
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  3. YiPing Chen
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  4. Yanding Zhang
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Correspondence to Yanding Zhang.

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Animals and procedures used in this study were approved by the Fujian Normal University Institutional Animal Care and Use Committee.

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The authors declare that they have no conflict of interest.

Electronic supplementary material

Figure S1.

The expression ofBmp2is significantly reduced in the palate, mandibular bone and Meckel’s cartilage of mutant mice. (a-c) Bmp2 is expressed in the anterior palatal (a) as well as in the nasal side palatal mesenchyme and the medial edge epithelium (MEE) region in the posterior palate (b) and Bmp2 is also detected in mandibular bone and Meckel’s cartilage in wild type controls at E13.5 (c). (d-f) In Wnt1-Cre;Bmp2f/f mice, the expression of Bmp2 is significantly reduced in the palate (d, e), mandibular bone and Meckel’s cartilage at E13.5 (f). Mb: mandibular bone; M: Meckel’s cartilage. Scale bars: 100 μm (PNG 2724 kb)

High resolution image (TIF 6476 kb)

Figure S2.

Expression of genes crucial to palate development in the wild type andWnt1-Cre;Bmp2f/fmice at E13.5. (a-d) Section in situ of Bmp7and Bmp4. Bmp7 is expressed in both the epithelium and mesenchyme in the anterior palatal shelves but only in the epithelium of the posterior palatal shelves. Bmp4 is expressed in the anterior palatal mesenchyme underlying MEE in both control and mutant (e, f). Whole mount in situ of Shh shows that the number and localization of rugae in the E13.5 mutant is comparable to wide type controls (g, h). Msx1 immunostaining(red) of wide type control (i) and mutant palate (j). Msx1 is detected in the anterior palatal mesenchyme of wide type and mutant embryos. Scale bars: 100 μm (a-f, i, j), 500 μm (g, h). (PNG 5202 kb)

High resolution image (TIF 4559 kb)

Figure S3.

Signal transduction effectors of BMP signaling in the developing palate. (a-d) At E13.5, pSmad1/5/8 are detected in both the anterior and posterior palatal shelves in the wild type and mutant. In the posterior palatal shelves of wild type, pSmad1/5/8 activity is mainly restricted in the mesenchyme of the future nasal side (c) but the pSmad1/5/8 signals become weakened in the mutant embryo (d). (e, g) In wild type control, the pp38 signal is at a high level in the epithelium but is sparse in the mesenchyme in the anterior palate (e); in the posterior palate, pp38 activity is detected in the epithelium and in the oral side palatal mesenchyme (g). (f, h) In the mutant palate, a comparable level of pp38 expression is found. The straight white line in c and d and the straight black line in g and h divides the palatal shelves into nasal and oral halves. Mb: Mandibular bone; M: Meckel’s cartilage. Scale bars: 100 μm. (PNG 2694 kb)

High resolution image (TIF 12497 kb)

Figure S4.

Myogenic differentiation is unaffected in Wnt1-Cre;Bmp2f/f tongues. (a-d) MF20 immunostaining of coronal sections of control and Wnt1-Cre;Bmp2f/f tongues at E13.5 and E14.5. Arrows point to the extrinsic muscles of the tongue, arrowheads point to the intrinsic muscles. Scale bars: 100 μm (PNG 2383 kb)

High resolution image (TIF 9458 kb)

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Chen, Y., Wang, Z., Chen, Y. et al. Conditional deletion of Bmp2 in cranial neural crest cells recapitulates Pierre Robin sequence in mice. Cell Tissue Res 376, 199–210 (2019). https://doi.org/10.1007/s00441-018-2944-5

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