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First published online 16 February 2005
doi: 10.1242/dev.01676

Development 132, 1453-1461 (2005)
Published by The Company of Biologists 2005
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Distinct functions for Bmp signaling in lip and palate fusion in mice

Wei Liu1, Xiaoxia Sun1, Alen Braut4, Yuji Mishina3, Richard R. Behringer2, Mina Mina4 and James F. Martin1,*

1 Alkek Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Boulevard, Houston, TX 77030, USA
2 Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030,USA
3 Laboratory of Reproductive and Developmental Toxicology, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
4 Department of Pediatric Dentistry, School of Dental Medicine, University of Connecticut Health Science Center, 263 Farmington Avenue, Farmington, CT 06030, USA



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  		Fig. 1. Spatiotemporal analysis of cre activity directed by the Nestin cre transgene. (A-F) Coronal sections through whole-mount ß-gal staining of E10.5 (A,B), 11.5 (C,D) and 13.5 (E,F) dpc Nestin cre+/-;R26R+/- mice showing cre activity in Nestin cre transgenic mice. Cre activity is denoted by the blue color (arrows). Boxed areas in A and E are shown at higher magnification in B and F, respectively. In E and F, ß-gal-positive cells are found throughout the palatal shelves. (G) PCR analysis of dissected tissues from Nestin cre;Bmpr1a n/f embryos at 10.5 dpc. Primers flanking the second LoxP site were used to determine the extent of recombination at the Bmpr1a locus. The upper band is the product of Bmpr1aflox allele (arrowhead). The lower band is the product of Bmpr1anull allele and serves as an internal control. M, DNA marker; Md, mandibular process; Mp, maxillary process; Ln, laternal nasal process; Mn, medial nasal process; ps. palatal shelf.

 


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  		Fig. 2. Cleft lip and palate in Nestin cre;Bmpr1a n/f mutant embryos. (A-D) Frontal (A,B) and lateral (C,D) views of 16.5 dpc wild-type (A,C) and (B,D) Nestin cre;Bmpr1a n/f mutants showing the bilateral cleft lip in mutant embryos (arrow). Star in A and C indicates area of lip fusion in wild-type embryos. (E-J) Hematoxylin and Eosin staining of coronal sections through the palatal region of wild-type (E,G,I) and Nestin cre;Bmpr1a n/f mutant embryos (F,H,J) at the labeled developmental stages. Arrows denote unfused palatal shelves. (K-N) Coronal sections stained with Hematoxylin and Eosin of wild-type (K,M) and Nestin cre;Bmpr1a n/f mutant (L,N) secondary palate explants cultured overnight. Palatal shelves were harvested at 14.5 dpc (K,L) and E13.5 (M,N). Arrows denote the degenerating medial edge epithelium (MEE) in K and L. ns, nasal septum; ps, palatal shelf; t, tongue.

 


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  		Fig. 3. Gene expression in secondary palates of Nestin cre;Bmpr1an/f mutant embryos. Whole-mount in situ analysis of 12.5 dpc embryos with the labeled probes. Arrows denote hybridization signal. Two arrows (C-F) mark the limits of the hybridization signal in the secondary palate.

 


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  		Fig. 4. Analysis of gene expression in the fusing lip of Nestin cre;Bmpr1a n/f mutant embryos. (A-D) Fgf8 expression in the fusing lip region of wild-type and Nestin cre;Bmpr1a n/f at the labeled stage. (E-L) Fgf8 (E,F), p63 (G-J) and pitx1 (K,L) expression in the edge epithelium of the MNP and MP of wild-type and Nestin cre;Bmpr1a n/f mutant 10.5 dpc embryos. Arrows denote areas of hybridization that are absent in the mutant embryos. (E,F,H,J-L) Ventral views; (G,I) lateral oblique views. (M-O) Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) studies on sections from wild-type and Nestin cre;Bmpr1a n/f mutant embryos. Arrows denote the area of elevated apoptosis in the mutant embryo. Two examples of mutant embryos are shown (N,O).

 


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  		Fig. 5. Tooth abnormalities in Nestin cre;Bmpr1a n/f mutant embryos compared with stage-matched wild-type embryos. (A-D) Coronal sections of 16.5 dpc maxillary and mandibular molars. Genotypes and tooth types are labeled. In wild-type embryos (A,B), the maxillary and mandibular molars are in the cap stage of development, whereas in Bmpr1a mutants, the maxillary molar is at the bud stage of tooth development (arrow in C). The Bmpr1a mutant mandibular molars (D) are at the same stage of development as in the wild-type (B). Scale bar: 100 µm. (E-H) Coronal sections of maxillary and mandibular incisors at E16.5. Genotypes and tooth type are labeled. The developing maxillary and mandibular incisors are seen in the wild-type embryos but in the mutant the maxillary incisors are absent. In addition, there is a cleft of the primary palate (asterisk, G). Scale bar: 100 µm. (I,J) At E18.5 in the wild-type embryos, the maxillary and mandibular molars have progressed to the more advanced (bell) stage of development. (K) At 18.5 dpc, in the Bmpr1a embryos, the maxillary molars are still in the bud stage of tooth development (arrow). (L) The mandibular molars in 18.5 dpc mutants are at the cap stage of tooth development (arrow). I, incisor; mo, molars; mc, Meckel's cartilage; ns, nasal septum; t, tongue. Scale bars in I-L: 100 µm.

 


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  		Fig. 6. Bmp4 function in lip fusion. (A,B) Whole-mount ß-gal staining (A) and whole-mount in situ hybridization with Bmp2 probe (B) in Bmp4lacZ+/- and wild-type embryos, showing expression of Bmp4 and Bmp2 (arrow) in the fusing region of the lip. (C,D) Whole-mount in situ hybridization with a Bmp4 exon 4 probe showing the absence of a hybridization signal in the Nestin cre;Bmp4 n/f mutant embryo (arrow). (E-H) ß-Gal stained embryos showing cleft lip in the Nestin cre;Bmp4 n/f embryos (arrows). In this experiment the Bmp4lacZ allele was used instead of the Bmp4null allele. Stages and genotypes are labeled.

 

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© The Company of Biologists Ltd 2005