Neural crest-specific deletion of Ldb1 leads to cleft secondary palate with impaired palatal shelf elevation

Abstract

Background: LIM domain binding protein 1 (LDB1) is a transcriptional co-factor, which interacts with multiple transcription factors and other proteins containing LIM domains. Complete inactivation of Ldb1 in mice resulted in early embryonic lethality with severe patterning defects during gastrulation. Tissue-specific deletions using a conditional knockout allele revealed additional roles of Ldb1 in the development of the central nervous system, hematopoietic system, and limbs. The goal of the current study was to determine the importance of Ldb1 function during craniofacial development in mouse embryos.

Results: We generated tissue-specific Ldb1 mutants using Wnt1-Cre, which causes deletion of a floxed allele in the neural crest; neural crest-derived cells contribute to most of the mesenchyme of the developing face. All examined Wnt1-Cre;Ldb1(fl/-) mutants suffered from cleft secondary palate. Therefore, we performed a series of experiments to investigate how Ldb1 regulated palate development. First, we examined the expression of Ldb1 during normal development, and found that Ldb1 was expressed broadly in the palatal mesenchyme during early stages of palate development. Second, we compared the morphology of the developing palate in control and Ldb1 mutant embryos using sections. We found that the mutant palatal shelves had abnormally blunt appearance, and failed to elevate above the tongue at the posterior domain. An in vitro head culture experiment indicated that the elevation defect was not due to interference by the tongue. Finally, in the Ldb1 mutant palatal shelves, cell proliferation was abnormal in the anterior, and the expression of Wnt5a, Pax9 and Osr2, which regulate palatal shelf elevation, was also altered.

Conclusions: The function of Ldb1 in the neural crest-derived palatal mesenchyme is essential for normal morphogenesis of the secondary palate.

Figure 1

Expression ofLdb1during palate development. (A-H) Coronal sections of the heads were processed by RNA in situ hybridization for Ldb1, using a probe against exons 5–9. Ldb1^fl/- embryos were used as controls in this figure. Brackets in A, oral half of the first pharyngeal arch (PA1). Arrows in E and G, condensed dental mesenchyme. Abbreviations: LM, lower molar; mdPA1, mandibular arch; mxPA1, maxillary arch; PS, palatal shelf; To, tongue; UM, upper molar. Bar, 0.5 mm.

Figure 2

Cleft secondary palate ofWnt1-Cre;Ldb1^fl/-mutants. All the samples are E18.5. A,B) Whole mount view of the palate. The nose is toward the top. C-F) Skeleton staining for bone (red) and cartilage (blue). C and D are lateral views of the head. E and F show the palate area after the removal of the lower jaw, in the same orientation as in A and B. Note that the vomer (Vo) and presphenoid (PSp) in F are not visible in E because they are underneath the maxilla (Max) and palatine (PL) normally. Abbreviations: BO, basioccipital; BS, basisphenoid; De, dentary; Fr, frontal bone; LO, lamina obturans; Pa, parietal bone; PMx, premaxilla; Pt, pterygoid. Bar, 1 mm.

Figure 3

Morphological and morphometric analyses of the palate inWnt1-Cre;Ldb1^fl/-mutants. A-R) Coronal sections of the heads were stained with cresyl violet. Arrows in A-D, the distal tip of the palatal shelf. Arrow in P, a protrusion on the medial side of the palatal shelf, considered an intermediate structure for palatal shelf elevation. Abbreviation: DT, diastema tooth. Bar, 0.5 mm. S and T) Quantitative analysis of the area of the palatal shelf. The boundary of the palatal shelf used for this measurement is indicated by the dotted lines in A-L. *: p < 0.05.

Figure 4

Removing the lower jaw does not restore the reorientation of the palatal shelves inWnt1-Cre;Ldb1^fl/-mutants. The upper jaw region from E13.5 embryos, before culture (A-D) and after 3-day culture in the absence of the lower jaw and the tongue (E-N). A-H are fluorescent pictures of the heads processed by whole mount DAPI staining. (A,C,E,G) Intra-oral views directly into the palate. (B,D,F,H) Oblique views showing the profile of the palatal shelf. Red arrows in F and H point to the posterior palatae. (I-N) Coronal sections were stained with Nuclear Fast Red. Abbreviations: ant, anterior; pos, posterior; No, nose. Bar, 0.5 mm.

Figure 5

Cell proliferation analysis. (A-F) Coronal sections of the heads from E13.5 embryos were processed with immunofluorescence for BrdU (red) and counter-stained with DAPI for nuclei (blue). (G-H) Quantitative analyses of the mitotic index in the palatal shelf, in the medial (M), lateral (L) or combined (I) areas as demarcated by white lines in A-F. *, p > 0.05. Bar, 0.2 mm.

Figure 6

Altered expression ofWnt5a, Osr2andPax9inWnt1-Cre;Ldb1^fl/-mutant palatal shelf. (A-R) Coronal sections of the heads from E13.5 embryos were processed by RNA in situ hybridization. Arrows in E and F, ectopic expression of Wnt5a in the posterior palate. Arrows in G-J, up-regulation of Osr2 in the distal tip of the Ldb1 mutant palatal shelf. Arrows in M-R, down-regulation of Pax9 in the Ldb1 mutant palatal shelf. Abbreviations: na, nasal domain of the palatal shelf; or, oral domain of the palatal shelf. Bar, 0.2 mm.