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First published online 1 September 2005
doi: 10.1242/dev.02013

Development 132, 4397-4406 (2005)
Published by The Company of Biologists 2005
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Shox2-deficient mice exhibit a rare type of incomplete clefting of the secondary palate

Ling Yu1,*, Shuping Gu1,*,{dagger}, Sylvia Alappat1, Yiqiang Song1, Mingquan Yan1, Xiaoyun Zhang1, Guozhong Zhang2, Yiping Jiang1, Zunyi Zhang1, Yanding Zhang2 and YiPing Chen1,2,{ddagger}

1 Division of Developmental Biology, Department of Cell and Molecular Biology, and Center for Bioenvironmental Research, Tulane University, New Orleans, LA 70118, USA
2 College of Bioengineering, Fujian Normal University, Fuzhou, Fujian 350007, PR China



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  		Fig. 1. Expression of Shox2 in the developing palate. (A-C) Shox2 expression (arrows) is detected by whole-mount (A) and section (B,C) in situ hybridization in the anterior (A,B) but not the posterior portion of palatal shelves at E11.5. The expression is restricted in the palatal mesenchyme (B). (D-F) At E12.5, Shox2 expression (arrows) domain expands but is still restricted in the anterior palatal mesenchyme, as detected by whole-mount (D) and section (E) in situ hybridization. (G-I) Shox2 expression (arrows) is still restricted in the anterior (G,H) but not in the posterior region (I) at E13.5. The expression at this stage expands to the palatal epithelium (H). (J-L) At E14.5, Shox2 expression exhibits a pattern of alternative stripes in the anterior half of the palate (J). The expression is detected in both the palatal mesenchyme and epithelium (K). (M) SHOX2 is also detected in the anterior palate of human embryo of 42 days post-conception (dpc), equivalent to mouse E11.5. (N) The posterior palate of human embryo of 42 dpc shows no SHOX2 expression. All sections shown were made through a coronal plane. M, the first molar; T, tongue; PS, palatal shelf. Scale bar: 200 µm.

 


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  		Fig. 2. Targeted disruption of the Shox2 gene in mice. (A) The mouse Shox2 genomic structure spans 8.3 kb, and contains 6 exons, as indicated by numbers. The targeting vector contains genomic fragments flanking the PGK-neo cassette which replaces exon 3 of Shox2 when correct homologous recombination occurs in ES cells. (B) Southern blot analysis of genomic DNA (digested with BamHI and probed with 3'-probe) derived from E11.5 embryos with different genotypes. (C) PCR analysis of DNA from DNA samples from yolk sac of embryos using W and M pairs of primers for wild-type and mutant alleles, respectively (see A). The W set of primers amplify the wild-type DNA fragment at the size of 520 bp, while the M set of primers amplify the mutant DNA fragment of 300 bp. (D) RT-PCR assays showing the absence of partial Shox2 transcripts in Shox2-deficient embryos.

 


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  		Fig. 3. Shox2 mutants exhibit clefting in the anterior palate. (A,B) Oral view of E15.0 wild-type (A) and Shox2-/- (B) palate shows an anterior clefting in the mutant. Arrows indicate the cleft. (C,D) Scanning electron microscopic images of oral view of E17.5 wild-type (C) and Shox2-/- (D) embryonic palate show an incomplete clefting (arrows) in the mutant palate. Arrow indicates the cleft, while arrowheads indicate the regions where the secondary palate fails to fuse with the primary palate and nasal septum. P, primary palate; PS, palatal shelf.

 


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  		Fig. 4. Shox2-/- mice exhibit impaired palatal growth in the anterior domain. Coronal sections were made in all panels. Those anterior (A,B,E,F,I,J) and posterior (C,D,G,H) to the first molar are indicated. (K and L) Sections were made through mid-level of the first molar. (A-D) At E13.5, the palatal shelves are vertically oriented in the wild type (A,C). The anterior palatal shelves (arrows) in the Shox2 mutants appear shorter and more rounded (B), but the posterior palatal shelves appear indistinguishable from the wild type (D). (E-H) At E14.5, the wild-type palatal shelves have elevated to the horizontal position above the tongue, closed and begun to fuse (E,G). The mutant palatal shelves have also elevated to the dorsum of the tongue, but have only made contact and fused in the posterior region (H). The anterior palatal shelves of the mutants appear too small to make contact at the midline, leaving an opening as indicated by a star (F). (I-L) At E15.0, palate fusion is still ongoing along the AP length in the wild-type embryo (I,K). A cleft (star) remains in the anterior palate of Shox2 mutants (J). A section through the mid-level of the first molar of the mutant shows the anterior contact point (arrow) of the palatal shelves (L). Inset in L shows disruption of the midline seam (arrowheads) in the posterior palate of the mutant at E15.0. T, tongue; PS, palatal shelves; th, tooth. Scale bar: 300 µm.

 


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  		Fig. 5. Altered cellular processes in the Shox2-/- palatal shelves. (A,B) PCNA staining on coronal sections of E12.5 wild-type (A) and Shox2-/- (B) palate shows significantly decreased level of cell proliferation in the anterior region of the Shox2-/- palate. (C,D) The TUNEL assay on coronal sections of E13.5 wild-type (C) and Shox2-/- (D) palate exhibits an increased level of cell apoptosis (arrows) in the anterior palatal epithelium of the Shox2 mutant. (E) Comparison of PCNA-positive cells in a fixed area of palate in wild-type and Shox2 mutant embryos. The number of PCNA-positive cells in the mutant type palatal apex epithelium (mean=22.7), as marked by arrows in A,B, is greatly reduced when compared with that in the wild type (mean=72.3) (P<0.0005). The number of PCNA-positive cells in the mutant palatal mesenchyme (mean=17.5), as counted within the large square in A,B, is reduced to about 30% when compared with that in the wild type (mean=65.6) (P<0.0005). However, the number of PCNA-positive cells in the laterally adjacent maxillary mesenchyme where Shox2 is not expressed, as counted within the small square in A,B, is similar between the wild type and the mutant (P>0.5). Standard deviation values were indicated for the error bars.

 


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  		Fig. 6. Gene expression in the Shox2-/- palatal shelves. (A) Msx1 expression in the anterior palatal mesenchyme of an E12.5 wild-type embryo. (B) Msx1 exhibits a wild-type pattern of expression in the anterior palatal mesenchyme of an E12.5 Shox2-/- embryo. (C,D) Pax9 expression is unaltered in the anterior palate of Shox2 mutant (D) when compared with wild-type control (C). (E,F) Ectopic Fgf10 expression is seen in the anterior palatal mesenchyme (arrows) of E13.5 mutant embryo (E) but not in the wild-type counterpart (F). (G,H) Fgfr2, which is restricted to the anterior palatal epithelium (arrowheads) in E13.5 wild-type embryo (G), is ectopically activated in the anterior palatal mesenchyme (arrows) of the age-matched Shox2 mutants. b, bead.

 


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  		Fig. 7. Fgf proteins exert different effects on cell proliferation in the developing palate. (A-C) Fgf10 inhibits cell proliferation (B), while Fgf2 stimulates cell proliferation (C) in E13.5 anterior palatal tissue when compared with tissue treated with BSA (A). (D-F) Cell proliferation levels are similar in the posterior palatal tissue implanted with either BSA bead (D) or Fgf10 bead (E). However, cell proliferation level is significantly increased in the posterior palatal tissue implanted with Fgf2 bead (F). b, bead.

 


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  		Fig. 8. Regulation of Shox2 expression in the developing palatal mesenchyme. (A-C) Shox2 expression (arrow) remains in the anterior palatal mesenchyme of an E11.5 palatal shelf after 24 hours in organ culture (A), but is not detected in the mesenchyme of an E11.5 de-epithelialized palatal shelf after 24 hours in organ culture (B). Shox2 expression (arrow) is maintained/induced in E11.5 palatal mesenchyme recombined a piece of anterior palatal epithelium from a donor palatal shelf of the same stage (C). (C, inset) A section through a recombinant sample shows Shox2 expression in the palatal mesenchyme immediately adjacent to the donor epithelium indicated by an asterisk. (D-F) At E12.5 Shox2 is expressed in the anterior palatal mesenchyme (arrowheads) of the palatal shelves with (D) or without (E) palatal epithelium after 24 hours in organ culture. Ectopic Shox2 expression (arrow) is induced in the mesenchyme of the posterior palatal shelf by an epithelium from the anterior region of the E12.5 palatal shelf after 24 hours in organ culture (F). Arrows in D-F indicate the posterior region of the cultured palatal shelves. (G-I) Shox2 expression is not altered by application of either BSA (G) or anti-Shh antibodies (H) in the anterior mesenchyme of the E11.5 palatal shelves, but is downregulated by Noggin beads after 24 hours in organ culture (I). b, bead.

 

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© The Company of Biologists Ltd 2005