spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 23 October 2008
doi: 10.1242/dev.025304

Development 135, 3959-3968 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, W.
Right arrow Articles by Jiang, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, W.
Right arrow Articles by Jiang, R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The Mn1 transcription factor acts upstream of Tbx22 and preferentially regulates posterior palate growth in mice

Wenjin Liu1, Yu Lan1, Erwin Pauws2, Magda A. Meester-Smoor3, Philip Stanier2, Ellen C. Zwarthoff3 and Rulang Jiang1,*

1 Department of Biomedical Genetics and Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
2 Institute of Child Health, University College London, London WC1N 1EH, UK.
3 Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.


Figure 1
View larger version (103K):
[in this window]
[in a new window]

  		Fig. 1. Expression pattern of Mn1 mRNA in developing mouse embryos. mRNA signals were detected by whole-mount in situ hybridization (A-C) or section in situ hybridization (D-F). (A) Mn1 mRNA expression was first detected in the developing brain tissues and in the craniofacial mesenchyme at E9.5. (B-E) Strong Mn1 mRNA expression was observed in the developing brain, frontonasal processes, maxillary processes, mandibular processes, the second branchial arch, the developing somites and limb buds at E10.5 (B,D) and E11.5 (C,E). (F) Section in situ hybridization showing strong Mn1 mRNA expression in the developing palatal mesenchyme and in the preossification mesenchymal cells in the mandible. ba1, first branchial arch; ba2, second branchial arch; e, eye; fb, forebrain; fl, forelimb; fnp, frontonasal process; hb, hindbrain; hl, hindlimb; man, mandibular process; max, maxillary process; mb, midbrain; ov, otic vesicle; p, palatal shelf; so, somite; t, tongue. Arrowheads in E indicate the initial palatal outgrowths.

 

Figure 2
View larger version (135K):
[in this window]
[in a new window]

  		Fig. 2. The expression pattern of Mn1 mRNA along the anteroposterior axis of the developing palate. Middle palate corresponds to the palatal region flanked by the maxillary first molar tooth germs. (A-C) At E12.5, Mn1 mRNA expression in the developing secondary palate exhibits an anteroposterior gradient, with low levels in the anterior palate (A), moderate levels in the middle palate (B) and highest levels in the posterior palate (C). (D-F) At E13.5, Mn1 mRNA expression is still high in the middle (E) and posterior (F) regions, and much weaker in the anterior region (D) of the developing palate. (G-I) At E14.5, the bilateral palatal shelves have elevated to the horizontal position above the tongue and have initiated contact and fusion in the anterior (G) and middle (H) regions. Mn1 mRNA expression remains strong in the middle and posterior (I) palatal regions, and relatively weak in the anterior palate. p, palatal shelf; t, tongue.

 

Figure 3
View larger version (155K):
[in this window]
[in a new window]

  		Fig. 3. Mn1-/- mutant mice exhibit palatal retardation and failure of palatal shelf elevation. All panels shown are from middle palate regions. (A,B) At E13.5, wild-type (A) and Mn1-/- homozygous mutant (B) embryos exhibited similar palatal shelf size and shape. (C,D) At E14.5, the wild-type (C) palatal shelves had elevated to the horizontal position above the tongue and initiated fusion by forming the midline epithelial seam, while the Mn1-/- homozygous mutant (D) palatal shelves were still vertically oriented. (E,F) At E15.5, the wild-type (E) palatal shelves had completed fusion, but the Mn1-/- homozygous mutant (F) palatal shelves remained vertically oriented. Arrows indicate the first molar tooth germs. p, palatal shelf; t, tongue.

 

Figure 4
View larger version (148K):
[in this window]
[in a new window]

  		Fig. 4. Histological analyses of palate development in Mn1-/- mutants at late stages. (A,B) At E16.5, the palatal shelves in Mn1-/- mutant embryos were still vertically oriented. In addition, the palatal shelves appeared to be significantly diminished in size in the middle to posterior regions (B). (C,D) At E18.5, although the palatal shelves in the anterior region in the Mn1-/- mutant mice were partially elevated (C), the middle and posterior regions of the palatal shelves were further retarded and retracted into the maxillary processes (D). Arrows in B and D indicate the upper first molar tooth germs. p, palatal shelf; t, tongue.

 

Figure 5
View larger version (118K):
[in this window]
[in a new window]

  		Fig. 5. Analyses of cell proliferation during palate development in wild-type and Mn1-/- mutant embryos. (A-F) In comparison with the wild-type littermates (A-C), the number of BrdU-labeled cells in the developing palatal shelves was greatly reduced in the middle (E) and posterior (F) palatal shelves, but not in the anterior (D) palatal mesenchyme in the Mn1-/- mutant embryos by E13.5. p, palatal shelf; t, tongue. (G,H) Comparison of the percentage of BrdU-labeled cells in the palatal mesenchyme (G) and epithelium (H), in the anterior, middle and posterior palatal regions in E13.5 wild-type (WT) and Mn1-/- mutant embryos. Error bars represent standard deviation and the asterisk indicates a significant difference (P<0.01) between the wild-type and Mn1-/- mutant samples.

 

Figure 6
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 6. Analyses of cell proliferation and cell apoptosis in wild-type (WT) and Mn1-/- mutant embryos at E15.5. (A-C) Cell proliferation in palatal mesenchyme detected using immunohistochemical staining of the Ki67 protein. In comparison with wild-type embryos (A), cell proliferation in the middle palatal region in Mn1-/- mutant embryos (B) was greatly reduced. Error bars in C represent standard deviation and the asterisk indicates a significant difference (P<0.01) between the wild-type and mutant samples of mesenchyme and epithelium. (D,E) TUNEL assays of mid-palatal sections of E15.5 palatal shelves of wild-type (D) and Mn1-/- mutant (E) embryos showed increased cell apoptosis in the palatal mesenchyme in the mutant. Small arrows indicate highly TUNEL-positive palatal mesenchymal cells and arrowheads indicate highly TUNEL-positive palatal epithelial cells.

 

Figure 7
View larger version (89K):
[in this window]
[in a new window]

  		Fig. 7. Expression of cyclin D2 was downregulated in Mn1-/- mutant palatal shelves at E13.5. (A,D) In the anterior region of the developing palate, expression of cyclin D2 was similarly weak in wild-type (A) and Mn1-/- mutant (D) embryos. (B,E) In the middle palate, cyclin D2 is highly expressed in both the epithelium and mesenchyme in the wild-type embryo (B) but its expression is much reduced in the Mn1-/- palatal mesenchyme (E). (C,F) In the posterior palate region, strong cyclin D2 expression was detected in both the epithelium and mesenchyme in the wild-type palate (C) but was much reduced in the Mn1-/- mutant embryo (F). Green arrows in C and F indicate the medial edge epithelium of the palatal shelves.

 

Figure 8
View larger version (113K):
[in this window]
[in a new window]

  		Fig. 8. The anteroposterior pattern of the developing palatal shelves is not disrupted in the Mn1-/- mutant embryos. (A-F) Expression of Fgf10 along the anteroposterior axis of the developing palatal shelves in wild-type (A-C) and Mn1-/- mutant (D-F) embryos at E13.5. Fgf10 showed similar restricted expression pattern in the anterior and middle palatal regions in both wild-type and Mn1-/- mutant embryos. (G-L) Expression of Shox2 along the anteroposterior axis of the developing palatal shelves in wild-type (G-I) and Mn1-/- mutant (J-L) embryos at E13.5. Shox2 mRNA is strongly expressed in the anterior and absent in the posterior palatal regions in wild-type embryos (G-I). In the middle palate, Shox2 mRNA expression is restricted in the proximolateral region (H). The expression pattern and levels of Shox2 mRNA are not altered in Mn1-/- mutant (J-L) embryos. (M-O) Meox2 mRNA expression is restricted to the middle and posterior regions, and is absent from the anterior region of the developing palate in wild-type embryos at E13.5. (P-R) The levels and anteroposterior pattern of Meox2 mRNA expression pattern in E13.5 Mn1-/- mutant embryo are comparable with that of the wild-type littermate (M-O). p, palatal shelf; t, tongue. Arrows indicate the mandibular first molar tooth germs and asterisks indicate the maxillary first molar tooth germs in the mid-palatal sections.

 

Figure 9
View larger version (93K):
[in this window]
[in a new window]

  		Fig. 9. Tbx22 is specifically downregulated in Mn1-/- mutant palatal shelves at E13.5. (A-C) Tbx22 mRNA is strongly expressed in the middle (B) and posterior (C) palatal mesenchyme, but barely expressed in the anterior (A) palatal mesenchyme in wild-type embryos. (D-F) Tbx22 expression is much reduced in the middle (E) and posterior (F) palatal mesenchyme in the Mn1-/- mutant littermates. Asterisks in B and E indicate the mandibular first molar tooth germ. p, palatal shelf; t, tongue.

 

Figure 10
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 10. Transcriptional regulation of Tbx22 by Mn1. (A) Tbx22 promoter-luciferase reporter activity in transfected NIH3T3 cells in the absence or presence of increasing amounts of co-transfected Mn1 expression vector. Luciferase activity was normalized against the control (promoter-less pGL3-basic) vector-transfected cells. Error bar represents standard deviation. hP0, hP0 promoter; hP1, hP1 promoter; mP0, mouse Tbx22 P0 promoter; mP1, mouse Tbx22 P1 promoter. (B,C) Effect of Mn1 overexpression on endogenous Tbx22 mRNA expression in NIH3T3 cells. Expression levels of Mn1 (B) and Tbx22 (C) mRNAs were quantified by real-time RT-PCR and normalized against mock-transfected cells. Error bar represents standard deviation.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008