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First published online 23 October 2008
doi: 10.1242/dev.025767

Development 135, 3871-3879 (2008)
Published by The Company of Biologists 2008
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Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development

Fenglei He1,2, Wei Xiong1,2, Xueyan Yu2, Ramon Espinoza-Lewis1,2, Chao Liu1,2, Shuping Gu2, Michiru Nishita3, Kentaro Suzuki4, Gen Yamada4, Yasuhiro Minami3 and YiPing Chen1,2,*

1 Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA.
2 Section of Oral Biology, The Ohio State University College of Dentistry, Columbus, OH 43210, USA.
3 Department of Physiology and Cell Biology, Faculty of Medical Sciences, Kobe University, Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
4 Center for Animal Resources and Development, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.


Figure 1
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  		Fig. 1. Differential gene expression along the AP axis of E13.5 palatal shelf. (A,E) Gradient Wnt5a expression is detected in palatal mesenchyme, higher in the anterior (A), and lower in the posterior (E) domain. (B,F) Fz4 expression is detected in the anterior palate (B), but not in the posterior region (F). (C,G) Ror2 exhibits a gradient expression, with higher level in the anterior palate (C) and lower in the posterior region (G). (D,H) Gradient Catnb expression is detected in the developing palate, higher in the anterior (D), while lower in the posterior (H) palate. There is strong Catnb expression in the MEE (arrow) of the anterior palate. T, tongue; PS, palatal shelf.

 

Figure 2
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  		Fig. 2. Cleft palate phenotype in Wnt5a mutant. (A) An E18.5 wild-type mouse shows an intact palate. (B) An E18.5 Wnt5a mutant shows wide open cleft secondary palate. (C,E) Coronal sections of an E13.5 control embryo show the normal palatal shelves at the anterior (C) and posterior domain (E). Arrows in C indicate the ventrolateral indention. (D) A coronal section of an E13.5 Wnt5a mutant shows the palatal shelves in the horizontal level lacking the ventrolateral indention (arrows). (F) The posterior palatal shelves of an E13.5 mutant exhibit an abnormal shape. (G,I) An E14.5 control embryo shows closure of the palate at the anterior (G) and posterior region (I). (H) The anterior palatal shelves of an E14.5 Wnt5a mutant remain at the horizontal level but appear too short to make contact at the midline. (J) The posterior palate of an E14.5 mutant fails to elevate, remaining at the vertical position. T, tongue; PS, palatal shelf.

 

Figure 3
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  		Fig. 3. BrdU labeling of cells in the developing palate. (A-D) Coronal sections through the anterior (A,B) and posterior (C,D) palates of an E13.5 wild-type control (A,C) and an E13.5 Wnt5a mutant (B,D) showing BrdU-labeled cells. Boxes in each panel indicate the area where the labeled cells were counted and compared. (E) Comparison of percentage of BrdU-labeled cells in the fixed areas of the palate in the wild-type control and mutant embryos. Standard deviation values are indicated as the error bars. *P<0.05.

 

Figure 4
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  		Fig. 4. Gene expression in E13.5 wild-type and Wnt5a-/- palate. (A) The anterior palate of a wild-type embryo shows Shh expression in the MEE (arrow) and rugal epithelium. (B) Shh expression becomes undetectable in the MEE but remains in the rugal epithelium (arrow) in the anterior palate of a Wnt5a mutant. (C,D) Bmp4 expression is detected in the anterior palate of a wild-type control (C), but is significantly reduced in the mutant (asterisk, D). (E,F) In the posterior palate of the control, Shh expression is detected in the rugal epithelium at the ventral (oral side) (E), whereas in the mutant, an ectopic Shh expression is found in the dorsal (nasal) epithelium (arrow) of the palate (F). (G,H) Bmp4 expression is absent in the posterior palate of the control (G), but is found ectopically in the mesenchyme of the posterior palate on the future nasal side (arrow) in the mutant (H). (I) Ptch1 expression is seen in the anterior palate of the control, mainly in the ventral (oral) side. (J) A downregulated Ptch1 is seen in the anterior palate of a Wnt5a mutant. (K,L) Msx1 expression is detected in the anterior palatal mesenchyme of a wild-type control (K), but is downregulated in the palatal mesenchyme (asterisk) adjacent to the MEE at the similar level of a mutant palate (L). (M,N) Ectopic Ptch1 expression is found in the dorsal (nasal) aspect (arrow) of the palate in the posterior region in a Wnt5a mutant (N), when compared with its normal expression in the control (M). (O,P) A downregulation of Bmp2 expression is found in the posterior palate of Wnt5a mutant (P), when compared with its normal expression pattern in the control (O). (Q) Sox9 expression in the anterior palatal mesenchyme of a control embryo. (R) A shifted Sox9 expression domain is seen in the anterior palate of a Wnt5a mutant. Broken lines in Q,R indicate the expression boundary. (S,T) Shox2 expression remains in the anterior palate of the mutant (T), when compared with that in the control (S). T, tongue; PS, palatal shelf; MNP, medial nasal process.

 

Figure 5
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  		Fig. 5. Wnt5a and Ror2 have an epistatic effect on palate development. (A) An E14.5 wild-type embryo shows the closure of the palate. (B) An E14.5 Ror2-/- embryo shows a failed palatal elevation. (C) Failed palatal elevation is observed in an E14.5 Ror2+/-;Wnt5a+/- embryo. (D-F) Sections through the anterior palate of E13.5 wild-type control (D), Ror2 mutant (E) and Ror2+/-;Wnt5a+/- embryo (F) show Shh expression in the MEE of the control palate, but a lack of Shh expression in the MEE of the Ror2-/- and Ror2+/-;Wnt5a+/- palate. Arrows indicate the MEE. (G) The anterior palate of an E13.5 control embryo shows BrdU-labeled cells. (H,I) The level of cell proliferation is similarly increased in the anterior palate of Ror2-/- (H) and Ror2+/-;Wnt5a+/- (I) embryos. The red lines in G-I separate the dorsal and ventral halves of the palate. T, tongue; PS, palatal shelf.

 

Figure 6
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  		Fig. 6. Regulation of cell proliferation in palatal mesenchyme by Wnt5a is mediated by Ror2. (A) An explanted wild-type palatal shelf implanted with BSA bead shows BrdU-labeled cells in the distal (the MEE side) mesenchyme. (B) An explanted wild-type palatal shelf implanted with a Wnt5a-soaked protein shows a reduced rate of cell proliferation in the distal mesenchyme. (C) A higher cell proliferation rate is seen in an explanted Wnt5a-/- palatal shelf implanted with a BSA bead. (D) Cell proliferation rate is rescued to about the normal level in an explanted Wnt5a-/- palate by a Wnt5a-soaked bead. (E,F) Elevated cell proliferation rate is seen in an explanted Ror2-/- palatal shelf implanted with a BSA bead (E), and remains in an explanted Ror2-/- palatal shelf implanted with a Wnt5a bead (F). (G) Comparison of percentage of BrdU-labeled cells in the fixed area (box) of the palate of different genotypes implanted with BSA- or Wnt5a-soaked beads. Standard deviation values were indicated as the error bars. B, bead. In all images, the MEE is towards the left. *P<0.01.

 

Figure 7
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  		Fig. 7. Aberrant cell migration in the Wnt5a-/- palate. (A-A'') Egfp-expressing mesenchymal cells from the anterior palate, when grafted to the anterior region of an explanted wild-type palatal shelf, exhibit a directional migration towards to the lateral side after 24 hours. Arrows indicate the migrating cells. (B-B'') Egfp-expressing mesenchymal cells from the posterior palate, when grafted to the posterior region of an explanted wild-type palatal shelf, show a directional migration towards the anterior end after 24 hours. (C-C'',D-D'') Directional migration by the Egfp-expressing cells is not observed when these cells are grafted to either the anterior (C-C'') or the posterior (D-D'') palatals shelves from Wnt5a mutants.

 

Figure 8
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  		Fig. 8. Ror2 mediates chemotactic action of Wnt5a in the developing palate. (A-A'') BSA bead fails to exert chemotactic effect on DiI-labeled mesenchymal cells and grafted Egfp-expressing cells (insert in A'). (B-B'') Wnt5a acts as a potent chemoattractant to attract DiI-labeled mesenchymal cells and grafted Egfp-expressing cells (inserts in B', 48 hours in culture). Arrows indicate DiI-labeled cells attracted to Wnt5a bead. (C-C'') Few DiI-labeled cells (arrows) are attracted to Wnt5a bead in an explanted palatal shelf from Ror2-/- embryo. (D-D'') Fgf10 acts as a chemoattractant for palatal mesenchymal cells. Heparin beads were used for Fgf10 soaking, which is invisible under UV light, and are outlined by the broken line. B, bead.

 

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© The Company of Biologists Ltd 2008