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First published online 22 February 2006
doi: 10.1242/dev.02298

Development 133, 1219-1229 (2006)
Published by The Company of Biologists 2006
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Multiple roles of mesenchymal ß-catenin during murine limb patterning

Theo P. Hill1, Makoto M. Taketo2, Walter Birchmeier3 and Christine Hartmann1,*

1 Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria.
2 Department of Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
3 Max Delbrück Center for Molecular Medicine, Robert-Roessle-Strasse 10, 13125 Berlin, Germany.


Figure 1
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  		Fig. 1. Loss- and gain-of ß-catenin activity in limb mesenchyme causes truncations of the limbs. (A-C) Normal wild-type limbs (A), and limb truncations in lof ß-cat{Delta}Prx1/- (B) and gof ß-cat{Delta}ex3Prx1/+ (C) E16.5 embryos. (D-G) In situ hybridization for ß-catenin using the mß-catenin-del probe. (D) Strong ubiquitous expression in flank and limb mesenchyme, and in the ectoderm and AER of wild-type E10.5 limbs. (E) Loss of ß-catenin expression at E10.5 in flank (black arrow) and forelimb (red arrow) mesenchyme in ß-cat{Delta}Prx1/- embryos. (F) Less deletion is detected in E10.5 ß-cat{Delta}Prx1/- hindlimbs (HL, red arrow). (G) Near complete loss of ß-catenin expression in the mesenchyme of ß-cat{Delta}Prx1/- E11.5 forelimbs, while ectodermal expression remains normal.

 

Figure 2
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  		Fig. 2. SEM and marker analysis of limb bud development in ß-cat{Delta}Prx1/- embryos. A-B') Scanning electron microscopy (SEM) images of wild-type (WT, A,B) and ß-cat{Delta}Prx1/- (A',B') forelimbs at E9.5 (A,A') and E10.5 (B,B'). (C-E') Analysis of Fgf8 expression in somite staged embryos. Fgf8 expression (arrow) in a narrow stripe along the body wall at 17 somites in wild type (C) and ß-cat{Delta}Prx1/- (C'). Fgf8 expression domain at 20 somites in wild type (D) and ß-cat{Delta}Prx1/- (D'). Extensive Fgf8 expression in wild type (E) at 25 somites, which is reduced in ß-cat{Delta}Prx1/- forelimbs (arrows in E'). (F,F') At E10.5, Fgf8 expression in wild type becomes refined to the AER (F); a dashed expression domain is observed in ß-cat{Delta}Prx1/- forelimbs, corresponding to notches in the AER (F'). (G,G') At 19-20 somites, similar Fgf10 expression is seen in wild type (G) and ß-cat{Delta}Prx1/- (G') forelimbs. (H,H') At E10.75, Fgf10 expression in the distal mesenchyme is reduced in ß-cat{Delta}Prx1/- forelimbs (arrow, H') compared with wild type (H). (I-J') Tbx5 expression at 19-20 somites and at E10.75, comparing wild-type (I,J) and ß-cat{Delta}Prx1/- (I',J') forelimbs. Embryos are oriented anterior to the right.

 

Figure 3
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  		Fig. 3. SEM and marker analysis of embryos with activated Wnt/ß-catenin signaling in the limb mesenchyme. (A,A') Whole-mount in situ hybridizations showing Fgf8 expression in wild-type (A) and ß-cat{Delta}ex3Prx1/+ (A', n=5) forelimbs at E11.5; note ectopic expression in the distal region (red arrow), as well as the loss from the posterior AER (white arrow). (B-B'') Fgf8 expression at E12.5 in wild-type (B) and ß-cat{Delta}ex3Prx1/+ (B',B'') embryos; note expression is lost from the posterior AER in the hindlimb (B') and is almost completely lost in the forelimb (B''). (C) Comparative quantification of the Fgf8-expressing regions (approximating the AER) in fore- and hindlimbs of wild-type and ß-cat{Delta}ex3Prx1/+ embryos at E10.5 (WT, n=6; ß-cat{Delta}ex3Prx1/+, n=12; *not significant) and E11.5 (WT, n=12; ß-cat{Delta}ex3Prx1/+, n=6; **significant: forelimb, P=0.01, hindlimb, P=0.02). (D-F') SEM images of wild-type (D-F) and ß-cat{Delta}ex3Prx1/+ (D'-F') forelimbs at E10.75 and E11.5. Brackets in D',E' indicate the flattened region of the AER; arrowhead in E indicates the necrotic region in the wild-type limb; arrowheads in F' indicate AP extension of the AER. (G,G') Wild-type Fgf4 expression in the posterior AER (G), which is downregulated in ß-cat{Delta}ex3Prx1/+ forelimbs (G'). (H,H') Wild-type Fgf9 expression in the AER (H), which is lost in ß-cat{Delta}ex3Prx1/+ forelimbs (H'). (I,I') Wild-type Fgf10 expression in the mesenchyme (I), which is lost in ß-cat{Delta}ex3Prx1/+ forelimbs (I'). (J,J') Wild-type Shh expression in the ZPA (J), which is absent in ß-cat{Delta}ex3Prx1/+ forelimbs (J'). (K,K') Wild-type Ptc1 expression (K), which is reduced in ß-cat{Delta}ex3Prx1/+ forelimbs (K'). Embryos are orientated anterior to the right. P, posterior; A, anterior. Scale bars: 100 µm in D-F'.

 

Figure 4
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  		Fig. 4. Upregulation of Bmps and Bmp target genes upon stabilization of ß-catenin in limb mesenchyme. (A-H) In situ hybridisation showing the expression of Bmps and Bmp target genes in wild-type and ß-cat{Delta}ex3Prx1/+ forelimbs at E11.5. (A) Wild-type Bmp4 expression in the distal mesenchyme and AER. (A') Expanded Bmp4 expression in ß-cat{Delta}ex3Prx1/+ forelimbs. (B) Wild-type Bmp7. (B') Upregulation of Bmp7 in ß-cat{Delta}ex3Prx1/+. (C) Wild-type Bmp2. (C') Distal upregulation of Bmp2 in ß-cat{Delta}ex3Prx1/+. (D) Wild-type gremlin. (D') Upregulated gremlin expression in ß-cat{Delta}ex3Prx1/+. (E) Wild-type Msx1. (E') Distal upregulation of Msx1 in ß-cat{Delta}ex3Prx1/+. (F) Wild-type Msx2. (F') Upregulation of Msx2 in distal ß-cat{Delta}ex3Prx1/+. (G-G'') Vibratome sections through whole-mount stained ß-cat{Delta}ex3Prx1/+ forelimbs demonstrating the mesenchymal upregulation of gremlin (G), Bmp2 (G') and Bmp7 (G''). (H) Section in situ hybridisation for Bmp4 on E11.5 forelimbs showing upregulation throughout the proximal mesenchyme. Whole-mount stained limbs are orientated anterior to the right and distal up.

 

Figure 5
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  		Fig. 5. TUNEL analysis of ß-cat{Delta}Prx1/- and ß-cat{Delta}ex3Prx1/+ mutant limb buds. Apoptotic cells are shown in green; red staining indicates autofluorescence. (A,A') E9.5 (25-26 somites) wild-type (A) and ß-cat{Delta}Prx1/- (A') forelimbs showing no significant apoptosis in the mesenchyme. (B,B') E10.25 forelimbs. A few apoptotic cells are visible in wild type (B), but there is a significant increase in apoptotic cells in the proximal mesenchyme in ß-cat{Delta}Prx1/- (B'). (C,C') E11 forelimb. Apoptosis is detected in the AER and in a central region of the mesenchyme in wild type (C), whereas an increased number of apoptotic cells is observed throughout the proximal mesenchyme in ß-cat{Delta}ex3Prx1/+ limbs (C').

 

Figure 6
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  		Fig. 6. Regulation of Lmx1b expression by ß-catenin activity. (A-B'') Expression of Wnt7a in the dorsal ectoderm (A-A'') and En1 in the ventral ectoderm (B-B'') at E10.5 in wild-type (A,B), ß-cat{Delta}Prx1/- (A',B'), and ß-cat{Delta}ex3Prx1/+ (A'',B'') fore- and hindlimbs (n=4). (C,C') Dorsal view of E9.5 embryos showing uniform expression of Lmx1b in wild type (C) and downregulation in ß-cat{Delta}Prx1/- (C', n=6) forelimbs. (D,D') Lateral view of E9.5 embryos, showing restricted expression of Lmx1b in the dorsal mesenchyme (arrow) in wild type (D), which expands into the ventral mesenchyme (arrow) in ß-cat{Delta}ex3Prx1/+ (D') forelimbs. (E,E') Lmx1b expression at E11.5 in wild-type (E) and ß-cat{Delta}Prx1/- (E') forelimbs. (F-K) Section in situ hybridizations. (F-F'') At E10.5, Lmx1b expression is dorsally restricted in wild-type forelimbs (F; delineated by a line running perpendicularly through the AER, red arrow), is reduced and lost in the distal limb mesenchyme adjoining the dorsal ectoderm in ß-cat{Delta}ex3Prx1/+ (F'; white arrow), but is expanded into the ventral mesenchyme in ß-cat{Delta}ex3Prx1/+ limbs (F''; note, expression is excluded from the ventral-most regions; black arrow; n=5). (G-G'') Distribution of ß-catenin protein in wild-type (G) and two different E11.5 ß-cat{Delta}ex3Prx1/+ (G',G'') hindlimbs at E11.5. (G-J'') Alternating sections of the same E11.5 wild-type (G-J) and ß-cat{Delta}ex3Prx1/+ (G'-J') hindlimbs. (H,H') Lmx1b expression in wild type (H) and ectopic expression in gof limbs (H'). (I,I') Lef1 expression in wild type (I) and ectopic expression in gof limbs (I'). (J,J') Tcf1 expression in wild type (J) and ectopic expression in gof limbs (J'). (H'',I'',K) Fluorescent double in situ hybridization on an E11.5 ß-cat{Delta}ex3Prx1/+ hindlimb section (same limb as is shown in G''), showing that Lmx1b- (H'') and Lef1 (I'')-positive regions overlap in the distal limb bud (K: Lef1 green; Lmx1b red). Embryos are orientated anterior to the right. Limb bud sections are dorsal up, distal to the right.

 

Figure 7
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  		Fig. 7. Shoulder girdle defects in ß-catenin loss- and gain-of mutants. (A-A'') Alcian Blue/Alizarin Red stained forelimbs from E18.5 wild-type (A), ß-cat{Delta}Prx1/- (A') and ß-cat{Delta}ex3Prx1/+ (A'') embryos (n=6), shown at different magnification. (B-C) In situ hybridisation for the chondrocyte marker Col2a1 on E12.5 wild-type (B) and ß-cat{Delta}Prx1/- (B') and E14.5 ß-cat{Delta}Prx1/- (C) forelimbs, showing partial loss of the scapula anlage (arrowhead in B') and the presence of a small element (black arrow) separated from the distal element by a zone of low Col2a1 expression (red arrows, C). (D-E) In situ hybridisation for the joint marker Gdf5 on E12.5 wild-type (D) and ß-cat{Delta}Prx1/- (D'), and E14.5 ß-cat{Delta}Prx1/- (E) forelimbs, showing that Gdf5 is still expressed (green arrows, D',E). (F-K'') Whole-mount in situ hybridisation for Hoxc6 (F-F''), Hoxa9 (G-G''), Hoxa10 (H-H''), Meis1 (I-I''), Pax1 (J-J'') and Emx2 (K-K'') on E10.5 embryos. ß-cat{Delta}Prx1/- embryos show downregulation of Pax1 (J') and Emx2 (K'; n=5). ß-cat{Delta}ex3Prx1/+ embryos show reduced Pax1 (J'') and expanded Emx2 expression (K''). Embryos are orientated anterior to the top, distal to the right. (L) Semi-quantitative RT-PCR from ß-cat ex3fl/ex3fl Adeno-Cre- and Adeno-GFP (control)-infected micromass cultures showing upregulation of Emx2 and no upregulation of Lmx1b 14 hours after infection.

 

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© The Company of Biologists Ltd 2006