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First published online 17 July 2008
doi: 10.1242/dev.020586

Development 135, 2815-2825 (2008)
Published by The Company of Biologists 2008
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Tissue-specific requirements of β-catenin in external genitalia development

Congxing Lin1,4, Yan Yin1, Fanxin Long2,3 and Liang Ma1,2,*

1 Division of Dermatology, Department of Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
2 Department of Developmental Biology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
3 Division of Bone and Mineral Diseases, Department of Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
4 Human Genetics Program, Tulane University School of Medicine, New Orleans, LA 70112, USA.


Figure 1
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  		Fig. 1. WNT signaling in the developing GT and tissue-specific Cre expression. (A-D) TOPGAL embryos stained with X-Gal at different stages. TOPGAL expression in the dUE persists from E10.5 to E14.5 (arrows, A-D). Preputial swellings (arrowheads, C) and labioscrotal swellings (arrowheads, D) are also positive for TOPGAL activity. (E,F) Coronal sections of E10.5 and E12.5 GTs showing distal urethral localization of TOPGAL positive cells (blue). (G,H) 35S in situ hybridization on E12.5 GT sections (plane of section is shown in M) using probes indicated. (I-L) Coronal sections (plane of section is shown in N) of X-Gal-stained E10.5 embryos showing tissue-specific Cre-mediated recombination in the GTs. No endogenous X-Gal activity was detected at this stage (I). Scale bars: 100 µm in F-H; 50 µm in E,I-L.

 

Figure 2
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  		Fig. 2. Endodermal WNT-β-catenin signaling is required for GT initiation. (A-C) SEM analysis showing an absence of GT outgrowth in the Shhcre/Gfp;β-Catc/c embryo (B) and a larger GT in the Shhcre/Gfp;β-CatloxEx3 embryo (C). (D-F) β-Catenin indirect immunoflouresence showing that the protein was detected mainly on the cell membranes of both GT ectoderm and UE, as well as weakly in the mesechyme (D). Complete removal of β-catenin in the UE was confirmed in Shhcre/Gfp;β-Catc/c GT (arrows, E), and ectopic accumulation of β-catenin was observed in Shhcre/Gfp;β-CatloxEx3 endoderm (arrow, F). (G-I) Whole-mount Fgf8 in situ hybridization showing expression in the distal cloacal endoderm in control embryos (G), but not in Shhcre/Gfp;β-Catc/c embryos (arrows, H); in Shhcre/Gfp;β-CatloxEx3 GT, Fgf8 expression is ectopically expanded (I). (J,K) Whole-mount Bmp4 in situ hybridization showing a reduction in Shhcre/Gfp;β-Catc/c cloacal mesenchyme (K). (M-O) TUNEL analysis showing increased cell death in Shhcre/Gfp;β-Catc/c cloacal endoderm and ectopic apoptotic cells in the surrounding mesenchyme (arrows in N). (P-R) PHH3 immunostaining revealing markedly reduced cell proliferation in E10.5 Shhcre/Gfp;β-Catc/c cloacal endoderm. (S-U,W,X) Fgf8 in situ hybridization showing an absence of expression in E10.5 Msx2-Cre;β-Catc/c limbs (T) and ectopic expression in the flank ectoderm and dorsal limb ectoderm in Msx2-cre;β-CatloxEx3 embryos (U). (V) Note that ectopic Fgf8 expression appears to correspond to Msx2-Cre expression (arrows in U,V). At E12.5, ectopic outgrowth was observed in the inter-limb region of Msx2-Cre;β-CatloxEx3 embryos (arrows, X). Scale bars: 100 µm in A-F; 50 µm in M,N,P,Q.

 

Figure 3
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  		Fig. 3. Endodermal β-catenin is required to maintain GT outgrowth. (A-E) SEM analysis showing reduced distal growth and ectopic proximal opening in ShhCre/esr;β-Catc/c embryos (arrowheads in B-D), and excessive distal growth with no proximal opening in ShhCre/esr;β-CatloxEx3 GT (arrowheads, E). Dashed lines in A, C and E indicate the plane of transverse sections used for histological/immunostaining analysis in Fig. 4. (A'-E') Fgf8 in situ hybridization on E12.5 GTs. Note the graded decrease in Fgf8 in LOF GTs (B'-D'). Fgf8 is both elevated and ectopically activated in GOF GT (E'). (F-F'') 35S Tcf-1 in situ hybridization revealed downregulation of Tcf1 in ShhCre/esr;β-catc/c dUE (F'), and upregulation and ectopic proximal UE expression in ShhCre/esr;β-CatloxEx3 GT (F''). (G-L'') Whole-mount in situ analysis using the probes indicated. Msx2 is expressed in the distal mesenchyme surrounding the dUE (H). Its expression domain is reduced in ShhCre/esr;β-Catc/c (H'), and is expanded proximally in ShhCre/esr;β-CatloxEx3 GT (H''). Lef1 and Wnt5a are strongly expressed in the distal mesenchyme, and this strong-expressing domain is also reduced in ShhCre/esr;β-Catc/c GT (I',J'), and expanded proximally in ShhCre/esr;β-CatloxEx3 GT (I'',J''). Hoxa13 and Hoxd13 expression remains unchanged in either mutant (K-K'' and L-L'', respectively). Scale bars: 100 µm in A-E; 100 µm in F-F''.

 

Figure 4
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  		Fig. 4. Urethral defects in endodermal β-catenin LOF and GOF mutants. (A-F') Hematoxylin and Eosin (H&E) staining (A-F) and indirect immunoflouresence for E-cadherin (A'-F') on distal and proximal GTs. Note that in wild-type GT urethral cells form well-organized urethral plate distally (A,A') but remain as a tube at the proximal end (D,D'). In ShhCre/esr;β-Catc/c GT, urethral plate fails to form distally (B,B'), and the proximal urethra is open (E,E'). In ShhCre/esr;β-CatloxEx3 GT, disorganized distal urethral plate is formed (C,C'), and the proximal urethra showed severe endodermal overgrowth (F,F'). (A''-F'') Immunostaining confirms that β-catenin protein is removed from ShhCre/esr;β-Catc/c UE (arrowheads, B'',E'') and accumulates in ShhCre/esr;β-CatloxEx3 UE (C'',F''). (A'''-F''') Immunostaining showing K14 expression was detected in both surface epithelium and UE in wild-type GTs (A''',D'''). The expression is maintained in LOF urethra (B''',E''') but is repressed in cells with high β-catenin expression in GOF urethra (C''',F'''). Scale bars: 100 µm; 200 µm in insets.

 

Figure 5
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  		Fig. 5. Altered cellular proliferation and gene expression in endodermal β-catenin LOF and GOF urethrae. All embryos were exposed to Tm on E10.5 and collected on E12.5. (A-A'') PHH3 staining showed reduced cell proliferation in ShhCre/esr;β-Catc/c UE (A'), and increased cell proliferation in proximal ShhCre/esr;β-CatloxEx3 UE (arrowheads, A''). UE is highlighted by white dashed lines. (B-C'') 35S in situ hybridization on adjacent coronal sections. Shh expression is downregulated in both ShhCre/esr;β-Catc/c UE (B') and distal ShhCre/esr;β-CatloxEx3 UE (arrows, B''), but is maintained in the proximal ShhCre/esr;β-CatloxEx3 UE (arrowheads, B''). Bmp4 is normally expressed in distal mesenchyme (C), is downregulated in ShhCre/esr;β-Catc/c GT (C'), and is ectopically activated in the dUE of ShhCre/esr;β-CatloxEx3 GT (C'', arrows). Note the complementary expression pattern of Shh and Bmp4 in ShhCre/esr;β-CatloxEx3 dUE (B'',C''). Scale bars: 100 µm.

 

Figure 6
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  		Fig. 6. Ectodermal defects in Msx2-Cre;β-Catc/c GTs. (A-H) SEM analysis of wild-type and Msx2-Cre;β-Catc/c GTs. Msx2-Cre;β-Catc/c GTs show absence of an urethral seam (arrowheads, A) at E12.5 (B), an ectopic opening in proximal GT at E13.5 (arrowheads, D), and a distal bifurcation at E14.5 (arrowhead, F). (I-P) Tissue lineage analysis revealed an ectodermal rupture in Msx2-cre;β-Catc/c GTs. The development of the ectodermal surface epithelium marked by Msx2-Cre;R26R was examined by X-Gal staining. β-Gal-positive ectodermal cells (blue) cover the entire GT surface throughout early development (I,K,M,O). By contrast, the mutant surface epithelium breaks down at the midline (arrowheads, J) and the disruption continues to expand (L,N). At E16.5, the ventral side of the GT is completely devoid of β-Gal-positive ectodermal epithelium (P). (Q,R) Shh in situ hybridization showing that Shh-expressing UE is covered by ventral ectoderm in wild-type GT (arrowheads, Q), but is exposed and expanded on the GT surface in Msx2-Cre;β-Catc/c GTs at E12.5 (arrowheads, R). The planes of section are indicated in I,J. (S,T) H&E staining showing an ectopic opening in the proximal region of Msx2-Cre;β-Catc/c GTs (T). (U,V) X-Gal staining showing that exposed epithelium in the mutant GT is Msx2-Cre negative. (W,X) Shh in situ hybridization showing that the exposed epithelium (arrowheads, X) expresses Shh. The planes of section in S-X are indicated in K and L. Scale bars: 100 µm in Q-X.

 

Figure 7
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  		Fig. 7. Ectodermal structural defects in Msx2-Cre;β-Catc/c embryos. (A-H) Toluidine Blue staining (A,B,E,F) and TEM analyses (C,D,G,H) on E10.5 coronal sections (A-D) and E12.5 transverse sections (E-H) reveal that the mutant surface epithelium is thinner (compare D to C, H to G). In addition, the ectoderm and endoderm maintain close contact in wild-type GTs (A,C), whereas the two layers appear to be separated in mutants (asterisks, D). (I-N) Immunostainings indicate an absence of β-catenin (J), an upregulation of plakoglobin (arrows, L) and normal E-cadherin expression in mutant ectoderm (N). (O-R) Immunostainings showing a total absence of K14 expression (R) but unchanged p63 expression (P) in E12.5 mutant ectoderm. Scale bars: 50 µm in I-N; 100 µm in O-R.

 

Figure 8
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  		Fig. 8. A model of signaling crosstalks regulating GT development. Evidence indicates that canonical WNT acts upstream of Fgf8 and Shh in the dUE. Fgf8 expression in turn is required for establishing distal mesenchymal gene expression in the GT. Shh expression is also dependent on WNT activity. Shh normally represses Bmp4 expression in the dUE. When WNT is constitutively activated, Bmp4 is ectopically turned on in dUE. The coordinated regulation of positive (e.g. Fgf8 and Shh, green) and negative (e.g. Bmp4, red) regulators of GT outgrowth is essential to maintain the homeostasis of the UE, as well as normal patterning of the GT. The pink region represents the distal mesenchyme; the green region represents the UE.

 

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© The Company of Biologists Ltd 2008