Hasebe T , Kajita M , Fu L , Shi YB , Ishizuya-Oka A .

Z01 HD008858-01 NICHD NIH HHS , Z01 HD008858-01 Intramural NIH HHS

	Figure 3. Localization of Ptc-1, Smo, Gli1, Gli2, and Gli3 mRNAs in the intestine during natural metamorphosis of X. laevis. a�u: Cross-sections of the intestine at premetamorphic stage 54 (a,d,g,j,n), at metamorphic climax stages 62 (b,e,h,k,l,o,q�u) and at the end of metamorphosis (stage 66; c,f,i,m,p) were hybridized with antisense Ptc-1 (a�c), Smo (d�f), Gli1 (g�i), Gli2 (j�m), or Gli3 (n�p) probes or their sense probes (q�u). Dark blue deposits indicate the sites of probe binding. Light or dark brown pigments in some pictures are melanin. The expression of all mRNAs becomes high at stage 62. They are expressed in the connective tissue (CT) and muscles (M), but not in the epithelium (Ep). The boxed area in the panel k is magnified to show Gli2 expression in both the circular (CM) and longitudinal (LM) muscle layers (l, arrowheads). A dashed line shows the boundary of the connective tissue and muscle layer. Scale bars = 100 μm in a�k,m�u, 20 μm in l.
	Figure 4. Comparison of the localization of Ptc-1, Smo, Gli1, Gli2, and Gli3 mRNAs with that of Sonic hedgehog (Shh) and/or bone morphogenetic protein-4 (BMP-4) mRNAs in the intestine at metamorphic climax. a�x: Cross-sections of the intestine at stage 61 (a,b,e,f,i,j,m,n,q,r,u,v) and stage 62 (c,d,g,h,k,l,o,p,s,t,w,x) were hybridized with antisense Ptc-1 (a,c,e,g), Smo (i,k), Gli1 (m,o), Gli2 (q,s), or Gli3 (u,w) probes. Their adjacent sections were hybridized with antisense Shh (b,d) or BMP-4 (f,h,j,l,n,p,r,t,v,x). a�d: Ptc-1 is expressed in the connective tissue (CT, arrows) just beneath the epithelial cells (Ep) expressing Shh (red arrowheads). e�h: Ptc-1 (arrows) is co-expressed with BMP-4 (dashed arrows). i�l: Smo (arrows) is co-expressed with BMP-4 (dashed arrows) in the connective tissue. In addition, Smo is weakly expressed in the circular muscle layer (CM, white arrowhead). m�p: Some cells in the connective tissue co-express Gli1 (arrows) and BMP-4 (dashed arrows). Gli1 is weakly expressed in the circular muscle layer (white arrowhead). q�t: Some cells in the connective tissue co-express Gli2 (arrows) and BMP-4 (dashed arrows). Gli2 is also expressed in both the circular and longitudinal muscle layers (LM; white arrowheads). u�x: Some cells in the connective tissue co�express Gli3 (arrows) and BMP-4 (dashed arrows). Gli3 is weakly expressed in the circular muscle layer (white arrowhead). Scale bars = 20 μm.
	Figure 5. Comparison of the localization of Ptc-1, Smo, Gli1, Gli2, and Gli3 mRNAs with that of Sonic hedgehog (Shh) and/or bone morphogenetic protein-4 (BMP-4) mRNAs in the intestine during TH-induced metamorphosis. a�x: Cross-sections of the intestine from premetamorphic tadpoles treated with 10 nM T3 for 3 days (a,b,e,f,i,j,m,n,q,r,u,v) and 5 days (c,d,g,h,k,l,o,p,s,t,w,x) were hybridized with antisense Ptc-1 (a,c,e,g), Smo (i,k), Gli1 (m,o), Gli2 (q,s), or Gli3 (u,w) probes. Their adjacent sections were hybridized with antisense Shh (b,d) or BMP-4 (f,h,j,l,n,p,r,t,v,x). a�d: Ptc-1 is expressed in the connective tissue (CT, arrows) just beneath the epithelial cells (Ep) expressing Shh (red arrowheads). e�h: Ptc-1 (arrows) is co-expressed with BMP-4 (dashed arrows). i�l: Smo (arrows) is co-expressed with BMP-4 (dashed arrows) in the connective tissue. In addition, Smo is weakly expressed in the circular muscle layer (CM, white arrowhead). m�p: Some cells in the connective tissue co-express Gli1 (arrows) and BMP-4 (dashed arrows). Gli1 is weakly expressed in the circular muscle layer (white arrowhead). q�t: Some cells in the connective tissue co-express Gli2 (arrow) and BMP-4 (dashed arrow). Gli2 is expressed in both the circular and longitudinal muscle layer (LM; white arrowheads). u�x: Some cells in the connective tissue co-express Gli3 (arrows) and BMP-4 (dashed arrows). Gli3 was detected also in the circular muscle layer (white arrowhead). Scale bars = 20 μm
	Figure 6. Overexpression of Sonic hedgehog (Shh) enhances the expression of genes involved in its signaling pathway. Intestinal fragments from stage 56/57 tadpoles were transfected with pIRES2_EGFP (c,f) or pIRES2_Shh-EGFP (d,g) or remained untreated (b,e), and then were cultured for 3 days in the absence of thyroid hormone (TH). a: Transfected gene expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). b�d: Transfected cells were detected by immunohistochemistry (IHC) using anti-green fluorescent protein (GFP) antibody. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). e�g: Effects of exogenous Shh on cell proliferation were examined by IHC using anti-proliferating cell nuclear antigen (PCNA) antibody. The percentage of nuclei strongly positive for PCNA (arrowheads) in the epithelium was shown as the mean � standard error in each panel. h: mRNA levels of indicated genes were analyzed by real-time RT-PCR. EGFP-transfected vs. Shh-transfected was analyzed by Student's t-test. All genes were significantly up-regulated by Shh. Ep, epithelium; CT, connective tissue. Scale bars = 100 μm in b�d; 50 μm in e�g.

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