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

Z01 HD001901-12 NICHD NIH HHS , Z01 HD001901-12 Intramural NIH HHS

Figure 2. Spatiotemporal localization of Sonic hedgehog (Shh) and hedgehog interacting protein (Hip) mRNAs in the Xenopus laevis intestine during natural and thyroid hormone (TH) -induced metamorphosis. Cross-sections of the intestine at premetamorphic stage 54 (a,b), at metamorphic climax stages 61 (c,d,j-l,n,o) and 62 (e,f,i) and at the end of metamorphosis (stage 66; g,h,m), and the intestine isolated from stage 54 tadpoles treated with 10 nM T3 for 3 days (p,q) were hybridized with antisense Shh (a,c,e,g,k,m,n,p) and Hip (b,d,f,h,l,q) probes. Some of the sections were stained by methyl green-pyronin Y (MGPY) to identify the adult primordia (dashed arrow in the panel o). To compare the localization of Shh with that of Hip and with the adult primordia, serial sections were used (compare c with d, e with f, k with l, n with o, and p with q). Dark blue deposits indicate the sites of probe binding. Light or dark brown pigments in e, f, g, and h are melanin. Arrowheads indicate the cells expressing Shh, while solid arrows indicate those expressing Hip. k-m: Higher magnification images of a boxed area in panels c, d and g, respectively. Shh and Hip mRNAs are hardly detectable at stages 54 (a,b). Signals for Shh become stronger at stage 61 (c), reach the maximal level at stage 62 (e) and then decrease at stage 66 (g). Shh mRNA is expressed only in the epithelium (Ep) but not in other tissues throughout metamorphosis (k and m). Particularly, at the climax, the cells expressing Shh correlate well with the adult primordia stained strongly red with MGPY (n and o). At stage 66 (m), signals for Shh are detectable in the trough region of the mature intestinal fold (right side) and the entire epithelium of the developing intestinal fold (left side). On the other hand, signals for Hip are detected at stage 61 (d) only in the connective tissue (CT) close to the muscle layer (M) but beneath Ep (l), which expresses Shh (k). At stage 62, signals for Hip decrease (f), and then become hardly detectable at stage 66 (h). Overall patterns of Shh (p) and Hip expression (q) induced by exogenous TH are essentially identical to those during natural metamorphosis. Sense Shh (i) and Hip probes (j) do not give any signal. L, lumen; Ty, typhlosole. Scale bars = 100 mu m in a-j, 20 mu m in k-q.

Figure 3. Comparison of hedgehog interacting protein (Hip) and bone morphogenetic protein (BMP) -4 localization. Cross-sections of the intestine at stage 61 (a-d) and stage 62 (e,f) were hybridized with antisense Hip (a,c,e) and BMP-4 (b,d,f) probes. To compare the localization of Hip with that of BMP-4, serial sections were used. Dark blue deposits indicate the sites of probe binding. Light or dark brown pigments are melanin. c,d: Higher magnification of a boxed area in a and b, respectively. c,d: The signals for BMP-4 mRNA (d, dashed arrow) are hardly detectable where Hip is strongly expressed (c, solid arrow). BMP-4 is highly expressed at stage 62 (f) when the expression of Hip decreases (e) and that of Shh peaks (Fig. 2c). Ep, epithelium; CT, connective tissue; M, muscle layer. Scale bars = 100 mu m in a,b,e,f, 20 mu m in c,d.

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