Ishizuya-Oka A et al. (2001), Thyroid hormone-induced expression of sonic hed...

XB-ART-7869

Differentiation 2001 Dec 01;691:27-37.

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Thyroid hormone-induced expression of sonic hedgehog correlates with adult epithelial development during remodeling of the Xenopus stomach and intestine.

Ishizuya-Oka A , Ueda S , Inokuchi T , Amano T , Damjanovski S , Stolow M , Shi YB .

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Sonic hedgehog (Shh) was isolated from the Xenopus laevis intestine as an early thyroid hormone (TH) response gene. To investigate possible roles of TH-upregulated expression of Shh during metamorphosis, we raised a polyclonal antibody against Xenopus Shh and immunohistochemically examined the relationship between Shh expression and the larval-to-adult intestinal remodeling at the cellular level. Our results indicate that the epithelial-specific expression of Shh in the intestine spatiotemporally correlates well with active proliferation and/or initial differentiation of the secondary (adult) epithelial primordia that originate from stem cells, but not with apoptosis of the primary (larval) epithelium. Given the similar transformations of the stomach during metamorphosis, we also analyzed Shh expression in this organ and found similar correlations in the stomach, although the position of the adult epithelial primordia and their final differentiation in the stomach are different from those in the intestine. Furthermore, we show here that Shh expression is organ-autonomously induced by TH and its correlation with the adult epithelial development is reproduced in vitro in both the intestine and the stomach. More importantly, addition of recombinant Shh protein to the culture medium results in developmental anomalies of both organs. However, differentiation of the adult epithelium is more severely inhibited by exogenous Shh in the intestine than in the stomach. These results suggest that TH-upregulated expression of Shh plays important roles in the postembryonic gastrointestinal remodeling, but its roles are at least partially different between the intestine and the stomach.

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Species referenced: Xenopus laevis
Genes referenced: fabp2 pcna shh

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	Fig. 1 Anti-Xenopus Shh antiserum specifically recognizes Shh proteins. The full-length Shh (Shh), its N-terminal fragment (N-Shh), or C-terminal fragment (C-Shh) were made in the presence of [35S] methionine (35S-Met) and were electrophoresed on SDS-protein gels. The gels were either autoradiographed directly (A) or subjected to Western blot analysis with the rabbit anti-Xenopus Shh antiserum (B). Note that all of Shh, N-Shh, and C-Shh (asterisks) were recognized by the antibody.
	Fig. 2 Shh is expressed in crude extract of the X. laevis stomach (lanes 2, 4) and small intestine (lanes 3, 5) at stage 60. The amount of protein applied to each lane was 20 mg. Molecular weight markers are indicated on the left (lane 1). Two immunoreactive bands (arrowheads), corresponding to full length and N-terminal half of the processed Shh, were clearly detected in the stomach. *Ω nonspecific bands.
	Fig. 3 A–I Shh expression correlates spatiotemporally with larval-to-adult remodeling of the Xenopus small intestine during metamorphosis. Crosssections were immunostained with Shh (A–F, H) and IFABP (G, I) antibodies. No immunoreactivity for Shh is detected in the larval intestine at stage 56 (A). Then only the larval epithelium (LE) facing the lumen (L) becomes positive for Shh at stage 59 (B). At early (C) and late (D) stage 60, primordia of the adult epithelium show a positive staining for Shh (arrowheads). The adult epithelial primordia (AE) remain positive for Shh, while the larval epithelium becomes almost negative at stage 61 (E). Thereafter, at stage 63, the Shh immunoreactivity in the adult epithelium rapidly decreases in intensity from the crest to the trough of newly formed intestinal folds (Fo) (F). In contrast, the immunoreactivity for IFABP rapidly increases in intensity from the crest to the trough of the folds (G). At the completion of metamorphosis (stage 66), the adult epithelium becomes negative for Shh (H) and is differentiated into the absorptive epithelium positive for IFABP (I). CTΩconnective tissue; MLΩmuscular layer; Bars: 20 mm.
	Fig. 4 A–I Development of the adult epithelium (AE) both in vivo and in vitro, stained with methyl green-pyronin Y. A–D Small intestine. Primordia of the adult epithelium appear as small islets between the larval epithelium (LE) and the connective tissue (CT) early stage 60 (A, arrows) and then rapidly grow in size (B). At stage 61 the adult epithelial primordia rapidly replace the degener- ating larval epithelium (C). At stage 63 the adult epithelium com- pletely replaces the larval epithelium and forms intestinal folds (Fo) (D). E–G Stomach. Primordia of the adult epithelium are localized in the basal region of larval glands (L-GE) at stage 60 (E). At stage 61 the adult epithelial primordia rapidly replace the degenerating larval epithelium (F). Thereafter, by stage 63, the adult epithelium completely replaces the larval epithelium and differenatiates into the surface (A-SE) and glandular epithelia (A-GE) (G). H, I Primordia of the adult epithelium in the intestine (H) and the stomach (I) organotypically cultured for 5 days in the presence of TH. L-SEΩlarval surface epithelium; MLΩmuscular layer; Bars: 20 mm.
	Fig. 5 A–D Primordia of the adult epithelium (AE) express Shh and actively proliferate in the intestine at early stage 61. (A, B) Adjacent sections stained with methyl green-pyronin Y (A) and immunostained with Shh antibody (B). A strong Shh immunoreac-tivity is localized in adult epithelial primordia but not in the larval epithelium (LE). (C, D) Adjacent sections immunostained with PCNA (C) and Shh (D) in the epithelium express Shh. Bars: 20 mm.antibodies. The actively proliferating cells
	Fig. 6 A–I Shh expression correlates spatiotemporally with larval-to-adult remodeling of the Xenopus stomach during metamorphosis. Cross-sections were immunostained with Shh (A–F, H) and Pg (G, I) antibodies. No immunoreactivity for Shh is detected in the larval stomach at stage 57 (A). Then only the larval surface epithelium (L-SE) becomes positive at stage 59 (B). At stage 60 adult epithelial primordia (AE) localized in the basal region of larval glands (L-GE) become strongly positive for Shh (C, arrowheads). Their immunoreactivity is stronger than that in the other region of the epithelium (D). Both the connective tissue (CT) and the muscular layer (ML) remain almost negative. At stage 61 the adult epithelial primordia remain strongly positive for Shh, while the larval epithelium (LE) becomes almost negative (E). At stage 63 the Shh immunoreactivity rapidly decreases in intensity in both the adult surface (A-SE) and glandular epithelia (A-GE) and is only weakly detected in the neck region of the adult glands (F, arrowheads). In contrast, the adult glands become positive for Pg (G). At stage 66 the adult epithelium is negative for Shh (H) and is fully differentiated into the surface mucous epithelium and the glandular epithelium positive for Pg (I). LΩlumen; Bars: 20 mm.
	Fig. 7 A–L Shh expression correlates with epithelial development and its addition to the medium caused developmental anomalies in vitro. The intestine (A, C, E, G, I–K) and the stomach (B, D, F, H, L) organotypically cultured in the presence (A–F, I–L) and absence (G, H) of TH. Cross-sections were immunostained with Shh (A–D, G, H), IFABP (E), Pg (F), and PCNA (I, J) antibodies, and stained with hematoxylin and PAS (K, L). A, B Day 5 in the presence of TH. Primordia of the adult epithelium (AE) are strongly positive for Shh. C–F Day 7 in the presence of TH. The adult epithelium becomes almost negative for Shh in both organs (C, D). The adult epithelium in the intestine is differentiated into the absorptive epithelium positive for IFABP in the intestine (E), whereas that in the stomach is differentiated into the surface epithelium (A-SE) and the glandular epithelium (AGE) positive for Pg (F). G, H Day 5 in the control medium deprived of TH. The larval epithelium (LE) remains intact and almost negative for Shh in both organs. I, J Day 5 in the presence of TH. Proliferating cells are mostly localized in the epithelium (arrowheads) in the absence of exogenous Shh (I), but are easily detectable in the connective tissue (arrows) when 0.5 mg/ml N-Shh protein is added to the medium (J). K, L Day 7 in the presence of both TH and 0.5 mg/ml NShh. The epithelium (E) has no lumen and fails to form a simple layer in the intestine (K). In contrast, in the stomach (L), the epithelium is differentiated into the PAS-positive surface mucous epithelium (arrow). L-SEΩlarval surface epithelium; L-GEΩlarval glandular epithelium; CTΩ connective tissue; Bars: 20 mm.
	Fig. 8 Addition of N-Shh protein to the medium changes the proliferating activity of intestinal tissues in vitro. Labeling indices of proliferating cells in the epithelium (hatched bars) and the nonepithelial tissues (filled bars) were measured after 5 days of treatment with 0, 0.5, or 5.0 mg/ml N-Shh. Each value represents the mean π SD of more than six explants. N-Shh significantly promotes cell proliferation both in the epithelium and in nonepithelial tissues in the absence of TH, but it does so only in the nonepithelial tissues in its presence. *P0.001 compared with control explants (0 mg/ml N-Shh) P 0.002 compared with control explants (0 mg/ml N-Shh)