Volume 116, Issue 6, Pages 1367-1378 (June 1999) Involvement of T3Rα- and β-receptor subtypes in mediation of T3 functions during postnatal murine intestinal development  Michelina Plateroti, Olivier Chassande, Alexandre Fraichard, Karine Gauthier, Jean–Noël Freund, Jacques Samarut, Michèle Kedinger  Gastroenterology  Volume 116, Issue 6, Pages 1367-1378 (June 1999) DOI: 10.1016/S0016-5085(99)70501-9 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of T3Rα1, T3Rα2, and T3Rβ in the gut. Longitudinal expression of the three transcripts in the different intestinal tissue layers of 12-day-old wild-type mice analyzed by semiquantitative RT-PCR. 36-B4 was used as internal control. Standard molecular masses for DNA size are in the first lane. E, epithelium; LP, lamina propria; M, muscle; PJ, proximal jejunum; DI, distal ileum; PC, proximal colon. Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Morphological features of (A and B) proximal jejunum and (C and D) distal ileum in 21-day-old (A and C) homozygous T3Rα−/− and (B and D) wild-type animals. Paraffin-embedded sections were stained with periodic acid–Schiff. v, villi; c, crypts; ml, muscle layers. Bars = 50 μm. Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunofluorescence detection of proliferating crypt epithelial cells 30 minutes after BrdU injection and staining of cryosections with anti-BrdU antibody. Samples were from 21-day-old (A) homozygous T3Rα knockout and (B) wild-type mice at the distal small intestinal level. Bars = 30 μm. Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Semithin and ultrathin sections of the distal small intestine from 21-day-old (A and C) homozygous T3Rα knockout and (B and D) wild-type mice. In the T3Rα knockout intestine, the intercellular spaces are dilated (arrow) and there is an accumulation of supranuclear vacuoles (*). Observations of the lamina propria and muscle cells did not show any obvious difference from those of control intestines. bb, brush borders. Bars = 20 μm (A and B); bars = 4 μm (C and D). Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Expression of digestive enzymes. (A) Analysis of lactase, sucrase, and aminopeptidase brush border activities (mU/mg brush border proteins; mean values ± SD; n = 3-5) at the proximal jejunum level in 2- and 3-week-old T3Rα knockout animals (2 w, 3 w); enzyme activities are represented as percentages of respective control levels. Statistical analysis of the differences between T3Rα−/− animals and their respective controls for each enzymatic activity was performed using the Student t test. *P < 0.05; **P < 0.001. (B and C) Histoenzymological staining of lactase in 3-week-old (B) homozygous T3Rα−/− and (C) wild-type animals. (D) Representative results of RT-PCR analysis of lactase and sucrase mRNA expression along the P-D axis (proximal jejunum [PJ], distal ileum [DI], proximal part of the colon [PC]) in 3-week-old T3Rα-deficient animals and in their controls. 36-B4 was used as internal control. Analysis of I-FABP mRNA shows that the effects resulting from inhibition of TR3α expression are not general to all intestinal epithelial cell markers. Note that the overall P-D expression patterns are maintained in the experimental animals compared with those of controls. Bars in B and C = 50 μm. Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Morphological analysis of (A, C, E, and G) proximal jejunum and (B, D, F, H, and I) distal ileum in 2-week-old (A and B) wild-type, (C and D) T3Rα−/−, (E and F) T3Rβ−/−, and (G-I) T3Rαβ−/− mice. Paraffin-embedded sections were stained with periodic acid–Shiff. Bars = 100 μm (A-H); bar = 40 μm (I). Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Representative results of RT-PCR analysis of lactase and Cdx-1 and Cdx-2 mRNAs along the P-D axis (a, proximal jejunum; b, distal ileum; c, proximal part of the colon) in 2-week-old T3Rα, T3Rβ, and T3Rαβ-deficient mice and in control animals (+/+). 36-B4 was used as internal control. Standard molecular masses for DNA size are in the first lane. (B and C) Histoenzymological staining of lactase at the proximal jejunum level in 2-week-old (B) homozygous T3Rβ−/− and (C) double knockout animals. T3Rβ−/− intestines showed staining for lactase similar to that of the control (not shown). Bars= 50 μm. Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Morphological and functional study of the intestinal mucosa in 2-month-old homozygous T3Rα−/− T3-injected and wild-type mice. (A and B) H&E staining of paraffin-embedded sections of (A) T3Rα-deficient and (B) control mice at the distal small intestinal level of the gut. Bars = 50 μm. (C) Analysis of lactase (■), sucrase (▩), and aminopeptidase (2) brush border activities (mU/mg brush border proteins; means ± SD; n = 3) at the proximal jejunum level; enzyme activities are expressed as percentages of respective control values. (D and E) Densitometric analysis of (D) Cdx-1 and (E) Cdx-2 signals after RT-PCR analysis. ■, Proximal jejunum; ▩, distal ileum; 2, proximal colon.The level of the two transcripts was compared in 3-week-old T3Rα knockout animals (−/− 3w) and in 2-month-old T3-injected knockout mice (−/− T3 inj 2m) with their respective controls of the same age (+/+ 3w and +/+ 2m). The relative amounts of Cdx-1 and Cdx-2 mRNAs along the P-D axis of the gut were unchanged in control intestines between 3 weeks and 2 months after birth. The specific bands (from two independent experiments) were scanned and normalized to the value of the 36-B4 internal control. Statistical analysis was performed using the Student t test. *P < 0.05; **P < 0.001. Gastroenterology 1999 116, 1367-1378DOI: (10.1016/S0016-5085(99)70501-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions