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First published online April 13, 2005
doi: 10.1242/10.1242/dev.01808

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COUP-TFII is essential for radial and anteroposterior patterning of the stomach

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
² Department of Cell Biology and Neurosciences, University of South Alabama, Mobile, AL 36688, USA
³ Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232,USA
⁴ Developmental Biological Program, Baylor College of Medicine, Houston, TX 77030, USA

View larger version (57K): [in a new window]   Fig. 1. Expression of COUP-TFII in the stomach using lacZ knock-in model. (A). Generation of the lacZ knock-in allele and generation of floxed COUP-TFII allele. Using homologous recombination, a targeting construct containing nuclear lacZ, Neo/TK and LoxP sites were inserted into the genomic COUP-TFII locus, generating a targeted allele in ES cells. Treatment with Cre recombinase and FIAU selection resulted in a lacZ knock-in allele in which lacZ gene expression was controlled by the endogenous COUP-TFII promoter when recombination took place between the first and the third loxP sites of the targeted allele. In addition, floxed COUP-TFII ES clones that retains COUP-TFII locus but lacks selection markers were generated when recombination took place between the second and the third loxP sites. B, BamHI; H, HindIII; S, SalI; X, XbaI. (B) Cryostat sections of E12.5 heterozygous COUP-TFII/lacZ knock-in embryo were stained (for 2 hours) for lacZ activity. There is relatively high expression in the mesenchymal cells just adjacent to the epithelium. (C). The stomach from a 3-day-old heterozygous knock-in animal was dissected and whole-mount X-gal staining was performed. The boundary between stomach and duodenum is indicated by arrowhead. (D) A cryostat section of stomach from adult heterozygous knock-in animal was stained for lacZ activity (blue) and counterstained with propidium iodide (red). DBA lectin immunostaining denotes the parietal cells (green). There is strong X-gal staining in the base layer and negligible staining in the surface pit layer of the adult Zymogenic unit. m, mesenchyme; e, epithelium; fs, fore-stomach; hs, hind-stomach; d, duodenum; oe, esophagus.

View larger version (74K): [in a new window]   Fig. 2. Ablation of COUP-TFII in the mesenchyme of the developing stomach using Nkx3-2Cre and COUP-TFII floxed mice. (A) Whole-mount X-gal staining of Nkx3-2Cre/+; COUP-TFIIflox/+ embryo, demonstrating the ablation of COUP-TFII by Nkx3-2Cre at E9.5 is shown. lacZ expression represents Cre-mediated recombination of floxed COUP-TFII. (B) The expression of Nkx3-2Cre at E9.5 was detected by whole-mount X-gal staining of Nkx3-2Cre/+; ROSA26R/+ embryo. X-gal staining represents Cre-mediated ablation of an interfering stop sequence in the ROSA26 reporter gene. Specific staining was observed in the developing stomach of both embryos. (C) A partially dissected Nkx3-2Cre/+; COUP-TFIIflox/+ embryo was stained by whole-mount X-gal staining, then paraffin embedded and serially sectioned. X-gal staining indicates Cre-mediated recombination and was detected throughout the gastric mesenchyme, but not in the epithelium, demonstrating the ablation of COUP-TFII in the gastric mesenchyme at E12.5. Scale bars: in B, 100 µm for A,B; in C, 100 µm for C.

View larger version (59K): [in a new window]   Fig. 3. Expansion of smooth muscle layers and enteric motoneurons in conditional mutant stomach. (A-D) Embryos were obtained by mating with COUP-TFII floxed homozygous males and Nkx3-2Cre/+; COUP-TFIIflox/+ females. COUP-TFII floxed homozygote served as controls, and Nkx3-2Cre/+; COUP-TFIIflox/flox were designated as conditional mutants. Histological analysis of stomach dissected from E12.5 controls (A) and conditional mutants (B) showed that the epithelium of the conditional mutant is thicker (marked by the black arrows) in Hematoxylin and Eosin stained sagittal sections. At E13.5, the smooth muscle layers of the mutant stomach are disorganized in comparison with the controls, as seen by -smooth muscle actin immunostaining (brown) (compare C with D). (E-H) -smooth muscle actin staining of sagittal sections of E16.5 stomach. Smooth muscle cells were immunoassayed for -smooth muscle actin (green), and counterstained with propidium iodide (red). White arrows indicate the extension of -smooth muscle actin staining in the submucosal mesenchyme of the conditional mutant (F). (E,G,H) The thickened circular smooth muscle layer formation was observed in both the fore-stomach (F) and the hind-stomach (H) of the conditional mutant in comparison with the control (E,G). (I,J) Semi-thin section semi-thin sections of the stomachs from E15.5 embryos were examined. The circular smooth muscle layer of the conditional mutant stomach (J) is disorganized in comparison with the control (I). (K,L) PGP9.5 staining of E16.5 stomach. Enteric neurons were stained by protein gene product 9.5 (PGP9.5) antiserum (brown) and counterstained with Hematoxylin. (M,N) TUJ1 staining of E13.5 stomach. Anti-TUJ1 antibody was employed in immunostaining. An increase of TUJ1-positive cells is shown in the conditional mutant (N) in comparison with the littermate control (M).

View larger version (103K): [in a new window]   Fig. 4. Analysis of AP patterning defects in conditional COUP-TFII mutant stomach. Controls and conditional mutant embryos were obtained by mating with COUP-TFII floxed homozygous males and Nkx3-2Cre/+; COUP-TFIIflox/+ females as described in Fig. 3. (A,B) Stomachs were dissected from E16.5 embryos and examined in whole mount under the dissecting microscope. The presumptive margin between fore-stomach and hind-stomach is indicated by dots and is shifted slightly anteriorly in the mutant. (C,D) Whole-mount postnatal day 28 stomachs were dissected and examined under the dissecting microscope. Position of limiting ridge is indicated by arrowhead. A clear anterior shift of the limited ridge is observed in the conditional mutant. (E,F) Dissected stomachs from E16.5 embryos were longitudinally sectioned and stained with Hematoxylin and Eosin. (G-H) Higher magnification of similar regions as indicated by lines in E,F. e, epithelium; sm, smooth muscle layer; fs, fore-stomach; hs, hind-stomach; oe, esophagus; d, duodenum.

View larger version (94K): [in a new window]   Fig. 5. Morphological changes in the posteriorized conditional mutant stomach: E16.5 whole embryos were sectioned sagittally and stained with Hematoxylin and Eosin for histological assessment. Corresponding regions in mutant and control were estimated by morphological characteristics of mesenchyme of each regions and relative location against other anatomical structures. (A,E) Duodenum. (B,F) Pyloric region. (C,G) Hind stomach. (D,H) Fore stomach. Oblique arrows across control and conditional mutant panels indicate changes of epithelial characteristics in the conditional mutants. e, epithelium; sm, smooth muscle layer.

View larger version (113K): [in a new window]   Fig. 6. Marker gene analyses confirm morphological changes in the conditional mutant stomach. (A,B) E16.5 sagittal sections were stained for stratified epithelial cell marker CK14. Intense staining was found in the basal cells in the control (A), while only faint staining was observed in the anterior edge of fore-stomach of the COUP-TFII conditional mutant (B). (C,D) Whole E11.5 embryos were cross-sectioned and Shh expressions in the fore-stomach were examined by section in situ hybridization using 35S-UTP labeled probes. Positive signals were pseudo-colored (red) and overlaid on bright-field image of Hematoxylin staining. (E-G) Paraffin sections of E18.5 stomachs were stained with anti-CK14 antibody. CK14 staining (dark brown) remained as intense throughout the fore-stomach of the control (E) and mutant (F). Again, it was barely detectable in the more caudal regions of the stomach of the mutant (G) in comparison with similar anatomical position of fore-stomach of the control (E).

View larger version (71K): [in a new window]   Fig. 7. (A,B) Dissected stomachs from E18.5 embryos were longitudinally sectioned and stained with Hematoxylin and Eosin. The thickness of the glandular epithelium is increased in the mutant stomach compared with the littermate control. (C-F) The adjacent tissue sections between fore-stomach and hind-stomach were stained by anti-H+/K+-ATPase ß-subunit antibody (C,D) and anti-GATA4 (E,F) antibodies. The transition zone between oxyntic mucosa (os) and stratified squamous epithelium (sse) of the fore-stomach are indicated by arrows. (G-T) Marker gene analyses in the adult stomachs: (G-L) Sections (4 µm) prepared from the stomachs of 35-day-old mice were staining with Hematoxylin and Eosin. The histological morphology of zymogenic region (G,H), mucoparietal zone (I,J) and pure mucus zone (K,L) showed no obvious difference in the conditional mutant stomach (H,J,L) and littermate control (G,I,K). (M,N) The secreted glycoprotein of the zymogenic zone of stomachs was visualized by PAS staining and no discernable difference was observed in the conditional mutant stomach (N) and littermate control (M). (O,P) The gastric parietal cells were stained with anti-H+/K+-ATPase ß-subunit antibody. (Q,R) The neck and pre-neck cells (red) at the gastric units showed no difference in the control (Q) and conditional mutant (R) stomachs. (S,T) Anti-GATA4 antibody was used to mark glandular gastric epithelium. Positive signals were found in base of glandular gastric epithelium at pure mucus zone in the control (S) and the mutant (T).

View larger version (92K): [in a new window]   Fig. 8. (A,B) Paraffin sections of E16.5 stomachs were stained for EAP activity. The bracket indicates corresponding regions of the caudal end of control and mutant stomach. oe, esophagus; Py, pylorus; d, duodenum. (C,D) The junction region of the caudal end of the stomach and the duodenum show slight morphological differences. The gastric parietal cells were stained with anti-H+/K+-ATPase ß-subunit antibody. (E,F) Anti-GATA4 antibody was used to mark glandular gastric epithelium. (G-J) Paraffin sections of E18.5 stomachs were stained for EAP activity. The bracket indicates corresponding regions of the caudal end of control and mutant stomach. Very low positive signals were found in glandular gastric epithelium of the control (G,I) while highly intense signals were seen in the mutant (H,J). High magnification of the boxed corresponding regions in the control (G) and mutant (H) were shown in I and J, respectively. Scale bar in B: 100 µm for A,B.

View larger version (86K): [in a new window]   Fig. 9. Effect of inhibition of Hh signaling in COUP-TFII expression. (A,B) Developing stomachs were dissected from E12.5 Shh wild-type and null mutant, and expression of COUP-TFII mRNA was detected by whole-mount in situ hybridization. Caudal margins of COUP-TFII expression are indicated by dots, and presumptive anatomical boundaries between stomach and duodenum are indicated by arrowheads. The relative size of fore-stomach is indicated by a white line at the top of the stomach. (C-F) Foregut explants were dissected from E10.5 knock-in embryos and cultured for 48 hours in increasing concentrations of cyclopamine (0-10 µM, D-F). Strong lacZ expression was detected in the mesenchyme between lung bud and fore-stomach, as indicated by arrowhead, and cyclopamine inhibited lacZ expression in a dose-dependent manner. Scale bars: in B, 0.5 mm for A,B; in F, 0.5 mm for C-F. oe, esophagus; fs, fore-stomach; hs, hind-stomach; dmg, dorsalmesogastrium; lb, lung bud; St, stomach; Py, pylorus; d, duodenum; e, hind-stomach epithelium.