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Stomach regional specification requires Hoxa5-driven mesenchymal-epithelial signaling

¹ Centre de recherche en cancérologie de l’Université Laval, Centre Hospitalier Universitaire de Québec, L’Hôtel-Dieu de Québec, Québec G1R 2J6, Canada
² Département d’Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Québec J1H 5N4, Canada

View larger version (79K): [in a new window]   Fig. 1. Hoxa5 expression in the developing stomach. In situ hybridization was performed on sections of E10.5 (A,B) and E12.5 (C,D) mouse embryos, and E15.5 (E), E17.5 (F-H) and P15 (I,J) stomachs. At E10.5, a widespread distribution of Hoxa5 transcripts was observed in the gastric mesenchyme (A,B). A gradient had formed by E12.5, with Hoxa5 being more expressed in the hindstomach (arrowheads; C,D). This gradient persisted at E15.5 (E). By E17.5, Hoxa5 transcripts were redistributed accompanying the formation of the infoldings (F-H). The signal became mainly confined to the submucosal cells underlying the epithelium and expression was observed in the muscular layer (G,H). Hoxa5 expression stopped around P15 (I,J). e, epithelium; f, forestomach; h, hindstomach; m, muscular layer; me, mesenchyme; s, submucosal layer. Scale bars: 100 µm.

View larger version (91K): [in a new window]   Fig. 2. Comparative histology of P15 wild-type and Hoxa5–/– gut specimens. Hematoxylin and Eosin stained sections of stomach (A,B), duodenum (C,D), jejunum (E,F), ileum (G,H), and proximal (I,J) and distal colon (K,L) from wild-type (A,C,E,G,I,K) and Hoxa5–/– (B,D,F,H,J,L) mice revealed a thinning of the gastric epithelium (B) and a reduction in villi length in the proximal colon (J) in Hoxa5–/– mutants. In both structures, the submucosa was hypertrophied. The duodenum (D), the jejunum (F), the ileum (H) and the distal colon (L) appeared morphologically normal. e, epithelium; m, muscular layer; s, submucosa. Scale bar: 100 µm.

View larger version (60K): [in a new window]   Fig. 3. Functional analysis of wild-type and Hoxa5–/– stomachs. (A) Postnatal ontogeny of pepsin activity in the stomach of wild-type (circles) and Hoxa5–/– (squares) mice at different time points. Pepsin activity in Hoxa5–/– mutants remained statistically lower (black squares) at P17 and P30 compared with wild-type samples. IF immunostaining (B,C) and silver staining (D,E) showed that reduced enzymatic activity correlated with a decrease in zymogenic cells (arrowheads) and enteroendocrine cells (arrows) in Hoxa5–/– mutants (C,E) compared with wild-type specimens (B,D). Some gastric units were deprived of zymogenic cells (asterisk). Scale bar: 100 µm.

View larger version (71K): [in a new window]   Fig. 4. Comparative histology of P15 stomach (A-L) and P30 proximal colon (M,N) of wild-type (A,C,E,G,I,K,M) and Hoxa5–/– mutants (B,D,F,H,J,L,N). Sections from zymogenic zone of the stomach were stained for representation of cell lineages: Hematoxylin and Eosin (A,B), Periodic acid/Schiff (C,D; mucus cells), and silver staining (G,H; enteroendocrine cells). IF immunostaining detected zymogenic cells (E,F). Proliferating cells were revealed by immunostaining with a pH3 antibody (I,J), and apoptotic cells by the TUNEL method (K,L). Hoxa5–/– stomach was characterized by a thinner epithelial layer and an hypertrophied submucosal layer (A,B), more mucus producing cells (C,D; arrowheads), an altered distribution of zymogenic cells along the gastric unit (E,F; arrows), and a decreased number of enteroendocrine cells (G,H; arrows). Proliferation in the isthmus (I,J; arrows) and apoptosis (K,L; arrows) were both reduced. In the proximal colon, abnormal distribution of goblet cells was noted in the Hoxa5–/– epithelium, as revealed by Alcian Blue staining (M,N; arrowheads). Scale bars: 100 µm.

View larger version (88K): [in a new window]   Fig. 5. Morphological differences during stomach development between wild type (A,C,E,G,I) and Hoxa5–/– mutants (B,D,F,H,J). Arrows delineate the prospective squamous (forestomach) and glandular (hindstomach) portions of the stomach. At E13.5 (A-D), a slight disorganization in the gastric submucosa was observed in mutants. By E15.5 (E,F), the reduced cellular density of Hoxa5–/– mesenchymal layer became obvious, as shown also for E17.5 (G,H). At E17.5, formation of foldings initiated properly but they were reduced in length at birth (I,J). f, forestomach; h, hindstomach. Scale bars: 100 µm.

View larger version (84K): [in a new window]   Fig. 6. Intestinal characteristics displayed by the Hoxa5–/– gastric mucosa. Alkaline phosphatase activity was tested on stomach sections from E18.5 wild-type (A) and Hoxa5–/– (B,C) fetuses. In wild-type specimens, a faint enzymatic activity was present in the intestine and in the most distal part of the hindstomach (A, arrowheads). By contrast, in Hoxa5–/– mutants, higher levels of reactivity that extended towards the forestomach were detected (B,C, arrows). d, duodenum. Scale bar: 100 µm.

View larger version (167K): [in a new window]   Fig. 7. Comparative expression pattern of signaling molecules in wild-type (A,C,E,G,I,K,M,O,Q,S) and Hoxa5–/– (B,D,F,H,J,L,N,P,R,T) E12.5 stomachs. Sagittal sections were oriented with hindstomach and forestomach from left to right. Arrowheads indicate the limits of the domain of high expression when appropriate. Shh (A,B) and Ihh (C,D) displayed reciprocal expression gradients in the gastric epithelium. They were expressed in the fore- and hindstomach, respectively. Compared with wild-type samples, high expression of Shh was more restricted in Hoxa5–/– stomachs. In contrast, Ihh expression extended more in the forestomach. Expression of Hh receptor Ptc (E,F) and its downstream effector Gli (G,H) was enhanced in Hoxa5–/– stomachs. Fgf10 transcripts were confined to the mesenchyme of the hindstomach in wild-type samples (I), while in Hoxa5–/– mutants (J), they spread into the forestomach. Bmp4 expression (K,L) was reduced particularly in the hindstomach of mutants. In controls, Tgfb1 expression was confined to the peri-epithelial zone of the stomach mesenchyme (M, inset). In mutants, Tgfb1 expression was more disseminated throughout the mesenchyme (N, inset). A gain of Tgfb3 expression was observed in the mutant mesenchyme (O,P). In contrast, Barx1 expression decreased in the mutants (Q,R). Nkx2.5 expression in the pylorus region was unaffected by the lack of Hoxa5 function (S,T, arrows). d, duodenum; e, esophagus. Scale bar: 100 µm.

View larger version (65K): [in a new window]   Fig. 8. Impact of the loss of Hoxa5 function on signaling pathways involved in stomach morphogenesis. The biological effect of FGF10-impregnated beads was tested on cultured embryonic stomach explants. Ihh expression was stimulated by FGF10 when the bead was implanted in the hindstomach (B), whereas the control bead had no effect (A). In contrast, FGF10-soaked beads were unable to induce Ihh expression when positioned in the forestomach (C), as shown after overexposure of the section. (D) Representation of the stomach mesenchyme (green) and endoderm (yellow) with the associated genes expressed during its ontogenesis. The gradient and domain of expression of Ihh, Shh, Fgf10 and Tgfb1 in the wt stomach as well as the changes in their expression pattern in Hoxa5–/– mutants are schematized. The lower panel represents a model of presumptive interactions between these signaling molecules in the developing stomach. Hypothetical links are indicated by dashed arrows. Shh and Ihh delimitate functional domains in the gastric endoderm. Fgf10 contributes to establish Ihh expression in the hindstomach. In absence of Hoxa5 function, Fgf10 expression domain extends rostrally while Tgfb1 is no longer restricted. Consequently, Ihh expression domain expands toward the forestomach, while that of Shh regresses. b, bead; d, duodenum; e, esophagus; f, forestomach; h, hindstomach. Scale bar: 100 µm.